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1
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Dash S, Ueda T, Komuro A, Honda M, Sugisawa R, Okada H. Deoxycytidine kinase inactivation enhances gemcitabine resistance and sensitizes mitochondrial metabolism interference in pancreatic cancer. Cell Death Dis 2024; 15:131. [PMID: 38346958 PMCID: PMC10861559 DOI: 10.1038/s41419-024-06531-x] [Citation(s) in RCA: 8] [Impact Index Per Article: 8.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/10/2023] [Revised: 01/28/2024] [Accepted: 02/01/2024] [Indexed: 02/15/2024]
Abstract
Pancreatic ductal adenocarcinoma (PDAC) is considered one of the most lethal forms of cancer. Although in the last decade, an increase in 5-year patient survival has been observed, the mortality rate remains high. As a first-line treatment for PDAC, gemcitabine alone or in combination (gemcitabine plus paclitaxel) has been used; however, drug resistance to this regimen is a growing issue. In our previous study, we reported MYC/glutamine dependency as a therapeutic target in gemcitabine-resistant PDAC secondary to deoxycytidine kinase (DCK) inactivation. Moreover, enrichment of oxidative phosphorylation (OXPHOS)-associated genes was a common property shared by PDAC cell lines, and patient clinical samples coupled with low DCK expression was also demonstrated, which implicates DCK in cancer metabolism. In this article, we reveal that the expression of most genes encoding mitochondrial complexes is remarkably upregulated in PDAC patients with low DCK expression. The DCK-knockout (DCK KO) CFPAC-1 PDAC cell line model reiterated this observation. Particularly, OXPHOS was functionally enhanced in DCK KO cells as shown by a higher oxygen consumption rate and mitochondrial ATP production. Electron microscopic observations revealed abnormal mitochondrial morphology in DCK KO cells. Furthermore, DCK inactivation exhibited reactive oxygen species (ROS) reduction accompanied with ROS-scavenging gene activation, such as SOD1 and SOD2. SOD2 inhibition in DCK KO cells clearly induced cell growth suppression. In combination with increased anti-apoptotic gene BCL2 expression in DCK KO cells, we finally reveal that venetoclax and a mitochondrial complex I inhibitor are therapeutically efficacious for DCK-inactivated CFPAC-1 cells in in vitro and xenograft models. Hence, our work provides insight into inhibition of mitochondrial metabolism as a novel therapeutic approach to overcome DCK inactivation-mediated gemcitabine resistance in PDAC patient treatment.
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Affiliation(s)
- Suman Dash
- Department of Biochemistry, Kindai University Faculty of Medicine, Osakasayama, Osaka, 589-8511, Japan
- Graduate School of Medical Sciences, Kindai University Faculty of Medicine, Osakasayama, Osaka, 589-8511, Japan
| | - Takeshi Ueda
- Department of Biochemistry, Kindai University Faculty of Medicine, Osakasayama, Osaka, 589-8511, Japan
- Graduate School of Medical Sciences, Kindai University Faculty of Medicine, Osakasayama, Osaka, 589-8511, Japan
| | - Akiyoshi Komuro
- Department of Biochemistry, Kindai University Faculty of Medicine, Osakasayama, Osaka, 589-8511, Japan
| | - Masahiko Honda
- Department of Biochemistry, Kindai University Faculty of Medicine, Osakasayama, Osaka, 589-8511, Japan
| | - Ryoichi Sugisawa
- Department of Biochemistry, Kindai University Faculty of Medicine, Osakasayama, Osaka, 589-8511, Japan
| | - Hitoshi Okada
- Department of Biochemistry, Kindai University Faculty of Medicine, Osakasayama, Osaka, 589-8511, Japan.
- Graduate School of Medical Sciences, Kindai University Faculty of Medicine, Osakasayama, Osaka, 589-8511, Japan.
- Anti-aging Center, Kindai University, Higashi-Osaka, Osaka, 577-8502, Japan.
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2
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Yoshida-Sakai N, Watanabe T, Yamamoto Y, Ureshino H, Kamachi K, Kurahashi Y, Fukuda-Kurahashi Y, Kimura S. Adult T-cell leukemia-lymphoma acquires resistance to DNA demethylating agents through dysregulation of enzymes involved in pyrimidine metabolism. Int J Cancer 2021; 150:1184-1197. [PMID: 34913485 PMCID: PMC9303000 DOI: 10.1002/ijc.33901] [Citation(s) in RCA: 8] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/12/2021] [Revised: 11/22/2021] [Accepted: 11/29/2021] [Indexed: 01/05/2023]
Abstract
Adult T-cell leukemia-lymphoma (ATL) is an aggressive neoplasm derived from T-cells transformed by human T-cell lymphotropic virus-1 (HTLV-1). Recently, we reported that regional DNA hypermethylation in HTLV-1-infected T-cells reflects the disease status of ATL and the anti-ATL effects of DNA demethylating agents, including azacitidine (AZA), decitabine (DAC) and a new DAC prodrug, OR-2100 (OR21), which we developed. Here, to better understand the mechanisms underlying drug resistance, we generated AZA-, DAC- and OR21-resistant (AZA-R, DAC-R and OR21-R, respectively) cells from the ATL cell line TL-Om1 and the HTLV-1-infected cell line MT-2 via long-term drug exposure. The efficacy of OR21 was almost the same as that of DAC, indicating that the pharmacodynamics of OR21 were due to release of DAC from OR21. Resistant cells did not show cellular responses observed in parental cells induced by treatment with drugs, including growth suppression, depletion of DNA methyltransferase DNMT1 and DNA hypomethylation. We also found that reduced expression of deoxycytidine kinase (DCK) correlated with lower susceptibility to DAC/OR21 and that reduced expression of uridine cytidine kinase2 (UCK2) correlated with reduced susceptibility to AZA. DCK and UCK2 catalyze phosphorylation of DAC and AZA, respectively; reconstitution of expression reversed the resistant phenotypes. A large homozygous deletion in DCK and a homozygous splice donor site mutation in UCK2 were identified in DAC-R TL-Om1 and AZA-R TL-Om1, respectively. Both genomic mutations might lead to loss of protein expression. Thus, inactivation of UCK2 and DCK might be a putative cause of phenotypes that are resistant to AZA and DAC/OR21, respectively.
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Affiliation(s)
- Nao Yoshida-Sakai
- Department of Drug Discovery and Biomedical Sciences, Faculty of Medicine, Saga University, Saga, Japan.,Division of Hematology, Respiratory Medicine and Oncology, Department of Internal Medicine, Faculty of Medicine, Saga University, Saga, Japan
| | - Tatsuro Watanabe
- Department of Drug Discovery and Biomedical Sciences, Faculty of Medicine, Saga University, Saga, Japan
| | - Yuta Yamamoto
- Department of Drug Discovery and Biomedical Sciences, Faculty of Medicine, Saga University, Saga, Japan
| | - Hiroshi Ureshino
- Department of Drug Discovery and Biomedical Sciences, Faculty of Medicine, Saga University, Saga, Japan.,Division of Hematology, Respiratory Medicine and Oncology, Department of Internal Medicine, Faculty of Medicine, Saga University, Saga, Japan
| | - Kazuharu Kamachi
- Department of Drug Discovery and Biomedical Sciences, Faculty of Medicine, Saga University, Saga, Japan.,Division of Hematology, Respiratory Medicine and Oncology, Department of Internal Medicine, Faculty of Medicine, Saga University, Saga, Japan
| | - Yuki Kurahashi
- Department of Drug Discovery and Biomedical Sciences, Faculty of Medicine, Saga University, Saga, Japan.,Division of Hematology, Respiratory Medicine and Oncology, Department of Internal Medicine, Faculty of Medicine, Saga University, Saga, Japan.,OHARA Pharmaceutical Co, Ltd, Tokyo, Japan
| | - Yuki Fukuda-Kurahashi
- Department of Drug Discovery and Biomedical Sciences, Faculty of Medicine, Saga University, Saga, Japan.,OHARA Pharmaceutical Co, Ltd, Tokyo, Japan
| | - Shinya Kimura
- Department of Drug Discovery and Biomedical Sciences, Faculty of Medicine, Saga University, Saga, Japan.,Division of Hematology, Respiratory Medicine and Oncology, Department of Internal Medicine, Faculty of Medicine, Saga University, Saga, Japan
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3
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Slusarczyk M, Serpi M, Ghazaly E, Kariuki BM, McGuigan C, Pepper C. Single Diastereomers of the Clinical Anticancer ProTide Agents NUC-1031 and NUC-3373 Preferentially Target Cancer Stem Cells In Vitro. J Med Chem 2021; 64:8179-8193. [PMID: 34085825 DOI: 10.1021/acs.jmedchem.0c02194] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/28/2022]
Abstract
A 3'-protected route toward the synthesis of the diastereomers of clinically active ProTides, NUC-1031 and NUC-3373, is described. The in vitro cytotoxic activities of the individual diastereomers were found to be similar to their diastereomeric mixtures. In the KG1a cell line, NUC-1031 and NUC-3373 have preferential cytotoxic effects on leukemic stem cells (LSCs). These effects were not diastereomer-specific and were not observed with the parental nucleoside analogues gemcitabine and FUDR, respectively. In addition, NUC-1031 preferentially targeted LSCs in primary AML samples and cancer stem cells in the prostate cancer cell line, LNCaP. Although the mechanism for this remains incompletely resolved, NUC-1031-treated cells showed increased levels of triphosphate in both LSC and bulk tumor fractions. As ProTides are not dependent on nucleoside transporters, it seems possible that the LSC targeting observed with ProTides may be caused, at least in part, by preferential accumulation of metabolized nucleos(t)ide analogues.
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Affiliation(s)
- Magdalena Slusarczyk
- Cardiff School of Pharmacy and Pharmaceutical Sciences, Cardiff University, King Edward VII Avenue, Redwood Building, Cardiff CF10 3NB, U.K
| | - Michaela Serpi
- Cardiff University, School of Chemistry, Main Building, Park Place, Cardiff CF10 3AT, U.K
| | - Essam Ghazaly
- Centre for Haemato-Oncology, Barts Cancer Institute, Queen Mary University of London, Charterhouse Square, London EC1M 6BQ, U.K
| | - Benson M Kariuki
- Cardiff University, School of Chemistry, Main Building, Park Place, Cardiff CF10 3AT, U.K
| | - Christopher McGuigan
- Cardiff School of Pharmacy and Pharmaceutical Sciences, Cardiff University, King Edward VII Avenue, Redwood Building, Cardiff CF10 3NB, U.K
| | - Chris Pepper
- Brighton and Sussex Medical School, University of Sussex, Medical Teaching Building, Brighton BN1 9PX, U.K
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4
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Fortuna A, Costa PJ, Piedade MFM, Conceição Oliveira M, Xavier NM. Synthesis of Triazole-Containing Furanosyl Nucleoside Analogues and Their Phosphate, Phosphoramidate or Phoshonate Derivatives as Potential Sugar Diphosphate or Nucleotide Mimetics. Chempluschem 2020; 85:1676-1691. [PMID: 32757384 DOI: 10.1002/cplu.202000424] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/30/2020] [Revised: 07/16/2020] [Indexed: 12/21/2022]
Abstract
The synthesis of stable and potentially bioactive xylofuranosyl nucleoside analogues and potential sugar diphosphate or nucleotide mimetics comprising a 1,2,3-triazole moiety is reported. 3'-O-Methyl-branched N-benzyltriazole isonucleosides were accessed in 5-7 steps and 42-54 % overall yields using a Cu(I)-catalyzed cycloaddition of 3-O-propargyl-1,2-O-isopropylidene-α-D-xylofuranose with benzyl azide as key step. Related isonucleotides were obtained by 5-O-phosphorylation of acetonide-protected 3-O-propargyl xylofuranose and further "click" cycloaddition or by Staudinger-phosphite reaction of a 5-azido N-benzyltriazole isonucleoside. Hydroxy-, amino- or bromomethyl triazole 5'-isonucleosides were synthesized by thermal cycloaddition of 5-azido 3-O-benzyl/dodecyl xylofuranoses with propargyl alcohol, propargylamine or propargyl bromide. Better yields (82-85 %) were obtained when using propargyl alcohol and a high 1,4-regioselectivity was attained with propargyl bromide. Further O/N-phosphorylation or Arbuzov reaction led to (triazolyl)methyl phosphates, phosphoramidates or phosphonates. The latter were converted into uracil nucleoside 5'-(triazolyl)methyl phosphonates as prospective nucleoside diphosphate mimetics.
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Affiliation(s)
- Andreia Fortuna
- Centro de Química Estrutural, Faculdade de Ciências, Universidade de Lisboa, Ed. C8, 5° Piso, Campo Grande, 1749-016, Lisboa, Portugal.,University of Lisboa, Faculty of Sciences, BioISI - Biosystems & Integrative Sciences Institute, Campo Grande, C8 bdg, 1749-016, Lisboa, Portugal
| | - Paulo J Costa
- University of Lisboa, Faculty of Sciences, BioISI - Biosystems & Integrative Sciences Institute, Campo Grande, C8 bdg, 1749-016, Lisboa, Portugal
| | - M Fátima M Piedade
- Centro de Química Estrutural, Faculdade de Ciências, Universidade de Lisboa, Ed. C8, 5° Piso, Campo Grande, 1749-016, Lisboa, Portugal.,Centro de Química Estrutural, Instituto Superior Técnico, Universidade de Lisboa, Av. Rovisco Pais, 1049-001, Lisboa, Portugal
| | - M Conceição Oliveira
- Centro de Química Estrutural, Instituto Superior Técnico, Universidade de Lisboa, Av. Rovisco Pais, 1049-001, Lisboa, Portugal
| | - Nuno M Xavier
- Centro de Química Estrutural, Faculdade de Ciências, Universidade de Lisboa, Ed. C8, 5° Piso, Campo Grande, 1749-016, Lisboa, Portugal
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5
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Lepeltier E, Rijo P, Rizzolio F, Popovtzer R, Petrikaite V, Assaraf YG, Passirani C. Nanomedicine to target multidrug resistant tumors. Drug Resist Updat 2020; 52:100704. [PMID: 32512316 DOI: 10.1016/j.drup.2020.100704] [Citation(s) in RCA: 84] [Impact Index Per Article: 16.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/02/2019] [Revised: 12/21/2019] [Accepted: 05/13/2020] [Indexed: 12/12/2022]
Abstract
Nanomedicine employs nanotechnologies to develop innovative applications, and more specifically nano-objects in the field of human health, through exploitation of the physical, chemical and biological properties of materials at the nanoscale. The use of nanovehicles capable of transporting and releasing the active therapeutic payload into target cells, particularly in the case of cancer or inflammatory diseases, can also enhance diagnosis. Therefore, nanomedicines improve the benefit/risk ratio of drugs by increasing their bioavailability, selectivity, and efficacy in the target tissue, while reducing the necessary doses and hence diminishing untoward toxicity to healthy tissues. Overcoming multidrug resistance (MDR) to antitumor agents is a central goal of cancer research and therapeutics, making it possible to treat these diseases more accurately and effectively. The adaptability of nanomedicines e.g. modulation of their components, surface functionalization, encapsulation of various active therapeutics as well as the possibility of combining several treatments using a single nanoparticle platform, are characteristics which are perfectly poised to address classical chemoresistance, a major obstacle towards curative cancer therapy. In this review, we discuss an assortment of nanomedicines along with those that should be developed in order to surmount cancer MDR; these include exosomes, natural compounds, lipid nanocapsules, prodrug self-assemblies, and gold nanoparticles.
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Affiliation(s)
- Elise Lepeltier
- Micro et Nanomédecines Translationnelles, MINT, UNIV Angers, UMR INSERM 1066, UMR CNRS 6021, Angers, France
| | - Patricia Rijo
- Research Center for Biosciences & Health Technologies (CBIOS), Lisboa, Portugal; iMed.ULisboa - Research Institute for Medicines, Lisboa, Portugal
| | - Flavio Rizzolio
- Department of Molecular Sciences and Nanosystems, Ca' Foscari University of Venice, 30123 Venezia, Italy; Pathology Unit, Centro di Riferimento Oncologico di Aviano (CRO) IRCCS, 33081 Aviano, Italy
| | - Rachela Popovtzer
- Faculty of Engineering and the Institute of Nanotechnology & Advanced Materials Bar-Ilan University, Ramat Gan, 5290002, Israel
| | - Vilma Petrikaite
- Laboratory of Drug Targets Histopathology, Institute of Cardiology, Lithuanian University of Health Sciences, Sukilėlių Av. 13, LT-50161 Kaunas, Lithuania; Institute of Physiology and Pharmacology, Faculty of Medicine, Lithuanian University of Health Sciences, A. Mickevičiaus 9, LT-44307 Kaunas, Lithuania
| | - Yehuda G Assaraf
- The Fred Wyszkowski Cancer Research Laboratory, Department of Biology, Technion-Israel Institute of Technology, Haifa, 3200003, Israel
| | - Catherine Passirani
- Micro et Nanomédecines Translationnelles, MINT, UNIV Angers, UMR INSERM 1066, UMR CNRS 6021, Angers, France.
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6
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Fatima M, Iqbal Ahmed MM, Batool F, Riaz A, Ali M, Munch-Petersen B, Mutahir Z. Recombinant deoxyribonucleoside kinase from Drosophila melanogaster can improve gemcitabine based combined gene/chemotherapy for targeting cancer cells. Bosn J Basic Med Sci 2019; 19:342-349. [PMID: 30903745 DOI: 10.17305/bjbms.2019.4136] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/26/2019] [Accepted: 03/03/2019] [Indexed: 11/16/2022] Open
Abstract
A recombinant deoxyribonucleoside kinase from Drosophila melanogaster with a deletion of the last 20 amino acid residues (named DmdNKΔC20) was hypothesized as a potential therapeutic tool for gene therapy due to its broad substrate specificity and better catalytic efficiency towards nucleosides and nucleoside analogs. This study was designed to evaluate the effect of DmdNKΔC20 for sensitizing human cancer cell lines to gemcitabine and to further investigate its role in reversal of acquired drug resistance in gemcitabine-resistant cancer cell line. The DmdNKΔC20 gene was delivered to three different cancer cell lines, including breast, colon and liver cancer cells, using lipid-mediated transfection reagent. After transfection, gene expression of DmdNKΔC20 was confirmed by quantitative reverse transcription PCR (qRT-PCR) and the combined effect of DmdNKΔC20 and gemcitabine based cytotoxicity was observed by cell viability assay. We further evolved a gemcitabine-resistant breast cancer cell line (named MCF7-R) through directed evolution in the laboratory, which showed 375-fold more resistance compared with parental MCF7 cells. Upon transfection with DmdNKΔC20 gene, MCF7-R cells showed 83-fold higher sensitivity to gemcitabine compared with the control group of MCF7-R cells. Moreover, we observed 79% higher expression of p21 protein in transfected MCF7-R cells, which may indicate induction of apoptosis. Our findings highlight the importance and therapeutic potential of DmdNKΔC20 in combined gene/chemotherapy approach to target a wide range of cancers, particularly gemcitabine-resistant cancers.
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Affiliation(s)
- Mahak Fatima
- Institute of Biochemistry and Biotechnology, University of the Punjab, Lahore, Pakistan.
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Filippi R, Lombardi P, Quarà V, Fenocchio E, Aimar G, Milanesio M, Leone F, Aglietta M. Pharmacotherapeutic options for biliary tract cancer: current standard of care and new perspectives. Expert Opin Pharmacother 2019; 20:2121-2137. [PMID: 31550186 DOI: 10.1080/14656566.2019.1667335] [Citation(s) in RCA: 7] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/06/2023]
Abstract
Introduction: Biliary tract cancer (BTC), which comprises gallbladder cancer, ampullary cancer, and cholangiocarcinoma, is a rare and heterogeneous entity, with limited approved therapeutic options. However, interest in this disease has grown exponentially in recent years, as a mounting body of evidence has shed light on the complex molecular and microenvironmental background of BTC, and clinical investigations have explored a variety of new agents and combinations, with promising results.Areas covered: This review describes the standard of care in advanced BTC and summarizes the most recent evidence available on the pharmacological treatment of resected and advanced disease, focusing on chemotherapy, targeted therapy, and immunotherapy.Expert opinion: The therapeutic armamentarium of BTC has made radical progress after almost a decade of very few positive results. Phase-III evidence now supports the use of adjuvant capecitabine after resection of localized disease, while investigations into improved regimens in the advanced setting are underway, exploring alternative options to the standard gemcitabine-cisplatin doublet. The first positive phase-III trial supports the use of the mFOLFOX6 regimen as a second-line chemotherapy. Targeted therapy against specific genomic alterations can combine with chemotherapy in specific subsets of patients. Despite recent advancements, conducting clinical trials for BTC is still a real challenge.
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Affiliation(s)
- Roberto Filippi
- Department of Oncology, University of Turin, Candiolo, Italy.,Department of Medical Oncology, Candiolo Cancer Institute, FPO-IRCCS, Candiolo, Italy
| | - Pasquale Lombardi
- Department of Oncology, University of Turin, Candiolo, Italy.,Department of Medical Oncology, Candiolo Cancer Institute, FPO-IRCCS, Candiolo, Italy
| | - Virginia Quarà
- Department of Oncology, University of Turin, Candiolo, Italy.,Department of Medical Oncology, Candiolo Cancer Institute, FPO-IRCCS, Candiolo, Italy
| | - Elisabetta Fenocchio
- Multidisciplinary Outpatient Oncology Clinic, Candiolo Cancer Institute, FPO-IRCCS, Candiolo, Italy
| | - Giacomo Aimar
- Department of Oncology, University of Turin, Candiolo, Italy.,Department of Medical Oncology, Candiolo Cancer Institute, FPO-IRCCS, Candiolo, Italy
| | - Michela Milanesio
- Department of Oncology, University of Turin, Candiolo, Italy.,Department of Medical Oncology, Candiolo Cancer Institute, FPO-IRCCS, Candiolo, Italy
| | - Francesco Leone
- Medical Oncology, Ospedale degli Infermi, Azienda Sanitaria Locale di Biella, Biella, Italy
| | - Massimo Aglietta
- Department of Oncology, University of Turin, Candiolo, Italy.,Department of Medical Oncology, Candiolo Cancer Institute, FPO-IRCCS, Candiolo, Italy
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8
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Bhise NS, Elsayed AH, Cao X, Pounds S, Lamba JK. MicroRNAs Mediated Regulation of Expression of Nucleoside Analog Pathway Genes in Acute Myeloid Leukemia. Genes (Basel) 2019; 10:genes10040319. [PMID: 31022985 PMCID: PMC6523677 DOI: 10.3390/genes10040319] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/26/2019] [Revised: 04/16/2019] [Accepted: 04/20/2019] [Indexed: 01/08/2023] Open
Abstract
Nucleoside analog, cytarabine (ara-C) is the mainstay of acute myeloid leukemia (AML) chemotherapy. Cytarabine and other nucleoside analogs require activation to the triphosphate form (ara-CTP). Intracellular ara-CTP levels demonstrate significant inter-patient variation and have been related to therapeutic response in AML patients. Inter-patient variation in expression levels of drug transporters or enzymes involved in their activation or inactivation of cytarabine and other analogs is a prime mechanism contributing to development of drug resistance. Since microRNAs (miRNAs) are known to regulate gene-expression, the aim of this study was to identify miRNAs involved in regulation of messenger RNA expression levels of cytarabine pathway genes. We evaluated miRNA and gene-expression levels of cytarabine metabolic pathway genes in 8 AML cell lines and The Cancer Genome Atlas (TCGA) data base. Using correlation analysis and functional validation experiments, our data demonstrates that miR-34a-5p and miR-24-3p regulate DCK, an enzyme involved in activation of cytarabine and DCDT, an enzyme involved in metabolic inactivation of cytarabine expression, respectively. Further our results from gel shift assays confirmed binding of these mRNA-miRNA pairs. Our results show miRNA mediated regulation of gene expression levels of nucleoside metabolic pathway genes can impact interindividual variation in expression levels which in turn may influence treatment outcomes.
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Affiliation(s)
- Neha S Bhise
- Department of Pharmacotherapy and Translational Research, Center for Pharmacogenomics, University of Florida, Gainesville, FL 32610, USA.
- Department of Experimental and Clinical Pharmacology, University of Minnesota, Minneapolis, MN 55455, USA.
| | - Abdelrahman H Elsayed
- Department of Pharmacotherapy and Translational Research, Center for Pharmacogenomics, University of Florida, Gainesville, FL 32610, USA.
| | - Xueyuan Cao
- Department of Acute and Tertiary Care, University of Tennessee Health Science Center, Memphis, TN 38163, USA.
| | - Stanley Pounds
- Department of Biostatistics, St. Jude Children's Research Hospital, Memphis, TN 38105, USA.
| | - Jatinder K Lamba
- Department of Pharmacotherapy and Translational Research, Center for Pharmacogenomics, University of Florida, Gainesville, FL 32610, USA.
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9
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Maity G, Ghosh A, Gupta V, Haque I, Sarkar S, Das A, Dhar K, Bhavanasi S, Gunewardena SS, Von Hoff DD, Mallik S, Kambhampati S, Banerjee SK, Banerjee S. CYR61/CCN1 Regulates dCK and CTGF and Causes Gemcitabine-resistant Phenotype in Pancreatic Ductal Adenocarcinoma. Mol Cancer Ther 2019; 18:788-800. [PMID: 30787177 DOI: 10.1158/1535-7163.mct-18-0899] [Citation(s) in RCA: 29] [Impact Index Per Article: 4.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/08/2018] [Revised: 10/30/2018] [Accepted: 01/30/2019] [Indexed: 02/03/2023]
Abstract
Pancreatic ductal adenocarcinoma (PDAC) develops extrinsic- and intrinsic-resistant phenotypes to prevent chemotherapies from entering into the cells by promoting desmoplastic reactions (DR) and metabolic malfunctions of the drugs. It is well established that these responses are also associated with pancreatic cancer cells' gemcitabine resistance. However, the mechanism by which these resistant pathways function in the pancreatic cancer cells remains poorly understood. In these studies, we show that CYR61/CCN1 signaling plays a vital role in making pancreatic cancer cells resistant to gemcitabine in vitro and also in a tumor xenograft model. We proved that the catastrophic effect of gemcitabine could significantly be increased in gemcitabine-resistant PDAC cells when CYR61/CCN1 is depleted, while this effect can be suppressed in gemcitabine-sensitive neoplastic cells by treating them with CYR61/CCN1 recombinant protein. Ironically, nontransformed pancreatic cells, which are sensitive to gemcitabine, cannot be resistant to gemcitabine by CYR61/CCN1 protein treatment, showing a unique feature of CYR61/CCN signaling that only influences PDAC cells to become resistant. Furthermore, we demonstrated that CYR61/CCN1 suppresses the expression of the gemcitabine-activating enzyme deoxycytidine kinase (dCK) while it induces the expression of a DR-promoting factor CTGF (connective tissue growth factor) in pancreatic cancer cells in vitro and in vivo Thus, the previously described mechanisms (dCK and CTGF pathways) for gemcitabine resistance may be two novel targets for CYR61/CCN1 to protect pancreatic cancer cells from gemcitabine. Collectively, these studies reveal a novel paradigm in which CYR61/CCN1regulates both extrinsic and intrinsic gemcitabine resistance in PDAC cells by employing unique signaling pathways.
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Affiliation(s)
- Gargi Maity
- Cancer Research Unit, VA Medical Center, Kansas City, Missouri
- Department of Pathology and Laboratory Medicine, University of Kansas Medical Center, Kansas City, Kansas
| | - Arnab Ghosh
- Cancer Research Unit, VA Medical Center, Kansas City, Missouri.
- Department of Pathology and Laboratory Medicine, University of Kansas Medical Center, Kansas City, Kansas
| | - Vijayalaxmi Gupta
- Cancer Research Unit, VA Medical Center, Kansas City, Missouri
- Department of Ob/Gyn, University of Kansas Medical Center, Kansas City, Kansas
| | - Inamul Haque
- Cancer Research Unit, VA Medical Center, Kansas City, Missouri
- Department of Pathology and Laboratory Medicine, University of Kansas Medical Center, Kansas City, Kansas
| | - Sandipto Sarkar
- Cancer Research Unit, VA Medical Center, Kansas City, Missouri
- Department of Pathology and Laboratory Medicine, University of Kansas Medical Center, Kansas City, Kansas
| | - Amlan Das
- Cancer Research Unit, VA Medical Center, Kansas City, Missouri
| | - Kakali Dhar
- Cancer Research Unit, VA Medical Center, Kansas City, Missouri
| | - Sneha Bhavanasi
- Cancer Research Unit, VA Medical Center, Kansas City, Missouri
| | - Sumedha S Gunewardena
- Department of Molecular and Integrative Physiology, University of Kansas Medical Center, Kansas City, Kansas
| | - Daniel D Von Hoff
- The Translational Genomics Research Institute (TGen), Phoenix, Arizona
| | - Sanku Mallik
- Department of Pharmaceutical Sciences, North Dakota State University, Fargo, North Dakota
| | - Suman Kambhampati
- Cancer Research Unit, VA Medical Center, Kansas City, Missouri
- The Sarah Cannon Cancer Center at HCA Midwest Health, Kansas City, Missouri
| | - Sushanta K Banerjee
- Cancer Research Unit, VA Medical Center, Kansas City, Missouri.
- Department of Pathology and Laboratory Medicine, University of Kansas Medical Center, Kansas City, Kansas
| | - Snigdha Banerjee
- Cancer Research Unit, VA Medical Center, Kansas City, Missouri.
- Department of Pathology and Laboratory Medicine, University of Kansas Medical Center, Kansas City, Kansas
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10
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Chen Z, Zheng Y, Shi Y, Cui Z. Overcoming tumor cell chemoresistance using nanoparticles: lysosomes are beneficial for (stearoyl) gemcitabine-incorporated solid lipid nanoparticles. Int J Nanomedicine 2018; 13:319-336. [PMID: 29391792 PMCID: PMC5768424 DOI: 10.2147/ijn.s149196] [Citation(s) in RCA: 28] [Impact Index Per Article: 4.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/13/2022] Open
Abstract
Despite recent advances in targeted therapies and immunotherapies, chemotherapy using cytotoxic agents remains an indispensable modality in cancer treatment. Recently, there has been a growing emphasis in using nanomedicine in cancer chemotherapy, and several nanomedicines have already been used clinically to treat cancers. There is evidence that formulating small molecular cancer chemotherapeutic agents into nanomedicines significantly modifies their pharmacokinetics and often improves their efficacy. Importantly, cancer cells often develop resistance to chemotherapy, and formulating anticancer drugs into nanomedicines also helps overcome chemoresistance. In this review, we briefly describe the different classes of cancer chemotherapeutic agents, their mechanisms of action and resistance, and evidence of overcoming the resistance using nanomedicines. We then emphasize on gemcitabine and our experience in discovering the unique (stearoyl) gemcitabine solid lipid nanoparticles that are effective against tumor cells resistant to gemcitabine and elucidate the underlying mechanisms. It seems that lysosomes, which are an obstacle in the delivery of many drugs, are actually beneficial for our (stearoyl) gemcitabine solid lipid nanoparticles to overcome tumor cell resistance to gemcitabine.
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Affiliation(s)
- Zhe Chen
- Inner Mongolia Key Lab of Molecular Biology, School of Basic Medical Sciences, Inner Mongolia Medical University, Hohhot, Inner Mongolia, China
| | - Yuanqiang Zheng
- Inner Mongolia Key Lab of Molecular Biology, School of Basic Medical Sciences, Inner Mongolia Medical University, Hohhot, Inner Mongolia, China
| | - Yanchun Shi
- Inner Mongolia Key Lab of Molecular Biology, School of Basic Medical Sciences, Inner Mongolia Medical University, Hohhot, Inner Mongolia, China
| | - Zhengrong Cui
- Inner Mongolia Key Lab of Molecular Biology, School of Basic Medical Sciences, Inner Mongolia Medical University, Hohhot, Inner Mongolia, China.,Division of Molecular Pharmaceutics and Drug Delivery, College of Pharmacy, The University of Texas at Austin, Austin, TX, USA
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11
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Feng F, Wang B, Sun X, Zhu Y, Tang H, Nan G, Wang L, Wu B, Huhe M, Liu S, Diao T, Hou R, Zhang Y, Zhang Z. Metuzumab enhanced chemosensitivity and apoptosis in non-small cell lung carcinoma. Cancer Biol Ther 2017; 18:51-62. [PMID: 28055291 PMCID: PMC5323017 DOI: 10.1080/15384047.2016.1276126] [Citation(s) in RCA: 16] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/13/2022] Open
Abstract
Targeted therapeutics is used as an alternative treatment of non-small cell lung cancer (NSCLC); however, treatment effect is far from being satisfactory, and therefore identification of new targets is needed. We have previously shown that metuzumab inhibit tumor growth in vivo. The present study was performed to investigate the anti-tumor efficacy of metuzumab combined with gemcitabine and cisplatin (GP), paclitaxel and cisplatin (TP) or navelbine and cisplatin (NP) regimens in multiple NSCLC cell lines. Our results demonstrate that, in comparison to single agent metuzumab or GP treated cells, metuzumab combined with GP display inhibitory effects on tumor growth. Furthermore, we found that metuzumab elevated the sensitivity of cell lines to gemcitabine, which was identified by MTT assay. Flow cytometric analysis showed that metuzumab combined with gemcitabine (GEM) treatment led to an obvious G1 arrest and an elevated apoptosis in A549, NCI-H460 and NCI-H520 cells. Western blot analysis also demonstrated a significantly reduced level of cyclin D1, Bcl-2, and an obviously increase level of Bax and full-length caspase-3 in A549, NCI-H460 and NCI-H520 cells treated with metuzumab/gemcitabine combination in comparison with single agent treated cells. In addition, metuzumab/gemcitabine treated A549, NCI-H460 and NCI-H520 cells also demonstrated a significantly increase in deoxycytidine kinase (dCK) protein level compared with single agent metuzumab or gemcitabine treated cells. Xenograft models also demonstrated that this metuzumab/gemcitabine combination led to upregulation of dCK. Taken together, the mechanisms of metuzumab combined with GP repress tumor growth were that the combined treatment significantly inhibited the tumor cell proliferation, apoptosis and cell cycle in vitro and in vivo and at least partially by induction of dCK expression. Our results suggested that metuzumab could significantly enhance chemosensitivity of human NSCLC cells to gemcitabine. Metuzumab/gemcitabine combination treatment may be a potentially useful therapeutic regimen for NSCLC patients.
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Affiliation(s)
- Fei Feng
- a National Translational Science Center for Molecular Medicine , Department of Cell Biology , Fourth Military Medical University , Xi'an , P.R. China
| | - Bin Wang
- a National Translational Science Center for Molecular Medicine , Department of Cell Biology , Fourth Military Medical University , Xi'an , P.R. China
| | - Xiuxuan Sun
- a National Translational Science Center for Molecular Medicine , Department of Cell Biology , Fourth Military Medical University , Xi'an , P.R. China
| | - Yumeng Zhu
- a National Translational Science Center for Molecular Medicine , Department of Cell Biology , Fourth Military Medical University , Xi'an , P.R. China
| | - Hao Tang
- b Pacific Meinuoke Biopharmaceutical Company , Changzhou , P.R. China
| | - Gang Nan
- a National Translational Science Center for Molecular Medicine , Department of Cell Biology , Fourth Military Medical University , Xi'an , P.R. China
| | - Lijuan Wang
- a National Translational Science Center for Molecular Medicine , Department of Cell Biology , Fourth Military Medical University , Xi'an , P.R. China
| | - Bo Wu
- a National Translational Science Center for Molecular Medicine , Department of Cell Biology , Fourth Military Medical University , Xi'an , P.R. China
| | - Muren Huhe
- a National Translational Science Center for Molecular Medicine , Department of Cell Biology , Fourth Military Medical University , Xi'an , P.R. China
| | - Shuangshuang Liu
- a National Translational Science Center for Molecular Medicine , Department of Cell Biology , Fourth Military Medical University , Xi'an , P.R. China
| | - Tengyue Diao
- a National Translational Science Center for Molecular Medicine , Department of Cell Biology , Fourth Military Medical University , Xi'an , P.R. China
| | - Rong Hou
- a National Translational Science Center for Molecular Medicine , Department of Cell Biology , Fourth Military Medical University , Xi'an , P.R. China
| | - Yang Zhang
- a National Translational Science Center for Molecular Medicine , Department of Cell Biology , Fourth Military Medical University , Xi'an , P.R. China
| | - Zheng Zhang
- a National Translational Science Center for Molecular Medicine , Department of Cell Biology , Fourth Military Medical University , Xi'an , P.R. China
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12
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Determination and quantification of intracellular fludarabine triphosphate, cladribine triphosphate and clofarabine triphosphate by LC-MS/MS in human cancer cells. J Chromatogr B Analyt Technol Biomed Life Sci 2017; 1053:101-110. [PMID: 28415014 DOI: 10.1016/j.jchromb.2017.03.024] [Citation(s) in RCA: 5] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [Key Words] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/24/2016] [Revised: 03/02/2017] [Accepted: 03/22/2017] [Indexed: 11/21/2022]
Abstract
Purine nucleoside analogues are widely used in the treatment of haematological malignancies, and their biological activity is dependent on the intracellular accumulation of their triphosphorylated metabolites. In this context, we developed and validated a liquid chromatography tandem mass spectrometry (LC-MS/MS) method to study the formation of 5'-triphosphorylated derivatives of cladribine, fludarabine, clofarabine and 2'-deoxyadenosine in human cancer cells. Br-ATP was used as internal standard. Separation was achieved on a hypercarb column. Analytes were eluted with a mixture of hexylamine (5 mM), DEA (0.4%, v/v, pH 10.5) and acetonitrile, in a gradient mode at a flow rate of 0.3mLmin-1. Multiple reactions monitoring (MRM) and electrospray ionization in negative mode (ESI-) were used for detection. The application of this method to the quantification of these phosphorylated cytotoxic compounds in a human follicular lymphoma cell line, showed that it was suitable for the study of relevant biological samples.
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13
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Rajabpour A, Rajaei F, Teimoori-Toolabi L. Molecular alterations contributing to pancreatic cancer chemoresistance. Pancreatology 2017; 17:310-320. [PMID: 28065383 DOI: 10.1016/j.pan.2016.12.013] [Citation(s) in RCA: 29] [Impact Index Per Article: 3.6] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 06/25/2016] [Revised: 12/27/2016] [Accepted: 12/28/2016] [Indexed: 02/06/2023]
Abstract
Pancreatic ductal adenocarcinoma (PDAC) is one of the most common causes of cancer-related death all over the world. This disease is difficult to treat and patients have an overall 5-year survival rate of less than 5%. Although two drugs, gemcitabine (GEM) and 5-fluorouracil (5-FU) have been shown to improve the survival rate of patients systematically, they do not increase general survival to a clinically acceptable degree. Lack of ideal clinical response of pancreatic cancer patients to chemotherapy is likely to be due to intrinsic and acquired chemoresistance of tumor cells. Various mechanisms of drug resistance have been investigated in pancreatic cancer, including genetic and epigenetic changes in particular genes or signaling pathways. In addition, evidence suggests that microRNAs (miRNAs) play significant roles as key regulators of gene expression in many cellular processes, including drug resistance. Understanding underlying genes and mechanisms of drug resistance in pancreatic cancer is critical to develop new effective treatments for this deadly disease. This review illustrates the genes and miRNAs involved in resistance to gemcitabine in pancreatic cancer.
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Affiliation(s)
- Azam Rajabpour
- Cellular and Molecular Research Center, Qazvin University of Medical Sciences, Qazvin, Iran; Department of Molecular Medicine, School of Medicine, Qazvin University of Medical Sciences, Qazvin, Iran; Department of Molecular Medicine, Pasteur Institute of Iran, Tehran, Iran
| | - Farzad Rajaei
- Cellular and Molecular Research Center, Qazvin University of Medical Sciences, Qazvin, Iran; Department of Molecular Medicine, School of Medicine, Qazvin University of Medical Sciences, Qazvin, Iran
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14
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Human pancreatic cancer progression: an anarchy among CCN-siblings. J Cell Commun Signal 2016; 10:207-216. [PMID: 27541366 DOI: 10.1007/s12079-016-0343-9] [Citation(s) in RCA: 16] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/23/2016] [Accepted: 07/27/2016] [Indexed: 02/07/2023] Open
Abstract
Decades of basic and translational studies have identified the mechanisms by which pancreatic cancer cells use molecular pathways to hijack the normal homeostasis of the pancreas, promoting pancreatic cancer initiation, progression, and metastasis, as well as drug resistance. These molecular pathways were explored to develop targeted therapies to prevent or cure this fatal disease. Regrettably, the studies found that majority of the molecular events that dictate carcinogenic growth in the pancreas are non-actionable (potential non-responder groups of targeted therapy). In this review we discuss exciting discoveries on CCN-siblings that reveal how CCN-family members contribute to the different aspects of the development of pancreatic cancer with special emphasis on therapy.
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15
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Gemcitabine resistance in pancreatic ductal adenocarcinoma. Drug Resist Updat 2015; 23:55-68. [PMID: 26690340 DOI: 10.1016/j.drup.2015.10.002] [Citation(s) in RCA: 296] [Impact Index Per Article: 29.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/26/2015] [Revised: 09/15/2015] [Accepted: 10/23/2015] [Indexed: 12/13/2022]
Abstract
Pancreatic ductal adenocarcinoma (PDA) ranks fourth among cancer related deaths. The disappointing 5-year survival rate of below 5% stems from drug resistance to all known therapies, as well as from disease presentation at a late stage when PDA is already metastatic. Gemcitabine has been the cornerstone of PDA treatment in all stages of the disease for the last two decades, but gemcitabine resistance develops within weeks of chemotherapy initiation. From a mechanistic perspective, gemcitabine resistance may result from alterations in drug metabolism until the point that the cytidine analog is incorporated into the DNA, or from mitigation of gemcitabine-induced apoptosis. Both of these drug resistance modalities can be either intrinsic to the cancer cell, or influenced by the cancer microenvironment. Mechanisms of intrinsic gemcitabine resistance are difficult to tackle, as many of the genes that drive the carcinogenic process itself also interfere with gemcitabine-induced apoptosis. In this regard, recent understanding of the involvement of microRNAs in gemcitabine resistance may offer new opportunities to overcome intrinsic gemcitabine resistance. The characteristically fibrotic and immune infiltrated stroma of PDA that accompanies tumor inception and expansion is a lush ground for treatments aimed at targeting tumor microenvironment-mediated drug resistance. In the last couple of years, drugs interfering with tumor microenvironment have matured to clinical trials. Although drugs inducing 'stromal depletion' have yet failed to improve survival, they have greatly increased our understanding of tumor microenvironment-mediated drug resistance. In this review we summarize the current knowledge on intrinsic and environment-mediated gemcitabine resistance, and discuss the impact of these pathways on patient screening, and on future treatments aimed to potentiate gemcitabine activity.
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16
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Potent Sensitisation of Cancer Cells to Anticancer Drugs by a Quadruple Mutant of the Human Deoxycytidine Kinase. PLoS One 2015; 10:e0140741. [PMID: 26485161 PMCID: PMC4618062 DOI: 10.1371/journal.pone.0140741] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.1] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/11/2015] [Accepted: 09/30/2015] [Indexed: 11/19/2022] Open
Abstract
Identifying enzymes that, once introduced in cancer cells, lead to an increased efficiency of treatment constitutes an important goal for biomedical applications. Using an original procedure whereby mutant genes are generated based on the use of conditional lentivector genome mobilisation, we recently described, for the first time, the identification of a human deoxycytidine kinase (dCK) mutant (G12) that sensitises a panel of cancer cell lines to treatment with the dCK analogue gemcitabine. Here, starting from the G12 variant itself, we generated a new library and identified a mutant (M36) that triggers even greater sensitisation to gemcitabine than G12. With respect to G12, M36 presents an additional mutation located in the region that constitutes the interface of the dCK dimer. The simple presence of this mutation halves both the IC50 and the proportion of residual cells resistant to the treatment. Furthermore, the use of vectors with self-inactivating LTRs leads to an increased sensitivity to treatment, a result compatible with a relief of the transcriptional interference exerted by the U3 promoter on the internal promoter that drives the expression of M36. Importantly, a remarkable effect is also observed in treatments with the anticancer compound cytarabine (AraC), for which a 10,000 fold decrease in IC50 occurred. By triggering the sensitisation of various cancer cell types with poor prognosis to two commonly used anticancer compounds M36 is a promising candidate for suicide gene approaches.
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17
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Di Cresce C, Figueredo R, Rytelewski M, Vareki SM, Way C, Ferguson PJ, Vincent MD, Koropatnick J. siRNA knockdown of mitochondrial thymidine kinase 2 (TK2) sensitizes human tumor cells to gemcitabine. Oncotarget 2015; 6:22397-409. [PMID: 26087398 PMCID: PMC4673171 DOI: 10.18632/oncotarget.4272] [Citation(s) in RCA: 6] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/27/2015] [Accepted: 06/03/2015] [Indexed: 11/25/2022] Open
Abstract
Nucleoside metabolism enzymes are determinants of chemotherapeutic drug activity. The nucleoside salvage enzyme deoxycytidine kinase (dCK) activates gemcitabine (2', 2'-difluoro-2'-deoxycytidine) and is negatively regulated by deoxycytidine triphosphate (dCTP). Reduction of dCTP in tumor cells could, therefore, enhance gemcitabine activity. Mitochondrial thymidine kinase 2 (TK2) phosphorylates deoxycytidine to generate dCTP. We hypothesized that: (1) TK2 modulates human tumor cell sensitivity to gemcitabine, and (2) antisense knockdown of TK2 would decrease dCTP and increase dCK activity and gemcitabine activation. siRNA downregulation of TK2 sensitized MCF7 and HeLa cells (high and moderate TK2) but not A549 cells (low TK2) to gemcitabine. Combined treatment with TK2 siRNA and gemcitabine increased dCK. We also hypothesized that TK2 siRNA-induced drug sensitization results in mitochondrial damage that enhances gemcitabine effectiveness. TK2 siRNA and gemcitabine decreased mitochondrial redox status, DNA content, and activity. This is the first demonstration of a direct role for TK2 in gemcitabine resistance, or any independent role in cancer drug resistance, and further distinguishes TK2 function from that of other dTMP-producing enzymes [cytosolic TK1 and thymidylate synthase (TS)]. siRNA knockdown of TK1 and/or TS did not sensitize cancer cells to gemcitabine indicating that, among the 3 enzymes, only TK2 is a candidate therapeutic target for combination with gemcitabine.
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Affiliation(s)
- Christine Di Cresce
- Department of Microbiology and Immunology, The University of Western Ontario, London, Ontario, Canada
- Cancer Research Laboratory Program, Lawson Health Research Institute and London Regional Cancer Program, London, Ontario, Canada
| | - Rene Figueredo
- Department of Oncology, The University of Western Ontario, London, Ontario, Canada
- Cancer Research Laboratory Program, Lawson Health Research Institute and London Regional Cancer Program, London, Ontario, Canada
| | - Mateusz Rytelewski
- Department of Microbiology and Immunology, The University of Western Ontario, London, Ontario, Canada
- Cancer Research Laboratory Program, Lawson Health Research Institute and London Regional Cancer Program, London, Ontario, Canada
| | - Saman Maleki Vareki
- Department of Microbiology and Immunology, The University of Western Ontario, London, Ontario, Canada
- Cancer Research Laboratory Program, Lawson Health Research Institute and London Regional Cancer Program, London, Ontario, Canada
| | - Colin Way
- Department of Microbiology and Immunology, The University of Western Ontario, London, Ontario, Canada
- Cancer Research Laboratory Program, Lawson Health Research Institute and London Regional Cancer Program, London, Ontario, Canada
| | - Peter J. Ferguson
- Department of Oncology, The University of Western Ontario, London, Ontario, Canada
- Cancer Research Laboratory Program, Lawson Health Research Institute and London Regional Cancer Program, London, Ontario, Canada
| | - Mark D. Vincent
- Department of Oncology, The University of Western Ontario, London, Ontario, Canada
- Cancer Research Laboratory Program, Lawson Health Research Institute and London Regional Cancer Program, London, Ontario, Canada
| | - James Koropatnick
- Department of Microbiology and Immunology, The University of Western Ontario, London, Ontario, Canada
- Department of Oncology, The University of Western Ontario, London, Ontario, Canada
- Department of Pathology, The University of Western Ontario, London, Ontario, Canada
- Department of Physiology and Pharmacology, The University of Western Ontario, London, Ontario, Canada
- Cancer Research Laboratory Program, Lawson Health Research Institute and London Regional Cancer Program, London, Ontario, Canada
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18
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Cividini F, Filoni DN, Pesi R, Allegrini S, Camici M, Tozzi MG. IMP-GMP specific cytosolic 5'-nucleotidase regulates nucleotide pool and prodrug metabolism. Biochim Biophys Acta Gen Subj 2015; 1850:1354-61. [PMID: 25857773 DOI: 10.1016/j.bbagen.2015.03.017] [Citation(s) in RCA: 14] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Abstract] [Key Words] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/03/2014] [Revised: 03/26/2015] [Accepted: 03/31/2015] [Indexed: 12/20/2022]
Abstract
BACKGROUND Type II cytosolic 5'-nucleotidase (cN-II) catalyzes the hydrolysis of purine and, to some extent, of pyrimidine monophosphates. Recently, a number of papers demonstrated the involvement of cN-II in the mechanisms of resistance to antitumor drugs such as cytarabine, gemcitabine and fludarabine. Furthermore, cN-II is involved in drug resistance in patients affected by hematological malignancies influencing the clinical outcome. Although the implication of cN-II expression and/or activity appears to be correlated with drug resistance and poor prognosis, the molecular mechanism by which cN-II mediates drug resistance is still unknown. METHODS HEK 293 cells carrying an expression vector coding for cN-II linked to green fluorescent protein (GFP) and a control vector without cN-II were utilized. A highly sensitive capillary electrophoresis method was applied for nucleotide pool determination and cytotoxicity exerted by drugs was determined with 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay. RESULTS Over-expression of cN-II causes a drop of nucleoside triphosphate concentration and a general disturbance of nucleotide pool. Over-expressing cells were resistant to fludarabine, gemcitabine and cytarabine independently of cN-II ability to hydrolyze their monophosphates. CONCLUSIONS An increase of cN-II expression is sufficient to cause both a general disturbance of nucleotide pool and an increase of half maximal inhibitory concentration (IC50) of the drugs. Since the monophosphates of cytarabine and gemcitabine are not substrates of cN-II, the protection observed cannot be directly ascribed to drug inactivation. GENERAL SIGNIFICANCE Our results indicate that cN-II exerts a relevant role in nucleotide and drug metabolism through not only enzyme activity but also a mechanism involving a protein-protein interaction, thus playing a general regulatory role in cell survival. SENTENCE Resistance to fludarabine, gemcitabine and cytarabine can be determined by an increase of cN-II both through dephosphorylation of active drugs and perturbation of nucleotide pool.
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Affiliation(s)
- Federico Cividini
- Dipartimento di Biologia, Unità di Biochimica, Università di Pisa, Via San Zeno 51, 56127, Pisa, Italy
| | - Daniela Nicole Filoni
- Dipartimento di Biologia, Unità di Biochimica, Università di Pisa, Via San Zeno 51, 56127, Pisa, Italy; Dipartimento di Chimica e Farmacia, Università di Sassari, Via Muroni 23A, 07100, Sassari, Italy
| | - Rossana Pesi
- Dipartimento di Biologia, Unità di Biochimica, Università di Pisa, Via San Zeno 51, 56127, Pisa, Italy
| | - Simone Allegrini
- Dipartimento di Chimica e Farmacia, Università di Sassari, Via Muroni 23A, 07100, Sassari, Italy.
| | - Marcella Camici
- Dipartimento di Biologia, Unità di Biochimica, Università di Pisa, Via San Zeno 51, 56127, Pisa, Italy
| | - Maria Grazia Tozzi
- Dipartimento di Biologia, Unità di Biochimica, Università di Pisa, Via San Zeno 51, 56127, Pisa, Italy
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19
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Acquired resistance to gemcitabine and cross-resistance in human pancreatic cancer clones. Anticancer Drugs 2015; 26:90-100. [DOI: 10.1097/cad.0000000000000165] [Citation(s) in RCA: 20] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/12/2023]
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20
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Kerr M, Scott HE, Groselj B, Stratford MRL, Karaszi K, Sharma NL, Kiltie AE. Deoxycytidine kinase expression underpins response to gemcitabine in bladder cancer. Clin Cancer Res 2014; 20:5435-45. [PMID: 25224279 PMCID: PMC4216732 DOI: 10.1158/1078-0432.ccr-14-0542] [Citation(s) in RCA: 18] [Impact Index Per Article: 1.6] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/16/2022]
Abstract
PURPOSE In a recent phase II clinical trial, low-dose (100 mg/m(2)) gemcitabine showed promise as a radiosensitizer in bladder cancer, but underlying mechanisms lack elucidation. Here, we investigated the mechanism of radiosensitization by low-dose gemcitabine in bladder cancer cell lines. EXPERIMENTAL DESIGN Four bladder cancer cell lines were screened for radiosensitization by low-dose gemcitabine using clonogenic assay, and gemcitabine-resistant RT112gem and CALgem cells created by exposure to increasing gemcitabine doses. Four key gemcitabine-regulatory genes were knocked down by transient siRNA. Nude mice carrying CALgem subcutaneous xenografts were exposed to 100 mg/kg gemcitabine ± ionizing radiation (IR) and response assessed by tumor growth delay. RESULTS Gemcitabine was cytotoxic in the low nanomolar range (10-40 nmol/L) in four bladder cancer cell lines and radiosensitized all four lines. Sensitizer enhancement ratios at 10% survival were: RT112 1.42, CAL29 1.55, T24 1.63, and VMCUB1 1.47. Transient siRNA knockdown of deoxycytidine kinase (dCK) significantly reduced radiosensitization by gemcitabine (P = 0.02). RT112gem and CALgem cells displayed robust decreases of dCK mRNA and protein levels; reexpression of dCK restored gemcitabine sensitivity. However, CALgem xenografts responded better to combination gemcitabine/IR than either treatment alone (P < 0.001) with dCK strongly expressed in the tumor vasculature and stroma. CONCLUSIONS Gemcitabine resistance in bladder cancer cell lines was associated with decreased dCK expression, but gemcitabine-resistant xenografts were responsive to combination low-dose gemcitabine/IR. We propose that dCK activity in tumor vasculature renders it gemcitabine sensitive, which is sufficient to invoke a tumor response and permit tumor cell kill in gemcitabine-resistant tumors.
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Affiliation(s)
- Martin Kerr
- Department of Oncology, CRUK/MRC Oxford Institute for Radiation Oncology and
| | - Helen E Scott
- Department of Oncology, CRUK/MRC Oxford Institute for Radiation Oncology and Nuffield Department of Surgical Sciences, University of Oxford, Oxford, United Kingdom
| | - Blaz Groselj
- Department of Oncology, CRUK/MRC Oxford Institute for Radiation Oncology and
| | | | - Katalin Karaszi
- Department of Oncology, CRUK/MRC Oxford Institute for Radiation Oncology and Nuffield Department of Surgical Sciences, University of Oxford, Oxford, United Kingdom
| | - Naomi L Sharma
- Department of Oncology, CRUK/MRC Oxford Institute for Radiation Oncology and Nuffield Department of Surgical Sciences, University of Oxford, Oxford, United Kingdom
| | - Anne E Kiltie
- Department of Oncology, CRUK/MRC Oxford Institute for Radiation Oncology and
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21
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Zhu C, Tang C, Cao Z, He W, Chen Y, Chen X, Guo K, Ying H. Fully Automated Continuous Meso-flow Synthesis of 5′-Nucleotides and Deoxynucleotides. Org Process Res Dev 2014. [DOI: 10.1021/op5002066] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/28/2022]
Affiliation(s)
- Chenjie Zhu
- College
of Biotechnology and Pharmaceutical Engineering, Nanjing University of Technology, Nanjing 210009, China
- National Engineering Technique Research Center for Biotechnology, Nanjing 211816, China
| | - Chenglun Tang
- College
of Biotechnology and Pharmaceutical Engineering, Nanjing University of Technology, Nanjing 210009, China
- National Engineering Technique Research Center for Biotechnology, Nanjing 211816, China
| | - Zhi Cao
- College
of Biotechnology and Pharmaceutical Engineering, Nanjing University of Technology, Nanjing 210009, China
- National Engineering Technique Research Center for Biotechnology, Nanjing 211816, China
| | - Wei He
- College
of Biotechnology and Pharmaceutical Engineering, Nanjing University of Technology, Nanjing 210009, China
| | - Yong Chen
- College
of Biotechnology and Pharmaceutical Engineering, Nanjing University of Technology, Nanjing 210009, China
- National Engineering Technique Research Center for Biotechnology, Nanjing 211816, China
| | - Xiaochun Chen
- College
of Biotechnology and Pharmaceutical Engineering, Nanjing University of Technology, Nanjing 210009, China
- National Engineering Technique Research Center for Biotechnology, Nanjing 211816, China
| | - Kai Guo
- College
of Biotechnology and Pharmaceutical Engineering, Nanjing University of Technology, Nanjing 210009, China
| | - Hanjie Ying
- College
of Biotechnology and Pharmaceutical Engineering, Nanjing University of Technology, Nanjing 210009, China
- National Engineering Technique Research Center for Biotechnology, Nanjing 211816, China
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22
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McAllister F, Pineda DM, Jimbo M, Lal S, Burkhart RA, Moughan J, Winter KA, Abdelmohsen K, Gorospe M, Acosta ADJ, Lankapalli RH, Winter JM, Yeo CJ, Witkiewicz AK, Iacobuzio-Donahue CA, Laheru D, Brody JR. dCK expression correlates with 5-fluorouracil efficacy and HuR cytoplasmic expression in pancreatic cancer: a dual-institutional follow-up with the RTOG 9704 trial. Cancer Biol Ther 2014; 15:688-98. [PMID: 24618665 DOI: 10.4161/cbt.28413] [Citation(s) in RCA: 28] [Impact Index Per Article: 2.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/21/2022] Open
Abstract
Deoxycytidine kinase (dCK) and human antigen R (HuR) have been associated with response to gemcitabine in small studies. The present study investigates the prognostic and predictive value of dCK and HuR expression levels for sensitivity to gemcitabine and 5-fluorouracil (5-FU) in a large phase III adjuvant trial with chemoradiation backbone in pancreatic ductal adenocarcinoma (PDA). The dCK and HuR expression levels were determined by immunohistochemistry on a tissue microarray of 165 resected PDAs from the Radiation Therapy Oncology Group (RTOG) 9704 trial. Association with overall survival (OS) and disease-free survival (DFS) status were analyzed using the log-rank test and the Cox proportional hazards model. Experiments with cultured PDA cells were performed to explore mechanisms linking dCK and HuR expression to drug sensitivity. dCK expression levels were associated with improved OS for all patients analyzed from RTOG 9704 (HR: 0.66, 95% CI [0.47-0.93], P = 0.015). In a subset analysis based on treatment arm, the effect was restricted to patients receiving 5-FU (HR: 0.53, 95% CI [0.33-0.85], P = 0.0078). Studies in cultured cells confirmed that dCK expression rendered cells more sensitive to 5-FU. HuR cytoplasmic expression was neither prognostic nor predictive of treatment response. Previous studies along with drug sensitivity and biochemical studies demonstrate that radiation interferes with HuR's regulatory effects on dCK, and could account for the negative findings herein based on the clinical study design (i.e., inclusion of radiation). Finally, we demonstrate that 5-FU can increase HuR function by enhancing HuR translocation from the nucleus to the cytoplasm, similar to the effect of gemcitabine in PDA cells. For the first time, in the pre-treatment tumor samples, dCK and HuR cytoplasmic expression were strongly correlated (chi-square P = 0.015). This dual-institutional follow up study, in a multi-institutional PDA randomized clinical trial, observed that dCK expression levels were prognostic and had predictive value for sensitivity to 5-FU.
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Affiliation(s)
- Florencia McAllister
- Departments of Medical Oncology and Pathology; Johns Hopkins University; Baltimore, MD USA; Department of Medicine; Division of Clinical Pharmacology; Johns Hopkins University; Baltimore, MD USA
| | - Danielle M Pineda
- Department of Surgery; Division of Surgical Research; The Jefferson Pancreas, Biliary, and Related Cancer Center; Jefferson Medical College; Thomas Jefferson University; Philadelphia, PA USA
| | - Masaya Jimbo
- Department of Surgery; Division of Surgical Research; The Jefferson Pancreas, Biliary, and Related Cancer Center; Jefferson Medical College; Thomas Jefferson University; Philadelphia, PA USA
| | - Shruti Lal
- Department of Surgery; Division of Surgical Research; The Jefferson Pancreas, Biliary, and Related Cancer Center; Jefferson Medical College; Thomas Jefferson University; Philadelphia, PA USA
| | - Richard A Burkhart
- Department of Surgery; Division of Surgical Research; The Jefferson Pancreas, Biliary, and Related Cancer Center; Jefferson Medical College; Thomas Jefferson University; Philadelphia, PA USA
| | | | | | - Kotb Abdelmohsen
- Laboratory of Genetics; National Institute on Aging Intramural Research Program; National Institutes of Health; Baltimore, MD USA
| | - Myriam Gorospe
- Laboratory of Genetics; National Institute on Aging Intramural Research Program; National Institutes of Health; Baltimore, MD USA
| | - Ana de Jesus Acosta
- Departments of Medical Oncology and Pathology; Johns Hopkins University; Baltimore, MD USA
| | - Rachana H Lankapalli
- Departments of Medical Oncology and Pathology; Johns Hopkins University; Baltimore, MD USA
| | - Jordan M Winter
- Department of Surgery; Division of Surgical Research; The Jefferson Pancreas, Biliary, and Related Cancer Center; Jefferson Medical College; Thomas Jefferson University; Philadelphia, PA USA
| | - Charles J Yeo
- Department of Surgery; Division of Surgical Research; The Jefferson Pancreas, Biliary, and Related Cancer Center; Jefferson Medical College; Thomas Jefferson University; Philadelphia, PA USA
| | - Agnieska K Witkiewicz
- Department of Pathology; The University of Texas Southwestern Medical Center; Dallas, TX USA
| | | | - Daniel Laheru
- Departments of Medical Oncology and Pathology; Johns Hopkins University; Baltimore, MD USA
| | - Jonathan R Brody
- Department of Surgery; Division of Surgical Research; The Jefferson Pancreas, Biliary, and Related Cancer Center; Jefferson Medical College; Thomas Jefferson University; Philadelphia, PA USA
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23
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Slusarczyk M, Lopez MH, Balzarini J, Mason M, Jiang WG, Blagden S, Thompson E, Ghazaly E, McGuigan C. Application of ProTide technology to gemcitabine: a successful approach to overcome the key cancer resistance mechanisms leads to a new agent (NUC-1031) in clinical development. J Med Chem 2014; 57:1531-42. [PMID: 24471998 DOI: 10.1021/jm401853a] [Citation(s) in RCA: 125] [Impact Index Per Article: 11.4] [Reference Citation Analysis] [Abstract] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/29/2022]
Abstract
Gemcitabine is a nucleoside analogue commonly used in cancer therapy but with limited efficacy due to a high susceptibility to cancer cell resistance. The addition of a phosphoramidate motif to the gemcitabine can protect it against many of the key cancer resistance mechanisms. We have synthesized a series of gemcitabine phosphoramidate prodrugs and screened for cytostatic activity in a range of different tumor cell lines. Among the synthesized compounds, one in particular (NUC-1031, 6f) was shown to be potent in vitro. Importantly, compared with gemcitabine, 6f activation was significantly less dependent on deoxycytidine kinase and on nucleoside transporters, and it was resistant to cytidine deaminase-mediated degradation. Moreover, 6f showed a significant reduction in tumor volumes in vivo in pancreatic cancer xenografts. The ProTide 6f is now in clinical development with encouraging efficacy signals in a Phase I/II study, which strongly supports the ProTide approach to generate promising new anticancer agents.
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Affiliation(s)
- Magdalena Slusarczyk
- Cardiff School of Pharmacy & Pharmaceutical Sciences, Cardiff University , King Edward VII Avenue, Cardiff CF10 3NB, United Kingdom
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24
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Mohelnikova-Duchonova B, Melichar B. Human equilibrative nucleoside transporter 1 (hENT1): do we really have a new predictive biomarker of chemotherapy outcome in pancreatic cancer patients? Pancreatology 2013; 13:558-63. [PMID: 24280569 DOI: 10.1016/j.pan.2013.09.005] [Citation(s) in RCA: 10] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 07/15/2013] [Revised: 09/28/2013] [Accepted: 09/30/2013] [Indexed: 02/06/2023]
Abstract
Although systemic chemotherapy significantly improves the overall survival of pancreatic cancer patients, the prognosis remains extremely poor. The development of a drug resistance, either de novo or induced resistance, significantly limits the effectiveness of chemotherapy. SLC29A1 gene encodes human equilibrative nucleoside transporter 1 (hENT1) protein that is mediating the transport of nucleotides, both purines and pyrimidines, into the tumor cells. The aim of this mini-review is to summarize the current information concerning the prognostic and predictive role of SLC29A1 transporter (hENT1) expression in pancreatic cancer. Increased expression of SLC29A1 in vitro has been described as a potential critical factor determining the sensitivity of pancreatic cancer cells to gemcitabine and 5-fluorouracil, the principal cytotoxic agents used in the treatment of pancreatic cancer. The reports on the relationship between SLC29A1 expression and prognosis of patients with pancreatic cancer are currently rather conflicting. However, majority of studies on patients with resected pancreatic cancer have suggested that high SLC29A1expression may be predictive of improved survival in patients treated with gemcitabine. SLC29A1 has not been shown to represent a predictive biomarker for patients treated by 5-fluorouracil. In conclusion, potential prognostic and predictive role of SLC29A1 has been demonstrated for selected subset of patients.
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Affiliation(s)
- Beatrice Mohelnikova-Duchonova
- Department of Oncology, Palacky University Medical School and Teaching Hospital, Olomouc, Czech Republic; Biomedical Centre, Faculty of Medicine in Plzen, Charles University in Prague, Plzen, Czech Republic.
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25
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Mezil L, Berruyer-Pouyet C, Cabaud O, Josselin E, Combes S, Brunel JM, Viens P, Collette Y, Birnbaum D, Lopez M. Tumor selective cytotoxic action of a thiomorpholin hydroxamate inhibitor (TMI-1) in breast cancer. PLoS One 2012; 7:e43409. [PMID: 23028451 PMCID: PMC3445597 DOI: 10.1371/journal.pone.0043409] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/21/2012] [Accepted: 07/23/2012] [Indexed: 02/07/2023] Open
Abstract
Background Targeted therapies, associated with standard chemotherapies, have improved breast cancer care. However, primary and acquired resistances are frequently observed and the development of new concepts is needed. High-throughput approaches to identify new active and safe molecules with or without an “a priori” are currently developed. Also, repositioning already-approved drugs in cancer therapy is of growing interest. The thiomorpholine hydroxamate compound TMI-1 has been previously designed to inhibit metalloproteinase activity for the treatment of rheumatoid arthritis. We present here the repositioning of TMI-1 drug in breast cancer. Methodology/Principal Findings We tested the effect of TMI-1 on luminal, basal and ERBB2-overexpressing breast tumor cell lines and on MMTV-ERBB2/neu tumor evolution. We measured the effects on i) cell survival, ii) cell cycle, iii) extrinsic and intrinsic apoptotic pathways, iv) association with doxorubicin, docetaxel and lapatinib, v) cancer stem cells compartment. In contrast with conventional cytotoxic drugs, TMI-1 was highly selective for tumor cells and cancer stem cells at submicromolar range. All non-malignant cells tested were resistant even at high concentration. TMI-1 was active on triple negative (TN) and ERBB2-overexpressing breast tumor cell lines, and was also highly efficient on human and murine “primary” ERBB2-overexpressing cells. Treatment of transgenic MMTV-ERBB2/neu mice with 100 mg/kg/day TMI-1 alone induced tumor apoptosis, inhibiting mammary gland tumor occurrence and development. No adverse effects were noticed during the treatment. This compound had a strong synergistic effect in association with docetaxel, doxorubicin and lapatinib. We showed that TMI-1 mediates its selective effects by caspase-dependent apoptosis. TMI-1 was efficient in 34/40 tumor cell lines of various origins (ED50: 0.6 µM to 12.5 µM). Conclusions/Significance This is the first demonstration of the tumor selective cytotoxic action of a thiomorpholin hydroxamate compound. TMI-1 is a novel repositionable drug not only for the treatment of adverse prognosis breast cancers but also for other neoplasms.
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Affiliation(s)
- Lynda Mezil
- Centre de Recherche en Cancérologie de Marseille (CRCM), Aix-Marseille Univ, Marseille, France
- Inserm-U1068 (laboratoire d'oncologie moléculaire), CNRS-UMR7258, Marseille, France
- Institut Paoli-Calmettes, Marseille, France
| | - Carole Berruyer-Pouyet
- Centre d'Immunologie de Marseille-Luminy (CIML), Aix-Marseille Univ, Campus de Luminy, case 906, Marseille, France
| | - Olivier Cabaud
- Centre de Recherche en Cancérologie de Marseille (CRCM), Aix-Marseille Univ, Marseille, France
- Inserm-U1068 (laboratoire d'oncologie moléculaire), CNRS-UMR7258, Marseille, France
- Institut Paoli-Calmettes, Marseille, France
| | - Emmanuelle Josselin
- Centre de Recherche en Cancérologie de Marseille (CRCM), Aix-Marseille Univ, Marseille, France
- Inserm-U1068 (laboratoire d'oncologie moléculaire), CNRS-UMR7258, Marseille, France
- Institut Paoli-Calmettes, Marseille, France
| | - Sébastien Combes
- Centre de Recherche en Cancérologie de Marseille (CRCM), Aix-Marseille Univ, Marseille, France
- Inserm-U1068 (laboratoire iSCB), CNRS-UMR7258, Marseille, France
- Institut Paoli-Calmettes, Marseille, France
| | - Jean-Michel Brunel
- Centre de Recherche en Cancérologie de Marseille (CRCM), Aix-Marseille Univ, Marseille, France
- Inserm-U1068 (laboratoire iSCB), CNRS-UMR7258, Marseille, France
- Institut Paoli-Calmettes, Marseille, France
| | - Patrice Viens
- Centre de Recherche en Cancérologie de Marseille (CRCM), Aix-Marseille Univ, Marseille, France
- Institut Paoli-Calmettes, Marseille, France
| | - Yves Collette
- Centre de Recherche en Cancérologie de Marseille (CRCM), Aix-Marseille Univ, Marseille, France
- Inserm-U1068 (laboratoire iSCB), CNRS-UMR7258, Marseille, France
- Institut Paoli-Calmettes, Marseille, France
| | - Daniel Birnbaum
- Centre de Recherche en Cancérologie de Marseille (CRCM), Aix-Marseille Univ, Marseille, France
- Inserm-U1068 (laboratoire d'oncologie moléculaire), CNRS-UMR7258, Marseille, France
- Institut Paoli-Calmettes, Marseille, France
| | - Marc Lopez
- Centre de Recherche en Cancérologie de Marseille (CRCM), Aix-Marseille Univ, Marseille, France
- Inserm-U1068 (laboratoire d'oncologie moléculaire), CNRS-UMR7258, Marseille, France
- Institut Paoli-Calmettes, Marseille, France
- * E-mail:
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26
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Huang TCJ, Kar S, Javle M. Personalized therapy for pancreatic cancer: Myth or reality in 2010? J Gastrointest Oncol 2012; 1:24-33. [PMID: 22811802 DOI: 10.3978/j.issn.2078-6891.2010.009] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 08/01/2010] [Accepted: 09/09/2010] [Indexed: 12/30/2022] Open
Affiliation(s)
- Tzu-Chuan Jane Huang
- Division of Cancer Medicine, University of Texas M.D. Anderson Cancer Center, 1515 Holcombe Blvd, G11.3315, Unit 10, Houston, TX 77030
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27
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Wang J, Guo J, Wu S, Feng H, Sun S, Pan J, Zhang J, Beebe SJ. Synergistic effects of nanosecond pulsed electric fields combined with low concentration of gemcitabine on human oral squamous cell carcinoma in vitro. PLoS One 2012; 7:e43213. [PMID: 22927951 PMCID: PMC3426536 DOI: 10.1371/journal.pone.0043213] [Citation(s) in RCA: 37] [Impact Index Per Article: 2.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/05/2012] [Accepted: 07/20/2012] [Indexed: 01/04/2023] Open
Abstract
Treatment of cancer often involves uses of multiple therapeutic strategies with different mechanisms of action. In this study we investigated combinations of nanosecond pulsed electric fields (nsPEF) with low concentrations of gemcitabine on human oral cancer cells. Cells (Cal-27) were treated with pulse parameters (20 pulses, 100 ns in duration, intensities of 10, 30 and 60 kV/cm) and then cultured in medium with 0.01 µg/ml gemcitabine. Proliferation, apoptosis/necrosis, invasion and morphology of those cells were examined using MTT, flow cytometry, clonogenics, transwell migration and TEM assay. Results show that combination treatments of gemcitabine and nsPEFs exhibited significant synergistic activities versus individual treatments for inhibiting oral cancer cell proliferation and inducing apoptosis and necrosis. However, there was no apparent synergism for cell invasion. By this we demonstrated synergistic inhibition of Cal-27 cells in vitro by nsPEFs and gemcitabine. Synergistic behavior indicates that these two treatments have different sites of action and combination treatment allows reduced doses of gemcitabine and lower nsPEF conditions, which may provide better treatment for patients than either treatment alone while reducing systemic toxicities.
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Affiliation(s)
- Jing Wang
- Department of Oral Medicine, School of Stomatology, Lanzhou University, Lanzhou Gansu, China
| | - Jinsong Guo
- Department of Oral Medicine, School of Stomatology, Lanzhou University, Lanzhou Gansu, China
- College of Engineering, Peking University, Beijing, China
| | - Shan Wu
- College of Engineering, Peking University, Beijing, China
| | - Hongqing Feng
- College of Engineering, Peking University, Beijing, China
| | - Shujun Sun
- Academy for Advanced Interdisciplinary Studies, Peking University, Beijing, China
| | - Jie Pan
- Department of General Dentistry, School of Stomatology, Peking University, Beijing, China
| | - Jue Zhang
- College of Engineering, Peking University, Beijing, China
- Academy for Advanced Interdisciplinary Studies, Peking University, Beijing, China
| | - Stephen J. Beebe
- Frank Reidy Research Center for Bioelectrics, Old Dominion University, Norfolk, Virginia, United States of America
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28
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Stålhandske P, Wang L, Westberg S, von Euler H, Groth E, Gustafsson SA, Eriksson S, Lennerstrand J. Homogeneous assay for real-time and simultaneous detection of thymidine kinase 1 and deoxycytidine kinase activities. Anal Biochem 2012; 432:155-64. [PMID: 22902741 DOI: 10.1016/j.ab.2012.08.004] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/18/2012] [Revised: 08/03/2012] [Accepted: 08/04/2012] [Indexed: 10/28/2022]
Abstract
Measurement of thymidine kinase-1 (TK1) and deoxycytidine kinase (dCK) activity may be useful in cancer disease management. Therefore, a one-step homogeneous assay for real-time determination of TK1 and dCK was developed by combining enzyme complementation with fluorescent signal generation using primer extension and a quenched probe oligodeoxyribonucleotide system at 37 °C. Complementation, for producing dCTP and TTP from nucleoside substrates, was carried out by dTMP kinase and/or UMP/CMP kinase and nucleoside diphosphate kinase. dNTP was continuously incorporated into a fixed oligodeoxyribonucleotide primer, template, and probe system, and the fluorescent signal was generated by using the combined actions of primer extension and 5' exonuclease activity of Thermophilus aquaticus (Taq) DNA polymerase for specific relief of fluorescent quenching. Fluorescence was captured at 1-min intervals using a real-time polymerase chain reaction (PCR) instrument. A horizontal threshold line, crossing all sample relative fluorescent units (RFU) values at the level of the RFU of the blank sample at the end of the assay (i.e., 90 min), was drawn, obtaining RFU measurement data in minutes for each sample. Duplex proof of principle was demonstrated by the independent determination of different amounts of dCK and TK1 in combination. R(2) values of 0.90 were demonstrated with Prolifigen TK-REA U/L reference values obtained from pathological canine and human serum samples.
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Affiliation(s)
- Per Stålhandske
- Section of Clinical Virology, Department of Medical Sciences, Uppsala University, SE-751 85 Uppsala, Sweden.
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29
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Saiki Y, Yoshino Y, Fujimura H, Manabe T, Kudo Y, Shimada M, Mano N, Nakano T, Lee Y, Shimizu S, Oba S, Fujiwara S, Shimizu H, Chen N, Nezhad ZK, Jin G, Fukushige S, Sunamura M, Ishida M, Motoi F, Egawa S, Unno M, Horii A. DCK is frequently inactivated in acquired gemcitabine-resistant human cancer cells. Biochem Biophys Res Commun 2012; 421:98-104. [PMID: 22490663 DOI: 10.1016/j.bbrc.2012.03.122] [Citation(s) in RCA: 86] [Impact Index Per Article: 6.6] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/24/2012] [Accepted: 03/27/2012] [Indexed: 12/11/2022]
Abstract
Although gemcitabine is the most effective chemotherapeutic agent against pancreatic cancer, a growing concern is that a substantial number of patients acquire gemcitabine chemoresistance. To elucidate the mechanisms of acquisition of gemcitabine resistance, we developed gemcitabine-resistant cell lines from six human cancer cell lines; three pancreatic, one gastric, one colon, and one bile duct cancer. We first analyzed gemcitabine uptake using three paired parental and gemcitabine resistant pancreatic cancer cell lines (PK-1 and RPK-1, PK-9 and RPK-9, PK-59 and RPK-59) and found that uptake of gemcitabine was rapid. However, no DNA damage was induced in resistant cells. We further examined the microarray-based expression profiles of the cells to identify genes associated with gemcitabine resistance and found a remarkable reduction in the expression of deoxycytidine kinase (DCK). DCK is a key enzyme that activates gemcitabine by phosphorylation. Genetic alterations and expression of DCK were studied in these paired parental and derived gemcitabine-resistant cell lines, and inactivating mutations were found only in gemcitabine-resistant cell lines. Furthermore, siRNA-mediated knockdown of DCK in the parental cell lines yielded gemcitabine resistance, and introduction of DCK into gemcitabine-resistant cell lines invariably restored gemcitabine sensitivities. Mutation analyses were expanded to three other different paired cell lines, DLD-1 and RDLD-1 (colon cancer cell line), MKN-28 and RMKN-28 (gastric cancer cell line), and TFK-1 and RTFK -1 (cholangiocarcinoma cell line). We found inactivating mutations in RDLD-1 and RTFK-1 and decreased expression of DCK in RMKN-28. These results indicate that the inactivation of DCK is one of the crucial mechanisms in acquisition of gemcitabine resistance.
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Affiliation(s)
- Yuriko Saiki
- Department of Molecular Pathology, Tohoku University Graduate School of Medicine, Sendai, Japan
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30
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Candelaria M, de la Cruz-Hernandez E, Taja-Chayeb L, Perez-Cardenas E, Trejo-Becerril C, Gonzalez-Fierro A, Chavez-Blanco A, Soto-Reyes E, Dominguez G, Trujillo JE, Diaz-Chavez J, Duenas-Gonzalez A. DNA methylation-independent reversion of gemcitabine resistance by hydralazine in cervical cancer cells. PLoS One 2012; 7:e29181. [PMID: 22427797 PMCID: PMC3299634 DOI: 10.1371/journal.pone.0029181] [Citation(s) in RCA: 40] [Impact Index Per Article: 3.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/16/2011] [Accepted: 11/22/2011] [Indexed: 11/18/2022] Open
Abstract
BACKGROUND Down regulation of genes coding for nucleoside transporters and drug metabolism responsible for uptake and metabolic activation of the nucleoside gemcitabine is related with acquired tumor resistance against this agent. Hydralazine has been shown to reverse doxorubicin resistance in a model of breast cancer. Here we wanted to investigate whether epigenetic mechanisms are responsible for acquiring resistance to gemcitabine and if hydralazine could restore gemcitabine sensitivity in cervical cancer cells. METHODOLOGY/PRINCIPAL FINDINGS The cervical cancer cell line CaLo cell line was cultured in the presence of increasing concentrations of gemcitabine. Down-regulation of hENT1 & dCK genes was observed in the resistant cells (CaLoGR) which was not associated with promoter methylation. Treatment with hydralazine reversed gemcitabine resistance and led to hENT1 and dCK gene reactivation in a DNA promoter methylation-independent manner. No changes in HDAC total activity nor in H3 and H4 acetylation at these promoters were observed. ChIP analysis showed H3K9m2 at hENT1 and dCK gene promoters which correlated with hyper-expression of G9A histone methyltransferase at RNA and protein level in the resistant cells. Hydralazine inhibited G9A methyltransferase activity in vitro and depletion of the G9A gene by iRNA restored gemcitabine sensitivity. CONCLUSIONS/SIGNIFICANCE Our results demonstrate that acquired gemcitabine resistance is associated with DNA promoter methylation-independent hENT1 and dCK gene down-regulation and hyper-expression of G9A methyltransferase. Hydralazine reverts gemcitabine resistance in cervical cancer cells via inhibition of G9A histone methyltransferase.
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Affiliation(s)
- Myrna Candelaria
- Division of Clinical Research, Instituto Nacional de Cancerologia, Mexico City, Mexico
| | | | - Lucia Taja-Chayeb
- Division of Basic Research, Instituto Nacional de Cancerologia, Mexico City, Mexico
| | | | | | | | - Alma Chavez-Blanco
- Division of Basic Research, Instituto Nacional de Cancerologia, Mexico City, Mexico
| | - Ernesto Soto-Reyes
- Division of Basic Research, Instituto Nacional de Cancerologia, Mexico City, Mexico
| | - Guadalupe Dominguez
- Division of Basic Research, Instituto Nacional de Cancerologia, Mexico City, Mexico
| | - Jaenai E. Trujillo
- Division of Basic Research, Instituto Nacional de Cancerologia, Mexico City, Mexico
| | - Jose Diaz-Chavez
- Division of Basic Research, Instituto Nacional de Cancerologia, Mexico City, Mexico
| | - Alfonso Duenas-Gonzalez
- Unit of Biomedical Research in Cancer. Instituto Nacional de Cancerologia/Instituto de Investigaciones Biomedicas UNAM, Mexico City, Mexico
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31
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Senanayake TH, Warren G, Vinogradov SV. Novel anticancer polymeric conjugates of activated nucleoside analogues. Bioconjug Chem 2011; 22:1983-93. [PMID: 21863885 DOI: 10.1021/bc200173e] [Citation(s) in RCA: 31] [Impact Index Per Article: 2.2] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/28/2022]
Abstract
Inherent or therapy-induced drug resistance is a major clinical setback in cancer treatment. The extensive usage of cytotoxic nucleobases and nucleoside analogues in chemotherapy also results in the development of specific mechanisms of drug resistance, such as nucleoside transport or activation deficiencies. These drugs are prodrugs; and being converted into the active mono-, di-, and triphosphates inside cancer cells following administration, they affect nucleic acid synthesis, nucleotide metabolism, or sensitivity to apoptosis. Previously, we actively promoted the idea that the nanodelivery of active nucleotide species, e.g., 5'-triphosphates of nucleoside analogues, can enhance drug efficacy and reduce nonspecific toxicity. In this study, we report the development of a novel type of drug nanoformulations, polymeric conjugates of nucleoside analogues, which are capable of the efficient transport and sustained release of phosphorylated drugs. These drug conjugates have been synthesized, starting from cholesterol-modified mucoadhesive polyvinyl alcohol or biodegradable dextrin, by covalent attachment of nucleoside analogues through a tetraphosphate linker. Association of cholesterol moieties in aqueous media resulted in intramolecular polymer folding and the formation of small nanogel particles containing 0.5 mmol/g of a 5'-phosphorylated nucleoside analogue, e.g., 5-fluoro-2'-deoxyuridine (floxuridine, FdU), an active metabolite of anticancer drug 5-fluorouracyl (5-FU). The polymeric conjugates demonstrated rapid enzymatic release of floxuridine 5'-phosphate and much slower drug release under hydrolytic conditions (pH 1.0-7.4). Among the panel of cancer cell lines, all studied polymeric FdU-conjugates demonstrated an up to 50× increased cytotoxicity in human prostate cancer PC-3, breast cancer MCF-7, and MDA-MB-231 cells, and more than 100× higher efficacy against cytarabine-resistant human T-lymphoma (CEM/araC/8) and gemcitabine-resistant follicular lymphoma (RL7/G) cells as compared to free drugs. In the initial in vivo screening, both PC-3 and RL7/G subcutaneous tumor xenograft models showed enhanced sensitivity to sustained drug release from polymeric FdU-conjugate after peritumoral injections and significant tumor growth inhibition. All these data demonstrate a remarkable clinical potential of novel polymeric conjugates of phosphorylated nucleoside analogues, especially as new therapeutic agents against drug-resistant tumors.
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Affiliation(s)
- Thulani H Senanayake
- Center for Drug Delivery and Nanomedicine and Department of Pharmaceutical Sciences, College of Pharmacy, University of Nebraska Medical Center, Omaha, Nebraska 68198-6025, United States
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Kim MK, Jeon YK, Woo JK, Choi Y, Choi DH, Kim YH, Kim CW. The C-terminal region of Bfl-1 sensitizes non-small cell lung cancer to gemcitabine-induced apoptosis by suppressing NF-κB activity and down-regulating Bfl-1. Mol Cancer 2011; 10:98. [PMID: 21843371 PMCID: PMC3166274 DOI: 10.1186/1476-4598-10-98] [Citation(s) in RCA: 13] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/06/2011] [Accepted: 08/16/2011] [Indexed: 12/16/2022] Open
Abstract
Gemcitabine is used to treat several cancers including lung cancer. However, tumor cells often escape gemcitabine-induced cell death via various mechanisms, which include modulating bcl-2 family members and NF-κB activation. We previously reported that the C-terminal region of Bfl-1 fused with GFP (BC) is sufficient to induce apoptosis in 293T cells. In the present study, we investigated the anti-tumor effect of combined BC gene therapy and gemcitabine chemotherapy in vitro and in vivo using non-small cell lung cancer cell lines and a xenograft model. Cell lines were resistant to low dose gemcitabine (4-40 ng/ml), which induced NF-κB activation and concomitant up-regulation of Bfl-1 (an NF-κB-regulated anti-apoptotic protein). BC induced the apoptosis of A549 and H157 cells with caspase-3 activation. Furthermore, co-treatment with BC and low dose gemcitabine synergistically and efficiently induced mitochondria-mediated apoptosis in these cells. When administered alone or with low dose gemcitabine, BC suppressed NF-κB activity, inhibited the nuclear translocation of p65/relA, and down-regulated Bfl-1 expression. Furthermore, direct suppression of Bfl-1 by RNA interference sensitized cells to gemcitabine-induced cell death, suggesting that Bfl-1 importantly regulates lung cancer cell sensitivity to gemcitabine. BC and gemcitabine co-treatment also showed a strong anti-tumor effect in a nude mouse/A549 xenograft model. These results suggest that lung cancer cells become resistant to gemcitabine via NF-κB activation and the subsequent overexpression of Bfl-1, and that BC, which has both pro-apoptotic and NF-κB inhibitory effects, could be harnessed as a gene therapy to complement gemcitabine chemotherapy in non-small cell lung cancer.
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Affiliation(s)
- Min-Kyoung Kim
- Department of Pathology, Cancer Research Institute, Seoul National University College of Medicine, 28 Yeongeon-dong, Jongno-gu, Seoul 110-799, South Korea
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Geutjes EJ, Tian S, Roepman P, Bernards R. Deoxycytidine kinase is overexpressed in poor outcome breast cancer and determines responsiveness to nucleoside analogs. Breast Cancer Res Treat 2011; 131:809-18. [PMID: 21465168 DOI: 10.1007/s10549-011-1477-3] [Citation(s) in RCA: 25] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/15/2010] [Accepted: 03/19/2011] [Indexed: 11/24/2022]
Abstract
Only a minority of breast cancer patients responds to chemotherapy and we lack predictive biomarkers that help to select a patient-tailored therapy that takes into consideration the molecular heterogeneity of the cancer type. Responsiveness to the clinically important nucleoside analogs gemcitabine and decitabine may be critically determined by Deoxycytidine kinase (DCK) expression as this enzyme is required to convert the inactive prodrugs into their pharmacologically active forms. Here, we examined whether DCK is differentially expressed in breast cancer and evaluated whether DCK expression levels control responsiveness to these nucleoside analogs in vitro by experimentally modulating DCK expression levels. We examined DCK expression in gene expression data sets of breast tumors including the series of 295 consecutive patients that have been classified into low or high risk for recurrence using the MammaPrint 70 gene profile. We found that DCK is expressed at higher levels in patients having poor clinical outcome as judged by the MammaPrint assay. As such, patients that have a poor prognosis may thus be susceptible to treatment with nucleoside analogs. In support of this, we found a causal relationship between DCK levels and sensitivity to these nucleoside analogs in breast cancer cell lines. The data indicate that breast cancers that are at high risk of recurrence express higher levels of DCK, which we find to be strongly correlated to a favorable response to nucleoside analogs. The data suggest that DCK expression in breast cancer could be exploited to select patients that are likely to respond to treatment with nucleoside analogs.
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Affiliation(s)
- Ernst-Jan Geutjes
- Division of Molecular Carcinogenesis, Center for Biomedical Genetics and Cancer Genomics Center, The Netherlands Cancer Institute, Plesmanlaan 121, 1066, Amsterdam, The Netherlands
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Rivero A, Rapado I, Tomás JF, Montalbán C, de Oña R, Paz-Carreira J, Canales M, Martínez R, Sánchez-Godoy P, de Sevilla AF, de la Serna J, Martínez-López J. Relationship between deoxycytidine kinase (DCK) genotypic variants and fludarabine toxicity in patients with follicular lymphoma. Leuk Res 2011; 35:431-7. [DOI: 10.1016/j.leukres.2010.09.026] [Citation(s) in RCA: 6] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/30/2010] [Revised: 09/23/2010] [Accepted: 09/29/2010] [Indexed: 02/02/2023]
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Caron J, Lepeltier E, Reddy LH, Lepêtre-Mouelhi S, Wack S, Bourgaux C, Couvreur P, Desmaële D. Squalenoyl Gemcitabine Monophosphate: Synthesis, Characterisation of Nanoassemblies and Biological Evaluation. European J Org Chem 2011. [DOI: 10.1002/ejoc.201100036] [Citation(s) in RCA: 21] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/09/2022]
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Tanaka M, Javle M, Dong X, Eng C, Abbruzzese JL, Li D. Gemcitabine metabolic and transporter gene polymorphisms are associated with drug toxicity and efficacy in patients with locally advanced pancreatic cancer. Cancer 2010; 116:5325-35. [PMID: 20665488 PMCID: PMC2966859 DOI: 10.1002/cncr.25282] [Citation(s) in RCA: 70] [Impact Index Per Article: 4.7] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/16/2022]
Abstract
BACKGROUND It has not been well established whether genetic variations can be biomarkers for clinical outcome of gemcitabine therapy. The purpose of this study was to identify single nucleotide polymorphisms (SNPs) of gemcitabine metabolic and transporter genes that are associated with toxicity and efficacy of gemcitabine-based therapy in patients with locally advanced pancreatic cancer. METHODS The authors evaluated 17 SNPs of the CDA,dCK, DCTD, RRM1, hCNT1-3, and hENT1 genes in 149 patients with locally advanced pancreatic cancer who underwent gemcitabine-based chemoradiotherapy. The association of genotypes with neutropenia, tumor response to therapy, overall survival, and progression-free survival (PFS) was analyzed by logistic regression, log-rank test, Kaplan-Meier plot, and Cox proportional hazards regression. RESULTS The CDA A-76C, dCK C-1205T, RRM1 A33G, and hENT1 C913T genotypes were significantly associated with grade 3 to 4 neutropenia (P = .020, .015, .003, and .017, respectively).The CDA A-76C and hENT1 A-201G genotypes were significantly associated with tumor response to therapy (P = .017 and P = .019). A combined genotype effect of CDA A-76C, RRM1 A33G, RRM1 C-27A, and hENT1 A-201G on PFS was observed. Patients carrying 0 to 1 (n = 64), 2 (n = 50), or 3 to 4 (n = 17) at-risk genotypes had median PFS times of 8.3, 6.0, and 4.2 months, respectively (P = .002). CONCLUSIONS The results indicated that some polymorphic variations of drug metabolic and transporter genes may be potential biomarkers for clinical outcome of gemcitabine-based therapy in patients with locally advanced pancreatic cancer.
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Affiliation(s)
- Motofumi Tanaka
- Department of Gastrointestinal Medical Oncology, The University of Texas M. D. Anderson Cancer Center, Houston, TX, USA.
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Galmarini CM, Warren G, Senanayake MT, Vinogradov SV. Efficient overcoming of drug resistance to anticancer nucleoside analogs by nanodelivery of active phosphorylated drugs. Int J Pharm 2010; 395:281-9. [PMID: 20580798 PMCID: PMC2904614 DOI: 10.1016/j.ijpharm.2010.05.028] [Citation(s) in RCA: 23] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/22/2010] [Revised: 05/11/2010] [Accepted: 05/15/2010] [Indexed: 12/12/2022]
Abstract
One of the major problems in cancer chemotherapy is the fast development of drug resistance to most anticancer therapeutics. Thus, an important cause of the eventual decline in clinical efficacy of cytotoxic nucleoside analogs was the selection of resistant cancer cells with deficiencies in the expression of nucleoside transporters or nucleoside-activating kinases. Here, we present an efficient strategy of overcoming this type of drug resistance by tumor-specific delivery of nanogel-encapsulated active triphosphates of nucleoside analogs (NATP). The small particles of biodegradable cationic nanogels loaded with anionic NATP efficiently interacted with cancer cells and released active drug compounds into the cytoplasm. The potential of novel drug formulations was evaluated in the nucleoside transport-deficient (CEM/araC/C8) or nucleoside activation-deficient (RL7/G) lymphogenic cancer cells. Compared to nucleoside analogs, NATP-loaded nanogels demonstrated increased cytotoxicity, reducing the drug resistance index 250- to 900-fold in CEM/araC/C8 cells and 70- to 100-fold in RL7/G cells. The strong cytotoxic effect of nanoformulations was accompanied by characteristic cell cycle perturbations, usually observed in drug-treated sensitive cells, and resulted in the induction of apoptosis in all studied drug-resistant cells. Efficient cellular accumulation of nanogels and the consequent increase in intracellular levels of NATP were found to be the major factors determining cytotoxic efficacy of nanoformulations. Decoration of nanogels with multiple molecules of tumor lymphatic-specific peptide (LyP1) enhanced the binding efficacy of nanocarriers with lymphogenic cancer cells. The targeted nanoformulation of activated gemcitabine (LyP1-NG-dFdCTP), when injected in subcutaneous RL7/G xenograft tumor model, demonstrated 2-fold more efficient tumor growth inhibition than gemcitabine at a higher dose. Nanogel-drug formulations exhibited no systemic toxicity during the treatment, hence extending the versatility of nucleoside analogs in the treatment of drug-resistant lymphogenic tumors.
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MESH Headings
- Animals
- Antimetabolites, Antineoplastic/chemistry
- Antimetabolites, Antineoplastic/metabolism
- Antimetabolites, Antineoplastic/pharmacology
- Apoptosis/drug effects
- Biological Transport
- Cell Cycle/drug effects
- Cell Line, Tumor
- Cell Survival/drug effects
- Chemistry, Pharmaceutical
- Cytarabine/chemistry
- Cytarabine/metabolism
- Cytarabine/pharmacology
- Deoxycytidine/analogs & derivatives
- Deoxycytidine/chemistry
- Deoxycytidine/metabolism
- Deoxycytidine/pharmacology
- Dose-Response Relationship, Drug
- Drug Carriers
- Drug Resistance
- Drug Resistance, Neoplasm
- Gels
- Humans
- Inhibitory Concentration 50
- Mice
- Mice, Inbred BALB C
- Mice, Nude
- Nanoparticles
- Nanotechnology
- Phosphorylation
- Protein Tyrosine Phosphatase, Non-Receptor Type 22/metabolism
- Technology, Pharmaceutical/methods
- Time Factors
- Tumor Burden/drug effects
- Xenograft Model Antitumor Assays
- Gemcitabine
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Affiliation(s)
- Carlos M. Galmarini
- ENS-CNRS UMR 5239; UFR Lyon-Sud, 165 Chemin du Grand Revoyet, BP12 – 69921, Oullins, France
| | - Galya Warren
- Department of Pharmaceutical Sciences, College of Pharmacy, and Center for Drug Delivery and Nanomedicine, University of Nebraska Medical Center, Omaha, NE 68198-6025, USA.
| | - Madapathage T. Senanayake
- Department of Pharmaceutical Sciences, College of Pharmacy, and Center for Drug Delivery and Nanomedicine, University of Nebraska Medical Center, Omaha, NE 68198-6025, USA.
| | - Serguei V. Vinogradov
- Department of Pharmaceutical Sciences, College of Pharmacy, and Center for Drug Delivery and Nanomedicine, University of Nebraska Medical Center, Omaha, NE 68198-6025, USA.
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Deville-Bonne D, El Amri C, Meyer P, Chen Y, Agrofoglio LA, Janin J. Human and viral nucleoside/nucleotide kinases involved in antiviral drug activation: structural and catalytic properties. Antiviral Res 2010; 86:101-20. [PMID: 20417378 DOI: 10.1016/j.antiviral.2010.02.001] [Citation(s) in RCA: 94] [Impact Index Per Article: 6.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/03/2009] [Revised: 01/31/2010] [Accepted: 02/01/2010] [Indexed: 12/11/2022]
Abstract
Antiviral nucleoside and nucleotide analogs, essential for the treatment of viral infections in the absence of efficient vaccines, are prodrug forms of the active compounds that target the viral DNA polymerase or reverse transcriptase. The activation process requires several successive phosphorylation steps catalyzed by different kinases, which are present in the host cell or encoded by some of the viruses. These activation reactions often are rate-limiting steps and are thus open to improvement. We review here the structural and enzymatic properties of the enzymes that carry out the activation of analogs used in therapy against human immunodeficiency virus and against DNA viruses such as hepatitis B, herpes and poxviruses. Four major classes of drugs are considered: thymidine analogs, non-natural L-nucleosides, acyclic nucleoside analogs and acyclic nucleoside phosphonate analogs. Their efficiency as drugs depends both on the low specificity of the viral polymerase that allows their incorporation into DNA, but also on the ability of human/viral kinases to provide the activated triphosphate active forms at a high concentration at the right place. Two distinct modes of action are considered, depending on the origin of the kinase (human or viral). If the human kinases are house-keeping enzymes that belong to the metabolic salvage pathway, herpes and poxviruses encode for related enzymes. The structures, substrate specificities and catalytic properties of each of these kinases are discussed in relation to drug activation.
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Affiliation(s)
- Dominique Deville-Bonne
- Enzymologie Moléculaire et Fonctionnelle, UR4 Université Pierre et Marie Curie, 7 quai St Bernard, 75252 Paris Cedex 05, France.
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Okazaki T, Javle M, Tanaka M, Abbruzzese JL, Li D. Single nucleotide polymorphisms of gemcitabine metabolic genes and pancreatic cancer survival and drug toxicity. Clin Cancer Res 2009; 16:320-9. [PMID: 20028759 DOI: 10.1158/1078-0432.ccr-09-1555] [Citation(s) in RCA: 81] [Impact Index Per Article: 5.1] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/06/2023]
Abstract
PURPOSE To show whether single nucleotide polymorphisms (SNP) of drug metabolic genes were associated with toxicity of 2',2'-difluoro 2'-deoxycytidine (gemcitabine)-based chemoradiotherapy and overall survival (OS) of patients with pancreatic cancer. EXPERIMENTAL DESIGN We evaluated 17 SNPs of the CDA, dCK, DCTD, RRM1, hCNT1, hCNT2, hCNT3, and hENT1 genes in 154 patients with potentially resectable pancreatic adenocarcinoma who were enrolled in clinical trials at The University of Texas M.D. Anderson Cancer Center (Houston, TX) from February 1999 to January 2006, with follow-up until April 2009. Patients received neoadjuvant concurrent gemcitabine and radiation therapy with or without gemcitabine-cisplatin induction therapy. The association of genotypes with toxicity or OS was tested, respectively, by logistic regression and Cox regression analysis. RESULTS None of the 17 SNPs, individually, had a significant association with OS. A combined genotype effect of CDA A-76C, dCK C-1205T, DCTD T-47C, hCNT3 C-69T, hENT1 T-549C, and hENT1 C913T on OS was observed. Patients carrying 0 to 1 (n = 43), 2 to 3 (n = 77), or 4 to 6 (n = 30) variant alleles had median survival time of 31.5, 21.4, and 17.5 months, respectively. The hazard ratio of dying was 1.71 (95% confidence interval, 1.06-2.76) and 3.16 (95% confidence interval, 1.77-5.63) for patients carrying two to three or four to six at-risk genotypes (P = 0.028 and P < 0.001), respectively, after adjusting for clinical predictors. CDA C111T, dCK C-1205T, dCK A9846G, and hCNT3 A25G, individually and jointly, had a significant association with neutropenia toxicity. CONCLUSIONS These observations suggest that polymorphic variations of drug metabolic genes were associated with toxicity of gemcitabine-based therapy and OS of patients with resectable pancreatic cancer.
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Affiliation(s)
- Taro Okazaki
- Department of Gastrointestinal Medical Oncology, The University of Texas MD Anderson Cancer Center, Houston, Texas 77030, USA
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Candelaria M, de la Cruz-Hernández E, Pérez-Cárdenas E, Trejo-Becerril C, Gutiérrez-Hernández O, Dueñas-González A. Pharmacogenetics and pharmacoepigenetics of gemcitabine. Med Oncol 2009; 27:1133-43. [PMID: 19902390 DOI: 10.1007/s12032-009-9349-y] [Citation(s) in RCA: 19] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/08/2009] [Accepted: 10/21/2009] [Indexed: 12/19/2022]
Abstract
Gemcitabine (2',2'-difluoro 2'deoxycytidine, dFdC) is an analog of cytosine with distinctive pharmacological properties and a wide antitumor-activity spectrum. The pharmacological characteristics of gemcitabine are unique because two main classes of genes are essential for its antitumor effects: membrane transporter protein-coding genes, whose products are responsible for drug intracellular uptake, as well as enzyme-coding genes, which catalyze its activation and inactivation. The study of the pharmacogenetics and pharmacoepigenetics of these two gene classes is greatly required to optimize the drug's therapeutic use in cancer. This review aims to provide an update of genetic and epigenetic bases that may account for interindividual variation in therapeutic outcome exhibited by gemcitabine.
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Affiliation(s)
- M Candelaria
- Division of Clinical Research, Instituto Nacional de Cancerología (INCan), Mexico City, Mexico
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Gemcitabine-based chemogene therapy for pancreatic cancer using Ad-dCK::UMK GDEPT and TS/RR siRNA strategies. Neoplasia 2009; 11:637-50. [PMID: 19568409 DOI: 10.1593/neo.81686] [Citation(s) in RCA: 63] [Impact Index Per Article: 3.9] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/02/2009] [Revised: 03/26/2009] [Accepted: 04/20/2009] [Indexed: 11/18/2022] Open
Abstract
Gemcitabine is a first-line agent for advanced pancreatic cancer therapy. However, its efficacy is often limited by its poor intracellular metabolism and chemoresistance. To exert its antitumor activity, gemcitabine requires to be converted to its active triphosphate form. Thus, our aim was to improve gemcitabine activation using gene-directed enzyme prodrug therapy based on gemcitabine association with the deoxycytidine kinase::uridine monophosphate kinase fusion gene (dCK::UMK) and small interference RNA directed against ribonucleotide reductase (RRM2) and thymidylate synthase (TS). In vitro, cytotoxicity was assessed by 3-[4,5-dimethylthiazol-2-yl]-3,5-diphenyl tetrazolium bromide and [(3)H]thymidine assays. Apoptosis-related gene expression and activity were analyzed by reverse transcription-polymerase chain reaction, Western blot, and ELISA. For in vivo studies, the treatment efficacy was evaluated on subcutaneous and orthotopic pancreatic tumor models. Our data indicated that cell exposure to gemcitabine induced a down-regulation of dCK expression and up-regulation of TS and RR expression in Panc1-resistant cells when compared with BxPc3- and HA-hpc2-sensitive cells. The combination of TS/RRM2 small interference RNA with Ad-dCK::UMK induced a 40-fold decrease of gemcitabine IC(50) in Panc1 cells. This strong sensitization was associated to apoptosis induction with a remarkable increase in TRAIL expression and a diminution of gemcitabine-induced nuclear factor-kappaB activity. In vivo, the gemcitabine-based tritherapy strongly reduced tumor volumes and significantly prolonged mice survival. Moreover, we observed an obvious increase of apoptosis and decrease of cell proliferation in tumors receiving the tritherapy regimens. Together, these findings suggest that simultaneous TS/RRM2-gene silencing and dCK::UMK gene overexpression markedly improved gemcitabine's therapeutic activity. Clearly, this combined strategy warrants further investigation.
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Reduced ribavirin antiviral efficacy via nucleoside transporter-mediated drug resistance. J Virol 2009; 83:4538-47. [PMID: 19244331 DOI: 10.1128/jvi.02280-08] [Citation(s) in RCA: 49] [Impact Index Per Article: 3.1] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/11/2022] Open
Abstract
Treatment for hepatitis C virus infection currently consists of pegylated interferon and ribavirin (RBV), a nucleoside analog. Although RBV clearly plays a role in aiding the treatment response, its antiviral mechanism is unclear. Regardless of the specific mechanism of RBV, we hypothesize that differences in levels of cellular uptake of RBV may affect antiviral efficacy and treatment success and that cells may become RBV resistant through reduced uptake. We monitored RBV uptake in various cell lines and determined the effect of uptake capacity on viral replication. RBV-resistant cells demonstrated reduced RBV uptake and increased growth of a model RNA virus, poliovirus, in the presence of RBV. Overexpression of equilibrative nucleoside transporter 1 (ENT1) or concentrative nucleoside transporter 3 (CNT3) increased RBV uptake in RBV-sensitive cell lines and restored the uptake defect in most RBV-resistant cell lines. However, CNT3 is not expressed in Huh-7 liver cells, and inhibition of concentrative transport did not affect RBV uptake. Blocking equilibrative transport using the inhibitor nitrobenzylmercaptopurine riboside recapitulated the RBV-resistant phenotype in RBV-sensitive cell lines, with a reduction in RBV uptake and increased poliovirus growth. Taken together, these results indicate that RBV uptake is restricted primarily to ENT1 in the cell lines examined. Interestingly, some RBV-resistant cell lines may compensate for reduced ENT1-mediated nucleoside uptake by increasing the activity of an alternative nucleoside transporter, ENT2. It is possible that RBV uptake affects the antiviral treatment response, either through natural differences in patients or through acquired resistance.
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43
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Noninvasive prediction of tumor responses to gemcitabine using positron emission tomography. Proc Natl Acad Sci U S A 2009; 106:2847-52. [PMID: 19196993 DOI: 10.1073/pnas.0812890106] [Citation(s) in RCA: 43] [Impact Index Per Article: 2.7] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/18/2022] Open
Abstract
Gemcitabine (2',2'-difluorodeoxycytidine, dFdC) and cytosine arabinoside (cytarabine, ara-C) represent a class of nucleoside analogs used in cancer chemotherapy. Administered as prodrugs, dFdC and ara-C are transported across cell membranes and are converted to cytotoxic derivatives through consecutive phosphorylation steps catalyzed by endogenous nucleoside kinases. Deoxycytidine kinase (DCK) controls the rate-limiting step in the activation cascade of dFdC and ara-C. DCK activity varies significantly among individuals and across different tumor types and is a critical determinant of tumor responses to these prodrugs. Current assays to measure DCK expression and activity require biopsy samples and are prone to sampling errors. Noninvasive methods that can detect DCK activity in tumor lesions throughout the body could circumvent these limitations. Here, we demonstrate an approach to detecting DCK activity in vivo by using positron emission tomography (PET) and (18)F-labeled 1-(2'-deoxy-2'-fluoroarabinofuranosyl) cytosine] ([(18)F]FAC), a PET probe recently developed by our group. We show that [(18)F]FAC is a DCK substrate with an affinity similar to that of dFdC. In vitro, accumulation of [(18)F]FAC in murine and human leukemia cell lines is critically dependent on DCK activity and correlates with dFdC sensitivity. In mice, [(18)F]FAC accumulates selectively in DCK-positive vs. DCK-negative tumors, and [(18)F]FAC microPET scans can predict responses to dFdC. We suggest that [(18)F]FAC PET might be useful for guiding treatment decisions in certain cancers by enabling individualized chemotherapy.
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Kocabas NA, Aksoy P, Pelleymounter LL, Moon I, Ryu JS, Gilbert JA, Salavaggione OE, Eckloff BW, Wieben ED, Yee V, Weinshilboum RM, Ames MM. Gemcitabine pharmacogenomics: deoxycytidine kinase and cytidylate kinase gene resequencing and functional genomics. Drug Metab Dispos 2008; 36:1951-9. [PMID: 18556440 PMCID: PMC3066084 DOI: 10.1124/dmd.108.020925] [Citation(s) in RCA: 21] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/22/2022] Open
Abstract
Gemcitabine and other cytidine antimetabolites require metabolic activation by phosphorylation. Deoxycytidine kinase (DCK) and cytidine monophosphate kinase (CMPK) catalyze these reactions. We have applied a genotype-to-phenotype strategy to study DCK and CMPK pharmacogenomics. Specifically, we resequenced DCK and CMPK using 240 DNA samples, 60 each from African-American, Caucasian-American, Han Chinese-American, and Mexican-American subjects. We observed 28 DCK polymorphisms and 28 polymorphisms in CMPK, 33 of which were novel. Expression in COS-1 cells showed that variant allozyme enzyme activities ranged from 32 to 105% of the wild type (WT) for DCK and from 78 to 112% of WT for CMPK--with no significant differences in apparent K(m) values for either enzyme except for a DCK Val24/Ser122 double variant allozyme. Relative levels of DCK and CMPK immunoreactive protein in the COS-1 cells paralleled relative levels of enzyme activity and were significantly correlated for DCK (R(p) = 0.89, P = 0.0004) but not for CMPK (R(p) = 0.82, P = 0.095). The results of an analysis of DCK and CMPK structural models were compatible with the observed functional consequences of sequence alterations in variant allozymes. We also confirmed that the CMPK protein expressed in COS-1 cells and in a rabbit reticulocyte lysate was 196 rather than 228 amino acids in length. In summary, we determined common sequence variations in DCK and CMPK and systematically evaluated their functional implications. These gene sequence differences may contribute to variations in the metabolic activation of gemcitabine and other cytidine antimetabolites.
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Affiliation(s)
- Neslihan Aygun Kocabas
- Department of Molecular Pharmacology and Experimental Therapeutics, Mayo Clinic, Rochester, Minnesota 55905, USA
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Reddy LH, Dubernet C, Mouelhi SL, Marque PE, Desmaele D, Couvreur P. A new nanomedicine of gemcitabine displays enhanced anticancer activity in sensitive and resistant leukemia types. J Control Release 2007; 124:20-7. [PMID: 17878060 DOI: 10.1016/j.jconrel.2007.08.018] [Citation(s) in RCA: 100] [Impact Index Per Article: 5.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/02/2007] [Revised: 07/24/2007] [Accepted: 08/17/2007] [Indexed: 11/22/2022]
Abstract
Gemcitabine is an anticancer nucleoside analogue active against various solid tumors. However, it possesses important drawbacks like a poor biological half-life and the induction of resistance. With the objective of overcoming the above drawbacks, we designed a new nanomedicine of gemcitabine and studied its anticancer efficacy against leukemia at preclinic. Gemcitabine has been covalently coupled with 1,1',2-tris-nor-squalenic acid to obtain the new anticancer nanomedicine 4-(N)-Tris-nor-squalenoyl-gemcitabine (SQdFdC NA). The SQdFdC NA exhibited, in comparison to gemcitabine, 3.26- and 3.22-folds higher cytotoxicity respectively, in murine resistant leukemia L1210 10K cells and in human leukemia resistant cell line CEM/ARAC8C. Following intravenous treatment of murine aggressive metastatic leukemia L1210 wt bearing mice, the SQdFdC NA caused significant increase in survival time compared to gemcitabine and also led to long-term survivals, which was not the case after gemcitabine treatment. This was attributed to significantly higher deposition of SQdFdC NA in spleen and liver (P<0.05), the major metastatic organs. In comparison to gemcitabine, SQdFdC NA displayed greater ability to induce S-phase arrest of the cancer cells followed by increased apoptotic induction. Interestingly, like gemcitabine, SQdFdC NA didn't induce appreciable differences in blood parameters even at doses higher than those used for anticancer evaluation. The preclinical data obtained in vitro and in vivo with SQdFdC NA demonstrate that this nanomedicine represents a new therapeutic system for the effective treatment of leukemia.
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Affiliation(s)
- L Harivardhan Reddy
- Université Paris-Sud XI, Faculté de Pharmacie, UMR CNRS 8612, IFR 141, 92296 Châtenay-Malabry Cedex, France
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Pham NA, Tsao MS, Cao P, Hedley DW. Dissociation of gemcitabine sensitivity and protein kinase B signaling in pancreatic ductal adenocarcinoma models. Pancreas 2007; 35:e16-26. [PMID: 17895832 DOI: 10.1097/mpa.0b013e318095a747] [Citation(s) in RCA: 12] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 12/12/2022]
Abstract
OBJECTIVE To understand the impact of protein kinase B (PKB; Akt) signaling on growth and protection from apoptosis in pancreatic ductal adenocarcinoma models demonstrating differences in PKB activity. METHODS Gemcitabine sensitivity was investigated in a panel of cell lines, characterized by differences in levels of activated PKB. Suppression of PKB activity was achieved with an inhibitor of phosphatidylinositol 3-kinase (wortmannin) and silencing RNA. RESULTS Enhanced gemcitabine (2',2'-difluoro-2'-deoxycytidine)-induced cytotoxicity in vitro was achieved with suppression of high PKB activity with wortmannin in BxPC-3, PK-1, and PK-8 cells and silencing RNA targeted to total PKB, rather than PKBbeta, in PANC-1 cells. Opposite to gemcitabine sensitivity levels in vitro, the growth of PANC-1 xenografts was inhibited with gemcitabine treatment, whereas BxPC-3 became drug resistant. Monolayer cell cultures reestablished from solid tumors behaved similarly to original cultures, suggesting that the tumor microenvironment has a critical role in determining drug sensitivity. A comparison of transcript profiles of the models indicated that PKB signaling might be modulated by a number of pathways responsive to the tumor hypoxia microenvironment. CONCLUSIONS These results suggested that gemcitabine efficacy involving the PKB pathway depends on PKB activity, its mechanisms of enhanced activity, as well as its function in a signaling network.
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MESH Headings
- Androstadienes/pharmacology
- Animals
- Antimetabolites, Antineoplastic/pharmacokinetics
- Antimetabolites, Antineoplastic/therapeutic use
- Apoptosis/drug effects
- Carcinoma, Pancreatic Ductal/drug therapy
- Carcinoma, Pancreatic Ductal/enzymology
- Carcinoma, Pancreatic Ductal/genetics
- Carcinoma, Pancreatic Ductal/pathology
- Cell Hypoxia
- Cell Line, Tumor/enzymology
- Cell Line, Tumor/transplantation
- Deoxycytidine/analogs & derivatives
- Deoxycytidine/pharmacokinetics
- Deoxycytidine/therapeutic use
- Drug Resistance, Neoplasm/drug effects
- Drug Resistance, Neoplasm/physiology
- Gene Expression Profiling
- Humans
- Male
- Mice
- Mice, SCID
- Neoplasm Proteins/genetics
- Neoplasm Proteins/physiology
- Pancreatic Neoplasms/drug therapy
- Pancreatic Neoplasms/enzymology
- Pancreatic Neoplasms/genetics
- Pancreatic Neoplasms/pathology
- Phosphoinositide-3 Kinase Inhibitors
- Proto-Oncogene Proteins c-akt/genetics
- Proto-Oncogene Proteins c-akt/physiology
- RNA, Small Interfering/pharmacology
- Signal Transduction/physiology
- Wortmannin
- Xenograft Model Antitumor Assays
- Gemcitabine
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Affiliation(s)
- Nhu-An Pham
- Ontario Cancer Institute and Princess Margaret Hospital, University Health Network, Toronto, Ontario, Canada
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Liu X, Zhou B, Mi S, Xue L, Shih J, Lee J, Chau J, Un F, Yen Y. An increase of cytochrome C oxidase mediated disruption of gemcitabine incorporation into DNA in a resistant KB clone. Biochem Pharmacol 2007; 73:1927-38. [PMID: 17428446 PMCID: PMC1950577 DOI: 10.1016/j.bcp.2007.03.014] [Citation(s) in RCA: 5] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/10/2007] [Revised: 03/14/2007] [Accepted: 03/15/2007] [Indexed: 12/15/2022]
Abstract
Mechanistic aberrations leading to Gemcitabine (2',2'-dFdCyd,2,2-difluorodeoxycytidine, Gem) resistance may include alteration in its transport, metabolism and incorporation into DNA. To explore the mechanism of Gem resistance, the restriction fragment differential display PCR (RFDD-PCR) was employed to compare the mRNA expression patterns of KBGem (Gem resistant), KBHURs (hydroxyurea resistant) and KBwt (parental KB cell). Nine gene fragments were overexpressed specifically in the KBGem clone. Sequencing and BLAST results showed that three fragments represent cytochrome C oxidase (CCOX, respiration complex IV) subunit III (CCOX3). The cDNA microarray confirmed that the mRNAs of CCOX and ATP synthase subunits were upregulated in KBGem as compared to KBwt and KBHURs. The increase in CCOX1 protein and activity led to the increase of free ATP concentration, which is consistent with the gene expression profile of KBGem. Furthermore, the sensitivity to Gem could be reversed by sodium azide, a CCOX inhibitor. Following the treatment of sodium azide, the cellular accumulation of [3H]-Gem increased in a dose (of azide)-dependent manner, which is associated with increase of [3H]-Gem incorporation into DNA in KBGem. In summary, an increase of CCOX activity and free ATP level may reduce the transport, metabolism and DNA incorporation of Gem, resulting in Gem resistance.
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Affiliation(s)
- Xiyong Liu
- Department of Clinical & Molecular Pharmacology, City of Hope National Medical Center, Duarte, CA 19010
| | - Bingsen Zhou
- Department of Clinical & Molecular Pharmacology, City of Hope National Medical Center, Duarte, CA 19010
| | - Shu Mi
- Department of Clinical & Molecular Pharmacology, City of Hope National Medical Center, Duarte, CA 19010
| | - Lijun Xue
- Department of Clinical & Molecular Pharmacology, City of Hope National Medical Center, Duarte, CA 19010
| | - Jennifer Shih
- Department of Clinical & Molecular Pharmacology, City of Hope National Medical Center, Duarte, CA 19010
| | - Janice Lee
- Department of Clinical & Molecular Pharmacology, City of Hope National Medical Center, Duarte, CA 19010
| | - Jennifer Chau
- Department of Clinical & Molecular Pharmacology, City of Hope National Medical Center, Duarte, CA 19010
| | - Frank Un
- Department of Clinical & Molecular Pharmacology, City of Hope National Medical Center, Duarte, CA 19010
| | - Yun Yen
- Department of Clinical & Molecular Pharmacology, City of Hope National Medical Center, Duarte, CA 19010
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Zhang J, Visser F, King KM, Baldwin SA, Young JD, Cass CE. The role of nucleoside transporters in cancer chemotherapy with nucleoside drugs. Cancer Metastasis Rev 2007; 26:85-110. [PMID: 17345146 DOI: 10.1007/s10555-007-9044-4] [Citation(s) in RCA: 184] [Impact Index Per Article: 10.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 12/21/2022]
Abstract
Nucleoside analogs are important components of treatment regimens for various malignancies. Nucleoside-specific membrane transporters mediate plasma membrane permeation of physiologic nucleosides and most nucleoside analogs, for which the initial event is cellular conversion of nucleosides to active agents. Understanding of the roles of nucleoside transporters in nucleoside drug toxicity and resistance will provide opportunities for potentiating anticancer efficacy and avoiding resistance. Because transportability is a possible determinant of toxicity and resistance of many nucleoside analogs, nucleoside transporter abundance might be a prognostic marker to assess drug resistance. Elucidation of the structural determinants of nucleoside analogs for interaction with transporter proteins as well as the structural features of transporter proteins required for permeant interaction and translocation will lead to "transportability guidelines" for the rational design and therapeutic application of nucleoside analogs as anticancer drugs. It should eventually be possible to develop clinical assays that predict sensitivity and/or resistance to nucleoside anti-cancer drugs and thus to identify those patient populations that will most likely benefit from optimal nucleoside analog treatments. This review discusses recent results from structure/function studies of human nucleoside transporters, the role of nucleoside transport processes in the cytotoxicity and resistance of several anticancer nucleoside analogs and strategies to improve the nucleoside transporter-related anticancer effects of nucleoside analogs.
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Affiliation(s)
- Jing Zhang
- Membrane Protein Research Group, University of Alberta, Edmonton, AB, Canada
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Nakahira S, Nakamori S, Tsujie M, Takahashi Y, Okami J, Yoshioka S, Yamasaki M, Marubashi S, Takemasa I, Miyamoto A, Takeda Y, Nagano H, Dono K, Umeshita K, Sakon M, Monden M. Involvement of ribonucleotide reductase M1 subunit overexpression in gemcitabine resistance of human pancreatic cancer. Int J Cancer 2007; 120:1355-63. [PMID: 17131328 DOI: 10.1002/ijc.22390] [Citation(s) in RCA: 161] [Impact Index Per Article: 8.9] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/12/2022]
Abstract
Pancreatic cancer is the most lethal of all solid tumors partially because of its chemoresistance. Although gemcitabine is widely used as a first selected agent for the treatment of this disease despite low response rate, molecular mechanisms of gemcitabine resistance in pancreatic cancer still remain obscure. The aim of this study is to elucidate the mechanisms of gemcitabine resistance. The 81-fold gemcitabine resistant variant MiaPaCa2-RG was selected from pancreatic cancer cell line MiaPaCa2. By microarray analysis between MiaPaCa2 and MiaPaCa2-RG, 43 genes (0.04%) were altered expression of more than 2-fold. The most upregulated gene in MiaPaCa2-RG was ribonucleotide reductase M1 subunit (RRM1) with 4.5-fold up-regulation. Transfection with RRM1-specific RNAi suppressed more than 90% of RRM1 mRNA and protein expression. After RRM1-specific RNAi transfection, gemcitabine chemoresistance of MiaPaCa2-RG was reduced to the same level of MiaPaCa2. The 18 recurrent pancreatic cancer patients treated by gemcitabine were divided into 2 groups by RRM1 levels. There was a significant association between gemcitabine response and RRM1 expression (p = 0.018). Patients with high RRM1 levels had poor survival after gemcitabine treatment than those with low RRM1 levels (p = 0.016). RRM1 should be a key molecule in gemcitabine resistance in human pancreatic cancer through both in vitro and clinical models. RRM1 may have the potential as predictor and modulator of gemcitabine treatment.
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Affiliation(s)
- Shin Nakahira
- Department of Surgery and Clinical Oncology, Graduate School of Medicine, Osaka University, Suita, Osaka, Japan
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50
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Nakano Y, Tanno S, Koizumi K, Nishikawa T, Nakamura K, Minoguchi M, Izawa T, Mizukami Y, Okumura T, Kohgo Y. Gemcitabine chemoresistance and molecular markers associated with gemcitabine transport and metabolism in human pancreatic cancer cells. Br J Cancer 2007; 96:457-63. [PMID: 17224927 PMCID: PMC2360025 DOI: 10.1038/sj.bjc.6603559] [Citation(s) in RCA: 248] [Impact Index Per Article: 13.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/04/2023] Open
Abstract
To identify predictive molecular markers for gemcitabine resistance, we investigated changes in the expression of four genes associated with gemcitabine transport and metabolism during the development of acquired gemcitabine resistance of pancreatic cancer cell lines. The expression levels of human equilibrative nucleoside transporter-1 (hENT1), deoxycytidine kinase (dCK), RRM1, and RRM2 mRNA were analysed by real-time light cycler-PCR in various subclones during the development of acquired resistance to gemcitabine. Real-time light cycler-PCR demonstrated that the expression levels of either RRM1 or RRM2 progressively increased during the development of gemcitabine resistance. Expression of dCK was slightly increased in cells resistant to lower concentrations of gemcitabine, but was decreased below the undetectable level in higher concentration-resistant subclones. Expression of hENT1 was increased in the development of gemcitabine resistance. As acquired resistance to gemcitabine seems to correlate with the balance of these four factors, we calculated the ratio of hENT1 × dCK/RRM1 × RRM2 gene expression in gemcitabine-resistant subclones. The ratio of gene expression decreased progressively with development of acquired resistance in gemcitabine-resistant subclones. Furthermore, the expression ratio significantly correlated with gemcitabine sensitivity in eight pancreatic cancer cell lines, whereas no single gene expression level correlated with the sensitivity. These results suggest that the sensitivity of pancreatic cancer cells to gemcitabine is determined by the ratio of four factors involved in gemcitabine transport and metabolism. The ratio of the four gene expression levels correlates with acquired gemcitabine-resistance in pancreatic cancer cells, and may be useful as a predictive marker for the efficacy of gemcitabine therapy in pancreatic cancer patients.
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Affiliation(s)
- Y Nakano
- Third Department of Internal Medicine, Asahikawa Medical College, Asahikawa, Japan
| | - S Tanno
- Department of General Medicine, Asahikawa Medical College, Asahikawa, Japan
- E-mail:
| | - K Koizumi
- Third Department of Internal Medicine, Asahikawa Medical College, Asahikawa, Japan
| | - T Nishikawa
- Third Department of Internal Medicine, Asahikawa Medical College, Asahikawa, Japan
| | - K Nakamura
- Third Department of Internal Medicine, Asahikawa Medical College, Asahikawa, Japan
| | - M Minoguchi
- Third Department of Internal Medicine, Asahikawa Medical College, Asahikawa, Japan
| | - T Izawa
- Third Department of Internal Medicine, Asahikawa Medical College, Asahikawa, Japan
| | - Y Mizukami
- Third Department of Internal Medicine, Asahikawa Medical College, Asahikawa, Japan
| | - T Okumura
- Department of General Medicine, Asahikawa Medical College, Asahikawa, Japan
| | - Y Kohgo
- Third Department of Internal Medicine, Asahikawa Medical College, Asahikawa, Japan
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