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Manohar SM, Joshi KS. Molecular Pharmacology of Multitarget Cyclin-Dependent Kinase Inhibitors in Human Colorectal Carcinoma Cells. Expert Opin Ther Targets 2023; 27:251-261. [PMID: 37015886 DOI: 10.1080/14728222.2023.2199924] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 04/06/2023]
Abstract
BACKGROUND Colorectal cancer (CRC) is a leading cause of cancer death. Certain signaling pathways are implicated in colorectal carcinogenesis. Cyclin-dependent kinases (CDKs) are commonly hyperactivated in CRC and hence multitarget CDK inhibitors serve as promising therapeutic drugs against CRC. OBJECTIVE Off-target effects of multitarget CDK inhibitors with differential CDK inhibitory spectrum viz. P276-00 (also known as riviciclib), roscovitine and UCN-01 on CRC cell lines of varied genetic background were delineated. METHOD Protein expression was analyzed for key signaling proteins by western blotting. β-catenin localization was assessed using immunofluorescence. HIF-1 transcriptional activity and target gene expression were studied by reporter gene assay and RT-PCR respectively. Anti-migratory and anti-angiogenic potential was evaluated by wound healing assay and endothelial tube formation assay. RESULTS CDK inhibitors modulated various signaling pathways in CRC and for certain proteins showed a highly cell line-dependent response. Riviciclib and roscovitine inhibited HIF-1 transcriptional activity and HIF-1α accumulation in hypoxic HCT116 cells. Both of these drugs also abrogated migration of HCT116 and in vitro angiogenesis in HUVECs. CONCLUSION Anticancer activity of multitarget CDK inhibitors can be certainly attributed to their off-target effects and should be analyzed while assessing their therapeutic utility against CRC.
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Affiliation(s)
- Sonal M Manohar
- Department of Biological Sciences, Sunandan Divatia of School of Science, NMIMS (Deemed-to-be) University, Vile Parle (West), Mumbai, India
| | - Kalpana S Joshi
- Discovery Engine, Cipla R and D, Cipla Ltd. Vikhroli (West), Mumbai, India
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2
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Sun J, Li M, Lin T, Wang D, Chen J, Zhang Y, Mu Q, Su H, Wu N, Liu A, Yu Y, Liu Y, Wang S, Yu X, Guo J, Yu W. Cell cycle arrest is an important mechanism of action of compound Kushen injection in the prevention of colorectal cancer. Sci Rep 2022; 12:4384. [PMID: 35288618 PMCID: PMC8921286 DOI: 10.1038/s41598-022-08336-4] [Citation(s) in RCA: 13] [Impact Index Per Article: 4.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/22/2021] [Accepted: 03/08/2022] [Indexed: 12/27/2022] Open
Abstract
Compound Kushen injection (CKI) is the most widely used traditional Chinese medicine preparation for the comprehensive treatment of colorectal cancer (CRC) in China, but its underlying molecular mechanisms of action are still unclear. The present study employed a network pharmacology approach, in which we constructed a "bioactive compound-target-pathway" network. Experimental RNA sequencing (RNA-Seq) analysis was performed to identify a key "bioactive compound-target-pathway" network for subsequent experimental validation. Cell cycle, proliferation, autophagy, and apoptosis assays and a model of azoxymethane/dextran sodium sulfate-induced colorectal carcinogenesis in mice were employed to detect the biological effect of CKI on CRC. Real-time reverse-transcription polymerase chain reaction, Western blot, and immunohistochemistry were performed to verify the selected targets and pathways. We constructed a predicted network that included 82 bioactive compounds, 34 targets, and 33 pathways and further screened an anti-CRC CKI "biological compound (hesperetin 7-O-rutinoside, genistein 7-O-rutinoside, and trifolirhizin)-target (p53 and checkpoint kinase 1 [CHEK1])" network that targeted the "cell cycle pathway". Validation experiments showed that CKI effectively induced the cell-cycle arrest of CRC cells in vitro and suppressed the development of CRC in vivo by downregulating the expression of p53 and CHEK1. Our findings confirmed that inducing cell-cycle arrest by CKI is an important mechanism of its anti-CRC action, which provides a direct and scientific experimental basis for the clinical application of CKI.
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Affiliation(s)
- Jie Sun
- Department of Central Laboratory and Institute of Clinical Molecular Biology, Peking University People's Hospital, Beijing, China
| | - Mei Li
- Department of Central Laboratory and Institute of Clinical Molecular Biology, Peking University People's Hospital, Beijing, China
| | - Tingru Lin
- Department of Central Laboratory and Institute of Clinical Molecular Biology, Peking University People's Hospital, Beijing, China
- Department of Gastroenterology, Peking University People's Hospital, Beijing, China
| | - Di Wang
- Department of Central Laboratory and Institute of Clinical Molecular Biology, Peking University People's Hospital, Beijing, China
| | - Jingyi Chen
- Department of Gastroenterology, Peking University People's Hospital, Beijing, China
| | - Yu Zhang
- Department of Gastroenterology, Peking University People's Hospital, Beijing, China
| | - Qing Mu
- Department of Central Laboratory and Institute of Clinical Molecular Biology, Peking University People's Hospital, Beijing, China
| | - Huiting Su
- Department of Central Laboratory and Institute of Clinical Molecular Biology, Peking University People's Hospital, Beijing, China
| | - Na Wu
- Department of Central Laboratory and Institute of Clinical Molecular Biology, Peking University People's Hospital, Beijing, China
| | - Aiyu Liu
- Department of Central Laboratory and Institute of Clinical Molecular Biology, Peking University People's Hospital, Beijing, China
| | - Yimeng Yu
- Department of Central Laboratory and Institute of Clinical Molecular Biology, Peking University People's Hospital, Beijing, China
| | - Yulan Liu
- Department of Gastroenterology, Peking University People's Hospital, Beijing, China
| | - Shaojie Wang
- Department of Traditional Chinese Medicine, Peking University People's Hospital, Beijing, China
| | - Xin Yu
- Department of Hepatobiliary Surgery, Peking University People's Hospital, Beijing, China
| | - Jingzhu Guo
- Department of Pediatric, Peking University People's Hospital, Beijing, China.
| | - Weidong Yu
- Department of Central Laboratory and Institute of Clinical Molecular Biology, Peking University People's Hospital, Beijing, China.
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5-aza-2'-deoxycytidine induces apoptosis and inhibits tumour growth in vivo of FaDu cells, a specific HPVnegative HNSCC cell line. PLoS One 2021; 16:e0253756. [PMID: 34534222 PMCID: PMC8448306 DOI: 10.1371/journal.pone.0253756] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/06/2020] [Accepted: 06/11/2021] [Indexed: 12/29/2022] Open
Abstract
Head and neck cancer squamous cell carcinoma (HNSCC) is the sixth most common cancer worldwide, resulting in over 600,000 new diagnoses annually. Traditionally, HNCC has been related to tobacco and alcohol exposure; however, over the past decade, a growing number of head and neck cancers are attributed to human papillomavirus (HPV) infection. 5-Aza-2'-deoxycytidine (5-AzaD) was demonstrated as an effective chemotherapeutic agent for acute myelogenous leukaemia. Preclinical data revealed that 5-aza inhibits growth and increases cell death of HPV(+) cancer cells. These effects are associated with reduced expression of HPV genes, stabilization of TP53, and activation of TP53-dependent apoptosis. The aim of the present study is to test the effect of 5-AzaD on growth of human squamous cell carcinoma (FaDu), a HPV(-) and p53 mutated cells, in vitro and in vivo. The effect of 5-AzaD on cell viability, cell cycle progression and induction of apoptosis was tested in vitro. The effect of 5-AzaD on tumour growth in vivo was tested using xenograft mice inoculated with FaDu cells. The results indicated that 5-AzaD reduced cell viability and induced apoptosis in FaDu cells in vitro. In vivo studies revealed that 5-AzaD suppresses the growth of tumours in xenograft mice inoculated with FaDu cells through inhibition of proliferation and induction of apoptosis. These findings may emphasis that 5-AzaD is effective in treatment of HPV(-) HNSCC tumours through TP53 independent pathway. Future studies are needed in order to clarify the molecular mechanism of action of 5-AzaD in HPV(-) cancer cells.
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4
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Colak DK, Egeli U, Eryilmaz IE, Aybastier O, Malyer H, Cecener G, Tunca B. The Anticancer Effect of Inula viscosa Methanol Extract by miRNAs' Re-regulation: An in vitro Study on Human Malignant Melanoma Cells. Nutr Cancer 2021; 74:211-224. [PMID: 33570434 DOI: 10.1080/01635581.2020.1869791] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/03/2023]
Abstract
Alternative and natural therapies are needed for malignant melanoma (MM), the most deadly skin cancer type due to chemotherapy's limited effect. In the present study, we evaluated the anticancer potentials of Inula viscosa methanol and water extracts (IVM and IVW) on MM cells, A2058 and MeWo, and normal fibroblasts. After the chromatographic and antioxidant activity analysis, their antiproliferative effects were determined with the increasing doses for 24-72 h. IVM induced more cell death in a dose and time-dependent manner in MM cells compared to IVW. This effect was probably due to the higher amount of phenolics in it. IVM significantly induced more apoptotic death in MM cells than fibroblasts (p < 0.01), which was also supported morphologically. IVM also caused cell cycle arrest at G0/G1 and G2/M phases in A2058 and MeWo, respectively, and suppressed the migration ability of MM cells (p < 0.01). Additionally, IVM was found to have significant potential in regulating MM-related miRNAs, upregulating miR-579 and miR-524, and downregulating miR-191 and miR-193, in MM cells (p < 0.05, p < 0.01). As a result, the anticancer effect of IVM via regulating miRNAs' expression has been demonstrated for the first time. Thus, IVM, with these potentials, may be a promising candidate for MM treatment.
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Affiliation(s)
| | - Unal Egeli
- Medical Biology Department, Bursa Uludag University, Bursa, Turkey
| | | | - Onder Aybastier
- Analytical Chemistry Department, Bursa Uludag University, Bursa, Turkey
| | - Hulusi Malyer
- Biology Department, Bursa Uludag University, Bursa, Turkey
| | - Gulsah Cecener
- Medical Biology Department, Bursa Uludag University, Bursa, Turkey
| | - Berrin Tunca
- Medical Biology Department, Bursa Uludag University, Bursa, Turkey
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Wang G, Han J, Wang G, Wu X, Huang Y, Wu M, Chen Y. ERO1α mediates endoplasmic reticulum stress-induced apoptosis via microRNA-101/EZH2 axis in colon cancer RKO and HT-29 cells. Hum Cell 2021; 34:932-944. [PMID: 33559868 DOI: 10.1007/s13577-021-00494-3] [Citation(s) in RCA: 7] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/15/2020] [Accepted: 01/18/2021] [Indexed: 01/15/2023]
Abstract
Although colon cancer is a leading and typical gastrointestinal tumor, there is little published data on the underlying molecular mechanisms of endoplasmic reticulum (ER) stress. Here, we investigated the role of ERO1α and its impact on microRNA (miR)-101 expression and ER stress in colon cancer cells. Cell ER stress was established by treating RKO or HT-29 cells with 1 μM thapsigargin (THG). Cell biological behaviors were detected using CCK-8, bromodeoxyuridine assay, flow cytometry and western blot. We also investigated the expression of ERO1α and miR-101 after THG treatment using RT-qPCR. Moreover, effects of ERO1α and miR-101 on ER stress of colon cancer cells were detected. Additionally, miR-101 impact on EZH2 expression and relevance of this regulation was confirmed by RT-qPCR and luciferase reporter. The regulation of miR-101/EZH2 axis and Wnt/β-catenin pathway in ER stress were investigated. Our results demonstrated that THG induced ER stress in colon cancer cells. Silencing ERO1α further promoted ER stress-induced cell apoptosis. ERO1α knockdown up-regulated miR-101 expression and promoted colon cancer cell apoptosis via regulating miR-101. Surprisingly, miR-101 negatively regulated EZH2 expression via miRNA-mRNA targeting. Moreover, ER stress promoted colon cancer cell apoptosis via regulating miR-101/EZH2 axis. Wnt/β-catenin pathway was also involved in the regulation of ERO1α/miR-101/EZH2 in ER stress of colon cancer cells. These findings illustrated that silencing ERO1α regulated ER stress-induced apoptosis via miR-101/EZH2 axis in RKO and HT-29 cells.
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Affiliation(s)
- Guoqin Wang
- Cancer Biotherapy Center, The Third Affiliated Hospital of Kunming Medical University, Yunnan Cancer Hospital, Kunming, 650118, Yunnan, China
| | - Jiangqiong Han
- Integrated Traditional Chinese and Western Medicine Department, The Third Affiliated Hospital of Kunming Medical University, Yunnan Cancer Hospital, Kunming, 650118, Yunnan, China
| | - Gaowei Wang
- Cancer Biotherapy Center, The Third Affiliated Hospital of Kunming Medical University, Yunnan Cancer Hospital, Kunming, 650118, Yunnan, China
| | - Xuesong Wu
- Department Gastrointestinal Surgery, The Third Affiliated Hospital of Kunming Medical University, Kunming, 650101, Yunnan, China
| | - Youguang Huang
- Tumor Institute of Yunnan Province, The Third Affiliated Hospital of Kunming Medical University, Kunming, 650101, Yunnan, China
| | - Min Wu
- Tumor Institute of Yunnan Province, The Third Affiliated Hospital of Kunming Medical University, Kunming, 650101, Yunnan, China
| | - Yunlan Chen
- Cadre Medical Department, The Third Affiliated Hospital of Kunming Medical University, Yunnan Cancer Hospital, No. 517 Kunzhou Road, Xishan District, Kunming, 650118, Yunnan, China.
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Bar-Shalom R, Bergman M, Grossman S, Azzam N, Sharvit L, Fares F. Inula Viscosa Extract Inhibits Growth of Colorectal Cancer Cells in vitro and in vivo Through Induction of Apoptosis. Front Oncol 2019; 9:227. [PMID: 31024836 PMCID: PMC6469364 DOI: 10.3389/fonc.2019.00227] [Citation(s) in RCA: 23] [Impact Index Per Article: 3.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/08/2019] [Accepted: 03/14/2019] [Indexed: 12/12/2022] Open
Abstract
Colorectal cancer (CRC) is the second most common cancer in females and the third in males worldwide. Conventional therapy of CRC is limited by severe side effects and by the development of resistance. Therefore, additional therapies are needed in order to combat the problem of selectivity and drug resistance in CRC patients. Inula viscosa (IV) is a well-known medicinal perennial herb in traditional medicine. It is used for different therapeutic purposes, such as; topical anti-inflammatic, diuretic, hemostatic, antiseptic, antiphlogistic, and in the treatment of diabetes. Several studies attempted to reveal the anti-cancer activity of different extracts prepared by different organic solvents from different parts of the IV plant. The aim of the present study is to examine the potential beneficial effects of IV leaf aqueous extract on the growth of colon cancer cells in vitro and in vivo. The results indicated that exposure of colorectal cancer cells to IV extract, significantly reduced cell viability in a dose and time dependent manner. Moreover, treatment of cells with 300 μg/ml of IV extract induced apoptosis, as it was detected by Annexin V/FITC/PI, TUNEL assay, and the activation of caspases. In vivo studies revealed that treatment with 150 or 300 mg/kg IV extract inhibited tumor growth in mice transplanted with MC38 cells. Tumors' weight and volume were significantly (P < 0.001) reduced when compared to untreated-control group. Staining of the paraffin section of tumors revealed that IV treatment inhibited cell proliferation and induced apoptosis. Additionally, no side effects such as; weight loss, behavior changes, ruffled fur or changes in kidney, and liver functions were observed. These results may indicate that active doses of IV extract are not toxic. Further studies are needed in order to identify the structure of the active compounds. Results from this study may contribute to the development of new and efficient strategies for treatment of human colon cancer.
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Affiliation(s)
- Rinat Bar-Shalom
- Department of Human Biology, Faculty of Natural Sciences, University of Haifa, Haifa, Israel
| | - Margalit Bergman
- Faculty of Life Sciences, Bar-Ilan University, Ramat-Gan, Israel
| | - Shlomo Grossman
- Faculty of Life Sciences, Bar-Ilan University, Ramat-Gan, Israel
| | - Naiel Azzam
- Department of Human Biology, Faculty of Natural Sciences, University of Haifa, Haifa, Israel
| | - Lital Sharvit
- Department of Human Biology, Faculty of Natural Sciences, University of Haifa, Haifa, Israel
| | - Fuad Fares
- Department of Human Biology, Faculty of Natural Sciences, University of Haifa, Haifa, Israel
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7
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Varghese V, Magnani L, Harada-Shoji N, Mauri F, Szydlo RM, Yao S, Lam EWF, Kenny LM. FOXM1 modulates 5-FU resistance in colorectal cancer through regulating TYMS expression. Sci Rep 2019; 9:1505. [PMID: 30728402 PMCID: PMC6365533 DOI: 10.1038/s41598-018-38017-0] [Citation(s) in RCA: 99] [Impact Index Per Article: 16.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/10/2018] [Accepted: 10/07/2018] [Indexed: 12/11/2022] Open
Abstract
Resistance to 5-Fluoruracil (5-FU) has been linked to elevated expression of the main target, thymidylate synthase (TYMS), which catalyses the de novo pathway for production of deoxythymidine monophosphate. The potent oncogenic forkhead box transcription factor, FOXM1 is is regulated by E2F1 which also controls TYMS. This study reveals a significant role of FOXM1 in 5-FU resistance. Overexpression and knock-down studies of FOXM1 in colon cancer cells suggest the importance of FOXM1 in TYMS regulation. ChIP and global ChIP-seq data also confirms that FOXM1 can also potentially regulate other 5-FU targets, such as TYMS, thymidine kinase 1 (TK-1) and thymidine phosphorylase (TYMP). In human colorectal cancer tissue specimens, a strong correlation of FOXM1 and TYMS staining was observed. Elevated FOXM1 and TYMS expression was also observed in acquired 5-FU resistant colon cancer cells (HCT116 5-FU Res). A synergistic effect was observed following treatment of CRC cells with an inhibitor of FOXM1, thiostrepton, in combination with 5-FU. The combination treatment decreased colony formation and migration, and induced cell cycle arrest, DNA damage, and apoptosis in CRC cell lines. In summary, this research demonstrated that FOXM1 plays a pivotal role in 5-FU resistance at least partially through the regulation of TYMS.
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Affiliation(s)
- Vidhya Varghese
- Department of Surgery and Cancer, Imperial College London, London, USA
| | - Luca Magnani
- Department of Surgery and Cancer, Imperial College London, London, USA
| | | | - Francesco Mauri
- Imperial College Healthcare NHS Trust, Imperial College London, London, USA
| | | | - Shang Yao
- Department of Surgery and Cancer, Imperial College London, London, USA
| | - Eric W-F Lam
- Department of Surgery and Cancer, Imperial College London, London, USA.
| | - Laura M Kenny
- Department of Surgery and Cancer, Imperial College London, London, USA.
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8
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Mechanisms of Chromosome Congression during Mitosis. BIOLOGY 2017; 6:biology6010013. [PMID: 28218637 PMCID: PMC5372006 DOI: 10.3390/biology6010013] [Citation(s) in RCA: 107] [Impact Index Per Article: 13.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Subscribe] [Scholar Register] [Received: 10/01/2016] [Revised: 01/07/2017] [Accepted: 01/28/2017] [Indexed: 12/13/2022]
Abstract
Chromosome congression during prometaphase culminates with the establishment of a metaphase plate, a hallmark of mitosis in metazoans. Classical views resulting from more than 100 years of research on this topic have attempted to explain chromosome congression based on the balance between opposing pulling and/or pushing forces that reach an equilibrium near the spindle equator. However, in mammalian cells, chromosome bi-orientation and force balance at kinetochores are not required for chromosome congression, whereas the mechanisms of chromosome congression are not necessarily involved in the maintenance of chromosome alignment after congression. Thus, chromosome congression and maintenance of alignment are determined by different principles. Moreover, it is now clear that not all chromosomes use the same mechanism for congressing to the spindle equator. Those chromosomes that are favorably positioned between both poles when the nuclear envelope breaks down use the so-called "direct congression" pathway in which chromosomes align after bi-orientation and the establishment of end-on kinetochore-microtubule attachments. This favors the balanced action of kinetochore pulling forces and polar ejection forces along chromosome arms that drive chromosome oscillatory movements during and after congression. The other pathway, which we call "peripheral congression", is independent of end-on kinetochore microtubule-attachments and relies on the dominant and coordinated action of the kinetochore motors Dynein and Centromere Protein E (CENP-E) that mediate the lateral transport of peripheral chromosomes along microtubules, first towards the poles and subsequently towards the equator. How the opposite polarities of kinetochore motors are regulated in space and time to drive congression of peripheral chromosomes only now starts to be understood. This appears to be regulated by position-dependent phosphorylation of both Dynein and CENP-E and by spindle microtubule diversity by means of tubulin post-translational modifications. This so-called "tubulin code" might work as a navigation system that selectively guides kinetochore motors with opposite polarities along specific spindle microtubule populations, ultimately leading to the congression of peripheral chromosomes. We propose an integrated model of chromosome congression in mammalian cells that depends essentially on the following parameters: (1) chromosome position relative to the spindle poles after nuclear envelope breakdown; (2) establishment of stable end-on kinetochore-microtubule attachments and bi-orientation; (3) coordination between kinetochore- and arm-associated motors; and (4) spatial signatures associated with post-translational modifications of specific spindle microtubule populations. The physiological consequences of abnormal chromosome congression, as well as the therapeutic potential of inhibiting chromosome congression are also discussed.
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Zeilstra J, Joosten SPJ, Vermeulen L, Koster J, Medema JP, Versteeg R, Spaargaren M, Pals ST. CD44 expression in intestinal epithelium and colorectal cancer is independent of p53 status. PLoS One 2013; 8:e72849. [PMID: 24009708 PMCID: PMC3756983 DOI: 10.1371/journal.pone.0072849] [Citation(s) in RCA: 17] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/01/2013] [Accepted: 07/15/2013] [Indexed: 11/18/2022] Open
Abstract
CD44 marks stem cell-like cells in a number of tumour types, including colorectal cancer (CRC), while aberrant CD44 expression conveys increased tumourigenic, invasive, and metastatic potential. Previous data indicate that CD44 is a direct target of p53-mediated transcriptional repression in breast cancer. Since inactivating p53 mutations are frequent genetic events in CRC these could unleash expression of CD44. In the present study, we therefore explored the relation between p53 mutational status and CD44 expression in a cohort of 90 localized primary CRCs and studied the effect of radiation-induced p53 activation on CD44 expression. Interestingly, we observed that, in contrast to breast cancer, loss of function p53 mutations were not associated with elevated CD44 expression in colon cancer. Moreover, DNA-damage induced p53 activation did not result in repression of CD44 expression, neither in colon cancer cells nor in normal intestinal epithelial cells. Our data demonstrate that CD44 expression in normal and malignant intestinal epithelial cells is not regulated by p53, implying that regulation of this potentially important therapeutic target is tissue and cancer-type specific.
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Affiliation(s)
- Jurrit Zeilstra
- Department of Pathology, Academic Medical Center, University of Amsterdam, Amsterdam, The Netherlands
| | - Sander P. J. Joosten
- Department of Pathology, Academic Medical Center, University of Amsterdam, Amsterdam, The Netherlands
| | - Louis Vermeulen
- Laboratory for Experimental Oncology and Radiobiology (LEXOR), Center for Experimental Molecular Medicine, Academic Medical Center, University of Amsterdam, Amsterdam, The Netherlands
| | - Jan Koster
- Department of Oncogenomics, Academic Medical Center, University of Amsterdam, Amsterdam, The Netherlands
| | - Jan Paul Medema
- Laboratory for Experimental Oncology and Radiobiology (LEXOR), Center for Experimental Molecular Medicine, Academic Medical Center, University of Amsterdam, Amsterdam, The Netherlands
| | - Rogier Versteeg
- Department of Oncogenomics, Academic Medical Center, University of Amsterdam, Amsterdam, The Netherlands
| | - Marcel Spaargaren
- Department of Pathology, Academic Medical Center, University of Amsterdam, Amsterdam, The Netherlands
| | - Steven T. Pals
- Department of Pathology, Academic Medical Center, University of Amsterdam, Amsterdam, The Netherlands
- * E-mail:
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Alami N, Paterson J, Belanger S, Juste S, Grieshaber C, Leyland-Jones B. Comparative Cytotoxicity of C-1311 in Colon CancerIn VitroandIn VivoUsing the Hollow Fiber Assay. J Chemother 2013; 19:546-53. [DOI: 10.1179/joc.2007.19.5.546] [Citation(s) in RCA: 6] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 10/31/2022]
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de la Cueva A, Ramírez de Molina A, Álvarez-Ayerza N, Ramos MA, Cebrián A, del Pulgar TG, Lacal JC. Combined 5-FU and ChoKα inhibitors as a new alternative therapy of colorectal cancer: evidence in human tumor-derived cell lines and mouse xenografts. PLoS One 2013; 8:e64961. [PMID: 23762272 PMCID: PMC3677921 DOI: 10.1371/journal.pone.0064961] [Citation(s) in RCA: 39] [Impact Index Per Article: 3.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/25/2011] [Accepted: 04/22/2013] [Indexed: 12/31/2022] Open
Abstract
Background Colorectal cancer (CRC) is the third major cause of cancer related deaths in the world. 5-fluorouracil (5-FU) is widely used for the treatment of colorectal cancer but as a single-agent renders low response rates. Choline kinase alpha (ChoKα), an enzyme that plays a role in cell proliferation and transformation, has been reported overexpressed in many different tumors, including colorectal tumors. ChoKα inhibitors have recently entered clinical trials as a novel antitumor strategy. Methodology/Principal Findings ChoKα specific inhibitors, MN58b and TCD-717, have demonstrated a potent antitumoral activity both in vitro and in vivo against several tumor-derived cell line xenografts including CRC-derived cell lines. The effect of ChoKα inhibitors in combination with 5-FU as a new alternative for the treatment of colon tumors has been investigated both in vitro in CRC-tumour derived cell lines, and in vivo in mouse xenografts models. The effects on thymidilate synthase (TS) and thymidine kinase (TK1) levels, two enzymes known to play an essential role in the mechanism of action of 5-FU, were analyzed by western blotting and quantitative PCR analysis. The combination of 5-FU with ChoKα inhibitors resulted in a synergistic effect in vitro in three different human colon cancer cell lines, and in vivo against human colon xenografts in nude mice. ChoKα inhibitors modulate the expression levels of TS and TK1 through inhibition of E2F production, providing a rational for its mechanism of action. Conclusion/Significance Our data suggest that both drugs in combination display a synergistic antitumoral effect due to ChoKα inhibitors-driven modulation of the metabolization of 5-FU. The clinical relevance of these findings is strongly supported since TCD-717 has recently entered Phase I clinical trials against solid tumors.
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Affiliation(s)
- Ana de la Cueva
- Traslational Oncology Unit, Instituto de Investigaciones Biomédicas, CSIC, Madrid, Spain
| | - Ana Ramírez de Molina
- Traslational Oncology Unit, Instituto de Investigaciones Biomédicas, CSIC, Madrid, Spain
| | - Néstor Álvarez-Ayerza
- Traslational Oncology Unit, Instituto de Investigaciones Biomédicas, CSIC, Madrid, Spain
| | - Ma Angeles Ramos
- Traslational Oncology Unit, Instituto de Investigaciones Biomédicas, CSIC, Madrid, Spain
| | - Arancha Cebrián
- Traslational Oncology Unit, Instituto de Investigaciones Biomédicas, CSIC, Madrid, Spain
| | | | - Juan Carlos Lacal
- Traslational Oncology Unit, Instituto de Investigaciones Biomédicas, CSIC, Madrid, Spain
- Instituto de Investigación Sanitaria IdiPAZ, Madrid, Spain
- * E-mail:
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12
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Ligabue A, Marverti G, Liebl U, Myllykallio H. Transcriptional activation and cell cycle block are the keys for 5-fluorouracil induced up-regulation of human thymidylate synthase expression. PLoS One 2012; 7:e47318. [PMID: 23056627 PMCID: PMC3467224 DOI: 10.1371/journal.pone.0047318] [Citation(s) in RCA: 18] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/17/2012] [Accepted: 09/14/2012] [Indexed: 12/27/2022] Open
Abstract
Background 5-fluorouracil, a commonly used chemotherapeutic agent, up-regulates expression of human thymidylate synthase (hTS). Several different regulatory mechanisms have been proposed to mediate this up-regulation in distinct cell lines, but their specific contributions in a single cell line have not been investigated to date. We have established the relative contributions of these previously proposed regulatory mechanisms in the ovarian cancer cell line 2008 and the corresponding cisplatin-resistant and 5-FU cross-resistant-subline C13*. Methodology/Principal Findings Using RNA polymerase II inhibitor DRB treated cell cultures, we showed that 70–80% of up-regulation of hTS results from transcriptional activation of TYMS mRNA. Moreover, we report that 5-FU compromises the cell cycle by blocking the 2008 and C13* cell lines in the S phase. As previous work has established that TYMS mRNA is synthesized in the S and G1 phase and hTS is localized in the nuclei during S and G2-M phase, the observed cell cycle changes are also expected to affect the intracellular regulation of hTS. Our data also suggest that the inhibition of the catalytic activity of hTS and the up-regulation of the hTS protein level are not causally linked, as the inactivated ternary complex, formed by hTS, deoxyuridine monophosphate and methylenetetrahydrofolate, was detected already 3 hours after 5-FU exposure, whereas substantial increase in global TS levels was detected only after 24 hours. Conclusions/Significance Altogether, our data indicate that constitutive TYMS mRNA transcription, cell cycle-induced hTS regulation and hTS enzyme stability are the three key mechanisms responsible for 5-fluorouracil induced up-regulation of human thymidylate synthase expression in the two ovarian cancer cell lines studied. As these three independent regulatory phenomena occur in a precise order, our work provides a feasible rationale for earlier observed synergistic combinations of 5-FU with other drugs and may suggest novel therapeutic strategies.
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Affiliation(s)
- Alessio Ligabue
- INSERM U696, Palaiseau, France
- Laboratoire d'Optique et Biosciences, CNRS, Ecole Polytechnique, Palaiseau, France
| | - Gaetano Marverti
- Dipartimento di Scienze Biomediche, Sezione di Chimica Biologica, University of Modena and Reggio Emilia, Modena, Italy
| | - Ursula Liebl
- INSERM U696, Palaiseau, France
- Laboratoire d'Optique et Biosciences, CNRS, Ecole Polytechnique, Palaiseau, France
| | - Hannu Myllykallio
- INSERM U696, Palaiseau, France
- Laboratoire d'Optique et Biosciences, CNRS, Ecole Polytechnique, Palaiseau, France
- * E-mail:
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13
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Abstract
Protein farnesylation and geranylgeranylation, together referred to as prenylation, are lipid post-translational modifications that are required for the transforming activity of many oncogenic proteins, including some RAS family members. This observation prompted the development of inhibitors of farnesyltransferase (FT) and geranylgeranyl-transferase 1 (GGT1) as potential anticancer drugs. In this Review, we discuss the mechanisms by which FT and GGT1 inhibitors (FTIs and GGTIs, respectively) affect signal transduction pathways, cell cycle progression, proliferation and cell survival. In contrast to their preclinical efficacy, only a small subset of patients responds to FTIs. Identifying tumours that depend on farnesylation for survival remains a challenge, and strategies to overcome this are discussed. One GGTI has recently entered the clinic, and the safety and efficacy of GGTIs await results from clinical trials.
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Affiliation(s)
- Norbert Berndt
- Drug Discovery Department, Moffitt Cancer Center, 12902 Magnolia Drive, Tampa, Florida 33612, USA
| | - Andrew D. Hamilton
- University of Oxford, Vice-Chancellor’s Office, Wellington Square, Oxford OX1 2JD, UK
| | - Saïd M. Sebti
- Drug Discovery Department, Moffitt Cancer Center, 12902 Magnolia Drive, Tampa, Florida 33612, USA
- Departments of Oncologic Sciences and Molecular Medicine, University of South Florida, 12901 Bruce B. Downs Boulevard, Tampa, Florida 33612, USA
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14
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Hu R, Lam W, Hsu CH, Cheng YC. UMP/CMPK is not the critical enzyme in the metabolism of pyrimidine ribonucleotide and activation of deoxycytidine analogs in human RKO cells. PLoS One 2011; 6:e19490. [PMID: 21559290 PMCID: PMC3086915 DOI: 10.1371/journal.pone.0019490] [Citation(s) in RCA: 6] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/11/2010] [Accepted: 04/07/2011] [Indexed: 11/26/2022] Open
Abstract
Background Human UMP/CMP kinase was identified based on its enzymatic activity in vitro. The role of this protein is considered critical for the maintenance of pyrimidine nucleotide pool profile and for the metabolism of pyrimidine analogs in cells, based on the in vitro study of partially purified enzyme and recombinant protein. However, no detailed study has yet addressed the role of this protein in nucleotide metabolism in cells. Methodology/Principal Findings Two stable cell lines in which UMP/CMP kinase (mRNA: AF087865, EC 2.7.4.14) can be either up-regulated or down-regulated were developed using Tet-On Gene Expression Systems. The amount and enzymatic activity of UMP/CMP kinase extracted from these two cell lines can be induced up by 500% or down by 95–98%. The ribonucleotides of endogenous pyrimidine as well as the metabolism of exogenous natural pyrimidine nucleosides and their analogs were not susceptible to the altered amount of UMP/CMP kinase in these two stable RKO cell lines. The level of incorporation of pyrimidine nucleoside analogs, such as gemcitabine (dFdC) and troxacitabine (L-OddC), into cellular DNA and their potency in inhibiting cell growth were not significantly altered by up-regulation or down-regulation of UMP/CMP kinase expression in cells. Conclusions/Significance The UMP/CMP kinase (EC 2.7.4.14) expressed in RKO cells is not critical for the phosphorylation of (d)CMP and the maintenance of natural nucleotide pools. It also does not play an important role in the activation of dFdC and L-OddC. The increase by 500% or decrease by 95–98% in the levels of UMP/CMP kinase do not affect steady state levels of dFdC and L-OddC in RKO cells. Overall, the activity and possible mechanisms of recombinant UMP/CMP kinase expressed in the in vitro system can not be extended to that of UMP/CMP kinase expressed in a cell system or an in vivo system.
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Affiliation(s)
- Rong Hu
- Department of Pharmacology, Yale University School of Medicine, New Haven, Connecticut, United States of America
| | - Wing Lam
- Department of Pharmacology, Yale University School of Medicine, New Haven, Connecticut, United States of America
| | - Chih-Hung Hsu
- Department of Pharmacology, Yale University School of Medicine, New Haven, Connecticut, United States of America
- Department of Oncology, National Taiwan University Hospital, Taipei, Taiwan, Republic of China
| | - Yung-Chi Cheng
- Department of Pharmacology, Yale University School of Medicine, New Haven, Connecticut, United States of America
- Department of Oncology, National Taiwan University Hospital, Taipei, Taiwan, Republic of China
- * E-mail:
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15
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Antitumor Activity of LB42907, a Potent and Selective Farnesyltransferase Inhibitor: Synergistic Effect in Combination with Other Anticancer Drugs. B KOREAN CHEM SOC 2008. [DOI: 10.5012/bkcs.2008.29.7.1303] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/20/2022]
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16
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Yang L, Hamilton SR, Sood A, Kuwai T, Ellis L, Sanguino A, Lopez-Berestein G, Boyd DD. The previously undescribed ZKSCAN3 (ZNF306) is a novel "driver" of colorectal cancer progression. Cancer Res 2008; 68:4321-30. [PMID: 18519692 DOI: 10.1158/0008-5472.can-08-0407] [Citation(s) in RCA: 62] [Impact Index Per Article: 3.6] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/03/2023]
Abstract
A relatively new view of colorectal cancer is that its development/progression reflects the contribution of a large set of altered gene products in varying combinations, each providing a "fitness advantage." In searching for novel contributing gene products using Unigene cluster data mining, we found overrepresentation of expressed sequence tags corresponding to a previously uncharacterized gene (ZKSCAN3) in colorectal tumors. ZKSCAN3 was pursued for several reasons: (a) its sequence similarity with bowl required for Drosophila hindgut development; (b) it lies in a chromosomal region (6p22.1) amplified in colorectal cancer; and (c) its coding sequence predicts tandem C(2)H(2) zinc finger domains present in a class of proteins gaining attention for their role in oncogenesis/tumor progression. Reverse transcription-PCR confirmed overexpression in colorectal tumor tissue compared with adjacent nonmalignant mucosa due in part to gene amplification determined by Southern blotting. Further, immunohistochemistry with an antibody generated to the predicted protein sequence revealed higher ZKSCAN3 expression in invasive compared with noninvasive tumors. Intriguingly, the ZKSCAN3 protein was also expressed in tumors wild-type for genes (APC, p53, K-Ras) commonly targeted in colorectal cancer. ZKSCAN3 knockdown in two independent colon cancer cell lines impaired anchorage-independent growth and orthotopic tumor growth, whereas overexpression in a third cell line had the opposite effect and increased 5-fluorouracil resistance. Liposomal delivery of a ZKSCAN3-targeting small interfering RNA reduced tumorigenicity of orthotopic colon cancer. Thus, the hitherto uncharacterized ZKSCAN3 adds to an expanding set of encoded products contributing to the progression of colorectal cancer.
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Affiliation(s)
- Lin Yang
- Department of Cancer Biology, The University of Texas M. D. Anderson Cancer Center, Houston, Texas 77030, USA
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17
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Konstantinopoulos PA, Karamouzis MV, Papavassiliou AG. Post-translational modifications and regulation of the RAS superfamily of GTPases as anticancer targets. Nat Rev Drug Discov 2007; 6:541-55. [PMID: 17585331 DOI: 10.1038/nrd2221] [Citation(s) in RCA: 357] [Impact Index Per Article: 19.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/31/2022]
Abstract
The involvement of the RAS superfamily of monomeric GTPases in carcinogenesis is increasingly being appreciated. A complex array of post-translational modifications and a highly sophisticated protein network regulate the spatio-temporal activation of these GTPases. Previous attempts to pharmacologically target this family have focused on the development of farnesyltransferase inhibitors, but the performance of such agents in cancer clinical trials has not been as good as hoped. Here, we review emerging druggable targets and novel therapeutic approaches targeting prenylation and post-prenylation modifications and the functional regulation of GDP/GTP exchange as exciting alternatives for anticancer therapy.
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18
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Jabbour E, Kantarjian H, Cortes J. Clinical activity of tipifarnib in hematologic malignancies. Expert Opin Investig Drugs 2007; 16:381-92. [PMID: 17302532 DOI: 10.1517/13543784.16.3.381] [Citation(s) in RCA: 10] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/05/2022]
Abstract
Farnesyltransferase inhibitors are a novel class of anticancer agents that competitively inhibit farnesyltransferase. Initially developed to inhibit the farnesylation that is necessary for Ras activation, their mechanism of action seems to be more complex, involving other proteins unrelated to Ras. Of the four classes of farnesyltransferase inhibitors, at least three agents have been investigated in hematologic malignancies. Tipifarnib (R-115777), an orally administered non-peptidomimetic farnesyltransferase inhibitor, has shown promising clinical activity. Preliminary results from clinical trials demonstrate enzyme target inhibition, an acceptable toxicity profile and promising evidence of clinical activity. Ongoing studies will better determine the mechanism of action of tipifarnib and the role of combination with other agents, defining its place in the therapeutic arsenal of hematologic disorders.
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Affiliation(s)
- Elias Jabbour
- MD Anderson Cancer Center, Department of Leukemia, Unit 428, 1515 Holcombe Boulevard, Houston, TX 77030, USA
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19
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Wang H, Yan C, Asangani I, Allgayer H, Boyd DD. Identification of an histone H3 acetylated/K4-methylated-bound intragenic enhancer regulatory for urokinase receptor expression. Oncogene 2006; 26:2058-70. [PMID: 17001307 DOI: 10.1038/sj.onc.1210003] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/09/2022]
Abstract
The transcriptionally regulated urokinase-type plasminogen activator receptor (u-PAR) contributes to cancer progression. Although previous studies have identified multiple 5' regulatory elements, these cis motifs cannot fully account for u-PAR expression prompting a search for hitherto uncharacterized regulatory elements. DNase I hypersensitivity and chromatin immunoprecipitation assays using u-PAR-expressing colon cancer cells indicated a hypersensitive region (+665/+2068) in intron 1 enriched with acetylated histone 3 (H3) and H3 methylated at lysine 4, markers of regulatory regions. The +665/+2068 region increased transcription from a u-PAR-promoter in an orientation- and distance-independent manner fulfilling the criteria of an enhancer. Optimal stimulation of the u-PAR promoter by phorbol ester required this enhancer. Systematic truncations combined with DNase I footprinting revealed two protected regions (+1060/+1099 and +1123/+1134) with deletion of the latter practically abolishing enhancer activity. The +1123/+1134 region harbored non-consensus activator protein-1 and Ets1 binding sites bound with c-Jun (and/or the related JunD/JunB) and c-Fos (and/or the related FosB/Fra-1/Fra-2) as revealed with chromatin immunoprecipitation. Further, nuclear extract from resected colon cancers showed elevated protein binding to a +1123/+1134-spanning probe coordinate with elevated u-PAR protein. Thus, we have defined a novel intragenic enhancer in the u-PAR gene required for constitutive and inducible expression.
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Affiliation(s)
- H Wang
- Department of Cancer Biology, MD Anderson Cancer Center, Houston, TX 77030, USA
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20
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Lebedeva IV, Su ZZ, Emdad L, Kolomeyer A, Sarkar D, Kitada S, Waxman S, Reed JC, Fisher PB. Targeting inhibition of K-ras enhances Ad.mda-7-induced growth suppression and apoptosis in mutant K-ras colorectal cancer cells. Oncogene 2006; 26:733-44. [PMID: 16924242 DOI: 10.1038/sj.onc.1209813] [Citation(s) in RCA: 21] [Impact Index Per Article: 1.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/23/2023]
Abstract
Melanoma differentiation-associated gene-7/interleukin-24 (mda-7/IL-24) is a cancer-specific, growth-suppressing and apoptosis-inducing gene with broad-spectrum antitumor activity. However, when administered by means of a replication-incompetent adenovirus, Ad.mda-7, several colorectal carcinoma cell lines are resistant to its antiproliferative and antisurvival effects. We have presently endeavored to determine if K-ras mutations, present in approximately 40-50% of colorectal cancers and which may mediate resistance to chemotherapy and radiotherapy, represent a predisposing genetic factor mitigating reduced sensitivity to Ad.mda-7. To suppress ras expression, three structurally different replication-incompetent adenoviral vectors were engineered that express (1) an intracellular, neutralizing single-chain antibody (scAb) to p21 ras (Ad.K-ras scAb), (2) an antisense (AS) K-ras gene (Ad.K-ras AS) or (3) both mda-7/IL-24 and a K-ras AS gene in a single bipartite virus (Ad.m7.KAS). Simultaneous inhibition of K-ras and expression of mda-7/IL-24 enhanced killing of colorectal carcinoma cells with mutated K-ras, but not with wild-type K-ras. The extent of killing depended on the degree of K-ras downregulation, with Ad.K-ras AS being generally more efficient than Ad.K-ras scAb in combination with Ad.mda-7. These findings support an effective dual-combinatorial approach for the therapy of colorectal cancers that employs a unique cancer-specific suppressor gene (mda-7/IL-24) with targeted inhibition of oncogene (ras) expression.
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Affiliation(s)
- I V Lebedeva
- Department of Pathology, Herbert Irving Comprehensive Cancer Center, Columbia University Medical Center, College of Physicians and Surgeons, New York, NY 10032, USA
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21
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Dang LH, Chen F, Ying C, Chun SY, Knock SA, Appelman HD, Dang DT. CDX2 has tumorigenic potential in the human colon cancer cell lines LOVO and SW48. Oncogene 2006; 25:2264-72. [PMID: 16314840 DOI: 10.1038/sj.onc.1209247] [Citation(s) in RCA: 42] [Impact Index Per Article: 2.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/07/2023]
Abstract
CDX2 is a Drosophila caudal-related homeobox transcription factor that is important for the establishment and maintenance of intestinal epithelial cells. CDX2 is a marker of colon cancer, with strong staining in up to 90% of colonic adenocarcinomas. CDX2 heterozygous-null mice develop colonic neoplasms, which have suggested that CDX2 is a tumor suppressor. However, CDX2 has not been reported to affect xenograft growth. Furthermore, CDX2 is rarely mutated in colon cancer, which has led to suggestions that it may play only a minor role as a tumor suppressor in colon cancer. To understand the functional contributions of CDX2 to colon cancer, we disrupted CDX2 in LOVO and SW48 human colon cancer cell lines by targeted homologous recombination. Consistent with the literature, disruption of CDX2 enhanced anchorage-dependent cell proliferation. However, homozygous loss of CDX2 led to significant inhibition of anchorage-independent growth in LOVO cells, and cell lethality in SW48 cells. Further analyses revealed that disruption of CDX2 led to anchorage-independent G1 to S growth arrest and anoikis. In vivo xenograft studies confirmed that disruption of CDX2 inhibited LOVO tumor growth. These data demonstrate that CDX2 mediates anchorage-independent growth and survival. Thus, CDX2 has tumorigenic potential in the human colon cancer cell lines LOVO and SW48.
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Affiliation(s)
- L H Dang
- Department of Internal Medicine, Division of Hematology/Oncology, University of Michigan, Ann Arbor, MI 48109-0682, USA
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22
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Dang DT, Chen F, Gardner LB, Cummins JM, Rago C, Bunz F, Kantsevoy SV, Dang LH. Hypoxia-inducible factor-1alpha promotes nonhypoxia-mediated proliferation in colon cancer cells and xenografts. Cancer Res 2006; 66:1684-936. [PMID: 16452228 DOI: 10.1158/0008-5472.can-05-2887] [Citation(s) in RCA: 98] [Impact Index Per Article: 5.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/16/2022]
Abstract
Hypoxia-inducible factor-1alpha (HIF-1alpha) is a transcription factor that directly transactivates genes important for the growth and metabolism of solid tumors. HIF-1alpha is overexpressed in cancer, and its level of expression is correlated with patient mortality. Increased synthesis or stability of HIF-1alpha can be induced by hypoxia-dependent or hypoxia-independent factors. Thus, HIF-1alpha is expressed in both nonhypoxic and hypoxic cancer cells. The role of HIF-1alpha in nonhypoxia-mediated cancer cell proliferation remains speculative. We have disrupted HIF-1alpha by targeted homologous recombination in HCT116 and RKO human colon cancer cells. Loss of HIF-1alpha significantly reduced nonhypoxia-mediated cell proliferation in vitro and in vivo. Paradoxically, loss of HIF-1alpha expression did not grossly affect the hypoxic compartments within tumor xenografts in vivo, although HIF-1alpha promoted cell proliferation and survival under hypoxia in vitro. To further test the role of HIF-1alpha within tumor compartments, we generated cells with combined disruptions of both HIF-1alpha and vascular endothelial growth factor (VEGF). In all xenografts, disruption of VEGF led to marked expansion of the hypoxic compartments and growth delay. Nonetheless, the presence or absence of HIF-1alpha did not grossly affect these expanded hypoxic compartments. These data provide compelling evidence that, in a subset of colon cancers, (a) HIF-1alpha is a positive factor for nonhypoxia-mediated cell proliferation in vitro and in vivo and (b) HIF-1alpha is a positive factor for cell proliferation and survival under hypoxic conditions in vitro, but does not grossly contribute to the tumor hypoxic compartments in vivo.
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Affiliation(s)
- Duyen T Dang
- Division of Gastroenterology, Department of Internal Medicine, University of Michigan Medical Center, 1150 West Medical Center Drive, Ann Arbor, MI 48109, USA
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23
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Basso AD, Kirschmeier P, Bishop WR. Thematic review series: Lipid Posttranslational Modifications. Farnesyl transferase inhibitors. J Lipid Res 2006; 47:15-31. [PMID: 16278491 DOI: 10.1194/jlr.r500012-jlr200] [Citation(s) in RCA: 237] [Impact Index Per Article: 12.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/31/2022] Open
Abstract
Some proteins undergo posttranslational modification by the addition of an isoprenyl lipid (farnesyl- or geranylgeranyl-isoprenoid) to a cysteine residue proximal to the C terminus. Protein isoprenylation promotes membrane association and contributes to protein-protein interactions. Farnesylated proteins include small GTPases, tyrosine phosphatases, nuclear lamina, cochaperones, and centromere-associated proteins. Prenylation is required for the transforming activity of Ras. Because of the high frequency of Ras mutations in cancer, farnesyl transferase inhibitors (FTIs) were investigated as a means to antagonize Ras function. Evaluation of FTIs led to the finding that both K- and N-Ras are alternatively modified by geranylgeranyl prenyltransferase-1 in FTI-treated cells. Geranylgeranylated forms of Ras retain the ability to associate with the plasma membrane and activate substrates. Despite this, FTIs are effective at inhibiting the growth of human tumor cells in vitro, suggesting that activity is dependent on blocking the farnesylation of other proteins. FTIs also inhibit the in vivo growth of human tumor xenografts and sensitize these models to chemotherapeutics, most notably taxanes. Several FTIs have entered clinical trials for various cancer indications. In some clinical settings, primarily hematologic malignancies, FTIs have displayed evidence of single-agent activity. Clinical studies in progress are exploring the antitumor activity of FTIs as single agents and in combination. This review will summarize the basic biology of FTIs, their antitumor activity in preclinical models, and the current status of clinical studies with these agents.
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Affiliation(s)
- Andrea D Basso
- Schering-Plough Research Institute, Kenilworth, NJ 07033, USA.
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24
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Kavgaci H, Ozdemir F, Ovali E, Yavuz A, Yavuz M, Aydin F. Effect of the Farnesyl Transferase Inhibitor L-744,832 on the Colon Cancer Cell Line DLD-1 and Its Combined Use with Radiation and 5-FU. Chemotherapy 2005; 51:319-23. [PMID: 16224182 DOI: 10.1159/000088954] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/10/2004] [Accepted: 03/11/2005] [Indexed: 11/19/2022]
Abstract
BACKGROUND Ras oncogenes are found in 25% of human tumors and they significantly affect prognosis. One of the major fields studied to improve anticancer drugs is blockade of the oncogenic ras protein function. One of the mechanisms to block the function of these proteins is to block farnesylation using a farnesyl transferase inhibitor (FTI) and thus to prevent the ras from anchoring to the cell membrane. METHODS In this study, we investigated the effects of FTI L-744,832 either alone or in combination with 5-fluorouracil (5-FU; 1 microM/l) and radiotherapy (2, 6, and 10 Gy) on the colon cancer cell line DLD-1 with mutations in K-, N- and H-ras, c-myb, c-myc, p53, fos, sis and DNA repair genes. Drugs were added 3 h after cultivation. Radiotherapy was performed on the 3rd day of the study. On the 3rd day, medium and drugs were changed. Evaluations were performed on the 6th day. RESULTS Administration of L-744,832, neither alone nor its combination with 5-FU and radiation, affected the number of DLD-1 cells and apoptosis rates. Regarding its effects on the cell cycle, L-744,832 was shown to lead to G(0)/G(1) and G(2)/M accumulation in a dose-dependent manner when administered alone. However, in combination with 5-FU, only a G(0)/G(1) accumulation was observed. CONCLUSION Our study showed that FTI L-744,832 does not effect the cell number and apoptosis rate of DLD-1 cells and it cannot overcome 5-FU and radiation resistance, although it is able to modify some phases of the cell cycle.
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Affiliation(s)
- Halil Kavgaci
- Department of Medical Oncology, Faculty of Medicine, Karadeniz Technical University, Trabzon, Turkey.
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25
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Cesario A, Catassi A, Festi L, Imperatori A, Pericelli A, Galetta D, Margaritora S, Porziella V, Cardaci V, Granone P, Dominioni L, Russo P. Farnesyltransferase inhibitors and human malignant pleural mesothelioma: a first-step comparative translational study. Clin Cancer Res 2005; 11:2026-37. [PMID: 15756029 DOI: 10.1158/1078-0432.ccr-04-1450] [Citation(s) in RCA: 5] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/16/2022]
Abstract
It is known that the potential clinical use of farnesyltransferase inhibitors (FTI) could be expanded to include cancers harboring activated receptor tyrosine kinases. Approximately 70% of malignant pleural mesotheliomas (MPM) overexpress epidermal growth factor receptors (EGFR) and a subset express both EGFR and transforming growth factor alpha (TGF-alpha), suggesting an autocrine role for EGFR in MPM. We checked on MPM cells (10 human cell lines, 11 primary cultures obtained by human biopsies, and 7 short-term normal mesothelial cell cultures) concerning the following: (a) the relative overexpression of EGFR (Western blotting, flow cytometry, immunohistochemistry), (b) the relative expression of EGFR ligands (EGF, amphiregulin, TGF-alpha, ELISA), (c) the relative increase of the activated form of Ras (Ras-bound GTP) after EGF stimulation (Ras activation assay), (d) the efficacy of five different FTIs (HDJ2 prenylation, cell cytotoxicity, and apoptosis using ApopTag and gel ladder). EGFR was overexpressed in MPM cells compared with normal pleural mesothelial cells in equivalent levels as in non-small cell lung cancer cells A459. MPM cells constitutively expressed EGFR ligands; however, Ras activation was attenuated at high EGF concentrations (100 ng/mL). Growth of MPM cells was substantially not affected by treatment with different FTIs (SCH66336, BMS-214662, R115777, RPR-115135, and Manumycin). Among these, BMS-214662 was the only one moderately active. BMS-214662 triggered apoptosis in a small fraction of cells (not higher than 30%) that was paralleled by a slight decrease in the levels of TGF-alpha secreted by treated MPM cells. Our data highlighted the concept that the same signaling pathway can be regulated in different ways and these regulations can differ between different cells of different origin.
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Affiliation(s)
- Alfredo Cesario
- Department of Surgical Science, Division of General Thoracic Surgery, Catholic University, Rome, Italy
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Fang JY, Chen YX, Lu J, Lu R, Yang L, Zhu HY, Gu WQ, Lu LG. Epigenetic modification regulates both expression of tumor-associated genes and cell cycle progressing in human colon cancer cell lines: Colo-320 and SW1116. Cell Res 2005; 14:217-26. [PMID: 15225415 DOI: 10.1038/sj.cr.7290222] [Citation(s) in RCA: 37] [Impact Index Per Article: 1.9] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/31/2023] Open
Abstract
The aim of this study is to assess the effects of DNA methylation and histone acetylation, alone or in combination, on the expression of several tumor-associated genes and cell cycle progression in two established human colon cancer cell lines: Colo-320 and SW1116. Treatments with 5-aza-2-deoxycytidine (5-aza-dC) and trichostatin A, alone or in combination, were applied respectively. The methylation status of the CDKN2A promoter was determined by methylation-specific PCR, and the acetylated status of the histones associated with the p21WAF1 and CDKN2A genes was examined by chromatin immunoprecipitation. The expression of the CDKN2A, p21WAF1, p53, p73, APC, c-myc, c-Ki-ras and survivin genes was detected by real-time RT-PCR and RT-PCR. The cell cycle profile was established by flow cytometry. We found that along with the demethylation of the CDKN2A gene promoter in both cell lines induced by 5-aza-dC alone or in combination with TSA, the expression of both CDKN2A and APC genes increased. The treatment of TSA or sodium butyrate up-regulated the transcription of p21WAF1 significantly by inducing the acetylation of histones H4 and H3, but failed to alter the acetylation level of CDKN2A-associated histones. No changes in transcription of p53, p73, c-myc, c-Ki-ras and survivin genes were observed. In addition, TSA or sodium butyrate was shown to arrest cells at the G1 phase. However, 5-aza-dC was not able to affect the cell cycle progression. In conclusion, regulation by epigenetic modification of the transcription of tumor-associated genes and the cell cycle progression in both human colon cancer cell lines Colo-320 and SW1116 is gene-specific.
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Affiliation(s)
- Jing Yuan Fang
- Shanghai Institute of Digestive Disease, Renji Hospital Shanghai Second Medical University, Shanghai 200001, China.
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Nishibori M, Mori S, Takahashi HK. Physiology and pathophysiology of proteinase-activated receptors (PARs): PAR-2-mediated proliferation of colon cancer cell. J Pharmacol Sci 2005; 97:25-30. [PMID: 15655297 DOI: 10.1254/jphs.fmj04005x5] [Citation(s) in RCA: 21] [Impact Index Per Article: 1.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 10/25/2022] Open
Abstract
Proteinase-activated receptor-2 (PAR-2) has been demonstrated to be highly expressed in the gastrointestinal tract. In the present minireview, we summarize the effects of PAR-1 and PAR-2 stimulation using their activating peptides and agonist proteinases on the calcium signaling and the cell proliferation in DLD-1 cell, a human colon cancer cell line. PAR-2 but not PAR-1 stimulation induced the enhancement of cell proliferation, whereas both PAR-1 and PAR-2 stimulation induced the transient increase in [Ca(2+)](i). PAR-2 stimulation induced the phosphorylation of MEK1/2 and ERK1/2, but PAR-1 stimulation did not. The inhibition of MEK1/2 by PD98059 completely abolished the proliferative response to PAR-2 stimulation. Thus, MEK-ERK activation plays major role in the PAR-2-mediated proliferative response. The coupling of PARs to calcium signaling and MEK-ERK activation may be independent, and varied dependent on cell types.
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Affiliation(s)
- Masahiro Nishibori
- Department of Pharmacology, Okayama University Graduate School of Medicine and Dentistry, Okayama, Japan.
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Trombino S, Cesario A, Margaritora S, Granone P, Motta G, Falugi C, Russo P. Alpha7-nicotinic acetylcholine receptors affect growth regulation of human mesothelioma cells: role of mitogen-activated protein kinase pathway. Cancer Res 2004; 64:135-45. [PMID: 14729617 DOI: 10.1158/0008-5472.can-03-1672] [Citation(s) in RCA: 86] [Impact Index Per Article: 4.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/16/2022]
Abstract
This study presents data suggesting that both human mesothelioma (cell lines and human mesothelioma biopsies) and human normal mesothelial cells express receptors for acetylcholine and that stimulation of these receptors by nicotine prompted cell growth via activation of nicotinic cholinergic receptors. Thus, these data demonstrate that: (a) human mesothelioma cells and human biopsies of mesothelioma as well as of normal pleural mesothelial cells express functionally alpha-7 nicotinic acethlycholine receptors, evaluated by alpha-bungarotoxin-FITC binding, receptor binding assay, Western blot, and reverse transcription-PCR; (b) choline acetyltransferase immunostaining is present in mesothelioma cells; (c) mesothelioma cell growth is modulated by the cholinergic system in which agonists (i.e., nicotine) has a proliferative effect, and antagonists (i.e., curare) has an inhibitory effect, evaluated by cell cloning, DNA synthesis and cell cycle; (d) nicotine induces Ca(+2) influx, evaluated by [(45)Ca(2+)] uptake, and consequently activation of mitogen-activated protein kinase pathway (extracellular signal-regulated kinase and p90(RSK) phosphorylation), evaluated by Western blot; and (e) apoptosis mechanisms in mesothelioma cells are under the control of the cholinergic system (nicotine antiapoptotic via induction of nuclear factor-kappaB complexes and phosphorylation of Bad at Ser(112); curare proapoptotic via G(0)-G(1) arrest p21(waf-1) dependent but p53 independent). The involvement of the nonneuronal cholinergic system in mesothelioma appears reasonable and open up new therapeutic strategies.
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Affiliation(s)
- Sonya Trombino
- Department of Biology, University of Genoa, Genoa, Italy
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Jikuhara A, Yoshii M, Iwagaki H, Mori S, Nishibori M, Tanaka N. MAP kinase-mediated proliferation of DLD-1 carcinoma by the stimulation of protease-activated receptor 2. Life Sci 2003; 73:2817-29. [PMID: 14511767 DOI: 10.1016/s0024-3205(03)00702-1] [Citation(s) in RCA: 25] [Impact Index Per Article: 1.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/08/2023]
Abstract
Protease-activated receptor-2 (PAR-2) has been demonstrated to be highly expressed in the gastrointestinal tract. In the present study, we investigated the effects of PAR-2 stimulation on the cell signaling and proliferation of DLD-1, a human colon carcinoma cell line, in comparison with the PAR-1 stimulation. PAR-2 stimulation by agonist peptide SLIGKV concentration-dependently induced the increase in [Ca2+]i and the proliferation of DLD-1 whereas the inverse peptide LSIGKV did not. Trypin (10(-9) M), an agonist protease for PAR-2, also enhanced the proliferation of DLD-1. The proliferative response of DLD-1 to PAR-2 stimulation was associated with the transient phosphorylation of MEK and MAP kinase, but not p38 MAP kinase and JNK. Inhibition of MEK by PD98059 (50 microM) completely inhibited the proliferation-stimulating effects as well as the phosphorylation of MAP kinase induced by PAR-2 agonist peptide (100 microM) and trypsin (10(-9) M). The prolonged treatment with PAR-2 agonist peptide for more than one hour was required for the enhanced proliferative response, suggesting the existence of unknown long-lasting cooperative signaling with MAP kinase cascade. PAR-1 stimulation by the agonist peptide SFLLRN (100 microM) or thrombin (10(-8) M) produced Ca2+ signaling, however, the stimulation neither produced the cell proliferative response nor the activation of MEK-MAP kinase cascade. These results indicated that Ca2+ signaling induced by PARs activation was not enough for inducing the cell proliferation in DLD-1 cells and that stimulation of PAR-2 can induce the activation of MEK-MAP kinase cascade, leading to the growth promoting response.
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Affiliation(s)
- Atsushi Jikuhara
- Department of Gastroenterological Surgery and Transplant, Okayama University Graduate School of Medicine and Dentistry, 2-5-1 Shikata-cho, Okayama 700-8558, Japan
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Fang JY, Lu J, Chen YX, Yang L. Effects of DNA methylation on expression of tumor suppressor genes and proto-oncogene in human colon cancer cell lines. World J Gastroenterol 2003; 9:1976-80. [PMID: 12970888 PMCID: PMC4656656 DOI: 10.3748/wjg.v9.i9.1976] [Citation(s) in RCA: 25] [Impact Index Per Article: 1.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 02/06/2023] Open
Abstract
AIM: To investigate the effects of DNA methylation on the expression of tumor suppressor genes and proto-oncogene in human colon cancer cell lines.
METHODS: Three colon cancer cell lines (HT-29, SW1116 and Colo-320) treated with different concentrations of DNA methyltransferase inhibitor, 5-aza-2’-deoxycytidine (5-aza-dC) were used to induce DNA demethylation. The expressions of p16INK4A, p21WAF1, APC and c-myc genes were observed by using RT-PCR. The methylation status of p16INK4A promoter in HT-29 cells was also determined by methylation-specific PCR (MSP).
RESULTS: Weak expressions of p16INK4A and APC in the three colon cancer cells were detected, and p21WAF1 expression was not found in SW1116 and Colo-320 cells before treatment. After treatment of 1 μmol/L but not 10 μmol/L of 5-aza-dC, the methylation level of p16INK4A gene promoter decreased significantly, and the hypomethylation led to the up-regulation of p16INK4A gene transcription in HT-29 cells. In the cell lines of SW1116 and Colo-320, p16INK4A and APC mRNA expressions were obviously enhanced after treatment of either 10 μmol/L or 5 μmol/L 5-aza-dC for 24 h. However, no evidence was found that methylation regulated the expression of p21WAF1 and c-myc genes in human colon cancer cell lines.
CONCLUSION: Expression of p16INK4A and APC genes is regulated by DNA methylation in three human colon cancer cell lines.
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Affiliation(s)
- Jing-Yuan Fang
- Shanghai Institute of Digestive Diseases, Renji Hospital, Shanghai Second Medical University, Shanghai 200001, China.
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Russo P, Arzani D, Trombino S, Falugi C. c-myc down-regulation induces apoptosis in human cancer cell lines exposed to RPR-115135 (C31H29NO4), a non-peptidomimetic farnesyltransferase inhibitor. J Pharmacol Exp Ther 2003; 304:37-47. [PMID: 12490573 DOI: 10.1124/jpet.102.042952] [Citation(s) in RCA: 22] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/22/2022] Open
Abstract
A therapeutic strategy that relies on the use of c-myc antisense in combination with a farnesyltransferase inhibitor, RPR-115135 (C31H29NO4), was studied in human cancer cell lines carrying different mutations (Ras, p53, myc amplification). Cell proliferation was strongly inhibited by the combination and was observed when c-myc oligo (at a concentration that down-regulates c-myc expression) was followed by RPR-115135. Cell cycle analysis demonstrated an accumulation in G0-G1 phase and a tendency to apoptosis (not detectable in cells treated with a single agent). Morphological examination and DNA fragmentation assays (filter binding and enzyme-linked immunosorbent assay DNA fragmentation) confirmed the induction of apoptosis. Apoptosis was not p53- and/or p21(waf-1)-dependent, and the key effector was caspase activation. The combination induced Bax expression and Bcl-2 inhibition. Down-regulation of c-myc amplification carried out a specific role exclusively when Ras was mutated. Exposure of human proliferating lymphocytes to combination did not result in cytotoxicity, suggesting that mechanisms regulating c-myc gene expression during normal T cell proliferation might not be involved. Because of the high percentage of human tumors overexpressing c-myc mRNA and/or protein and, simultaneously, harboring oncogenic Ras mutants (i.e., colon cancers), interrupting the myc- and Ras-signaling pathway would be one of the major focuses on therapy of these types of tumors.
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Affiliation(s)
- Patrizia Russo
- Laboratory of Experimental Oncology, Molecular Pathology Section, National Institute for Research on Cancer, Genoa, Italy.
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