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Sgobba MN, Musio B, Pastrana CI, Todisco S, Schlosserovà N, Mastropirro F, Favia M, Radesco A, Duarte IF, De Grassi A, Volpicella M, Gallo V, Pierri CL, Ciani E, Guerra L. Serum Starvation Enhances the Antitumor Activity of Natural Matrices: Insights into Bioactive Molecules from Dromedary Urine Extracts. Molecules 2025; 30:821. [PMID: 40005133 PMCID: PMC11858132 DOI: 10.3390/molecules30040821] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/30/2024] [Revised: 01/31/2025] [Accepted: 02/05/2025] [Indexed: 02/27/2025] Open
Abstract
Natural matrices have historically been a cornerstone in drug discovery, offering a rich source of structurally diverse and biologically active compounds. However, research on natural products often faces significant challenges due to the complexity of natural matrices, such as urine, and the limitations of bioactivity assessment assays. To ensure reliable insights, it is crucial to optimize experimental conditions to reveal the bioactive potential of samples, thereby improving the validity of statistical analyses. Approaches in metabolomics further strengthen this process by identifying and focusing on the most promising compounds within natural matrices, enhancing the precision of bioactive metabolite prioritization. In this study, we assessed the bioactivity of 17 dromedary urine samples on human renal cells under serum-reduced conditions (1%FBS) in order to minimize possible FBS-derived interfering factors. Using viability assays and Annexin V/PI staining, we found that the tumor renal cell lines Caki-1 and RCC-Shaw were more sensitive to the cytotoxic effects of the small molecules present in dromedary urine compared to non-tumor HK-2 cells. Employing NMR metabolomics analysis combined with detected in vitro activity, our statistical model highlights the presence of bioactive compounds in dromedary urine, such as azelaic acid and phenylacetyl glycine, underscoring its potential as a sustainable source of bioactive molecules within the framework of green chemistry and circular economy initiatives.
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Affiliation(s)
- Maria Noemi Sgobba
- Department of Biosciences, Biotechnologies and Environment, University of Bari “Aldo Moro”, Via Orabona 4, 70125 Bari, Italy (F.M.); (A.D.G.); (M.V.); (E.C.); (L.G.)
| | - Biagia Musio
- Department of Civil, Environmental, Land, Building Engineering and Chemistry (DICATECh), Polytechnic University of Bari, Via Orabona 4, 70125 Bari, Italy; (B.M.); (S.T.); (V.G.)
| | - Carlos Iglesias Pastrana
- Faculty of Veterinary Sciences, Department of Genetics, University of Córdoba, 14071 Córdoba, Spain;
| | - Stefano Todisco
- Department of Civil, Environmental, Land, Building Engineering and Chemistry (DICATECh), Polytechnic University of Bari, Via Orabona 4, 70125 Bari, Italy; (B.M.); (S.T.); (V.G.)
| | - Nikola Schlosserovà
- Department of Biosciences, Biotechnologies and Environment, University of Bari “Aldo Moro”, Via Orabona 4, 70125 Bari, Italy (F.M.); (A.D.G.); (M.V.); (E.C.); (L.G.)
| | - Federica Mastropirro
- Department of Biosciences, Biotechnologies and Environment, University of Bari “Aldo Moro”, Via Orabona 4, 70125 Bari, Italy (F.M.); (A.D.G.); (M.V.); (E.C.); (L.G.)
| | - Maria Favia
- Department of Translational Biomedicine and Neurosciences (DiBraiN), University of Bari “Aldo Moro”, Piazza Giulio Cesare, 70124 Bari, Italy;
| | - Antonio Radesco
- Istituto Tumori “Giovanni Paolo II” I.R.C.C.S., Viale Orazio Flacco 65, 70124 Bari, Italy
| | - Iola F. Duarte
- Department of Chemistry, CICECO—Aveiro Institute of Materials and LAQV-REQUIMTE, University of Aveiro, 3810-193 Aveiro, Portugal;
| | - Anna De Grassi
- Department of Biosciences, Biotechnologies and Environment, University of Bari “Aldo Moro”, Via Orabona 4, 70125 Bari, Italy (F.M.); (A.D.G.); (M.V.); (E.C.); (L.G.)
| | - Mariateresa Volpicella
- Department of Biosciences, Biotechnologies and Environment, University of Bari “Aldo Moro”, Via Orabona 4, 70125 Bari, Italy (F.M.); (A.D.G.); (M.V.); (E.C.); (L.G.)
| | - Vito Gallo
- Department of Civil, Environmental, Land, Building Engineering and Chemistry (DICATECh), Polytechnic University of Bari, Via Orabona 4, 70125 Bari, Italy; (B.M.); (S.T.); (V.G.)
- Innovative Solutions S.r.l.—Spin-Off Company of the Polytechnic University of Bari, Zona H 150/B, 70015 Noci, Italy
| | - Ciro Leonardo Pierri
- Department of Pharmacy—Pharmaceutical Sciences, University of Bari Aldo Moro, Via Orabona 4, 70125 Bari, Italy
| | - Elena Ciani
- Department of Biosciences, Biotechnologies and Environment, University of Bari “Aldo Moro”, Via Orabona 4, 70125 Bari, Italy (F.M.); (A.D.G.); (M.V.); (E.C.); (L.G.)
| | - Lorenzo Guerra
- Department of Biosciences, Biotechnologies and Environment, University of Bari “Aldo Moro”, Via Orabona 4, 70125 Bari, Italy (F.M.); (A.D.G.); (M.V.); (E.C.); (L.G.)
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Downregulation of c-Myc expression confers sensitivity to CHK1 inhibitors in hematologic malignancies. Acta Pharmacol Sin 2022; 43:220-228. [PMID: 33782542 PMCID: PMC8724279 DOI: 10.1038/s41401-021-00652-1] [Citation(s) in RCA: 6] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 12/26/2020] [Accepted: 03/12/2021] [Indexed: 01/03/2023]
Abstract
Checkpoint kinase 1 inhibitors (CHK1i) have shown impressive single-agent efficacy in treatment of certain tumors, as monotherapy or potentiators of chemotherapy in clinical trials, but the sensitive tumor types and downstream effectors to dictate the therapeutic responses to CHK1i remains unclear. In this study we first analyzed GDSC (Genomics of Drug Sensitivity in Cancer) and DepMap database and disclosed that hematologic malignancies (HMs) were relatively sensitive to CHK1i or CHK1 knockdown. This notion was confirmed by examining PY34, a new and potent in-house selective CHK1i, which exhibited potent anti-HM effect in vitro and in vivo, as single agent. We demonstrated that the downregulation of c-Myc and its signaling pathway was the common transcriptomic profiling response of sensitive HM cell lines to PY34, whereas overexpressing c-Myc could partially rescue the anticancer effect of PY34. Strikingly, we revealed the significant correlations between downregulation of c-Myc and cell sensitivity to PY34 in 17 HM cell lines and 39 patient-derived cell (PDC) samples. Thus, our results demonstrate that HMs are more sensitive to CHK1i than solid tumors, and c-Myc downregulation could represent the CHK1i efficacy in HMs.
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Isono M, Okubo K, Asano T, Sato A. Inhibition of checkpoint kinase 1 potentiates anticancer activity of gemcitabine in bladder cancer cells. Sci Rep 2021; 11:10181. [PMID: 33986399 PMCID: PMC8119486 DOI: 10.1038/s41598-021-89684-5] [Citation(s) in RCA: 11] [Impact Index Per Article: 2.8] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/06/2020] [Accepted: 04/26/2021] [Indexed: 12/13/2022] Open
Abstract
Checkpoint kinases (CHKs) are involved in the DNA damage response in many cancer cells. CHK inhibitors have been used in clinical trials in combination with chemotherapeutics; however, their effect against bladder cancer remains unclear. Here, we investigated the efficacy of combining gemcitabine with MK-8776, a novel CHK1 inhibitor, in four bladder cancer cell lines. The effects of gemcitabine and MK-8776 on cell viability, clonogenicity, cell cycle, and apoptosis were examined alongside in vivo efficacy using murine xenograft tumor models. Combined treatment inhibited the viability and colony formation of bladder cancer cells compared to either single treatment. Although gemcitabine (10 nM) alone increased the cell number in S-phase, it increased the cell number in sub-G1 phase when combined with MK-8776 (0.5 µM). Combined treatment enhanced cleaved poly[ADP-ribose]-polymerase expression alongside the number of annexin-V-positive cells, indicating the induction of apoptosis. In vivo, administration of gemcitabine and MK-8776 was well tolerated and suppressed tumor growth. Mechanistically, the combined treatment elevated γH2A.X and suppressed Rad51 expression. Our study demonstrates that MK-8776 and gemcitabine combined induces apoptosis and suppresses proliferation in bladder cancer cells by inhibiting CHKs and DNA repair. Therefore, CHK1 inhibition combined with gemcitabine may be a potential treatment for bladder cancer.
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Affiliation(s)
- Makoto Isono
- Department of Urology, National Defense Medical College, 3-2 Namiki, Tokorozawa, Saitama, 359-8513, Japan.
| | - Kazuki Okubo
- Department of Urology, National Defense Medical College, 3-2 Namiki, Tokorozawa, Saitama, 359-8513, Japan
| | - Takako Asano
- Department of Urology, National Defense Medical College, 3-2 Namiki, Tokorozawa, Saitama, 359-8513, Japan
| | - Akinori Sato
- Department of Urology, National Defense Medical College, 3-2 Namiki, Tokorozawa, Saitama, 359-8513, Japan
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Chronic Sulforaphane Administration Inhibits Resistance to the mTOR-Inhibitor Everolimus in Bladder Cancer Cells. Int J Mol Sci 2020; 21:ijms21114026. [PMID: 32512849 PMCID: PMC7312500 DOI: 10.3390/ijms21114026] [Citation(s) in RCA: 15] [Impact Index Per Article: 3.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/28/2020] [Revised: 05/28/2020] [Accepted: 06/03/2020] [Indexed: 02/06/2023] Open
Abstract
Progressive bladder cancer growth is associated with abnormal activation of the mammalian target of the rapamycin (mTOR) pathway, but treatment with an mTOR inhibitor has not been as effective as expected. Rather, resistance develops under chronic drug use, prompting many patients to lower their relapse risk by turning to natural, plant-derived products. The present study was designed to evaluate whether the natural compound, sulforaphane (SFN), combined with the mTOR inhibitor everolimus, could block the growth and proliferation of bladder cancer cells in the short- and long-term. The bladder cancer cell lines RT112, UMUC3, and TCCSUP were exposed short- (24 h) or long-term (8 weeks) to everolimus (0.5 nM) or SFN (2.5 µM) alone or in combination. Cell growth, proliferation, apoptosis, cell cycle progression, and cell cycle regulating proteins were evaluated. siRNA blockade was used to investigate the functional impact of the proteins. Short-term application of SFN and/or everolimus resulted in significant tumor growth suppression, with additive inhibition on clonogenic tumor growth. Long-term everolimus treatment resulted in resistance development characterized by continued growth, and was associated with elevated Akt-mTOR signaling and cyclin-dependent kinase (CDK)1 phosphorylation and down-regulation of p19 and p27. In contrast, SFN alone or SFN+everolimus reduced cell growth and proliferation. Akt and Rictor signaling remained low, and p19 and p27 expressions were high under combined drug treatment. Long-term exposure to SFN+everolimus also induced acetylation of the H3 and H4 histones. Phosphorylation of CDK1 was diminished, whereby down-regulation of CDK1 and its binding partner, Cyclin B, inhibited tumor growth. In conclusion, the addition of SFN to the long-term everolimus application inhibits resistance development in bladder cancer cells in vitro. Therefore, sulforaphane may hold potential for treating bladder carcinoma in patients with resistance to an mTOR inhibitor.
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Fang X, Liu X, Weng C, Wu Y, Li B, Mao H, Guan M, Lu L, Liu G. Construction and Validation of a Protein Prognostic Model for Lung Squamous Cell Carcinoma. Int J Med Sci 2020; 17:2718-2727. [PMID: 33162799 PMCID: PMC7645351 DOI: 10.7150/ijms.47224] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 04/20/2020] [Accepted: 09/14/2020] [Indexed: 12/13/2022] Open
Abstract
Lung squamous cell carcinoma (LUSCC), as the major type of lung cancer, has high morbidity and mortality rates. The prognostic markers for LUSCC are much fewer than lung adenocarcinoma. Besides, protein biomarkers have advantages of economy, accuracy and stability. The aim of this study was to construct a protein prognostic model for LUSCC. The protein expression data of LUSCC were downloaded from The Cancer Protein Atlas (TCPA) database. Clinical data of LUSCC patients were downloaded from The Cancer Genome Atlas (TCGA) database. A total of 237 proteins were identified from 325 cases of LUSCC patients based on the TCPA and TCGA database. According to Kaplan-Meier survival analysis, univariate and multivariate Cox analysis, a prognostic prediction model was established which was consisted of 6 proteins (CHK1_pS345, CHK2, IRS1, PAXILLIN, BRCA2 and BRAF_pS445). After calculating the risk values of each patient according to the coefficient of each protein in the risk model, the LUSCC patients were divided into high risk group and low risk group. The survival analysis demonstrated that there was significant difference between these two groups (p= 4.877e-05). The area under the curve (AUC) value of the receiver operating characteristic (ROC) curve was 0.699, which suggesting that the prognostic risk model could effectively predict the survival of LUSCC patients. Univariate and multivariate analysis indicated that this prognostic model could be used as independent prognosis factors for LUSCC patients. Proteins co-expression analysis showed that there were 21 proteins co-expressed with the proteins in the risk model. In conclusion, our study constructed a protein prognostic model, which could effectively predict the prognosis of LUSCC patients.
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Affiliation(s)
- Xisheng Fang
- Department of Medical Oncology, Guangzhou First People's Hospital, School of Medicine, South China University of Technology, Guangzhou, Guangdong, China, 510180.,Department of Medical Oncology, Guangzhou First People's Hospital, Guangzhou Medical University, Guangzhou, Guangdong, China, 510180
| | - Xia Liu
- Department of Medical Oncology, Guangzhou First People's Hospital, School of Medicine, South China University of Technology, Guangzhou, Guangdong, China, 510180.,Department of Medical Oncology, Guangzhou First People's Hospital, Guangzhou Medical University, Guangzhou, Guangdong, China, 510180
| | - Chengyin Weng
- Department of Medical Oncology, Guangzhou First People's Hospital, School of Medicine, South China University of Technology, Guangzhou, Guangdong, China, 510180.,Department of Medical Oncology, Guangzhou First People's Hospital, Guangzhou Medical University, Guangzhou, Guangdong, China, 510180
| | - Yong Wu
- Department of Medical Oncology, Guangzhou First People's Hospital, School of Medicine, South China University of Technology, Guangzhou, Guangdong, China, 510180.,Department of Medical Oncology, Guangzhou First People's Hospital, Guangzhou Medical University, Guangzhou, Guangdong, China, 510180
| | - Baoxiu Li
- Department of Medical Oncology, Guangzhou First People's Hospital, School of Medicine, South China University of Technology, Guangzhou, Guangdong, China, 510180.,Department of Medical Oncology, Guangzhou First People's Hospital, Guangzhou Medical University, Guangzhou, Guangdong, China, 510180
| | - Haibo Mao
- Department of Medical Oncology, Guangzhou First People's Hospital, School of Medicine, South China University of Technology, Guangzhou, Guangdong, China, 510180.,Department of Medical Oncology, Guangzhou First People's Hospital, Guangzhou Medical University, Guangzhou, Guangdong, China, 510180
| | - Mingmei Guan
- Department of Medical Oncology, Guangzhou First People's Hospital, School of Medicine, South China University of Technology, Guangzhou, Guangdong, China, 510180.,Department of Medical Oncology, Guangzhou First People's Hospital, Guangzhou Medical University, Guangzhou, Guangdong, China, 510180
| | - Lin Lu
- Department of Medical Oncology, Guangzhou First People's Hospital, School of Medicine, South China University of Technology, Guangzhou, Guangdong, China, 510180.,Department of Medical Oncology, Guangzhou First People's Hospital, Guangzhou Medical University, Guangzhou, Guangdong, China, 510180
| | - Guolong Liu
- Department of Medical Oncology, Guangzhou First People's Hospital, School of Medicine, South China University of Technology, Guangzhou, Guangdong, China, 510180.,Department of Medical Oncology, Guangzhou First People's Hospital, Guangzhou Medical University, Guangzhou, Guangdong, China, 510180
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Cheng C, Yun F, He J, Ullah S, Yuan Q. Design, synthesis and biological evaluation of novel thioquinazolinone-based 2-aminobenzamide derivatives as potent histone deacetylase (HDAC) inhibitors. Eur J Med Chem 2019; 173:185-202. [DOI: 10.1016/j.ejmech.2019.04.017] [Citation(s) in RCA: 10] [Impact Index Per Article: 1.7] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/19/2018] [Revised: 03/15/2019] [Accepted: 04/08/2019] [Indexed: 10/27/2022]
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Wu YC, Wang WT, Huang LJ, Cheng RY, Kuo YR, Hou MF, Lai CS, Yu J. Differential Response of Non-cancerous and Malignant Breast Cancer Cells to Conditioned Medium of Adipose tissue-derived Stromal Cells (ASCs). Int J Med Sci 2019; 16:893-901. [PMID: 31337963 PMCID: PMC6643111 DOI: 10.7150/ijms.27125] [Citation(s) in RCA: 5] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 05/07/2018] [Accepted: 04/03/2019] [Indexed: 01/22/2023] Open
Abstract
Background: The application of adipose tissue-derived stromal cells (ASCs) in regenerative medicine has become a growing trend due to its abundance and differentiation potentials. However, several breast cancer studies indicated that ASCs promote tumor progression, therefore, the use of ASCs for reconstruction after oncological surgery poses potential risks. In this study, we aimed to examine whether cancerous or non-cancerous breast cells will exhibit different responses to ASC-derived CM. Methods: ASCs were isolated from residuals of subcutaneous adipose tissue obtained from patients undergoing surgery. Cancerous MCF-7, MDA-MB231, and MDA-MB468 cell lines and one non-cancerous M10/H184B5F5 cell line were cultured with variant concentrations of ASC-derived conditioned medium (CM) for analysis. Results: ASC-derived CM significantly reduced cell viability by triggering apoptosis in MCF-7, MDA-MB231, and MDA-MB468 cell lines. ATM-Chk2-dependent DNA damage response was activated early in cancer cells when exposed to ASC-derived CM. By contrast, prompted cell proliferation instead of cell death was detected in M10/H184B5F5 cells under the treatment of lower CM concentration. Even when exposed to the highest concentration of CM, only cell cycle arrest accompanied by a weak DNA damage response were detected in M10/H184B5F5 cells, no cell deaths were observed. Conclusions: Overall, this study demonstrated that cancerous and non-cancerous breast cells respond differently to ASC-derived CM. ASC-derived CM triggered significant cell death in breast cancer cell lines, however non-cancerous breast cells exhibited dissimilar response to ASC-derived CM.
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Affiliation(s)
- Yi-Chia Wu
- Division of Plastic Surgery, Department of Surgery, Kaohsiung Medical University Hospital, Kaohsiung, Taiwan.,Department of Plastic Surgery, Kaohsiung Municipal Ta-Tung Hospital, Kaohsiung, Taiwan.,Graduate Institute of Medicine, Kaohsiung Medical University, Kaohsiung, Taiwan.,PhD. Programme in Translational Medicine, Kaohsiung Medical University, Kaohsiung, and Academia Sinica, Taipei, Taiwan
| | - Wei-Ting Wang
- Center of Teaching and Research, Kaohsiung Municipal Ta-Tung Hospital, Kaohsiung, Taiwan
| | - Li-Ju Huang
- Center of Teaching and Research, Kaohsiung Municipal Ta-Tung Hospital, Kaohsiung, Taiwan
| | - Ruo-You Cheng
- Division of Plastic Surgery, Department of Surgery, Kaohsiung Medical University Hospital, Kaohsiung, Taiwan
| | - Yur-Ren Kuo
- Division of Plastic Surgery, Department of Surgery, Kaohsiung Medical University Hospital, Kaohsiung, Taiwan.,Graduate Institute of Medicine, Kaohsiung Medical University, Kaohsiung, Taiwan.,Faculty of Medicine, College of Medicine, Kaohsiung Medical University, Kaohsiung, Taiwan
| | - Ming-Feng Hou
- Graduate Institute of Medicine, Kaohsiung Medical University, Kaohsiung, Taiwan.,Faculty of Medicine, College of Medicine, Kaohsiung Medical University, Kaohsiung, Taiwan
| | - Chung-Sheng Lai
- Division of Plastic Surgery, Department of Surgery, Kaohsiung Medical University Hospital, Kaohsiung, Taiwan.,Graduate Institute of Medicine, Kaohsiung Medical University, Kaohsiung, Taiwan.,Faculty of Medicine, College of Medicine, Kaohsiung Medical University, Kaohsiung, Taiwan
| | - John Yu
- Institute of Stem Cell and Translational Cancer Research, Chang Gung Memorial Hospital at Linko, Taoyuan, Taiwan.,Institute of Cellular and Organismic Biology, Academia Sinica, Taipei, Taiwan
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Alternative Chk1-independent S/M checkpoint in somatic cells that prevents premature mitotic entry. Med Oncol 2017; 34:70. [PMID: 28349497 DOI: 10.1007/s12032-017-0932-3] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/23/2017] [Accepted: 03/23/2017] [Indexed: 12/22/2022]
Abstract
Genomic instability is the hallmark of cancer. Checkpoint kinase-1 (Chk1) is required for cell cycle delay after DNA damage or blocked DNA replication. Chk1-depleted tumor cells undergo premature mitosis and apoptosis. Here we analyzed the depletion of Chk1 in normal somatic cells in the absence of DNA damage in order to investigate alternative cell cycle checkpoint mechanism(s). By means of adenoviruses, flow cytometry, immunofluorescence and Western blotting, Chk1-depleted mouse embryonic fibroblasts (MEFs) were investigated. Chk1-/- MEFs arrested at the S/G2 boundary of the cell cycle with decreased protein levels of many cell cycle key players. Cyclin B1 was predominantly cytoplasmic. Interestingly, overexpression of nuclear dominant Cyclin B1 leads to nuclear translocation and premature mitosis. Chk1-/- MEFs exhibited the absence of double-strand breaks, yet cells showed delayed DNA damage recovery with pan-nuclear immunostaining pattern of Histone H2AX. Activation of this checkpoint would elicit a senescent-like phenotype. Taken together, our elaborated data revealed the existence of an additional S/M checkpoint functioning via γH2AX signaling and cytoplasmic retention of Cyclin B1 in somatic cells.
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Isono M, Hoffmann MJ, Pinkerneil M, Sato A, Michaelis M, Cinatl J, Niegisch G, Schulz WA. Checkpoint kinase inhibitor AZD7762 strongly sensitises urothelial carcinoma cells to gemcitabine. JOURNAL OF EXPERIMENTAL & CLINICAL CANCER RESEARCH : CR 2017; 36:1. [PMID: 28049532 PMCID: PMC5209915 DOI: 10.1186/s13046-016-0473-1] [Citation(s) in RCA: 72] [Impact Index Per Article: 9.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Subscribe] [Scholar Register] [Received: 08/25/2016] [Accepted: 12/12/2016] [Indexed: 01/22/2023]
Abstract
Background More effective chemotherapies are urgently needed for bladder cancer, a major cause of morbidity and mortality worldwide. We therefore explored the efficacy of the combination of gemcitabine and AZD7762, a checkpoint kinase 1/2 (CHK1/2) inhibitor, for bladder cancer. Methods Viability, clonogenicity, cell cycle distribution and apoptosis were assessed in urothelial cancer cell lines and various non-malignant urothelial cells treated with gemcitabine and AZD7762. DNA damage was assessed by γH2A.X and 53-BP1 staining and checkpoint activation was followed by Western blotting. Pharmacological inhibition of CHK1 and CHK2 was compared to downregulation of either CHK1 or CHK2 using siRNAs. Results Combined use of gemcitabine and AZD7762 synergistically reduced urothelial carcinoma cell viability and colony formation relative to either single treatment. Non-malignant urothelial cells were substantially less sensitive to this drug combination. Gemcitabine plus AZD7762 inhibited cell cycle progression causing cell accumulation in S-phase. Moreover, the combination induced pronounced levels of apoptosis as indicated by an increase in the fraction of sub-G1 cells, in the levels of cleaved PARP, and in caspase 3/7 activity. Mechanistic investigations showed that AZD7762 treatment inhibited the repair of gemcitabine-induced double strand breaks by interference with CHK1, since siRNA-mediated depletion of CHK1 but not of CHK2 mimicked the effects of AZD7762. Conclusions AZD7762 enhanced sensitivity of urothelial carcinoma cells to gemcitabine by inhibiting DNA repair and disturbing checkpoints. Combining gemcitabine with CHK1 inhibition holds promise for urothelial cancer therapy. Electronic supplementary material The online version of this article (doi:10.1186/s13046-016-0473-1) contains supplementary material, which is available to authorized users.
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Affiliation(s)
- Makoto Isono
- Department of Urology, Medical Faculty, Heinrich-Heine-University Düsseldorf, Universitätsstraße 1, 40225, Düsseldorf, Germany
| | - Michèle J Hoffmann
- Department of Urology, Medical Faculty, Heinrich-Heine-University Düsseldorf, Universitätsstraße 1, 40225, Düsseldorf, Germany
| | - Maria Pinkerneil
- Department of Urology, Medical Faculty, Heinrich-Heine-University Düsseldorf, Universitätsstraße 1, 40225, Düsseldorf, Germany
| | - Akinori Sato
- Department of Urology, National Defense Medical College, Namiki 3-2, 359-8513, Tokorozawa, Japan
| | - Martin Michaelis
- Centre for Molecular Processing and School of Biosciences, University of Kent, Canterbury, CT2 7NJ, UK
| | - Jindrich Cinatl
- Institut für Medizinische Virologie, Klinikum der Goethe-Universität, Paul-Ehrlich‑Str. 40, 60596, Frankfurt am Main, Germany
| | - Günter Niegisch
- Department of Urology, Medical Faculty, Heinrich-Heine-University Düsseldorf, Universitätsstraße 1, 40225, Düsseldorf, Germany
| | - Wolfgang A Schulz
- Department of Urology, Medical Faculty, Heinrich-Heine-University Düsseldorf, Universitätsstraße 1, 40225, Düsseldorf, Germany.
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10
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Desmarais JA, Unger C, Damjanov I, Meuth M, Andrews P. Apoptosis and failure of checkpoint kinase 1 activation in human induced pluripotent stem cells under replication stress. Stem Cell Res Ther 2016; 7:17. [PMID: 26810087 PMCID: PMC4727355 DOI: 10.1186/s13287-016-0279-2] [Citation(s) in RCA: 24] [Impact Index Per Article: 2.7] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/20/2015] [Revised: 04/27/2015] [Accepted: 01/12/2016] [Indexed: 12/16/2022] Open
Abstract
Background Human induced pluripotent stem (hiPS) cells have the ability to undergo self-renewal and differentiation similarly to human embryonic stem (hES) cells. We have recently shown that hES cells under replication stress fail to activate checkpoint kinase 1 (CHK1). They instead commit to apoptosis, which appears to be a primary defense mechanism against genomic instability. It is not known whether the failure of CHK1 activation and activation of apoptosis under replication stress is solely a feature of hES cells, or if it is a feature that can be extended to hiPS cells. Methods Here we generated integration-free hiPS cell lines by mRNA transfection, and characterised the cell lines. To investigate the mechanism of S phase checkpoint activation, we have induced replication stress by adding excess thymidine to the cell culture medium, and performed DNA content analysis, apoptosis assays and immunoblottings. Results We are showing that hiPS cells similarly to hES cells, fail to activate CHK1 when exposed to DNA replication inhibitors and commit to apoptosis instead. Our findings also suggest the Ataxia Telangiectasia Mutated pathway might be responding to DNA replication stress, resulting in apoptosis. Conclusion Together, these data suggest that the apoptotic response was properly restored during reprogramming with mRNA, and that apoptosis is an important mechanism shared by hiPS and hES cells to maintain their genomic integrity when a replication stress occurs.
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Affiliation(s)
- Joelle A Desmarais
- Centre for Stem Cell Biology, Department of Biomedical Science, University of Sheffield, Western Bank, Sheffield, S10 2TN, UK. .,Institute for Cancer Studies, Department of Oncology, School of Medicine, University of Sheffield, Beech Hill Road, Sheffield, S10 2RX, UK.
| | - Christian Unger
- Centre for Stem Cell Biology, Department of Biomedical Science, University of Sheffield, Western Bank, Sheffield, S10 2TN, UK.
| | - Ivan Damjanov
- Department of Pathology, University of Kansas Hospital, 3901 Rainbow Blvd, Kansas City, KS, 66160, USA.
| | - Mark Meuth
- Institute for Cancer Studies, Department of Oncology, School of Medicine, University of Sheffield, Beech Hill Road, Sheffield, S10 2RX, UK.
| | - Peter Andrews
- Centre for Stem Cell Biology, Department of Biomedical Science, University of Sheffield, Western Bank, Sheffield, S10 2TN, UK.
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Juengel E, Thomas A, Rutz J, Makarevic J, Tsaur I, Nelson K, Haferkamp A, Blaheta RA. Amygdalin inhibits the growth of renal cell carcinoma cells in vitro. Int J Mol Med 2015; 37:526-32. [PMID: 26709398 DOI: 10.3892/ijmm.2015.2439] [Citation(s) in RCA: 27] [Impact Index Per Article: 2.7] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/05/2015] [Accepted: 12/04/2015] [Indexed: 11/06/2022] Open
Abstract
Although amygdalin is used by many cancer patients as an antitumor agent, there is a lack of information on the efficacy and toxicity of this natural compound. In the present study, the inhibitory effect of amygdalin on the growth of renal cell carcinoma (RCC) cells was examined. Amygdalin (10 mg/ml) was applied to the RCC cell lines, Caki-1, KTC-26 and A498, for 24 h or 2 weeks. Untreated cells served as controls. Tumor cell growth and proliferation were determined using MTT and BrdU tests, and cell cycle phases were evaluated. Expression of the cell cycle activating proteins cdk1, cdk2, cdk4, cyclin A, cyclin B, cyclin D1 and D3 as well as of the cell cycle inhibiting proteins p19 and p27 was examined by western blot analysis. Surface expression of the differentiation markers E- and N-cadherin was also investigated. Functional blockade by siRNA was used to determine the impact of several proteins on tumor cell growth. Amygdalin treatment caused a significant reduction in RCC cell growth and proliferation. This effect was correlated with a reduced percentage of G2/M-phase RCC cells and an increased percentage of cells in the G0/1-phase (Caki-1 and A498) or cell cycle arrest in the S-phase (KTC-26). Furthermore, amygdalin induced a marked decrease in cell cycle activating proteins, in particular cdk1 and cyclin B. Functional blocking of cdk1 and cyclin B resulted in significantly diminished tumor cell growth in all three RCC cell lines. Aside from its inhibitory effects on growth, amygdalin also modulated the differentiation markers, E- and N-cadherin. Hence, exposing RCC cells to amygdalin inhibited cell cycle progression and tumor cell growth by impairing cdk1 and cyclin B expression. Moreover, we noted that amygdalin affected differentiation markers. Thus, we suggest that amygdalin exerted RCC antitumor effects in vitro.
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Affiliation(s)
- Eva Juengel
- Department of Urology, Goethe-University Hospital, D-60590 Frankfurt am Main, Germany
| | - Anita Thomas
- Department of Urology, Goethe-University Hospital, D-60590 Frankfurt am Main, Germany
| | - Jochen Rutz
- Department of Urology, Goethe-University Hospital, D-60590 Frankfurt am Main, Germany
| | - Jasmina Makarevic
- Department of Urology, Goethe-University Hospital, D-60590 Frankfurt am Main, Germany
| | - Igor Tsaur
- Department of Urology, Goethe-University Hospital, D-60590 Frankfurt am Main, Germany
| | - Karen Nelson
- Department of Vascular and Endovascular Surgery, Goethe-University Hospital, D-60590 Frankfurt am Main, Germany
| | - Axel Haferkamp
- Department of Urology, Goethe-University Hospital, D-60590 Frankfurt am Main, Germany
| | - Roman A Blaheta
- Department of Urology, Goethe-University Hospital, D-60590 Frankfurt am Main, Germany
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12
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Meuth M. Inhibitors of cell cycle checkpoints and DNA replication cause different responses in normal versus malignant urothelial cells. Mol Cell Oncol 2014; 1:e968508. [PMID: 27308366 PMCID: PMC4905207 DOI: 10.4161/23723548.2014.968508] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.1] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/20/2014] [Revised: 08/20/2014] [Accepted: 08/20/2014] [Indexed: 11/23/2022]
Abstract
S-phase checkpoints are triggered in tumor cells in response to DNA replication stress caused by the tumor microenvironment or oncogenes. A recent report from our laboratory showed that tumor cells and more normal epithelial cells have a very different response to replication stress. In this Author's View, the implications of this finding are discussed.
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Affiliation(s)
- Mark Meuth
- Molecular Oncology Unit; Department of Oncology; University of Sheffield School of Medicine ; Sheffield, UK
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