DOI QR코드

DOI QR Code

No Relationship between the Amount of DNA Damage and the Level of hMLH1 and RASSF1A Gene Expression in Bladder Cancer Cells Treated with Cisplatin and Gemcitabine

  • de Camargo, Elaine Aparecida (Department of Pathology, Sao Paulo State University, Botucatu Medical School) ;
  • da Silva, Glenda Nicioli (Department of Pathology, Sao Paulo State University, Botucatu Medical School) ;
  • Gobette, Camila Pereira (Department of Pathology, Sao Paulo State University, Botucatu Medical School) ;
  • de Castro Marcondes, Joao Paulo (Department of Pathology, Sao Paulo State University, Botucatu Medical School) ;
  • Salvadori, Daisy Maria Favero (Department of Pathology, Sao Paulo State University, Botucatu Medical School)
  • Published : 2013.10.30

Abstract

Tumor response to antineoplastic drugs is not always predictable. This is also true for bladder carcinoma, a highly recurrent neoplasia. Currently, the combination of cisplatin and gemcitabine is well accepted as a standard protocol for treating bladder carcinoma. However, in some cases, this treatment protocol causes harmful side effects. Therefore, we investigated the roles of the genes TP53, RASSF1A (a tumor suppressor gene) and hMLH1 (a gene involved in the mismatch repair pathway) in cell susceptibility to cisplatin/gemcitabine treatment. Two bladder transitional carcinoma cell (TCC) lines, RT4 (wild-type TP53) and 5637 (mutated TP53), were used in this study. First, we evaluated whether the genotoxic potential of cisplatin/gemcitabine was dependent on TP53 status. Then, we evaluated whether the two antineoplastic drugs modulated RASSF1A and hMLH1 expression in the two cell lines. Increased DNA damage was observed in both cell lines after treatment with cisplatin or gemcitabine and with the two drugs simultaneously, as depicted by the comet assay. A lack of RASSF1A expression and hypermethylation of its promoter were observed before and after treatment in both cell lines. On the other hand, hMLH1 downregulation, unrelated to methylation status, was observed in RT4 cells after treatment with cisplatin or with cisplatin and gemcitabine simultaneously (wild-type TP53); in 5637 cells, hMLH1 was upregulated only after treatment with gemcitabine. In conclusion, the three treatment protocols were genotoxic, independent of TP53 status. However, cisplatin was the most effective, causing the highest level of DNA damage in both wild-type and mutated TP53 cells. Gemcitabine was the least genotoxic agent in both cell lines. Furthermore, no relationship was observed between the amount of DNA damage and the level of hMLH1 and RASSF1A expression. Therefore, other alternative pathways might be involved in cisplatin and gemcitabine genotoxicity in these two bladder cancer cell lines.

Keywords

DNA damage;DNA repair;gene expression profile;genotoxicity

References

  1. Aebi S, Fink D, Gordon R, et al (1997). Resistance to cytotoxic drugs in DNA mismatch repair-deficient cells. Clin Cancer Res, 3, 1763-7.
  2. Andrews GA, Xi S, Pomerantz RG, et al (2004). Mutation of p53 in head and neck squamous cell carcinoma correlates with BCL-2 expression and increased susceptibility to cisplatin-induced apoptosis. Head Neck, 26, 870-7. https://doi.org/10.1002/hed.20029
  3. Bernstein C, Bernstein H, Payne CM, Garewal H (2002). DNA repair/pro-apoptotidual-role proteins in five major DNA repair pathways: fail-safe protectionagainst carcinogenesis. Mutat Res, 511, 145-78. https://doi.org/10.1016/S1383-5742(02)00009-1
  4. Basu A, Krishnamurthy S (2010). Cellular responses to cisplatin-induced DNA damage. J Nucleic Acids, 2, 201367.
  5. Batista LFZ, Roos WP, Christmann M, Menck CFM, Kaina B (2007). Differential sensitivity of malignant glioma cells to methylating and chloroethylating anticancer drugs: p53 determines the switch by regulating xpc, ddb2, and dna double-strand breaks. Cancer Res, 67, 11886-95. https://doi.org/10.1158/0008-5472.CAN-07-2964
  6. Bellmut J, Albiol S, Ramirez de Olano A, Pujadas J, Maroto P (2006). On behalf the Spanish oncology genitourinary group (SOGUG). Gemcitabine in the treatment of advanced transitional cell carcinoma of the urothelium. Ann Oncol, 17, 113-7. https://doi.org/10.1093/annonc/mdj964
  7. Blasiak J, Kowalik J, Trzeciak A, Wojewodzka M (1999). Cytotoxicity and DNA damage and repair in human lymphocytes exposed to three anticancer platinum drugs. Neoplasma, 46, 61-3.
  8. Brooks CL, Gu W (2003). Ubiquitination, phosphorylation and acetylation: the molecular basis for p53 regulation. Curr Opin Cell Biol, 15, 164-71. https://doi.org/10.1016/S0955-0674(03)00003-6
  9. Burbee DG, Forgacs E, Zochbauer-Muller S, et al (2001). Epigenetic inactivation of RASSF1A in lung and breast cancers and malignant phenotype suppression. J Natl Cancer Inst, 93, 691-9. https://doi.org/10.1093/jnci/93.9.691
  10. Brown R, Hirst GL, Gallagher WM, et al (1997). hMLH1 expression and cellular responses of ovarian tumour cells to treatment with cytotoxic anticancer agents. Oncogene, 15, 45-52. https://doi.org/10.1038/sj.onc.1201167
  11. Chen D, Kon N, Li M, et al (2005). ARFBP1/Mule is a critical mediator of the ARF tumor suppressor. Cell, 121, 1071-83. https://doi.org/10.1016/j.cell.2005.03.037
  12. Cote RJ, Esrig D, Groshen S, Jones PA, Skinne DG (1997). P53 and treatment of bladder cancer. Nature, 385, 124-5. https://doi.org/10.1038/385124a0
  13. Chen Z, Yang J, Wang G, et al (2007). Attenuated expression of xeroderma pigmentosum group C is associated with critical events in human bladder cancer carcinogenesis and progression. Cancer Res, 67, 4578-85. https://doi.org/10.1158/0008-5472.CAN-06-0877
  14. Cordon-Cardo C (2008). Molecular alterations associated with bladder cancer initiation and progression. Scand J Urol Nephro, 218, 154-65.
  15. Cooper MJ, Haluschak JJ, Johsond D, et al (1994). p53 mutations in bladder carcinoma cell lines. Oncol Res, 6, 569-79.
  16. Crul M, van Waardenburg RC, Bocxe S (2003). DNA repair mechanisms involved in gemcitabine cytotoxicity and in the interaction between gemcitabine and cisplatin. Biochem Pharmacol, 65, 275-82. https://doi.org/10.1016/S0006-2952(02)01508-3
  17. Da Silva GN, Marcondes JPC, Camargo EA, et al (2010). Cell cycle arrest and apoptosis in TP53 subtypes of bladder carcinoma cell lines treated with cisplatin and gemcitabine. Exp Med Biol, 235, 814-24. https://doi.org/10.1258/ebm.2010.009322
  18. Ding X, Mohd AB, Huang Z, et al (2009). MLH1 expression sensitises ovarian cancer cells to cell death mediated by XIAP inhibition. Br J Cancer, 101, 269-77. https://doi.org/10.1038/sj.bjc.6605180
  19. Dulaimi E, Uzzo RG, Greenberg RE, Al-Saleem T, Cairns P (2004). Detection of bladder cancer in urine by a tumor suppressor gene hypermethylation panel. Clin Cancer Res, 10, 1887-93. https://doi.org/10.1158/1078-0432.CCR-03-0127
  20. Fink D, Aebi S, Howell SB (1998). The role of DNA mismatch repair in drug resistance. Clin Cancer Res, 4, 1-6.
  21. Gallagher DJ, Milowsky MI, Bajorin DF (2009). Advanced bladder cancer: status of first-line chemotherapy and the search for active agents in the second-line setting. Cancer, 113, 1284-93.
  22. Kim HG, Lee S, Kim DY, et al (2010). Aberrant methylation of DNA Mismatch repair genes in elderly patients with sporadic gastric carcinoma: a comparision with younger patients. J Surg Oncol, 101, 28-35. https://doi.org/10.1002/jso.21432
  23. Hall PA, McCluggage WG (2006). Assessing p53 in clinical contexts: unlearned lessons and new perspectives. J Pathol, 208, 1-6. https://doi.org/10.1002/path.1913
  24. Hamilton G, Yee KS, Scrace S, O'Neill E (2009). ATM regulates a RASSF1A-dependent DNA damage response. Current Biol, 19, 2020-5. https://doi.org/10.1016/j.cub.2009.10.040
  25. Heringova P, Woods J, Mackay FS, et al (2006). Transplatin is cytotoxic when photoactivated: enhanced formation of DNA cross-links. J Med Chem, 49, 7792-8. https://doi.org/10.1021/jm0606692
  26. Kosmider B, Osiecka R, Zyner E, Ochocki J (2005). Comparison between the genotoxicity of cis'Pt(II) complex of 3-aminoflavone and cis-DDP in lymphocytes evaluated by the comet assay. Drug Chem Toxicol, 28, 231-44. https://doi.org/10.1081/DCT-52555
  27. Kufe DW, Weichselbaum R, Egan EM, Dahlberg W, Fram RJ (1984). Lethal effects of 1-${\beta}$-D-arabinofuranosyleytosine incorporation into deoxyribonucleic acid during ultraviolet repair. Mol Pharmacol, 25, 322-6.
  28. LaConti JJ, Shivapurkar N, Preet A, et al (2011). Tissue and serum microRNAs in the Kras (G12D) transgenic animal model and in patients with pancreatic cancer. PLoS One, 6, 20687. https://doi.org/10.1371/journal.pone.0020687
  29. Larson ED, Drummond JT (2001). Human mismatch repair and G*T mismatch binding by hMutSalpha in vitro is inhibited by adriamycin, actinomycin D, and nogalamycin. J Biol Chem, 276, 9775-83. https://doi.org/10.1074/jbc.M006390200
  30. Marsit CJ, Karagas MR, Danaee H, et al (2006). Carcinogen exposure and gene promoter hypermethylation in bladder cancer. Carcinogenesis, 27, 112-6.
  31. Pagliarone AC, Orsatti CL, Bufalo MC, et al (2009). Propolis effects on pro-inflammatory cytokine production and Toll-like receptor 2 and 4 expression in stressed mice. Int Immunopharmacol, 9, 1352-6. https://doi.org/10.1016/j.intimp.2009.08.005
  32. Nadin SB, Vargas-Roig LM, Drago G, Ibarra J, Ciocca DR (2006). DNA damage and repair in peripheral blood lymphocytes from healthy individuals and cancer patients: a pilot study on the implications in the clinical response to chemotherapy. Cancer Lett, 23, 84-97.
  33. Negraes PD, Favaro FP, Camargo JLV, et al (2008). DNA methylation patterns in bladder cancer and washing cell sediments: a perspective for tumor recurrence detection. BMC Cancer, 8, 238-49. https://doi.org/10.1186/1471-2407-8-238
  34. Orsatti CL, Missima F, Pagliarone AC, et al (2010). Propolis immunomodulatory action in vivo on Toll-like receptors 2 and 4 expression and on pro-inflammatory cytokines production in mice. Phytother Res, 24, 1141-6.
  35. Park JW, Baek IH, Kim YT (2012). Preliminary study analyzing the methylated genes in the plasma of patients with pancreatic cancer. Scand J Surg, 101, 38-44. https://doi.org/10.1177/145749691210100108
  36. Paulwels B, Korst AE, Andriessen V, et al (2005). Unraveling the mechanism of radiosensitization by gemcitabine: the role of TP53. Radiat Res, 164, 642-50. https://doi.org/10.1667/RR3445.1
  37. Phe V, Cussenot O, Roupret M (2009). Interest of methylated genes as biomarkers in urothelial cell carcinomas of the urinary tract. Bju Int, 104, 896-901. https://doi.org/10.1111/j.1464-410X.2009.08696.x
  38. Plumb JA, Strathdee G, Sludden J, Kaye SB, Brown R (2000). Reversal of drug resistance in human tumor xenografts by 2_-deoxy- 5-azacytidine-induced demethylation of the MLH1 gene promoter. Cancer Res, 60, 6039-44.
  39. Sanchez-Carbayo M, Socci ND, Richstone L, et al (2007). Genomic and proteomic profiles reveal the association of gelsolin to TP53 status and bladder cancer progression. Am J Pathol, 171, 1650-8. https://doi.org/10.2353/ajpath.2007.070338
  40. Song MS, Song SJ, Kim SY, Oh HJ, Lim DS (2008). The tumour suppressor RASSF1A promotes MDM2 self-ubiquitination by disrupting the MDM2-DAXX-HAUSP complex. Embo J, 27, 1863-74. https://doi.org/10.1038/emboj.2008.115
  41. Schafer A, Schomacher L, Barreto G, Doderlein G, Niehrs C (2010). Gemcitabine functions epigenetically by inhibiting repair mediated DNA demethylation. PLoS One, 19, 14060.
  42. Scrace SF, O'Neill E (2012). RASSF signalling and DNA damage: monitoring the integrity of the genome? Mol Biol Int, 2012, 141732.
  43. Shimabukuro F, Neto CF, Sanches Jr JA, Gattas GJF (2011). DNA damage and repair in leukocytes of melanoma patients exposed in vitro to cisplatin. Melanoma Res, 21, 99-105. https://doi.org/10.1097/CMR.0b013e3283426839
  44. Stadler WM, Lerner SP, Groshen S, et al (2011). Phase III study of molecularly targeted adjuvant therapy in locally advanced urothelial cancer of the bladder based on p53 status. J Clin Oncol, 29, 3443-49. https://doi.org/10.1200/JCO.2010.34.4028
  45. Suzuki HI, Yamagata K, Sugimoto K, et al (2009). Modulation of microRNA processing by p53. Nature, 460, 529-33. https://doi.org/10.1038/nature08199
  46. Tajima A, Iwaizumi M, Tseng-Rogenski S, Cabrera BL, Carethers JM (2011). Both $hMutS{\alpha}$ and $hMutS{\beta}$ DNA mismatch repair complexes participate in 5-fluorouracil cytotoxicity. PLoS One, 6, 28117. https://doi.org/10.1371/journal.pone.0028117
  47. Tian Y, Hou Y, Zhou X, Cheng H, Zhou R (2011). Tumor suppressor RASSF1A promoter: p53 binding and methylation. PLoS One, 6, 17017. https://doi.org/10.1371/journal.pone.0017017
  48. Tice RR, Agurell E, Anderson D, et al (2000). Single cell gel/comet assay: guidelines for in vitro and in vivo genetic toxicology testing. Environ Mol Mutagen, 35, 206-21. https://doi.org/10.1002/(SICI)1098-2280(2000)35:3<206::AID-EM8>3.0.CO;2-J
  49. Toschi L, Finocchiaro G, Gioia V (2005). Role of gemcitabine in cancer therapy. Future Oncol, 1, 7-17. https://doi.org/10.1517/14796694.1.1.7
  50. Wang D, Lippard SJ (2005). Cellular processing of platinum anticancer drugs. Nature Rev, 4, 307-19.
  51. Wolff EM, Liang G, Jones PA (2005). Mechanisms of disease: genetic and epigenetic alterations that drive bladder cancer. Nat Clin Pract Urol, 2, 502-10.
  52. Xu XS, Wang L, Abrams J, Wang G (2011). Histone deacetylases (HDACs) in XPC gene silencing and bladder cancer. J Hematol Oncol, 4, 17. https://doi.org/10.1186/1756-8722-4-17
  53. Yang LY, Li L, Jiang H, Shen Y, Plunkett W (2000). Expression of ERCC1 antisense RNA abrogates gemcitabine-mediated cytotoxic synergism with cisplatin in human colon tumor cells defective in mismatch repair but proficient in nucleotide excision repair. Clin Cancer Res, 6, 773-81.
  54. Yu J, Zhu T, Wang Z, et al (2007). A Novel set of DNA methylation markers in urine sedments for sensitive/specific detection of bladder cancer. Clin Cancer Res, 13, 7296-304. https://doi.org/10.1158/1078-0432.CCR-07-0861

Cited by

  1. MRE11A and SKP2 genes are associated with the increased cytotoxicity induced by the synergistic effects of cisplatin and gemcitabine in bladder cancer cells vol.41, pp.7, 2014, https://doi.org/10.1007/s11033-014-3332-1
  2. New insight for metformin against bladder cancer vol.39, pp.1, 2017, https://doi.org/10.1186/s41021-017-0074-z
  3. Gemcitabine/Cisplatin Treatment Induces Concomitant SERTAD1, CDKN2B and GADD45A Modulation and Cellular Changes in Bladder Cancer Cells Regardless of the Site of TP53 Mutation pp.1532-2807, 2017, https://doi.org/10.1007/s12253-017-0255-x