Advertisement

Tumor Biology

, Volume 36, Issue 5, pp 3197–3207 | Cite as

Gemcitabine impacts differentially on bladder and kidney cancer cells: distinct modulations in the expression patterns of apoptosis-related microRNAs and BCL2 family genes

  • Emmanuel I. Papadopoulos
  • George M. Yousef
  • Andreas Scorilas
Research Article

Abstract

Bladder and renal cancer are two representative cases of tumors that respond differentially to gemcitabine. Previous studies have shown that gemcitabine can trigger apoptosis in various cancer cells. Herein, we sought to investigate the impact of gemcitabine on the expression levels of the BCL2 family members BCL2, BAX, and BCL2L12 and the apoptosis-related microRNAs miR-182, miR-96, miR-145, and miR-16 in the human bladder and kidney cancer cell lines T24 and Caki-1, respectively. Cancer cells’ viability as well as the IC50 doses of gemcitabine were estimated by the MTT assay, while the detection of cleaved PARP via Western blotting was used as an indicator of apoptosis. Furthermore, T24 and Caki-1 cells’ ability to recover from treatment was also monitored. Two different highly sensitive quantitative real-time RT-PCR methodologies were developed in order to assess the expression levels of BCL2 family genes and microRNAs. Exposure of cancer cells to gemcitabine produced the IC50 values of 30 and 3 nM for Caki-1 and T24 cells, correspondingly, while cleaved PARP was detected only in Caki-1 cells. T24 cells demonstrated the ability to recover from gemcitabine treatment, whereas Caki-1 cells’ recovery capability was dependent on the initial time of exposure. BCL2 and BAX were significantly modulated in treated Caki-1 cells. Instead, T24 cells exhibited alterations only in the latter, as well as in all studied microRNAs. Therefore, according to our data, bladder and renal cancer cells’ response to gemcitabine is accompanied by distinct alterations in the expression levels of their apoptosis-related genes and microRNAs.

Keywords

T24 urinary bladder cancer cell line Caki-1 renal cancer cell line BCL2L12 Gemcitabine MicroRNAs BCL2 family 

Notes

Acknowledgments

This work has been carried out with the financial support of the European Commission of the European Community through the INsPiRE project (EU-FP7-REGPOT-2011-1).

Conflicts of interest

None

References

  1. 1.
    Elmore S. Apoptosis: a review of programmed cell death. Toxicol Pathol. 2007;35:495–516.CrossRefPubMedPubMedCentralGoogle Scholar
  2. 2.
    Hanahan D, Weinberg RA. Hallmarks of cancer: the next generation. Cell. 2011;144:646–74.CrossRefPubMedGoogle Scholar
  3. 3.
    Fulda S. Tumor resistance to apoptosis. Int J Cancer. 2009;124:511–5.CrossRefPubMedGoogle Scholar
  4. 4.
    Galluzzi L, Vitale I, Abrams JM, Alnemri ES, Baehrecke EH, Blagosklonny MV, et al. Molecular definitions of cell death subroutines: recommendations of the Nomenclature Committee on Cell Death 2012. Cell Death Differ. 2012;19:107–20.CrossRefPubMedGoogle Scholar
  5. 5.
    Thomadaki H, Scorilas A. Bcl2 family of apoptosis-related genes: functions and clinical implications in cancer. Crit Rev Clin Lab Sci. 2006;43:1–67.CrossRefPubMedGoogle Scholar
  6. 6.
    Pegoraro L, Palumbo A, Erikson J, Falda M, Giovanazzo B, Emanuel BS, et al. A 14;18 and an 8;14 chromosome translocation in a cell line derived from an acute B-cell leukemia. Proc Natl Acad Sci U S A. 1984;81:7166–70.CrossRefPubMedPubMedCentralGoogle Scholar
  7. 7.
    Yang J, Liu X, Bhalla K, Kim CN, Ibrado AM, Cai J, et al. Prevention of apoptosis by Bcl-2: release of cytochrome c from mitochondria blocked. Science. 1997;275:1129–32.CrossRefPubMedGoogle Scholar
  8. 8.
    Oltvai ZN, Milliman CL, Korsmeyer SJ. Bcl-2 heterodimerizes in vivo with a conserved homolog, Bax, that accelerates programmed cell death. Cell. 1993;74:609–19.CrossRefPubMedGoogle Scholar
  9. 9.
    Scorilas A, Kyriakopoulou L, Yousef GM, Ashworth LK, Kwamie A, Diamandis EP. Molecular cloning, physical mapping, and expression analysis of a novel gene, bcl2l12, encoding a proline-rich protein with a highly conserved bh2 domain of the bcl-2 family. Genomics. 2001;72:217–21.CrossRefPubMedGoogle Scholar
  10. 10.
    Kontos CK, Scorilas A. Molecular cloning of novel alternatively spliced variants of BCL2L12, a new member of the BCL2 gene family, and their expression analysis in cancer cells. Gene. 2012;505:153–66.CrossRefPubMedGoogle Scholar
  11. 11.
    Korbakis D, Scorilas A. Quantitative expression analysis of the apoptosis-related genes BCL2, BAX and BCL2L12 in gastric adenocarcinoma cells following treatment with the anticancer drugs cisplatin, etoposide and taxol. Tumour Biol. 2012;33:865–75.CrossRefPubMedGoogle Scholar
  12. 12.
    Thomadaki H, Scorilas A. Breast cancer cells response to the antineoplastic agents cisplatin, carboplatin, and doxorubicin at the mRNA expression levels of distinct apoptosis-related genes, including the new member, BCL2L12. Ann N Y Acad Sci. 2007;1095:35–44.CrossRefPubMedGoogle Scholar
  13. 13.
    Ziegler DS, Kung AL. Therapeutic targeting of apoptosis pathways in cancer. Curr Opin Oncol. 2008;20:97–103.CrossRefPubMedGoogle Scholar
  14. 14.
    Bartel DP. MicroRNAs: target recognition and regulatory functions. Cell. 2009;136:215–33.CrossRefPubMedPubMedCentralGoogle Scholar
  15. 15.
    Subramanian S, Steer CJ. MicroRNAs as gatekeepers of apoptosis. J Cell Physiol. 2010;223:289–98.PubMedGoogle Scholar
  16. 16.
    Sarkar FH, Li Y, Wang Z, Kong D, Ali S. Implication of microRNAs in drug resistance for designing novel cancer therapy. Drug Resist Updat. 2010;13:57–66.CrossRefPubMedPubMedCentralGoogle Scholar
  17. 17.
    Mini E, Nobili S, Caciagli B, Landini I, Mazzei T. Cellular pharmacology of gemcitabine. Ann Oncol. 2006;17 Suppl 5:v7–v12.CrossRefPubMedGoogle Scholar
  18. 18.
    Siegel R, Naishadham D, Jemal A. Cancer statistics, 2013. CA Cancer J Clin. 2013;63:11–30.CrossRefPubMedGoogle Scholar
  19. 19.
    Ljungberg B, Campbell SC, Choi HY, Jacqmin D, Lee JE, Weikert S, et al. The epidemiology of renal cell carcinoma. Eur Urol. 2011;60:615–21.CrossRefPubMedGoogle Scholar
  20. 20.
    Figlin R, Sternberg C, Wood CG. Novel agents and approaches for advanced renal cell carcinoma. J Urol. 2012;188:707–15.CrossRefPubMedGoogle Scholar
  21. 21.
    Ismaili N, Amzerin M, Flechon A. Chemotherapy in advanced bladder cancer: current status and future. J Hematol Oncol. 2011;4:35.CrossRefPubMedPubMedCentralGoogle Scholar
  22. 22.
    De Mulder PH, Weissbach L, Jakse G, Osieka R, Blatter J. Gemcitabine: a phase II study in patients with advanced renal cancer. Cancer Chemother Pharmacol. 1996;37:491–5.CrossRefPubMedGoogle Scholar
  23. 23.
    Mertens WC, Eisenhauer EA, Moore M, Venner P, Stewart D, Muldal A, et al. Gemcitabine in advanced renal cell carcinoma. A phase II study of the National Cancer Institute of Canada Clinical Trials Group. Ann Oncol. 1993;4:331–2.CrossRefPubMedGoogle Scholar
  24. 24.
    Shi R, Chiang V. Facile means for quantifying microRNA expression by real-time PCR. Biotechniques. 2005;39:519–25.CrossRefPubMedGoogle Scholar
  25. 25.
    Kozomara A, Griffiths-Jones S. miRBase: integrating microRNA annotation and deep-sequencing data. Nucleic Acids Res. 2011;39 Suppl 1:D152–7.CrossRefPubMedGoogle Scholar
  26. 26.
    Schmittgen TD, Livak KJ. Analyzing real-time PCR data by the comparative C(T) method. Nat Protoc. 2008;3:1101–8.CrossRefPubMedGoogle Scholar
  27. 27.
    Bradford MM. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem. 1976;72:248–54.CrossRefPubMedGoogle Scholar
  28. 28.
    Laemmli UK. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970;227:680–5.CrossRefPubMedGoogle Scholar
  29. 29.
    Batteiger B, Newhall 5th WJ, Jones RB. The use of Tween 20 as a blocking agent in the immunological detection of proteins transferred to nitrocellulose membranes. J Immunol Methods. 1982;55:297–307.CrossRefPubMedGoogle Scholar
  30. 30.
    Huang P, Plunkett W. Induction of apoptosis by gemcitabine. Semin Oncol. 1995;22 Suppl 11:19–25.PubMedGoogle Scholar
  31. 31.
    Chandler NM, Canete JJ, Callery MP. Caspase-3 drives apoptosis in pancreatic cancer cells after treatment with gemcitabine. J Gastrointest Surg. 2004;8:1072–8.CrossRefPubMedGoogle Scholar
  32. 32.
    Ferreira CG, Span SW, Peters GJ, Kruyt FA, Giaccone G. Chemotherapy triggers apoptosis in a caspase-8-dependent and mitochondria-controlled manner in the non-small cell lung cancer cell line NCI-H460. Cancer Res. 2000;60:7133–41.PubMedGoogle Scholar
  33. 33.
    D'Amours D, Sallmann FR, Dixit VM, Poirier GG. Gain-of-function of poly(ADP-ribose) polymerase-1 upon cleavage by apoptotic proteases: implications for apoptosis. J Cell Sci. 2001;114:3771–8.PubMedGoogle Scholar
  34. 34.
    Soldani C, Lazzè MC, Bottone MG, Tognon G, Biggiogera M, Pellicciari CE, et al. Poly(ADP-ribose) polymerase cleavage during apoptosis: when and where? Exp Cell Res. 2001;269:193–201.CrossRefPubMedGoogle Scholar
  35. 35.
    Gobeil S, Boucher CC, Nadeau D, Poirier GG. Characterization of the necrotic cleavage of poly(ADP-ribose) polymerase (PARP-1): implication of lysosomal proteases. Cell Death Differ. 2001;8:588–94.CrossRefPubMedGoogle Scholar
  36. 36.
    da Silva GN, de Castro Marcondes JP, de Camargo EA, da Silva Passos Júnior GA, Sakamoto-Hojo ET, Salvadori DM. Cell cycle arrest and apoptosis in TP53 subtypes of bladder carcinoma cell lines treated with cisplatin and gemcitabine. Exp Biol Med (Maywood). 2010;235:814–24.CrossRefGoogle Scholar
  37. 37.
    Tannock IF, Lee C. Evidence against apoptosis as a major mechanism for reproductive cell death following treatment of cell lines with anti-cancer drugs. Br J Cancer. 2001;84:100–5.CrossRefPubMedPubMedCentralGoogle Scholar
  38. 38.
    Scheltema JM, Romijn JC, van Steenbrugge GJ, Schröder FH, Mickisch GH. Inhibition of apoptotic proteins causes multidrug resistance in renal carcinoma cells. Anticancer Res. 2001;21:3161–6.PubMedGoogle Scholar
  39. 39.
    Cho HJ, Kim JK, Kim KD, Yoon HK, Cho MY, Park YP, et al. Upregulation of Bcl-2 is associated with cisplatin-resistance via inhibition of Bax translocation in human bladder cancer cells. Cancer Lett. 2006;237:56–66.CrossRefPubMedGoogle Scholar
  40. 40.
    Yu DS, Chang SY. The expression of oncoproteins in transitional cell carcinoma: its correlation with pathological behavior, cell cycle and drug resistance. Urol Int. 2002;69:46–50.CrossRefPubMedGoogle Scholar
  41. 41.
    Nana-Sinkam SP, Croce CM. Clinical applications for microRNAs in cancer. Clin Pharmacol Ther. 2013;93:98–104.CrossRefPubMedGoogle Scholar
  42. 42.
    Hirata H, Ueno K, Shahryari V, Tanaka Y, Tabatabai ZL, Hinoda Y, et al. Oncogenic miRNA-182-5p targets Smad4 and RECK in human bladder cancer. PLoS One. 2012;7:e51056.CrossRefPubMedPubMedCentralGoogle Scholar
  43. 43.
    Guo Y, Liu H, Zhang H, Shang C, Song Y. miR-96 regulates FOXO1-mediated cell apoptosis in bladder cancer. Oncol Lett. 2012;4:561–5.PubMedPubMedCentralGoogle Scholar
  44. 44.
    Wang Y, Luo H, Li Y, Chen T, Wu S, Yang L. hsa-miR-96 up-regulates MAP4K1 and IRS1 and may function as a promising diagnostic marker in human bladder urothelial carcinomas. Mol Med Rep. 2012;5:260–5.PubMedGoogle Scholar
  45. 45.
    Sachdeva M, Mo YY. miR-145-mediated suppression of cell growth, invasion and metastasis. Am J. Transl Res. 2010;2:170–80.Google Scholar
  46. 46.
    Song T, Xia W, Shao N, Zhang X, Wang C, Wu Y, et al. Differential miRNA expression profiles in bladder urothelial carcinomas. Asian Pac J Cancer Prev. 2010;11:905–11.PubMedGoogle Scholar
  47. 47.
    Huang Y, Dai Y, Yang J, Chen T, Yin Y, Tang M, et al. Microarray analysis of microRNA expression in renal clear cell carcinoma. Eur J Surg Oncol. 2009;35:1119–23.CrossRefPubMedGoogle Scholar
  48. 48.
    Cimmino A, Calin GA, Fabbri M, Iorio MV, Ferracin M, Shimizu M, et al. miR-15 and miR-16 induce apoptosis by targeting BCL2. Proc Natl Acad Sci U S A. 2005;102:13944–9.CrossRefPubMedPubMedCentralGoogle Scholar
  49. 49.
    Ostenfeld MS, Bramsen JB, Lamy P, Villadsen SB, Fristrup N, Sørensen KD, et al. miR-145 induces caspase-dependent and -independent cell death in urothelial cancer cell lines with targeting of an expression signature present in Ta bladder tumors. Oncogene. 2010;29:1073–84.CrossRefPubMedGoogle Scholar
  50. 50.
    Sachdeva M, Zhu S, Wu F, Wu H, Walia V, Kumar S, et al. p53 represses c-Myc through induction of the tumor suppressor miR-145. Proc Natl Acad Sci U S A. 2009;106:3207–12.CrossRefPubMedPubMedCentralGoogle Scholar
  51. 51.
    Grimm MO, Jürgens B, Schulz WA, Decken K, Makri D, Schmitz-Dräger BJ. Inactivation of tumor suppressor genes and deregulation of the c-myc gene in urothelial cancer cell lines. Urol Res. 1995;23:293–300.CrossRefPubMedGoogle Scholar
  52. 52.
    Xia L, Zhang D, Du R, Pan Y, Zhao L, Sun S, et al. miR-15b and miR-16 modulate multidrug resistance by targeting BCL2 in human gastric cancer cells. Int J Cancer. 2008;123:372–9.CrossRefPubMedGoogle Scholar

Copyright information

© International Society of Oncology and BioMarkers (ISOBM) 2015

Authors and Affiliations

  • Emmanuel I. Papadopoulos
    • 1
  • George M. Yousef
    • 2
  • Andreas Scorilas
    • 1
  1. 1.Department of Biochemistry and Molecular Biology, Faculty of BiologyUniversity of AthensAthensGreece
  2. 2.Department of Laboratory Medicine and PathobiologyUniversity of TorontoTorontoCanada

Personalised recommendations