Abstract
We studied alterations in the mRNA expression levels of BCL2 (Bcl-2), BCL2L12, BAX, FAS and CASPASE-9 genes in the MCF-7 breast cancer cell line in response to treatment with two anticancer drugs. Cell toxicity was evaluated by the MTT method, trypan blue staining and DNA laddering, whereas the expression levels of the apoptosis-related genes were analysed by RT-PCR using gene-specific primers. In the case of etoposide, down-regulation of the BCL2L12-A gene variant and of CASPASE-9, as well as upregulation of BAX, was observed, whereas treatment of MCF-7 cells with taxol led to down-regulation of the mRNA levels of all genes examined. Our results support the idea that after long-term clinical studies, mRNA expression analysis of BCL2L12 and other members of the BCL2 gene family may serve as useful molecular markers predicting chemotherapy response in breast cancer.
References
Baldwin, E.L. and Osheroff, N. (2005). Etoposide, topoisomerase II and cancer. Curr. Med. Chem. Anti-Cancer Agents5, 363–372.10.2174/1568011054222364Search in Google Scholar
Benjamin, C.W., Hiebsch, R.R., and Jones, D.A. (1998). Caspase activation in MCF7 cells responding to etoposide treatment. Mol. Pharmacol.53, 446–450.10.1124/mol.53.3.446Search in Google Scholar
Bewick, M., Conlon, M., Parissenti, A.M., Lee, H., Zhang, L., Gluck, S., and Lafrenie, R.M. (2001). Soluble Fas (CD95) is a prognostic factor in patients with metastatic breast cancer undergoing high-dose chemotherapy and autologous stem cell transplantation. J. Hematother. Stem Cell Res.10, 759–768.10.1089/152581601317210854Search in Google Scholar
Boldt, S., Weidle, U.H., and Kolch, W. (2002). The role of MAPK pathways in the action of chemotherapeutic drugs. Carcinogenesis23, 1831–1838.10.1093/carcin/23.11.1831Search in Google Scholar
Bontenbal, M., Planting, A.S., Verweij, J., de Wit, R., Kruit, W.H., Stoter, G., and Klijn, J.G. (1995). Second-line chemotherapy with long-term low-dose oral etoposide in patients with advanced breast cancer. Breast Cancer Res. Treat.34, 185–189.10.1007/BF00665790Search in Google Scholar
Botti, C., Buglioni, S., Benevolo, M., Giannarelli, D., Papaldo, P., Cognetti, F., Vici, P., Di Filippo, F., Del Nonno, F., Venanzi, F.M., et al. (2004). Altered expression of FAS system is related to adverse clinical outcome in stage I-II breast cancer patients treated with adjuvant anthracycline-based chemotherapy. Clin. Cancer Res.10, 1360–1365.10.1158/1078-0432.CCR-1092-03Search in Google Scholar
Duffy, M.J. (2001). Biochemical markers in breast cancer: which ones are clinically useful? Clin. Biochem.34, 347–352.10.1016/S0009-9120(00)00201-0Search in Google Scholar
Dwarakanath, B.S., Khaitan, D., and Mathur, R. (2004). Inhibitors of topoisomerases as anticancer drugs: problems and prospects. Indian J. Exp. Biol.42, 649–659.Search in Google Scholar
Eastman, A. (1995). Assays for DNA fragmentation, endonucleases, and intracellular pH and Ca2+ associated with apoptosis. Methods Cell Biol.46, 41–55.10.1016/S0091-679X(08)61923-8Search in Google Scholar
Evan, G.I. and Vousden, K.H. (2001). Proliferation, cell cycle and apoptosis in cancer. Nature411, 342–348.10.1038/35077213Search in Google Scholar PubMed
Fitzgibbons, P.L., Page, D.L., Weaver, D., Thor, A.D., Allred, D.C., Clark, G.M., Ruby, S.G., O'Malley, F., Simpson, J.F., Connolly, J.L., et al. (2000). Prognostic factors in breast cancer. College of American Pathologists Consensus Statement 1999. Arch. Pathol. Lab. Med.124, 966–978.10.5858/2000-124-0966-PFIBCSearch in Google Scholar PubMed
Floros, K.V., Thomadaki, H., Lallas, G., Katsaros, N., Talieri, M., and Scorilas, A. (2003). Cisplatin-induced apoptosis in HL-60 human promyelocytic leukaemia cells: differential expression of BCL2 and novel apoptosis-related gene BCL2L12. Ann. NY Acad. Sci.1010, 153–158.10.1196/annals.1299.025Search in Google Scholar
Floros, K.V., Thomadaki, H., Katsaros, N., Talieri, M., and Scorilas, A. (2004). mRNA expression analysis of a variety of apoptosis-related genes, including the novel gene of the BCL2-family, BCL2L12, in HL-60 leukemia cells after treatment with carboplatin and doxorubicin. Biol. Chem.385, 1099–1103.10.1515/BC.2004.143Search in Google Scholar
Fried, G., Stein, M.E., and Haim, N. (2000). Clinical activity of cisplatin and prolonged oral administration of etoposide in previously treated, anthracycline-resistant, metastatic breast cancer patients: a phase II study. Med. Pediatr. Oncol.34, 10–13.10.1002/(SICI)1096-911X(200001)34:1<10::AID-MPO2>3.0.CO;2-ASearch in Google Scholar
Ghersi, D., Wilcken, N., Simes, J., and Donoghue, E. (2005). Taxane containing regimens for metastatic breast cancer. Cochrane Database Syst Rev.2, CD003366.10.1002/14651858.CD003366.pub2Search in Google Scholar
Gibson, L.F., Fortney, J., Magro, G., Ericson, S.G., Lynch, J.P., and Landreth, K.S. (1999). Regulation of BAX and BCL-2 expression in breast cancer cells by chemotherapy. Breast Cancer Res. Treat.55, 107–117.10.1023/A:1006175811676Search in Google Scholar
Horwitz, S.B. (1994). Taxol (paclitaxel): mechanisms of action. Ann. Oncol.5, S3–S6.Search in Google Scholar
Howell, A. and Wardley, A.M. (2005). Overview of the impact of conventional systemic therapies on breast cancer. Endocr. Relat. Cancer12, S9–S16.10.1677/erc.1.01003Search in Google Scholar
Icli, F., Akbulut, H., Uner, A., Yalcin, B., Baltali, E., Altinbas, M., Coskun, S., Komurcu, S., Erkisi, M., Demirkazik, A., et al. (2005). Cisplatin plus oral etoposide (EoP) combination is more effective than paclitaxel in patients with advanced breast cancer pretreated with anthracyclines: a randomised phase III trial of Turkish Oncology Group. Br. J. Cancer92, 639–644.10.1038/sj.bjc.6602388Search in Google Scholar
Jeffrey, S.S., Lonning, P.E., and Hillner, B.E. (2005). Genomics-based prognosis and therapeutic prediction in breast cancer. J. Natl. Compr. Canc. Netw.3, 291–300.10.6004/jnccn.2005.0016Search in Google Scholar
Johnson, V.L., Ko, S.C., Holmstrom, T.H., Eriksson, J.E., and Chow, S.C. (2000). Effector caspases are dispensable for the early nuclear morphological changes during chemical induced apoptosis. J. Cell Sci.113, 2941–2953.10.1242/jcs.113.17.2941Search in Google Scholar
Kishi, T., Grass, L., Soosaipillai, A., Scorilas, A., Harbeck, N., Schmalfeldt, B., Dorn, J., Mysliwiec, M., Schmitt, M., and Diamandis, E.P. (2003). Human kallikrein 8, a novel biomarker for ovarian carcinoma. Cancer Res.63, 2771–2774.Search in Google Scholar
Kurokawa, H., Nishio, K., Fukumoto, H., Tomonari, A., Suzuki, T., and Saijo, N. (1999). Alteration of caspase-3 (CPP32/ Yama/apopain) in wild-type MCF-7 breast cancer cells. Oncol. Rep.6, 33–37.10.3892/or.6.1.33Search in Google Scholar
Leung, L.K. and Wang, T.T. (1999). Differential effects of chemotherapeutic agents on the Bcl-2/Bax apoptosis pathway in human breast cancer cell line MCF-7. Breast Cancer Res. Treat.55, 73–83.10.1023/A:1006190802590Search in Google Scholar
Mathioudaki, K., Scorilas, A., Papadokostopoulou, A., Xynopoulos, D., Arnogianaki, N., Agnanti, N., and Talieri, M. (2004). Expression analysis of BCL2L12, a new member of apoptosis-related genes, in colon cancer. Biol. Chem.385, 779–783.10.1515/BC.2004.101Search in Google Scholar
Mosmann, T. (1983). Rapid colorimetric assay for the cellular growth and survival: application to proliferation and cyctotoxic assays. J. Immunol. Methods65, 55–63.10.1016/0022-1759(83)90303-4Search in Google Scholar
Murphy, C.N., Millar, E., and Lee, C.S. (2005). Gene expression profiling in breast cancer: towards individualising patient management. Pathology37, 271–277.10.1080/00313020500169586Search in Google Scholar
Neskovic-Konstantinovic, Z.B., Bosnjak, S.M., Radulovic, S.S., and Mitrovic, L.B. (1996). Daily oral etoposide in metastatic breast cancer. Anticancer Drugs7, 543–547.10.1097/00001813-199607000-00009Search in Google Scholar
Ogretmen, B. and Safa, A.R. (1996). Down-regulation of apoptosis-related bcl-2 but not bcl-xL or bax proteins in multidrug-resistant MCF-7/Adr human breast cancer cells. Int. J. Cancer.67, 608–614.10.1002/(SICI)1097-0215(19960904)67:5<608::AID-IJC3>3.0.CO;2-YSearch in Google Scholar
Pusztai, L., Walters, R.S., Valero, V., Theriault, R.L., and Hortobagyi, G.N. (1998). Daily oral etoposide in patients with heavily pretreated metastatic breast cancer. Am. J. Clin. Oncol.21, 442–446.10.1097/00000421-199810000-00004Search in Google Scholar
Razandi, M., Pedram, A., and Levin, E.R. (2000). Plasma membrane estrogen receptors signal to antiapoptosis in breast cancer. Mol. Endocrinol.14, 1434–1447.10.1210/mend.14.9.0526Search in Google Scholar
Sakakura, C., Sweeney, E.A., Shirahama, T., Igarashi, Y., Hakomori, S., Tsujimoto, H., Imanishi, T., Ogaki, M., Ohyama, T., Yamazaki, J., et al. (1997). Overexpression of bax sensitizes breast cancer MCF-7 cells to cisplatin and etoposide. Surg. Today27, 676–679.10.1007/BF02388231Search in Google Scholar
Schorr, K., Li, M., Krajewski,S., Reed, J.C., and Furth, P.A. (1999). Bcl-2 gene family and related proteins in mammary gland involution and breast cancer. J. Mammary Gland Biol. Neoplasia4, 153–164.10.1023/A:1018773123899Search in Google Scholar
Scorilas, A., Diamandis, E.P., Levesque, M.A., Papanistasiou-Diamandi, A., Khosravi, M.J., Giai, M., Ponzone, R., Roagna, R., Sismondi, P., and Lopez-Otin, C. (1999a). Immunoenzymatically determined pepsinogen C concentration in breast tumor cytosols: an independent favorable prognostic factor in node-positive patients. Clin. Cancer Res.5, 1778–1785.Search in Google Scholar
Scorilas, A., Trangas, T., Yotis, J., Pateras, C., and Talieri, M. (1999b). Determination of c-myc amplification and overexpression in breast cancer patients. Evaluation of its prognostic value against c-erbB-2, cathepsin-D and clinicopathologic characteristics using univariate and multivariate analysis. Br. J. Cancer81, 1385–1391.10.1038/sj.bjc.6693404Search in Google Scholar
Scorilas, A., Kyriakopoulou, L., Yousef, G.M., Ashworth, L.K., Kwamie, A., and Diamandis, E.P. (2001). 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 BCL2 family. Genomics72, 217–221.10.1006/geno.2000.6455Search in Google Scholar
Talieri, M., Diamandis, E.P., Katsaros N., Gourgiotis, D., and Scorilas, A. (2003). Expression of BCL2L12, a new member of apoptosis-related genes, in breast tumors. Thromb. Haemost.89, 1081–1088.10.1055/s-0037-1613411Search in Google Scholar
Thomadaki, H. and Scorilas, A. (2006). BCL2 family of apoptosis-related genes: functions and clinical implications in cancer. Crit. Rev. Clin. Lab. Sci.43, 1–67.Search in Google Scholar
Thomadaki, H., Tsiapalis, C.M., and Scorilas A (2005). Poly-adenylate polymerase modulations in human epithelioid cervix and breast cancer cell lines, treated with etoposide or cordycepin, follow cell cycle rather than apoptosis induction. Biol. Chem.386, 471–480.10.1515/BC.2005.056Search in Google Scholar
van Maanen, J.M., Retel, J., de Vries, J., and Pinedo, H.M. (1988). Mechanism of action of antitumor drug etoposide: a review. J. Natl. Cancer Inst.80, 1526–1533.10.1093/jnci/80.19.1526Search in Google Scholar
Xue, L.Y., Chiu, S.M., and Oleinick, N.L. (2003). Staurosporine-induced death of MCF-7 human breast cancer cells: a distinction between caspase-3-dependent steps of apoptosis and the critical lethal lesions. Exp. Cell Res.283, 135–145.10.1016/S0014-4827(02)00032-0Search in Google Scholar
Yardley, D.A. (2005). Gemcitabine plus paclitaxel in breast cancer. Semin. Oncol.32, S14–S21.10.1053/j.seminoncol.2005.06.025Search in Google Scholar PubMed
Yousef, G.M., Scorilas, A., Magklara, A., Memari, N., Ponzone, R., Sismondi, P., Biglia, N., Abd Ellatif, M., and Diamandis, E.P. (2002). The androgen-regulated gene human kallikrein 15 (KLK15) is an independent and favourable prognostic marker for breast cancer. Br. J. Cancer87, 1294–3000.10.1038/sj.bjc.6600590Search in Google Scholar PubMed PubMed Central
Yousef, G.M., Polymeris, M.E., Grass, L., Soosaipillai, A., Chan, P.C., Scorilas, A., Borgono, C., Harbeck, N., Schmalfeldt, B., Dorn, J., et al. (2003). Human kallikrein 5: a potential novel serum biomarker for breast and ovarian cancer. Cancer Res.63, 3958–3965.Search in Google Scholar
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