Determination of mechanisms of multiple drug resistance in MCF-7 cell line developed against paclitaxel and vincristine by microarray analysis

Abstract

OBJECTIVES Acquired or intrinsic multiple drug resistance (MDR) is one of the limitations for successful chemotherapy. In this study, MDR mechanisms developed after paclitaxel and vincristine application in MCF-7 cell line were investigated. METHODS Paclitaxel and vincristine were applied to MCF-7 cell line in stepwise manner. The resistance levels of the cells were determined by XTT test. The levels of all of the genes in resistant and sensitive cells were determined by microarray assay. RESULTS MCF-7/400nMPac and MCF-7/120nMVinc cells are 150- and 30-fold resistant compared to sensitive MCF-7. Upregulation of MDR1 gene is found to be important in resistance. The expression levels of the genes encoding detoxifying enzymes were upregulated and some apoptotic gene levels were downregulated. Changes in expression levels of metastatic genes, some oncogenes, cell cycle, and tubulin-related genes were found. CONCLUSION According to these results, change in expression level of MDR1 gene seems to be important in resistance in MCF-7 cells.

Keywords

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Meme kanseri MCF-7 hücre hattında paklitaksel ve vinkristin'e karşı gelişmiş çoklu ilaç direnci mekanizmalarının mikrodizin analizi ile belirlenmesi

Öz

AMAÇ Sonradan kazanılan veya tedavi öncesi varolan çoklu ilaç dirençliliği (ÇİD) kemoterapide başarıyı engelleyen nedenlerden biridir. Bu çalışmada, paklitaksel ve vinkristin'e karşı gelişen ÇİD mekanizmaları MCF-7 hücre hattında araştırıldı. GEREÇ VE YÖNTEM MCF-7 hücreleri önce kademeli olarak paklitaksel ve vinkristin doz artışlarına maruz bırakıldı. Hücrelerin direnç seviyeleri XTT testi ile belirlendi. Tüm dirençli ve duyarlı hücrelerde genlerin düzeyleri mikrodizin yöntemiyle belirlendi. BULGULAR MCF-7/400nMPak ve MCF-7/120nMVink hücre hatlarının orjinal hücrelere göre 150 ve 30 kat daha dirençli oldukları bulundu. MDR1 genin ifadesindeki artışın dirençlilik mekanizmaları arasında önemli olduğu bulundu. Hücrelerde toksik maddelerin metabolize edilmesiyle ilgili genlerin ifade düzeylerinde artışlar gözlenirken, apoptozla ilgili bazı genlerin ifadelerinde azalmalar olduğu görüldü. Metastaz genlerinde, bazı onkojenlerde, hücre döngüsünü düzenleyen ve tübülin metabolizmasıyla ilgili genin ifade düzeylerinde değişimler saptandı. SONUÇ ÇİD'de MDR1 genindeki değişikliğin MCF-7'de paklitaksel ve vinkristin dirençliliğinde temel mekanizma olduğu görülmüştür.

Anahtar Kelimeler

• Apoptotik yolak, beta tübülin, çoklu ilaç dirençliliği, MCF-7, mikrodizin analizi

Kaynakça

1. 1. Krishan A, Fitz CM, Andritsch I. Drug retention, efflux, and resistance in tumor cells. Cytometry 1997;29(4):279-85.
2. 2. Ueda K, Cardarelli C, Gottesman MM, Pastan I. Expression of a full-length cDNA for the human “MDR1” gene confers resistance to colchicine, doxorubicin, and vinblastine. Proc Natl Acad Sci U S A 1987;84(9):3004-8.
3. 3. Fitzpatrick FA, Wheeler R. The immunopharmacology of paclitaxel (Taxol), docetaxel (Taxotere), and related agents. Int Immunopharmacol 2003;3(13-14):1699-714.
4. 4. Drukman S, Kavallaris M. Microtubule alterations and resistance to tubulin-binding agents (review). Int J Oncol 2002;21(3):621-8.
5. 5. Liu Y, Peng H, Zhang JT. Expression profiling of ABC transporters in a drug-resistant breast cancer cell line using AmpArray. Mol Pharmacol 2005;68(2):430-8.
6. 6. Reed JC. Bcl-2: prevention of apoptosis as a mechanism of drug resistance. Hematol Oncol Clin North Am 1995;9(2):451-73.
7. 7. Kavallaris M, Tait AS, Walsh BJ, He L, Horwitz SB, Norris MD, et al. Multiple microtubule alterations are associated with Vinca alkaloid resistance in human leukemia cells. Cancer Res 2001;61(15):5803-9.
8. 8. Huzil JT, Luduena RF, Tuszynski J. Comparative modelling of human ß tubulin isotypes and implications for drug binding. Nanotechnology 2006;17:90-100.

9. 9. Kars MD, Iseri OD, Gündüz U, Ural AU, Arpaci F, Molnár J. Development of rational in vitro models for drug resistance in breast cancer and modulation of MDR by selected compounds. Anticancer Res 2006;26(6B):4559-68.
10. 10. İşeri ÖD, Kars MD, Eroğlu S, Gündüz U, Drug Resistant MCF-7 Cell Lines Also Developed Cross-resistance to Structurally Unrelated Anticancer Agents. Int J Hematol Oncol 2009;19(1):1-8.
11. 11. Işeri OD, Kars MD, Arpaci F, Gündüz U. Gene expression analysis of drug-resistant MCF-7 cells: implications for relation to extracellular matrix proteins. Cancer Chemother Pharmacol 2009 Jun 19. DOI 10.1007/ s00280-009-1048-z.
12. 12. Lage H. Drug resistance in breast cancer. Cancer Therapy 2003;1:81-91.
13. 13. Rouzier R, Rajan R, Wagner P, Hess KR, Gold DL, Stec J, et al. Microtubule-associated protein tau: a marker of paclitaxel sensitivity in breast cancer. Proc Natl Acad Sci U S A 2005;102(23):8315-20.
14. 14. Lee JH, Rho SB, Chun T. Programmed cell death 6 (PDCD6) protein interacts with death-associated protein kinase 1 (DAPk1): additive effect on apoptosis via caspase-3 dependent pathway. Biotechnol Lett 2005;27(14):1011-5.
15. 15. Baron BW, Anastasi J, Thirman MJ, Furukawa Y, Fears S, Kim DC, et al. The human programmed cell death-2 (PDCD2) gene is a target of BCL6 repression: implications for a role of BCL6 in the down-regulation of apoptosis. Proc Natl Acad Sci U S A 2002;99(5):2860- 5.
16. 16. Shih SC, Claffey KP. Role of AP-1 and HIF-1 transcription factors in TGF-beta activation of VEGF expression. Growth Factors 2001;19(1):19-34.
17. 17. Sabbah M, Emami S, Redeuilh G, Julien S, Prévost G, Zimber A, et al. Molecular signature and therapeutic perspective of the epithelial-to-mesenchymal transitions in epithelial cancers. Drug Resist Updat 2008;11(4-5):123-51.
18. 18. Akira S, Taga T, Kishimoto T. Interleukin-6 in biology and medicine. Adv Immunol 1993;54:1-78.
19. 19. Hazlehurst LA, Landowski TH, Dalton WS. Role of the tumor microenvironment in mediating de novo resistance to drugs and physiological mediators of cell death. Oncogene 2003;22(47):7396-402.
20. 20. O’Loughlin C, Heenan M, Coyle S, Clynes M. Altered cell cycle response of drug-resistant lung carcinoma cells to doxorubicin. Eur J Cancer 2000;36(9):1149-60.