Karaciğer, prostat ve kolon kanseri hücrelerinde cisplatin ve 5FU uygulamasına çoklu ilaç direnci gen ifadesi yanıtı

Abstract

Tümör hücrelerinin plazma membranında ilaç taşıyıcıların değişken ekspresyonu, ilaç direncine katkıda bulunmuştur. Bu çalışmanın amacı Hep3B, PC3 ve HT29 hücre hatlarında Cisplatin ve 5-florourasil (5FU) uygulamasının MRP2 ve MRP3 genlerinin ekspresyon seviyelerine etkilerini araştırmaktır. Cisplatin ve 5FU'nun sitotoksik etkilerini belirlemek için MTT metodu kullanıldı. MTT testinden elde edilen sonuçlar PC3 hücre hattının, 48 saat cisplatin uygulamasına en duyarlı hücre hattı olduğunu göstermiştir ve bu zaman aralığı için en düşük IC50 değerini (0.5 µg / ml) göstermiştir. 5FU uygulaması PC3 hücrelerinin çoğalmasını farklı şekillerde değiştirmiştir, 48 saat için 6.0 µg / ml ve 72 saat için8.2 µg / ml IC50 değerleri elde edilmiştir. MRP2 ve MRP3'ün mRNA ekspresyon seviyeleri, kantitatif ters transkripsiyon polimeraz zincir reaksiyonu ile belirlendi. qRT-PCR sonuçları, MRP3 mRNA ekspresyon seviyesinin cisplatin ve 5FU ile muamele edilmiş tüm hücre hatlarında genel olarak azalmaya neden olmasına ragmen, MRP2'nin PC3 ve Hep3B hücrelerinde genel olarak artışa neden olduğu belirlenmiştir.

Keywords

• sitotoksisite,cisplatin,5FU,çoklu ilaç direnci

Multidrug resistance gene expression response to cisplatin and 5FU treatment in hepatoma, prostate and colon cancer cells

Abstract

Variable expression of drug transporters at the plasma membrane of tumor cells contributed to drug resistance. The aim of the present study was to explore the expression levels of MRP2 and MRP3genes in Cisplatin and 5 fluorouracil (5FU) treated Hep3B, PC3 and HT29 cell lines. MTT assay was used to determine the cytotoxic effects of cisplatin and 5FU. The results from the MTT assay revealed that PC3 cell line is the most sensitive for cisplatin treatment presenting the lowest IC50 value (0.5 µg/ml) for 48h. 5FU treatment altered the proliferation of PC3 cells with the IC50 values 6.0 µg/ml for 48 h and 8.2 µg/ml for 72h. The mRNA expression levels of MRP2 and MRP3 were determined by quantitative reverse transcription polymerase chain reaction. qRT-PCR results revealed that while the expression levels of MRP3 mRNA was generally down regulated in all cell lines treated with cisplatin and 5FU, MRP2 was upregulated in PC3 and Hep3B cells.

Keywords

• cytotoxicity,cisplatin,5FU,multidrug resistance

References

1. Wakamatsu, T., Nakahashı, Y., Hachımıne, D., Sekı, T., Okazak, K., The combination of glycyrrhizin and lamivudine can reverse the cisplatin resistance in hepatocellular carcinoma cells through inhibition of multidrug resistance-associated proteins, Internatıonal Journal of Oncology, 31, 1465-1472, (2007).
2. Fojo, A.T., Ueda, K., Slamon, D.J., Poplack, D.G., Gottesman, M.M., Pastan I., Expression of a multidrug-resistance gene in human tumors and tissues. Medical Sciences, 84, 265-269, (1987).
3. Ma, S., Hu, Y., Wang, F., Huang, Z., Chen, Y., Wang, X., Fu, L., Lapatinib Antagonizes Multidrug Resistance–Associated Protein 1–Mediated Multidrug Resistance by Inhibiting Its Transport Function, Molecular Medicine, 20, 390-399, (2014).
4. Kawase, A., Norıkane, S., Okada, A., Adachı, M., Kato, Y., Iwakı, M., Distinct Alterations in ATP-Binding Cassette Transporter Expression in Liver, Kidney, Small Intestine, and Brain in Adjuvant-Induced Arthritic Rats, Pharmacokinetics Pharmacodynamics Drug Transport and Metabolism, 103,2556–2564, (2014).
5. Warta, R., Theile, D., Mogler ,C., Herpel, E., Grabe, N., Association of Drug Transporter Expression with Mortality and Progression-Free Survival in Stage IV Head and Neck Squamous Cell Carcinoma, PLOS ONE, 9, 10, e108908, (2014).
6. Namisaki, T., Schaeffeler, E., Fukui, H., Yoshiji, H., Nakajima, Y., Fritz, P., Schwab, M., Nies, A.T., Differential Expression of Drug Uptake and Efflux Transporters in Japanese Patients with Hepatocellular Carcinoma, Drug Metabolism and Disposition, 42, 2033–2040, (2014).
7. Zollner, G., Wagner, M., Fickert, P., Silbert, D., Fuchsbichler , A., Zatloukal, K., Denk, H., Trauner, M., Hepatobiliary transporter expression in human hepatocellular carcinoma, Liver International, 25, 367–379, (2005).
8. Prashanth, R., Tagore, S., Pradhyumna, M., Jeffrey, P,R., Update in global trends and aetiology of hepatocellular carcinoma, Contemporary Oncology , 22, 3, 141–150, (2018).

9. Mosmann, T.R., Coffman ,R.L., Two types of mouse helper T cell clone:implications for immune regulation, Immunology Today, 8, 7-8, 223-227, (1987).
10. Tepsiri, N., Chaturat, L., Sripa, B., Namwat, W., Wongkham, S., Bhudhisawasdi, V., Tassaneeyakul, W., Drug sensitivity and drug resistance profiles of human intrahepatic cholangiocarcinoma cell lines, World Journal of Gastroenterology, 11, 18, 2748-2753, (2005).
11. Livak, K.J., Schmittgen, T.D., Analysis of relative gene expression data using real-time quantitative PCR and the 2(- DeltaDeltaC(T)) method, Methods, 25, 402–408, (2001).
12. Kang, B.W., Kim, J.G., Kwon, O.K., Chung, H.Y., Yu, W., Non-platinum-based chemotherapy for treatment of advanced gastric cancer: 5-fluorouracil, taxanes, and irinotecan, World Journal of Gastroenterology, 14, 20, 5396-5402, (2014).
13. Price, T.J., Shapiro, J.D., Segelov, E., Karapetis, C.S., Pavlakis, N., Van Cutsem, E., Shah, M.A., Kang, Y.K., Tebbutt, N.C., Management of advanced gastric cancer, Expert Review of Gastroenterology and Hepatology, 6, 199-208, (2012).
14. Koike, K., Kawabe, T., Tanaka, T., Toh, S., Uchiumi, T., Wada, M., Akiyama, S.I., Ono ,M., Kuwano, M. A., Canalicular multispecific organic anion transporter (cMOAT) antisense cDNA enhances drug sensitivity in human hepatic cancer cells, Cancer Research, 15, 57, 5475-5479, (1997).
15. Nies, A.T., König, J., Pfannschmidt, M., Klar, E., Hofmann, W.J. Keppler, D., Expression of the multidrug resistance proteins MRP2 and MRP3 in human hepatocellular carcinoma, International Journalf of Cancer, 94, 492–499, (2001).
16. Sun, Z., Zhao, Z., Li, G., Dong, S., Huang, Z., Ye, L., Liang, H., Qu, J., Ai, X., Zhang, W., et al., Relevance of two genes in the multidrug resistance of hepatocellular carcinoma: in vivo and clinical studies, Tumori, 96, 90–96, (2010).
17. Brenes ,O., Arce, F., Gätjens-Boniche, O., Díaz, C., Characterization of cell death events induced by anti-neoplastic drugs cisplatin, paclitaxel and 5-fluorouracil on human hepatoma cell lines: Possible mechanisms of cell resistance, Biomedicine and Pharmacotherapy, 61, 6, 347-355, (2007).
18. Boku, N., Yamamoto, S., Fukuda, H., Shirao, K., Doi, T., Sawaki, A., Koizumi, W., Saito, H., Yamaguchi, K., Takiuchi, H., Nasu, J., Ohtsu, A., Fluorouracil versus combination of irinotecan plus cisplatin versus S-1 in metastatic gastric cancer: a randomised phase 3 study, The Lancet Oncology, 10, 1063-1069, (2009).
19. Ohtsu, A., Shimada, Y., Shirao, K., Boku, N., Hyodo, I., Saito, H., Yamamichi, N., Miyata, Y., Ikeda, N., Yamamoto, S, Fukuda, H., Yoshida, S., Randomized phase III trial of fluorouracil alone versus fluorouracil plus cisplatin versus uracil and tegafur plus mitomycinin patients with unresectable, advanced gastric cancer: The Japan Clinical Oncology Group Study, Journal of Clinical Oncology, 21, 54-59, (2003).
20. Gillet, J.P., and Gottesman, M.M., Mechanisms of multidrug resistance in cancer in Methods in Molecular Biology (Zhou J ed), 47–76, Springer, New York, (2010).
21. Gottesman, M.M., Fojo, T., Bates, S.E., Multidrug resistance in cancer: role of ATP-dependent transporters, Nature Reviews Cancer, 2, 48–58, (2002).
22. Yu, Z., Zhang, C., Wang, H., Xing, J., Gong, H., Yu, E., Zhang, W., Zhang, X., Cao, G., Fu, C., Multidrug resistance-associated protein 3 confers resistance to chemoradiotherapy for rectal cancer by regulating reactive oxygen species and caspase-3-dependent apoptoticpathway, Cancer Letters, 353, 2, 182-193, (2014).
23. König, J., Rost, D., Cui, Y., Keppler, D., Characterization of the human multidrug resistance protein isoform MRP3 localized to the basolateral hepatocyte membrane, Hepatology, 29, 1156-1163, (1999).
24. Kool, M., van der Linden, M., de Haas, M., Scheffer, G.L., de Vree, J.M., Smith, A.J., Jansen G., Peters, G.J., Ponne, N., Scheper, R.J., Elferink, R.P., Baas, F., Borst P., MRP3, an organic anion transporter able to transport anti-cancer drugs, Proceedings of the National Academy of Sciences of the United States of America , 96, 6914–6919, (1999).
25. Young, L.C., Campling, B.G., Cole, S.P., Deeley, R.G., Gerlach, J.H., Multidrug resistance proteins MRP3, MRP1, and MRP2 in lung cancer: Correlation of protein levels with drug response and messenger RNA levels, Clinical Cancer Research, 7, 1798–1804, (2001).
26. Liu, Y.H., Di, Y.M., Zhou, Z.W., Mo, S.L., Zhou, S.F., Multidrug resistance associated proteins and implications in drug development, Clinical and Experimantal Pharmacology and Physiology, 37, 115–120, (2009).