HEPG2 Hücrelerinde Apoptotik Proteinlerin ve Çoklu İlaç Direnci Geninin Ekspresyon Düzeyleri

Abstract

Bu çalışmada, kanser hücrelerinde işleyişi hasarlı olan apoptotik, anti-apoptotik mekanizmaların ve çoklu ilaç direnci yolakların incelenmesi amacıyla, karaciğer kanseri hücre hattı HepG2 kullanılmıştır. Bu çalışmanın amacı: HepG2 hücre hattını en etkili antikanser ilaçlardan biri olan Doksorubisin ile muamele ederek, apoptozun uyarılması ve apoptozda önemli rol oynayan birbiriyle ilişkili p53, Bcl-2 ve MDR-1 genlerinin ekspresyon düzeylerinin incelenmesidir. Bu amaçla hücreler ilaç ile muamele edilerek büyütülmüş, sonrasında RNA’ları saflaştırılmış, cDNA’ya dönüştürülmüş ve elde edilen cDNA’lar üç genin ekspresyon düzeyini belirlemek amacıyla RT-PCR cihazında/yönteminde kullanılmıştır. HepG2 hücrelerinde 50 nM, 200 nM, 400 nM ve 800 nM ilaç uygulanmasından sonra kontrol gruplarına kıyasla deney gruplarında, p53 geninin ekspresyon düzeylerinin sırasıyla 2,176, 1,718, 1,524, 1,225 kat arttığı, Bcl-2 gen ekspresyon düzeylerinin sırasıyla 4,182, 6,512, 5,641, 3,910 kat arttığı ve MDR-1 gen ekspresyon düzeyinin ise uygulanan ilaç dozundan bağımsız olarak değişmediği gözlenmiştir.

Keywords

• Gen ekspresyonu,p53,bcl-2,mdr-1,HepG2

Gene Expression Levels of Apoptotic Proteins and Multidrug Resistance Genes in HEPG2 Cells

Abstract

In this study, liver cancer cell line HepG2 were used with the aim of examining the apoptotic-antiapoptotic mechanisms and multi drug resistance pathways which are defective in cancer cells. The aim of this study: is to trigger apoptosis by treatment HepG2 cancer cell line with one of the most effective anticancer drug Doxorubicin and to investigate the expression levels of p53, Bcl-2 and MDR-1 genes which are related with each other and play an important role in apoptosis. With this purpose, the cells were grown by treating with increasing doses of Doxorubicin. Their RNA’s were purified, converted into cDNAs and used in Real Time PCR device in order to determine the expression levels of these genes. It is observed in HepG2 cells, compared to the control groups after 50 nM, 200 nM, 400 nM ve 800 nM drug treatment, p53 gene expression level is respectively 2,176, 1,718, 1,524, 1,225 fold increased, Bcl-2 gene expression level is respectively 4,182, 6,512, 5,641, 3,910 fold increased and MDR-1 gene expression level is not dependent to the treated drug dose.

Keywords

• Gene expression,p53,bcl-2,mdr-1,HepG2

References

1. Reed, J.C.. Bcl-2 family proteins. Oncogene, 17 (25), 3225 (1998)
2. Levine, A.J., Oren, M.. The first years of p53: growing ever more complex. Nat Rev Can- cer, 9 (10), 749 (2009)
3. Lane D.P., Crawford, L.V.. T antigen is bound to a host protein in SV40-transformed cells. Nature, 278 (5701), 261 (1979)
4. Linzer, D.I., Maltzman W., Levine A.J.. The SV40 A gene product is required for the production of a 54,000 MW cellular tumor antigen. Virology, 98 (2), 308 (1979)
5. Hollstein, M., Hainaut P.. Massively regulated genes: the example of TP53. J Pathol, 220 (2), 164 (2010)
6. Semczuk, A., Schneider-Stock, R., Szewczuk, W.. Prevalence of allelic loss at TP53 in endometrial carcinomas. Oncology, 78 (3-4), 220 (2010)
7. Fischer, D.E. Pathways of apoptosis and the modulation of cell death in cancer. Hema- tol Oncol Clin North Am, 15 (5), 931 (2001)
8. Boehme, K.A., Blattner, C. Regulation of p53-insights into a complex process. Crit Rev Biochem Mol Biol, 44 (6), 367 (2009)

9. Polager, S., Ginsberg, D. p53 and E2f: partners in life and death. Nat Rev Cancer, 9 (10), 738, (2009)
10. Bitomsky, N., Hofmann, T.G. Apoptosis and autophagy: Regulation of apoptosis by DNA damage signalling - roles of p53, p73 and HIPK2. FEBS J, 276 (21), 6074 (2009)
11. Wood, N.B., Kotelnikov, V., Caldarelli, D.D., Hutchinson, J., Panje, W.R., Hegde, P. et al. Mutation of p53 in squamous cell cancer of the head and neck: relationship to tumor cell proliferation. Laryngoscope, 107 (6), 827 (1997)
12. Kiuru, A., Servomaa, K., Grenman, R., Pulkkinen, J., Rytomaa, T. p53 mutations in human head and neck cancer cell lines. Acta Otolaryngol Suppl, 529, 237 (1997)
13. Ho, C.C., Hau, P.M., Marxer, M., Poon, R.Y. The requirement of p53 for maintaining choromosomal stability during tetraploidization. Oncotarget, 1 (7), 583 (2010)
14. Lumachi, F., Basso, S. Apoptosis: life through planned cellular death regulating mech- anisms, control systems, and relations with thyroid diseases. Thyroid, 12 (1), 27 (2002)
15. Kerr, J.F., Wyllie, A.H., Currie, A.R. Apoptosis: a basic biological phenomenon with wide-ranging implications in tissue kinetics. Br J Cancer, 26 (4), 239 (1972)
16. Duke, R.C., Chervenak, R., Cohen, J.J. Endogenous endonuclease-induced DNA frag- mentation: an early event in cell-mediated cytolysis. Proc Natl Acad Sci U S A, 80 (20), 6361 (1983)
17. Kerr, J.F. Shrinkage necrosis of adrenal cortical cells. J Pathol, 107 (3), 217 (1972).
18. Wyllie, A.H. Apoptosis (the 1992 Frank Rose Memorial Lecture). Br J Cancer, 67 (2), 205 (1993)
19. Liu, H., Li, H., Xu, A., Kan, Q., Liu, B.. Role of phosphorylated ERK in amygdala neu- ronal apoptosis in single-prolonged stress rats. Mol Med Report, 3 (6), 1059 (2010)
20. Eberle, J., Fecker, L.F., Forschner, T., Ulrich, C., Rowert-Huber, J., Stockfleth, E. Apop- tosis pathways as promising targets for skin cancer therapy. Br J Dermatol, 156 Suppl 3, 18 (2007)
21. Renehan, A.G., Booth, C., Potten, C.S. What is apoptosis, and why is it important? BMJ, 322 (7301), 1536 (2001)
22. Saikumar, P., Dong, Z., Mikhailov, V., Denton, M., Weinberg, J.M., Venkatachalam, M.A. Apoptosis: definition, mechanisms, and relevance to disease. Am J Med, 107 (5), 489 (1999)
23. Adams, S.M., de Rivero Vaccari, J.C., Corriveau, R.A. Pronounced cell death in the absence of NMDA receptors in the developing somatosensory thalamus. J Neurosci, 24 (42), 9441 (2004)
24. Mason, R.P. Calcium channel blockers, apoptosis and cancer: is there a biologic rela- tionship? J Am Coll Cardiol, 34 (7), 1857 (1999)
25. Elmore, S. Apoptosis: a review of programmed cell death. Toxicol Pathol, 35 (4), 495 (2007)
26. Nanji, A.A., Hiller-Sturmhofel, S. Apoptosis and necrosis: two types of cell death in alcoholic liver disease. Alcohol Health Res World, 21 (4), 325 (1997)
27. Zeiss, C.J. The apoptosis-necrosis continuum: insights from genetically altered mice. Vet Pathol, 40 (5), 481 (2003)
28. O'Brien, V. Viruses and apoptosis. J Gen Virol, 79 (Pt 8), 1833 (1998)
29. Zhang, M., Chen, Z.C., Liu, F., You, Y., Liu, Z.P., Zou, P. Effects of PLK1 gene silence on apoptosis of K562 cells. Zhonghua Xue Ye Xue Za Zhi, 26 (12), 715 (2005)
30. Rai, N.K., Tripathi, K., Sharma, D., Shukla, V.K. Apoptosis: a basic physiologic process in wound healing. Int J Low Extrem Wounds, 4 (3), 138 (2005)
31. Gulbins, E., Jekle, A., Ferlinz, K., Grassme, H., Lang, F. Physiology of apoptosis. Am J Physiol Renal Physiol, 279 (4), F605 (2000)
32. Taheri, M., Mahjoubi, F., Omranipour, R. Effect of MDR1 polymorphism on multidrug resistance expression in breast cancer patients. Genet Mol Res, 9 (1), 34 (2010)
33. Weinstein, R.S., Kuszak, J.R., Kluskens, L.F., Coon, J.S. P-glycoproteins in pathology: the mutlidrug resistance gene family in humans. Hum Pathol, 21 (1), 34 (1990).
34. Roninson, I.B., Chin, J.E., Choi, K.G., Gros, P., Housman, D.E., Fojo, A. et al. Isolation of human mdr DNA sequences amplified in multidrug-resistant KB carcinoma cells. Proc Natl Acad Sci U S A, 83 (12), 4538 (1986)
35. Chen, T., Wong, Y.S. Selenocystine induces caspase-independent apoptosis in MCF-7 human breast carcinoma cells with involvement of p53 phosphorylation and reactive oxygen species generation. Int J Biochem Cell Biol, 41 (3), 666 (2009)
36. Sukhai, M., Piquette-Miller, M. Regulation of the multidrug resistance genes by stress signals. J Pharm Pharm Sci, 3 (2), 268 (2000)
37. Chen, C.J., Chin, J.E., Ueda, K., Clark, D.P., Pastan, I., Gottesman, M.M. et al. Internal duplication and homology with bacterial transport proteins in the mdr1 (P-glycopro- tein) gene from multidrug-resistant human cells. Cell, 47 (3), 381 (1986)
38. Youle, R.J., Strasser, A. The BCL-2 protein family: opposing activities that mediate cell death. Nat Rev Mol Cell Biol, 9 (1), 47 (2008)
39. Adams, J.M., Cory, S. Bcl-2-regulated apoptosis: mechanism and therapeutic potential. Curr Opin Immunol, 19 (5), 488 (2007)
40. Chipuk, J.E., Bouchier-Hayes, L., Green, D.R. Mitochondrial outer membrane permea- bilization during apoptosis: the innocent bystander scenario. Cell Death Differ, 13 (8), 1396 (2006)
41. Henderson, I.C., Frei, E., 3rd. Testing for doxorubicin cardiotoxicity. N Engl J Med, 300 (24), 1393 (1979)
42. Singal, P.K., Iliskovic, N. Doxorubicin-induced cardiomyopathy. N Engl J Med, 339 (13), 900 (1998)
43. Shi, Y., Moon, M., Dawood, S., McManus, B., Liu, P.P. Mechanisms and management of doxorubicin cardiotoxicity. Herz, 36 (4), 296 (2011)
44. Swain, S.M., Whaley, F.S., Ewer, M.S. Congestive heart failure in patients treated with doxorubicin: a retrospective analysis of three trials. Cancer, 97 (11), 2869 (2003)
45. Robert Souhami, J.T. Cancer and its Management. Wiley-Blackwell (2008)
46. Momparler, R.L., Karon, M., Siegel, S.E., Avila, F. Effect of adriamycin on DNA, RNA, and protein synthesis in cell-free systems and intact cells. Cancer Res, 36 (8), 2891 (1976)
47. Arcamone, F., Franceschi, G., Penco, S., Selva, A. Adriamycin (14-hydroxydaunomy- cin), a novel antitumor antibiotic. Tetrahedron Lett (13), 1007 (1969)
48. Ludke, A.R., Al-Shudiefat, A.A., Dhingra, S., Jassal, D.S., Singal, P.K. A concise de- scription of cardioprotective strategies in doxorubicin-induced cardiotoxicity. Can J Physiol Pharmacol, 87 (10), 756 (2009)
49. May, P., May, E. Twenty years of p53 research: structural and functional aspects of the p53 protein. Oncogene, 18 (53), 7621 (1999)
50. Hainaut, P., Hollstein, M. p53 and human cancer: the first ten thousand mutations. Adv Cancer Res, 77, 81 (2000)
51. Adimoolam, S., Ford, J.M. p53 and DNA damage-inducible expression of the xero- derma pigmentosum group C gene. Proc Natl Acad Sci U S A, 99 (20), 12985 (2002)
52. Hofseth, L.J., Robles, A.I., Yang, Q., Wang, X.W., Hussain, S.P., Harris, C. p53: at the crossroads of molecular carcinogenesis and molecular epidemiology. Chest, 125 (5 Suppl), 83S (2004)
53. Chekhun, V.F., Lukyanova, N.Y., Urchenko, O.V., Kulik, G.I. The role of expression of the components of proteome in the formation of molecular profile of human ovarian carcinoma A2780 cells sensitive and resistant to cisplatin. Experimental oncology, 27 (3), 191 (2005)
54. Kanagasabai, R., Krishnamurthy, K., Druhan, L.J., Ilangovan, G. Forced expression of Hsp27 reverses P-glycoprotein (ABCB1) mediated drug efflux and MDR1 gene ex- pression in adriamycin resistant human breast cancer cells. The Journal of biological chemistry. (2011)
55. Tsang, W.P., Ho, F.Y., Fung, K.P., Kong, S.K., Kwok, T.T. p53-R175H mutant gains new function in regulation of doxorubicin-induced apoptosis. International journal of can- cer. Journal international du cancer, 114 (3), 331 (2005)
56. Takara, K., Sakaeda, T., Okumura, K. An update on overcoming MDR1-mediated mul- tidrug resistance in cancer chemotherapy. Current pharmaceutical design, 12 (3), 273 (2006)