Venhar GÜRBÜZ, AKIN YILMAZ, Özlem GÖKÇE, ECE KONAÇ

İnsan Kolon Kanser Hücre Hattında (HT29) Sisplatin'in Apoptotik Etkisi

Amaç: Çalışmamızda, sisplatinin 72 saatlik farklı dozlarda insan kolon kanseri olan HT29 hücre hattındaki etkilerinin araştırılması hedeflenmiştir. Gereç ve Yöntem: 5-200 μM' lık dozlar arasında sisplatin eklenen hücreler 72 saat süresince kültüre edilmiştir. EtBr-AO boyama tekniği apoptozun değerlendirilmesinde kullanılmıştır. BCL2L1, CASP3 ve CASP8 genlerinin mRNA ifade düzeyleri RTPCR yöntemi ile değerlendirilmiştir. Bulgular: Sisplatinin 72 saatte doza bağlı sitotoksik etki gösterdiği ve uygun apoptotik dozunun yine bu saat için 50μM olduğu tespit edilmiştir. Araştırılan üç genin mRNA düzeylerinin, 5μM sisplatin uygulandığında kontrole göre istatistiksel olarak anlamlı artış gösterdiği belirlenmiştir (p<0.05). 50μM sisplatin uygulamasının ise BCL2L1 mRNA düzeyinde kontrole göre değişiklik oluşturmadığı (p>0.05), ancak aynı doz ve sürede CASP3 mRNA düzeyinin 2.2 kat, CASP8 mRNA düzeyinin 3.0 kat arttığı bulunmuştur (p<0.05). Sonuç: Çalışmamızda insan kolon kanser hücrelerinde sisplatinin 72'inci saatte ve 50μM dozda en etkin değerde apoptozu uyardığı, sinyal iletim yolaklarından kaspaz kaskadını kullandığı ve antiapoptotik yolağı baskıladığı görülmüştür.Anahtar Kelimeler: Apoptoz, HT29, Hücre kültürü, Kolon kanseri, Sisplatin

Anahtar Kelimeler:

Apoptoz, HT29, Hücre kültürü, Kolon kanseri, Sisplatin

-

Keywords:

-,

PDF

___

1. Jemal A, Siegel R, Ward E, Hao Y, Xu J, Thun MJ. Cancer statistics, 2009. CA Cancer J Clin 2009; 59: 225-49. doi: 10.3322/caac.20006
2. Ekmekçi A, Konaç E, Önen EI. Gen polimorfizmi ve kansere yatkınlık. Marmara Med J 2008; 21: 282-95.
3. Aral C, Özer A. Mitochondrial DNA and cancer. Marmara Med J 2007; 20: 127-36.
4. Segal NH, Saltz LB. Evolving treatment of advanced colon cancer. Annu Rev Med 2009; 60: 207-19. doi: 10.1146/annurev.med.60.041807.132435
5. Lee CK, Park KK, Lim SS, Park JH, Chung WY. Effects of the licorice extract against tumor growth and cisplatininduced toxicity in a mouse xenograft model of colon cancer. Biol Pharm Bull 2007; 30: 2191-5. doi: 10.1248/bpb.30.2191
6. Stubbert LJ, Smith JM, McKay BC. Decreased transcription-coupled nucleotide excision repair capacity is associated with increased p53- and MLH1- independent apoptosis in response to cisplatin. BMC Cancer 2010;10:207-10. doi: 10.1186/1471-2407-10- 207
7. Ho YP, Au-Yeung SC, To KK. Platinum-based anticancer agents: innovative design strategies and biological perspectives. Med Res Rev 2003;23:633-55. doi: 10.1002/med.10038
8. Siddik Z H. Cisplatin: mode of cytotoxic action and molecular basis of resistance. Oncogene 2003;22:7265–79. doi: 10.1038/sj.onc.1206933
9. Ho YP, Au-Yeung SC, To KK. Platinum-based anticancer agents: innovative design strategies and biological perspectives. Med Res Rev 2003;23:633-55. doi: 10.1002/med.10038
10. Bose RN. Biomolecular targets for platinum antitumor drugs. Mini Rev Med Chem 2002;2:103-11.
11. Salles B, Butour JL, Lesca C, Macquet JP. CisPt(NH3)2Cl2 and trans- Pt(NH3)2Cl2 inhibit DNA synthesis in cultured L1210 leukemia cells. Biochem Biophys Res Commun 1983;112:555– 63. doi:10.1016/0006-291X(83)91500-0
12. Ormerod MG, Orr RM, Peacock JH. The role of apoptosis in cell killing by ciplatin: a flow cytometric study. Br J Cancer 1994;69:93–100.
13. Nguyen HN, Sevin BU, Averette HE, et al. Cell cycle perturbations of platinum derivatives on two ovarian cancer cell lines. Cancer Invest 1993;11:264–75.
14. Voland C, Bord A, Péleraux A, et al. Repression of cell cycle-related proteins by oxaliplatin but not cisplatin in human colon cancer cells. Mol Cancer Ther 2006;5: 2149-57. doi: 10.1158/1535-7163.MCT-05-0212
15. Pfaffl MW, Horgan GW, Dempfle L. Relative expression software tool (REST) for group-wise comparison and statistical analysis of relative expression results in realtime PCR. Nucleic Acids Res 2002;30:e36.
16. Rabik CA, Dolan ME. Molecular mechanisms of resistance and toxicity associated with platinating agents. Cancer Treat Rev 2007;33:9- 23. doi: 10.1016/j.ctrv.2006.09.006
17. Fernández de Mattos S, Villalonga P, Clardy J, Lam EW. FOXO3a mediates the cytotoxic effects of cisplatin in colon cancer cells. Mol Cancer Ther 2008;7:3237- 46. doi: 10.1158/1535-7163.MCT-08-0398
18. Suzuki K, Kokuryo T, Senga T, Yokoyama Y, Nagino M, Hamaguchi M. Novel combination treatment for colorectal cancer using Nek2 siRNA and cisplatin. Cancer Sci 2010;101(5):1163-9. doi: 10.1111/j.1349- 7006.2010.01504.x
19. Kirkin V, Joos S, Zörnig M. The role of Bcl-2 family members in tumorigenesis. Biochim Biophys Acta 2004; 1644 (2-3): 229-49. doi: 10.1016/j.bbamcr.2003.08.009
20. Minn AJ, Rudin CM, Boise LH, Thompson CB. Expression of bcl-xL can confer a multidrug resistance phenotype. Blood 1995;86:1903-10.
21. Dole MG, Jasty R, Cooper MJ, Thompson CB, Nuñez G, Castle VP. Bcl-xL is expressed in neuroblastoma cells and modulates chemotherapy-induced apoptosis. Cancer Res 1995;55:2576-82.
22. Simonian PL, Grillot DA, Nuñez G. Bcl-2 and Bcl-XL can differentially block chemotherapyinduced cell death. Blood 1997;90:1208-16.
23. Lebedeva I, Rando R, Ojwang J, Cossum P, Stein CA. Bcl-xL in prostate cancer cells: effects of overexpression and down-regulation on chemosensitivity. Cancer Res 2000; 60:6052-60.
24. Luo D, Cheng SC, Xie H, Xie Y. Effects of Bcl-2 and Bcl-XL protein levels on chemoresistance of hepatoblastoma HepG2 cell line. Biochem Cell Biol 2000;78: 119-26.
25. Kyprianou N, King ED, Bradbury D, Rhee JG. bcl-2 over-expression delays radiation-induced apoptosis without affecting the clonogenic survival of human prostate cancer cells. Int J Cancer 1997;70:341-8. doi: 10.1002/(SICI)1097- 0215(19970127)70:3<341::AIDIJC16> 3.0.CO;2- I
26. Konopleva M, Zhao S, Hu W, et. al. The antiapoptotic genes Bcl-X(L) and Bcl-2 are overexpressed and contribute to chemoresistance of non-proliferating leukaemic CD34+ cells. Br J Haematol 2002;118:521- 34.
27. Villedieu M, Louis MH, Dutoit S, et. al. Absence of BclxL down-regulation in response to cisplatin is associated with chemoresistance in ovarian carcinoma cells. Gynecol Oncol 2007;105:31-44. doi: 10.1016/j.ygyno.2006.12.011.
28. Fulda S, Sieverts H, Friesen C, Herr I, Debatin KM. The CD95 (APO-1/Fas) system mediates drug-induced apoptosis in neuroblastoma cells. Cancer Res 1997;57: 3823-9.
29. Rehemtulla A, Hamilton CA, Chinnaiyan AM, Dixit VM. Ultraviolet radiation-induced apoptosis is mediated by activation of CD-95 (Fas/APO-1). J Biol Chem 1997;272:25783-6. doi: 10.1074/jbc.272.41.25783.
30. Duale N, Lindeman B, Komada M, et. al. Molecular portrait of cisplatin induced response in human testis cancer cell lines based on gene expression profiles. Mol Cancer 2007;6:53. doi: 10.1186/1476-4598-6-53.