Rumeysa ÖZYURT, Mete OZKURT, Abdullah KARADAĞ, Cemile MERAL, Nılufer ERKASAP

Ceranib-2, HIF1-α gen ekspresyonunu inhibe eder ve HepG2 hücrelerinde apoptozu indükler

Amaç: Bu çalışmanın amacı, yeni bir anti-kanser ilaç olan seranib-2' nin apoptotik etkisini ve HIF-lα düzeylerinin HepG2 üzerindeki etkisini araştırmaktır. Gereç ve Yöntem: Hücreler 0, 1, 1, 5, 10, 25 ve 50 µM seranib-2 ile 24 ve 48 saat süreyle muamele edildi ve hücre canlılığı belirlendi. Asit seramidaz, kaspaz-3, kaspaz-8, kaspaz-9, Sitokrom C1, HIF-1α,ve TNF-α mRNA seviyeleri qPCR ile ölçüldü. Bulgular: 10 µM konsantrasyonda Ceranib-2; 24 ve 48 saat tedaviden sonra hücre canlılığını yaklaşık % 58 azalttı. Aynı doz, kaspaz-3'ün mRNA seviyesini arttırdı ve 24 saat sonra kontrol grubuna kıyasla kaspaz-8 üzerinde herhangi bir değişiklik tespit edilmedi. Kaspaz-3'te herhangi bir değişiklik saptanmazken kaspaz-8 mRNA seviyesi, 48 saat sonra 10 µM seranib-2 konsantrasyonunda arttı. Kaspaz-9 mRNA seviyeleri 24 ve 48 saat sonra değişmedi. 10 µM konsantrasyonda Ceranib-2, 24 saatlik tedaviden sonra kontrol grubuna karşı Sitokrom C1'in mRNA seviyesini düşürdü. ASAH mRNA seviyesi, 10 µM seranib-2 ile 48 saatlik tedaviden sonra azaltılmıştır. ASAH' ın azaltılması, 10 µM seranib-2'nin 48 saat sonra seramidazı inhibe edebileceğini ve bunun seramid konsantrasyonunu artırabildiğini gösterdi. TNF-α mRNA seviyesi hem 24 hem de 48 saat sonra arttı, ancak HIF-lα mRNA seviyesi, 24 saat sonra kontrol grubuna kıyasla düşüktü. Sonuç: Seranib-2' nin HepG2'de apoptozu indüklediğini bulduk, bu nedenle seranib-2’ nin 10 µM konsantrasyonda anti-kanser bir rolü olabilir.

Ceranib-2 inhibits HIF1-α gene expression and induces apoptosis in HepG2 cells

Purpose: The aim of this study is to investigate the apoptotic effect of a novel anti-cancer drug, ceranib-2 and impact on HIF-1α levels on HepG2. Materials and Methods: The cell line was treated in vitro with 0,1, 1, 5, 10, 25 and 50 µM ceranib-2 for 24 and 48 hours and cell viabilitiy was determined. mRNA levels of acid ceramidase, caspase-3, caspase-8, caspase-9, Cyc1, HIF-1α and TNF-α were measured by qPCR. Results: Ceranib-2 at 10 µM concentration reduced the viability by about 58 % after 24 and 48 hours. The same dose increased mRNA level of caspase-3 and no change was detected on caspase-8 when compared to the control group after 24 hours. No difference was detected on caspase-3, but caspase-8 mRNA level increased after 48 hours with ceranib-2 at 10 µM concentration. Caspase-9 mRNA levels did not differ after 24 and 48 hours. Ceranib-2 at 10 µM concentration lowered mRNA level of Cyc1 against the control group after the 24- hour treatment. ASAH mRNA level was reduced after the 48-hour treatment with 10 µM ceranib-2. Reduction of ASAH indicated that 10 µM ceranib-2 could inhibit ceramidase after 48 hours and this may elavate ceramide concentration. TNF-α mRNA increased after 24 and 48 hours, but HIF-1α expression was low after 24 hours when compared to the control group. Conclusion: We have found that ceranib-2 induces apoptosis in HepG2, thus ceranib-2 may play an anti-cancer role at 10 µM concentration.

Keywords:

ceranib-2, apoptosis, HepG2,

PDF

___

1. Waller LP, Deshpande V, Pyrsopoulos N. Hepatocellular carcinoma: A comprehensive review. World J Hepatol. 2015;7:2648-63.
2. Furuya H, Shimizu Y, Kawamori T. Sphingolipids in cancer. Cancer Metastasis Rev. 2011;30:567-76.
3. Maurer BJ, Metelitsa LS, Seeger RC, Cabot MC, Reynolds CP. Increase of ceramide and induction of mixed apoptosis/necrosis by N-(4-hydroxyphenyl)- retinamide in neuroblastoma cell lines. J Natl Cancer Inst. 1999;91:1138-46.
4. Cuvillier O, Pirianov G, Kleuser B, Vanek PG, Coso OA, Gutkind S et al. Suppression of ceramide - mediated programmed cell death by sphingosine-1- phosphate. Nature. 1996;381:800-3.
5. Norris JS, Bielawska A, Day T, El -Zawahri A, ElOjeimy S, Hannun Y, et al. Combined therapeutic use of AdGFPFasL and small molecule inhibitors of ceramide metabolism in prostate and head and neck cancers: a status report. Cancer Gene Ther. 2006;13:1045-51.
6. Draper JM, Xia Z, Smith RA, Zhuang Y, Wang W, Smith CD. Discovery and evaluation of inhibitors of human ceramidase. Mol Cancer Ther. 2011;10:2052- 61.
7. Schuchman EH. Acid ceramidase and the treatment of ceramide diseases: The expanding role of enzyme replacement therapy. Biochim Biophys Acta. 2016;1862:1459-71.
8. Bielawska A, Linardic CM, Hannun YA. Ceramide - mediated biology. Determination of structural and stereospecific requirements through the use of N - acyl-phenylaminoalcohol analogs. J Biol Chem. 1992;267:18493-7.
9. Selzner M, Bielawska A, Morse MA, Rudiger HA, Sindram D, Hannun YA et al. Induction of apoptotic cell death and prevention of tumor growth by ceramide analogues in metastatic human colon cancer. Cancer Res. 2001;61:1233-40.
10. Kolesnick R. The therapeutic potential of modulating the ceramide/sphingomyelin pathway. J Clin Invest. 2002;110:3-8.
11. Kus G, Kabadere S, Uyar R, Kutlu HM. Induction of apoptosis in prostate cancer cells by the novel ceramidase inhibitor ceranib-2. In Vitro Cell Dev Biol Anim. 2015;51:1056-63.
12. Raisova M, Goltz G, Bektas M, Bielawska A, Riebeling C, Hossini AM et al. Bcl-2 overexpression prevents apoptosis induced by ceramidase inhibitors in malignant melanoma and HaCaT keratinocytes. FEBS Lett. 2002;516:47-52.
13. Zhu Q, Yang J, Zhu R, Jiang X, Li W, He S et al. Dihydroceramide-desaturase-1-mediated caspase 9 activation through ceramide plays a pivotal role in palmitic acid-induced HepG2 cell apoptosis. Apoptosis. 2016;21:1033-44.
14. Sawada M, Nakashima S, Banno Y, Yamakawa H, Hayashi K, Takenaka K et al. Ordering of ceramide formation, caspase activation, and Bax/Bcl-2 expression during etoposide-induced apoptosis in C6 glioma cells. Cell Death Differ. 2000;7:761-72.
15. Susin SA, Zamzami N, Larochette N, Dallaporta B, Marzo I, Brenner C et al. A cytofluorometric assay of nuclear apoptosis induced in a cell-free system: application to ceramide-induced apoptosis. Exp Cell Res. 1997;236:397-403.
16. Wiesner DA, Kilkus JP, Gottschalk AR, Quintans J, Dawson G. Anti-immunoglobulin-induced apoptosis in WEHI 231 cells involves the slow formation of ceramide from sphingomyelin and is blocked by bcl- XL. J Biol Chem. 1997;272:9868-76.
17. Soans E, Evans SC, Cipolla C, Fernandes E. Characterizing the sphingomyelinase pathway triggered by PRIMA-1 derivatives in lung cancer cells with differing p53 status. Anticancer Res. 2014;34:3271-83.
18. Wali JA, Masters SL, Thomas HE. Linking metabolic abnormalities to apoptotic pathways in Beta cells in type 2 diabetes. Cells. 2013;2:266-83.
19. Dakroub Z, Kreydiyyeh SI. Sphingosine-1-phosphate is a mediator of TNF -alpha action on the Na+/K+ ATPase in HepG2 cells. J Cell Biochem. 2012;113:2077-85.
20. Wilson WR, Hay MP. Targeting hypoxia in cancer therapy. Nat Rev Cancer. 2011;11:393-410.
21. Zhang FJ, Tang WX, Wu CH, Yan W, Gu H. Expression and significance of hypoxia inducible factor-1 alpha in hepatocellular carcinoma tissues. Zhonghua Gan Zang Bing Za Zhi. 2006;14:281-4.
22. Chen J, Kobayashi M, Darmanin S, Qiao Y, Gully C, Zhao R et al. Pim -1 plays a pivotal role in hypoxia- induced chemoresistance. Oncogene. 2009;28:2581- 92.