Proceedings of The Physiological Society

Europhysiology 2018 (London, UK) (2018) Proc Physiol Soc 41, PCA091

Poster Communications

Targeting Inhibition of Ceramidase Induces Apoptosis in Hepatocellular Carcinoma in vitro

N. erkasap1, R. ÖZYURT1, M. OZKURT1, A. KARADAĞ1, C. MERAL EVIS1

1. physiology, eskisehir osmangazi university medical faculty, Eskisehir, Turkey.


Ceramide is a bioactive lipid, synthesized by hidrolization of sphingomyelin. Over-expression of ceramidases is involved in increased resistance to apoptosis which results in poor prognosis in several types of cancer including melanoma, prostate cancer and colon cancer whereas exposure to ceramides or sphingomyelinase, which generates ceramide, has been shown to induce apoptosis in cancer cells. Ceranib-2 is a novel anti-cancer drug targeting the enzyme acid ceramidase. However the possible effects of ceranib-2 on hepatocellular cancer cell line HepG-2 is not studied yet. The present study was designed to investigate the effects of ceranib-2 on the viability of human HepG-2 cells as well as Caspase-3, Cytochrome c1 (cyc1) and ASAH (acid ceramidase) expressions. The study shows that ceranib-2 increased apoptosis by decreasing ASAH expression and inducing caspase-3 expression in human hepatocellular cancer cell line in a dose and time-dependent manner. We studied the apoptotic affect of ceranib-2 in 0,1, 1, 5, 10, 25 and 50 µM concentrations on HepG-2 cells for 24 and 48 hours by 3-(4,5-D-methylthiazol-2-yl)-2,5-diphenyltetrazolium bromide thyazolyl blue (MTT). In 10 µM concentration of ceranib-2 we detected Caspase-3, Cyc-1 ASAH mRNA expressions by Real-Time PCR (RT-PCR). Statistical analysis was performed by using the GraphPad software program. Following the determination of Caspase-3, Cyc-1 and ASAH mRNA expression using beta-actin as a reference gene, the data obtained from RT PCR were calculated using the formula 2−ΔΔCt. In our study ceranib-2 decreased the viability of HepG-2 cells in10 µM concentration for 24 and 48 hours. This impact shows %58 difference compared to control. According to RT-PCR results, while mRNA levels of Caspase-3 increased (p<0.05), mRNA levels of Cyc1 were not changed in 24th hour and 10 µM concentration compared to control group and no difference for Caspase-3 in 48th hour. mRNA levels of ASAH showed a reduction in 10 µM concentration in 48th hour compared to control group (p<0.05). Our Study shows that ceranib-2 has strong anti-cancer effect as time and dose dependent. Low levels of mRNA of ASAH in treatment groups showed that 10 µM ceranib-2 could inhibit ceramidase at 48th hour and this may suggest that ceramide levels are increasing. PCR data of the current study indicated that while cytochrome c mRNA expression level was not change, mRNA levels of Caspase-3 increased in 10 µM concentration. Due to this contradictory results, we will focus on possible different apoptotic pathways to explain the effects of seranib-2 in our future studies.

Where applicable, experiments conform with Society ethical requirements