J. -F. Zhang, P. -Q. Liu, G. -H. Chen, M. -Q. Lu, C. -J. Cai, Y

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Ponicidin inhibits cell growth on hepatocellular carcinoma cells by induction of apoptosis J.-F. Zhang, P.-Q. Liu, G.-H. Chen, M.-Q. Lu, C.-J. Cai, Y. Yang, H. Li Digestive and Liver Disease Volume 39, Issue 2, Pages 160-166 (February 2007) DOI: 10.1016/j.dld.2006.09.011 Copyright © 2006 Editrice Gastroenterologica Italiana S.r.l. Terms and Conditions

Fig. 1 Chemical structure of ponicidin (C20H26O6). Digestive and Liver Disease 2007 39, 160-166DOI: (10.1016/j.dld.2006.09.011) Copyright © 2006 Editrice Gastroenterologica Italiana S.r.l. Terms and Conditions

Fig. 2 Cell viability caused by ponicidin. Cells were treated with different concentrations of ponicidin for 24, 48 and 72h. Ponicidin (over 10μmol/L) had significant growth inhibition effects on the two kinds of cells in a dose-and time-dependent manner. The two kinds of hepatocellular carcinoma cells showed somewhat different sensitivity to different concentrations of ponicidin measured by the MTT test, and HepG-2 cells were a bit more sensitive to ponicidin comparing with QGY7701 cells. After treatment by 40μmol/L ponicidin for 72h, the cell viability of HepG-2 and QGY7701 cells were 12.7 and 9.3%, respectively, and 40μmol/L ponicidin caused cell viability were much more lower that of other concentrations (p<0.05). Digestive and Liver Disease 2007 39, 160-166DOI: (10.1016/j.dld.2006.09.011) Copyright © 2006 Editrice Gastroenterologica Italiana S.r.l. Terms and Conditions

Fig. 3 Cell apoptosis caused by poncidin. After cells treated with ponicidin, flow cytometry (FCM) analysis was used to detect apoptotic rate. The cells were stained with PI and then analysed by FCM, the percentage of sub-G1 cells in the two kinds of cell lines was increased in both time- and dose-dependent manner. After treatment by 40μmol/L ponicidin for 72h, the cell apoptotic rate of HepG-2 and QGY7701 cells were 51.5 and 63.3%, respectively. The cell apoptotic rate of ponicidin at 40μmol/L is much higher than that of lower concentrations of ponicidin (p<0.05). Digestive and Liver Disease 2007 39, 160-166DOI: (10.1016/j.dld.2006.09.011) Copyright © 2006 Editrice Gastroenterologica Italiana S.r.l. Terms and Conditions

Fig. 4 Expressions of pro-and anti-apoptotic genes detected by RT-PCR. After treatment with 40μmol/L ponicidin for 48 and 72h, RT-PCR was used to detect the variation of Bcl-2, Bax and Survivin as described in Section 2. The data showed that the mRNA expressions of both Survivin and Bcl-2 were down-regulated, while mRNA expression of Bax up-regulated. (1) QGY7701 cell and (2) HepG-2 cell. Digestive and Liver Disease 2007 39, 160-166DOI: (10.1016/j.dld.2006.09.011) Copyright © 2006 Editrice Gastroenterologica Italiana S.r.l. Terms and Conditions

Fig. 5 Western blot analysis of apoptosis related gene. After treatment with 40μmol/L ponicidin for 48 and 72h, Western blot analysis was used to detect the variation of Bcl-2, Bax and Survivin as described in Section 2. The results showed that both Bcl-2 and Survivin were down-regulated while Bax up-regulated remarkably especially after the cells were treated for 72h. (1) QGY7701 cell and (2) HepG-2 cell. Digestive and Liver Disease 2007 39, 160-166DOI: (10.1016/j.dld.2006.09.011) Copyright © 2006 Editrice Gastroenterologica Italiana S.r.l. Terms and Conditions

Fig. 6 Apoptosis observed by Hoechst 33258 staining (100×). After cells treated with 40μmol/L ponicidin for 48h, marked morphological changes of cell apoptosis such as condensation of chromatin and nuclear fragmentations were found clearly using Hoechst 33258 staining. (A) Control (B) Cells treated for 48h. Digestive and Liver Disease 2007 39, 160-166DOI: (10.1016/j.dld.2006.09.011) Copyright © 2006 Editrice Gastroenterologica Italiana S.r.l. Terms and Conditions

Fig. 7 DNA fragmentation analysis. Incubation of QGY7701 and HepG-2 cells with 40μmol/L ponicidin for 48 and 72h elicited a characteristic ‘ladder’ of DNA fragments representing integer multiples of the internucleosomal DNA length. (A) Control (no reagents were used); (B) cells treated for 48h; (C) cells treated for 72h; lane 1: QGY-7701 cell; lane 2: HepG-2 cell; M: Marker. Digestive and Liver Disease 2007 39, 160-166DOI: (10.1016/j.dld.2006.09.011) Copyright © 2006 Editrice Gastroenterologica Italiana S.r.l. Terms and Conditions