Effects Of Extracts From Seeds Of Cerbera Manghas L. On Cell Proliferation, Cell Cycle Progression And Apoptosis Of Human Hepatocellular Carcinoma HepG2 Cells And Their Mechanisms

Hepatocellular carcinoma (HCC) is generally acknowledged as the sixth most prevalent cancer in the word and is currently the third most common cause of cancer death with a 5-year survival rate of 7%. Hepatic resection and liver transplantation are the two mainstays of curative treatment for HCC, but can only be applied to the early stage of HCC. The majority of patients with HCC are diagnosed at a late stage when curative treatment options are not applicable. Thus, developing new therapeutic and preventive strategies targeted at apoptosis inducing and cell cycle arrest could be effective in controlling the proliferation and invasiveness as well as in the prognosis advanced stages of HCC.β-D-glucosyl-(1-4)-α-L-thevetosides of 17β-digitoxigenin (GHSC-73) and 2'-epi-2'-O-Acetylthevetin B (GHSC-74) are isolated from the seeds of Cerbera manghas L., belonging to the class of steroid-like compounds designated as cardiac glycosides. Their continued efficacy in the treatment of congestive heart failure and dysrhythmia is well appreciated. However, there is little knowledge about the role of this category of compounds in the prevention and/or treatment of proliferative diseases such as cancer. New findings in recent five years have demonstrated that these compounds are involved in complex cell-signal transduction mechanisms, inducing selective control of human tumors rather than normal cellular proliferation, and as such represent a promising candidate for targeted cancer chemotherapy. In this study, we investigated in vitro effects of GHSC-73 and GHSC-74 on cell growth, cell cycle regulation, and apoptosis in HepG2 cells, and explored their cellular mechanisms.SectionⅠ:β-D-glucosyl-(1-4)-а?-L-thevetosides of 17β-digitoxigenin extracted from seeds of Cerbera manghas L. induces cell cycle arrest and apoptosis in human hepatocellular carcinoma HepG2 cellsβ-D-glucosyl-(1-4)-а?-L-thevetosides of 17β-digitoxigenin (GHSC-73) is a cardiac glycoside isolated from the seeds of Cerbera manghas L. The aim of this study is to investigate in vitro effects of GHSC-73 on cell growth, cell cycle regulation, and apoptosis in HepG2 cells. It was found that GHSC-73 reduced viability of HepG2 cells in a time- and dose-dependent manner without decreasing the viability of Chang human liver cells and Swiss albino 3T3 fibroblasts. GHSC-73 induced S phase arrest of the cell cycle. S phase arrest was accompanied with down-regulation of Cyclin A2, DHFR and TYMS gene expression, and up-regulation of p21, GADD153 and Cyclin D1 gene expression. GHSC-73 efficiently stimulated apoptosis in HepG2 cells as evidenced by DNA fragmentation, flow cytometry of sub-G1 DNA content, DAPI staining, AnnexinⅤ/PI binding assay and Hoechst 33342 staining. This apoptotic process was accompanied by the loss of mitochondrial membrane potential (ΔΨm), translocation of apoptosis-inducing factor (AIF) from the mitochondrion to the nucleus, a transient increase in intracellular reactive oxygen species (ROS), activation of caspase-3 and caspase-8, and inactivation of nuclear factor-kappaB (NF-κB). However, exposure to GHSC-73 did not produce significant up-regulation of Fas and FasL in HepG2 cells as detected by flow cytometric analysis. In addition, a broad-spectrum caspase inhibitor (z-VAD-fmk), a caspase-3 inhibitor (Ac-DEVD-CHO) and an antioxidant N-acetyl-L-cysteine (NAC) tested in this experiment failed to rescue HepG2 cells from GHSC-73-induced cell death and did not affect translocation of AIF from the mitochondrion to the nucleus after GHSC-73 treatment, suggesting that caspase and ROS-independent pathway was involved in GHSC-73-induced apoptosis. GHSC-73 combined with a specific inhibitor of IκBаphosphorylation BAY 11-7082 significantly increased the apoptotic rate of HepG2 cells, and GHSC-73 inhibited p65 nuclear translocation as analyzed by immunofluorescence microscopy and Western blotting. In conclusion, our results provide the first evidence for a molecular mechanism of cytotoxicity of GHSC-73, showing that GHSC-73 inhibited growth of HepG2 cells by inducing S arrest of the cell cycle and by triggering apoptosis via caspase-independent AIF release from mitochondria, inactivation of NF-κB, independent of Fas/FasL interaction and ROS generation. We also demonstrated that GHSC-73 induced selective control of human tumors rather than normal cellular proliferation, thus GHSC-73 may be a potential candidate of anti-cancer drugs.SectionⅡ: 2'-epi-2'-O-Acetylthevetin B extracted from seeds of Cerbera manghas L. induces cell cycle arrest and apoptosis in human hepatocellular carcinoma HepG2 cells2'-epi-2'-O-Acetylthevetin B (GHSC-74), a cardiac glycoside, is isolated from the seeds of Cerbera manghas L. The aim of this study is to investigate in vitro effects of GHSC-74 on cell growth, cell cycle regulation, and apoptosis in HepG2 cells. It was found that GHSC-74 reduced viability of HepG2 cells in a time- and dose-dependent manner without decreasing the viability of Chang human liver cells and Swiss albino 3T3 fibroblasts. Cell cycle flow cytometry demonstrated that HepG2 cells treated with GHSC-74 (4μM) resulted in S and G2 phase arrest in a time-dependent manner, as confirmed by mitotic index analysis. G2 phase arrest was accompanied with down-regulation of CDC2 and Cyclin B1 protein. GHSC-74 efficiently induced apoptosis in HepG2 cells as evidenced by AnnexinⅤ/PI binding assay, Hoechst 33342 staining, DNA fragmentation, DAPI staining, and flow cytometric detection of sub-G1 DNA content. This apoptotic process was accompanied by dissipation of mitochondrial membrane potential, sustained elevation of intracellular [Ca2+], down-regulation of Bcl-2, a significant increase in reactive oxygen species (ROS), and translocation of apoptosis-inducing factor (AIF) from the mitochondrion to the nucleus. Nevertheless, after GHSC-74 exposure, no significant Fas and FasL up-regulation was observed in HepG2 cells by flow cytometry. In addition, treatment with antioxidant N-acetyl-L-cysteine (NAC) largely prevented apoptosis but did not abrogate GHSC-74-induced nuclear translocation of AIF, indicating that oxidative stress was involved in GHSC-74-mediated cell death. Treatment with Pan-caspase inhibitor z-VAD-fmk did not abrogate GHSC-74-induced nuclear translocation of AIF, indicating that AIF released from the mitochondrion is caspase-independent. BAPTA-AM, an intracellular Ca2+ chelator, partly suppressed cell death and prevented mitochondrial membrane potential from losing in GHSC-74-treated HepG2 cells. In contrast, EGTA, an extracellular Ca2+ chelator, exhibited a weaker effect as compared to that of BAPTA-AM. In conclusion, we have demonstrated that GHSC-74 inhibited growth of HepG2 cells by inducing S and G2 arrest of the cell cycle and by triggering apoptosis via caspase-independent AIF released from the mitochondrion, Ca2+-mediated mitochondrial pathway and ROS generation, independent of Fas/FasL interaction. We also demonstrated that GHSC-74 induced selective control of human tumors rather than normal cellular proliferation, thus GHSC-74 may be a potential candidate of anti-cancer drugs.