hormone-dependent cancer and non-hormone dependent cancer such as breast, prostate, ovarian, gastric cancer and choriocarcinoma via a possible mechanism of promoting apoptosis of cancer cells

We previously reported that purified vitexin compound 1 (VB1, a neolignan from the seed of Chinese herb Vitex negundo) exhibited antitumor activity in cancer cell lines and xenograft models. In the present study, we examined the molecular mechanisms by which activation of the FOXO3a transcription factor mediated VB1-induced apoptosis in hepatocellular carcinoma (HCC) cells. The effects of VB1 on the proliferation of HCC cell lines HepG2, Hep3B, Huh-7 and human embryo liver L-02 cells were investigated using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Apoptotic death in HepG2 cells was examined using an enzyme-linked immunosorbent assay (ELISA) detection kit, flow cytometry after propidium iodide (PI) staining, and by DNA agarose gel electrophoresis. Caspase activity was measured using ELISA. The AKT/FOXO3a and ERK/ FOXO3a pathways were analyzed using western blotting. VB1 inhibited human HCC cell proliferation in a concentrationdependent manner and increased the percentage of sub-G1 population HepG2 cells. Histone/DNA fragmentation and active caspase-3, -8 and -9 levels increased in a concentrationdependent manner and a DNA ladder was formed. The phosphorylation of AKT and ERK1/2 were inhibited and FOXO3a transcription factor was activated, resulting in apoptotic death. Knockdown of AKT1 by small interfering RNA (siRNA) and the MEK1/2 inhibitor, PD98059, enhanced VB1-induced apoptosis and FOXO3a transcriptional activity. Suppression of FOXO3a expression by siRNA inhibited VB1-induced apoptosis. VB1 induced expression of Bim, TRAIL, DR4 and DR5. Activation of the FOXO3a transcription factor appears to mediate pro-apoptotic effects of VB1 by inhibiting the AKT and ERK pathways. Introduction Liver cancer is the fifth most common cancer worldwide and the third most common cause of cancer mortality (1,2). Hepatocellular carcinoma (HCC), which accounts for 80-90% of primary liver tumors, is characterized by a poor prognosis and high mortality (1,2). Despite therapeutic advances, the overall survival of patients with HCC has not significantly improved in the last two decades. Surgical resection, radiofrequency ablation (RFA) and percutaneous ethanol injection (PEI) are accompanied by high recurrence rates (3-5). Transarterial chemoembolization (TACE), the most commonly used non-surgical alternative, is usually reserved for palliative treatment (6), and chemotherapy and radiotherapy generally offer unsatisfactory response rates. Thus, new therapeutic strategies are urgently required for the treatment of HCC. Vitexins are a group of complex polyphenolic antioxidants found in plants. Vitexin inhibits α-glucosidase, restrains rotavirus infection and has significant protective effects against CCl4-induced hepatotoxicity (7-9). Several vitexins have been shown to induce apoptosis of human leukemia and colon cancer cells (10,11). In previous studies, we demonstrated that a mixture of vitexin lignans, known as EVn-50, exhibits obvious in vitro and in vivo anticancer activity against hormone-dependent cancer and non-hormone dependent cancer such as breast, prostate, ovarian, gastric cancer and choriocarcinoma via a possible mechanism of promoting apoptosis of cancer cells (12). The purified vitexin compound 1 (VB1, a neolignan compound), which is the most abundant Vitexin compound in EVn-50, has potent cytotoxic effects in several cancer cell lines, including human HCC cells (13,14). The broad-ranging antitumor effects and cytotoxicity of VB1 may be mediated by alternation of Bax/Bcl-2 ratio in favor of Bax, by activation of caspases, and by suppression of the mTOR pathway (13,14). These studies strongly suggest that VB1 can be developed as a cancer preventive agent. However, to date, no studies address the effects of VB1 on the induction of apoptosis in HCC cells. The PI3K/AKT and MEK/ERK pathways have profound effects on proliferative, apoptotic and differentiation pathways. Dysregulation of components of these cascades can contribute to resistance to other pathway inhibitors, chemotherapeutic Purified vitexin compound 1 induces apoptosis through activation of FOXO3a in hepatocellular carcinoma JIAN-GANG WANG1, XING-XING ZHENG1, GUANG-YAO ZENG2, YING-JUN ZHOU2 and HONG YUAN1,3 1Department of Cardiology, The Third Xiangya Hospital of Central South University; 2School of Pharmaceutical Science, Central South University; 3Center of Clinical Pharmacology, The Third Xiangya Hospital, Central South University, Changsha, Hunan 410013, P.R. China Received September 13, 2013; Accepted October 29, 2013 DOI: 10.3892/or.2013.2855 Correspondence to: Dr Hong Yuan, Department of Cardiology, The Third Xiangya Hospital of Central South University, Changsha, Hunan 410013, P.R. China E-mail: [email protected]