Tetrandrine-induced cell cycle arrest and apoptosis in Hep G2 cells
Introduction
Hepatocellular carcinoma (HCC) is one of the most common malignancies, responsible for over one million deaths annually worldwide [1], [2]. Risk factors associated with HCC include not only chronic infection with hepatitis B virus (HBV) and/or hepatitis C virus (HCV) with subsequent cirrhosis, but also metabolic and alcoholic chronic liver diseases. Most patients diagnosed with HCC have low recovery rates, and conventional and modified therapies currently available are rarely beneficial [2], [3]. Therefore, it is imperative to search for a more effective anti-hepatoma drug.
Tetrandrine (TET), a bis–benzylisoquinoline alkaloid, is isolated from the root of Stephania tetrandra (S. Moore) which has been used in traditional Chinese medicine as an antirheumatic, antiinflammation, and antihypertensive agent [4]. Previous studies have shown that TET possesses anti-inflammatory, antiplatelet aggregation, Ca2+ channel block, immunosuppressive, free radical scavenging effects [5], [6], [7], [8], and inhibits the proliferation of several human cancer cell lines, including U937, HL-60 and Hela cells [9], [10], [11]. Many recent studies have reported that TET can prevent and improve liver-related disease [12], [13], [14], [15]. It has been shown to possess a potent protective effect against the development of liver fibrosis which subsequent progression of liver cirrhosis. TET has been reported as exhibiting an antifibrotic effect by a direct inhibiting effect on hepatic stellate cells, and as blocking collagen synthesis in hepatic fibrosis induced by carbon tetrachloride [12], [13]. In addition, TET can also exhibit antifibrotic activity in rat models of silicosis, effectively block the ability of quartz to stimulate oxidant release from pulmonary phagocytes, and protects hepatocytes against CCl4-injury [14], [15].
In this study, we examined the effects of TET on cell proliferation, cell cycle distribution, and apoptosis in a well-characterized human hepatoblastoma cell line, Hep G2. Furthermore, both p53 and Fas/Fas ligand (Fas/APO-1/FasL) system participate in triggering of apoptosis and are strongly associated with the chemosensitivity of liver tumors to anticancer agents [16], [17]. Therefore, we also assayed the effects of TET on the molecular levels of p53 pathway (p53 and p21/WAF1 protein) and Fas/FasL apoptotic system (Fas/APO-1 and Fas ligand).
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Materials
Fetal calf serum (FCS), penicillin G, streptomycin, and amphotericin B were obtained from GIBCO BRL (Gaithersburg, MD). Dulbecco's modified Eagle's medium (DMEM), ribonuclease (RNase), and propidium iodide (PI) were purchased from Sigma Chemical (St. Louis, MO). XTT and p53 pan ELISA kits were obtained from Roche Diagnostics GmbH (Germany). Nucleosome ELISA, WAF1 ELISA, Fas/APO-1, and Fas Ligand ELISA kits were purchased from Calbiochem (Cambridge, MA).
Preparation of TET
TET (C38H42O6N2) was purchased from
Effect of TET on Hep G2 cell proliferation
We first tested the antiproliferative effect of TET in the liver cancer cell line, Hep G2. As shown in Fig. 1, the growth inhibitory effect of TET was observed in a dose-dependent manner. Its IC50 value was 1.42 μM. The maximum growth inhibition of TET was 81.35%, which occurred at 72 h using 3.2 μM TET.
TET-induced cell cycle arrest and apoptosis in Hep G2 cells
The effect of TET on cell cycle progression of Hep G2 was determined by flow cytometry. As shown in Fig. 2, the results indicated that, compared with the control, 1.6 μM TET increased the
Discussion
TET is a bis–benzyl isoquinoline alkaloid derived from Stephania tetrandra (S. Moore). In previous studies, TET was reported as being effective in preventing the proliferation of several cancer cell lines, including U937, HL-60, Hela and neuro 2a mouse neuroblastoma cells [9], [10], [11], [25]. These studies demonstrated that TET could inhibit cancer cell proliferation by inducing apoptosis. In neuro 2a mouse neuroblastoma cells, TET's arrest of the proliferation of cancer cells is associated
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