Antitumor Effect Of Matrine In Human Hepatoma G2 Cells: Induction Of Apoptosis And Autophagy

Aims Matrine, one of the main alkaloid components extracted from the sophora root, was known to have a wide range of pharmacological effects. This study was performed to investigate matrine's antitumor effect in human hepatoma G2 (Hep G2) cells, and further clarify the molecular mechanism involved in the antineoplastic activities.Methods MTT assay was used to examine cell viability of Hep G2 cells. The effect of matrine on cell cycle was measured by flow cytometry and Annexin-V-FITC/PI double staining assay was used to detect cellular apoptosis. Cellular morphological changes were observed under inverted phase contrast microscope and Transmission Electron Microscopy was employed to further examine ultrastructural structure of the cells treated with matrine. Monodansylcadaverine (MDC) staining was used to detect autophagy; and simultaneously, we evaluated whether autophagy was blocked by 3-methyladenine (3-MA, an autophagy inhibitor). The expression levels of Bax and Beclin 1 in Hep G2 cells were monitored by real-time quantitative reverse transcription PCR.Results The results showed that matrine significantly inhibited the proliferation of Hep G2 cells in dose-and time-dependent manner and induced G1-phase cell cycle arrest. Matrine induced apoptosis of Hep G2 cells in a dose-dependent manner. The total apoptosis rate was 0.14%in untreated Hep G2 cells, in contrast, the percentages of apoptosis sharply increased to 28.91,34.36 and 38.80%, respectively, when Hep G2 cells were treated with matrine at concentrations of 0.5,1.0,2.0 mg/ml. The remarkable morphological changes were observed by inverted phase contrast microscope. It was visible that there were abundant cytoplasmic vacuoles of varying sizes in Hep G2 cells treated with matrine. Furthermore, with the increase of matrine's concentration, vacuolization in cytoplasm progressively became larger and denser. What is more important, electron microscopy further demonstrated formation of abundant autophagic vacuoles in Hep G2 cells followed matrine treatment. When the specific autophagic inhibitor 3-MA was applied, the number of autophagic vacuoles greatly decreased. MDC staining presented that matrine-treated group displayed higher fluorescent density and more MDC-labeled particles in Hep G2 cells compared with the untreated group. With the addition of 3-MA before matrine treatment, fewer autophagic vacuoles were evident in the 3-MA and matrine treatment group. The real-time quantitative RT-PCR revealed that the expression of Bax gene, an apoptosis related molecule, and Beclin 1 gene which plays a key role in autophagy were at high levels in matrine-treated group.Conclusion These findings suggested that matrine has potent antitumor activities in Hep G2 cells. Matrine significantly inhibited the proliferation of Hep G2 cells and induced G1-phase cell cycle arrest. Both autophagy and apoptosis were activated during the matrine-induced death of hepatoma G2 cells. Beclin 1 was involved in matrine-induced autophagy and the pro-apoptotic mechanisms of matrine might be related to its upregulation on Bax expression. Therefore, matrine may be useful as a novel effective reagent in treating hepatocellular carcinoma.