Molecular Mechanisms Of Matrine-induced Autophagy And Apoptosis In Human Gastric Cancer Cells

AimsGastric cancer is one of the most frequent malignancies worldwide. Although adjuvant chemotherapy for gastric cancer has been made progress, the existing chemotherapeutic drugs do not have ideal curative effects and meanwhile have many undesirable side effects. Thus, it is necessary to develop new active agents with little toxicity or few side effects, particularly drugs from natural products. Matrine, one of the main alkaloid components extracted from a traditional Chinese herb, Sophora flavescens Ait, has a wide range of pharmacological effects. The aim of this study was to investigate the antitumor effect of matrine on human gastric cancer SGC-7901cells, and to further elucidate the roles of matrine-induced autophagy and apoptosis and the molecular mechanism by which matrine induces autophagy in gastric cancer cells, which would provide a theoretical basis for the clinical application of matrine in gastric cancer.MethodsSulphorhodamine B (SRB) assay was used to examine matrine’s cytotoxicity against SGC-7901gastric cancer cells. Effects of matrine on cell cycle and apoptosis were measured by flow cytometry, and cellular morphology was observed under an inverted phase contrast microscope and transmission electron microscope. Monodansylcadaverine (MDC) staining was used to detect autophagy. To test autophagy at the molecular level, the expression of microtubule-associated protein1light chain3(LC3) was detected by western blot analysis. Cell death in response to matrine was quantified by propidium iodide (PI) staining and flow cytometry, and PI positive cells were counted as dead cells. To further explore the interconnection between matrine-induced autophagy and apoptosis, we use autophagy inhibitor3-methyladenine (3-MA) or bafilomycin Al, autophagy inducer rapamycin, or apoptosis inhibitor zVAD-fmk to manipulate autophagy and apoptosis induced by matrine. In addition, we examined whether the PI3K/Akt/mTOR/p70S6K signaling pathways are involved in the regulation of matrine-induced autophagy. The expression levels of Bax and Beclin1in SGC-7901cells were monitored by real-time quantitative reverse transcription-polymerase chain reaction (RT-PCR). ResultsThe results showed that matrine significantly inhibited the proliferation of SGC-7901gastric cancer cells and induced G0/G1-phase cell cycle arrest. After treatment with matrine at the concentration of0,0.5,1.0and2.0mg/ml for24h, the apoptosis rate of SGC-7901cells was0.20±0.13%,72.92±3.41%,77.75±2.19%,83.28±2.75%, respectively, indicating matrine induced apoptosis of SGC-7901cells in a dose-dependent manner. The remarkable morphological changes were observed under an inverted phase contrast microscope. The cells treated with matrine displayed abundant cytoplasmic vacuoles with varying sizes. Furthermore, vacuolization in cytoplasm progressively became larger and denser when the concentration of matrine was increased. Electron microscopy demonstrated formation of abundant autophagic vacuoles in SGC-7901cells after matrine treatment. MDC staining showed that the fluorescent density was higher and the number of MDC-labeled particles in SGC-7901cells was greater in matrine treatment group than in control group. Western blot analysis showed that exposure of gastric cancer cells to matrine resulted in the extent of autophagy increasing in a dose-and time-dependent manner by detecting LC3-Ⅱ. This induction was due to activation of autophagic flux, as supported using the lysosome inhibitor, bafilomycin Al, which produced an accumulation of LC3-Ⅱ. PI staining demonstrated that matrine induced cell death in a dose-dependent manner and the autophagy inhibitor3-MA or bafilomycin Al enhanced lethality of matrine against gastric cancer cells. Moreover, after pretreatment with3-MA, some of the gastric cancer cells treated with matrine exhibited prototypical characteristics of apoptosis by transmission electron microscopy. The ability of3-MA to increase matrine-induced apoptosis was further confirmed by Annexin V-FITC/PI staining. Also, the combination of matrine and3-MA was more potent than matrine alone in inhibiting the proliferation of SGC-7901cells assessed by SRB assay. Furthermore, administration of the pan-caspase inhibitor zVAD-fmk or autophagy inducer rapamycin decreased the matrine-induced cell death. In addition, matrine treatment increases the phosphorylation of Akt and its downstream effectors mammalian target of rapamycin (mTOR) as well as p70ribosomal protein S6kinase (p70S6K) in a dose-dependent manner, although the levels of the total Akt and mTOR were decreased. Beclin1is involved in matrine-induced autophagy and the pro-apoptotic mechanisms of matrine may be associated with its up-regulation of Bax expression.ConclusionThese findings indicate that matrine is a potent antitumor agent for treating gastric cancer. Both autophagy and apoptosis were activated during the matrine-induced death of SGC-7901cells. Matrine-induced autophagy is an adaptive response and functions as a protective mechanism in gastric cancer cells, and blockade of autophagy increase matrine-induced apoptosis. Therefore, inhibition of autophagy may be an attractive strategy for enhancing the antitumor potential of matrine in gastric cancer. In addition, the PI3K/Akt/mTOR/p70S6K signaling pathway might not be involved in the induction of autophagy in the matrine-treated SGC-7901cells.