Study On The Autophagy And Apoptosis Induced By Berberine In Human Hepatoma Cells With The Molecular Mechanism Research

Hepatocellular carcinoma (HCC) is the sixth most common malignancy worldwide and the third leading cause of death from cancer because of its very poor prognosis. More than one million cases of HCC occur in the world each year. HCC is highly resistant to conventional systemic therapies and the prognosis for patients with advanced HCC remains poor. Although much progress has been made in terms of chemotherapy, which provides significant survival benefits for patients with HCC, it is associated with significant side effects, highlighting the need for therapeutic strategies that target tumor cells without compromising normal tissue function. Thus, the development of novel systemic agents from natural products with low toxicity and few side effects is being actively pursued.Berberine is an isoquinoline alkaloid found in a number of important medicinal plant species such as Berberis aristata and Berberis aquifolium, and has antibacterial, anti-hypertensive, anti-inflammatory, anti-diabetic, and anti-hyperlipidemic effects. Recently, researchers have become interested in the anti-neoplastic activities of berberine and have demonstrated its anticancer effects against a variety of human cancer cells both in vitro and in vivo through suppression of tumor cell proliferation, induction of tumor cell apoptosis, and inhibition of both tumor invasion and metastasis. These findings suggest that berberine is a promising candidate for clinical use in cancer chemotherapy.CD147, a glycosylated immunoglobulin super family transmembrane protein, is highly expressed by HCC cells. Several in vitro studies suggest that CD147 promotes tumor invasion and metastasis, inhibits apoptosis and anoikis, promotes tumor angiogenesis, and confers resistance to some chemotherapeutic drugs. These findings indicate that CD147 may be a chemotherapeutic target for treating hepatoma. Our previous study shows that CD147 may play an important role in the inhibitory regulation of autophagy and autophagic cell death in HCC cells; however, whether CD147 also plays a role in mediating the anti-cancer effects of berberine remains unclear.Previous studies confirm the anti-tumor effects of berberine on HCC. Berberine acts by inhibiting proliferation and inducing apoptosis in HCC cells. It can also inhibit the migration of HCC cells by downregulating the Rho/ROCK signaling pathway. However, the exact mechanisms underlying the anti-tumor effects of berberine are still unknown.In this study, we investigated the molecular mechanisms involved in berberine-induced cell death in the human hepatoma carcinoma cell (HCC) lines, HepG2 and SMMC7721. Our results showed that berberine inhibited tumor cell viability in a dose- and time-dependent manner, and induced cell death via apoptosis and autophagy. Moreover, berberine treatment significantly inhibited CD147 expression by HCC cells in a dose-dependent manner. Overexpression of CD147 protein markedly reduced berberine-induced cell death. Our data provide the first experimental evidence that berberine induces cell death in HCC cells via downregulation of CD147 and suggest a new mechanism to explain its anti-tumor effects.Three sections for this research as follow:Section 1 Berberine reduced cell viability in hepatoma cell lines both in dose-effect and time dependent effectTo verify the effect of berberine on viabilities in SMMC7721 and HepG2 cells, at first, we treated cells with 0, 31.25, 62.5, 125, 250 or 500μM berberine for 24 h respectively. The MTT assay was carried out. A significant decrease in cell viability was noted at 31.25μM (P<0.05), with a further decrease at 62.5, 125, 250 and 500μM of berberine in both SMMC7721 and HepG2 cells, which indicated dose-effect of berberine on HCC cells. On the basis of this result, we further assayed whether berberine reduced cell viability in time-dependent. The dose of 62.5μM berberine was selected for subsequent experiment. The result showed that 62.5μM berberine inhibited cell viability in time-dependent in both SMMC7721 and HerpG2 cells line.Section 2 Study on the cell autophagy and apoptosis induced by berberineSMMC7721 cells were transfected with pEGFP-LC3 for 24 hours transfection and were subjected to culture with different concentration berberine (0, 31, 62, 125, 250, or 500μM). Then autophagy was analyzed by using an Olympus BX60 fluorescence microscope (Olympus, Tokyo, Japan). The percentage of cells with LC3 punctate dots was determined as previously described29. Briefly, a minimum of 100 cells was counted for each sample in three independent experiments. The percentage of cells with LC3 punctate dots was calculated by dividing the number of cells with punctate dots by total cells counted. To further confirm the inducing effect of berberine on autophagy, we evaluated the level of berberine-induced autophagy in SMMC7721 and HepG2 cells using transmission Electron Microscope . Our findings indicated that there was a significantly higher autophagy level in both SMMC7721 and HepG2 cells treated with 500μM berberine than control (0μM). Moreover, we also observed that berberine notably induced apoptosis . The results showed that berberine not only induced autophagy but also apoptosis in SMMC7721 and HepG2 cells. We subsequently investigated the berberine-induced cell death in SMMC7721 and HepG2 cells by using trypan blue exclusion assay. The 3-methyladenine (3-MA) is a common autophagy inhibitor. The z-DEVD-fmk is an apoptosis inhibitor. In our study, 3-MA was used to inhibit autophagy, and thus decrease the autophagic cell death, but not other cell deaths; z-DEVD-fmk was used to inhibit apoptosis, but not other cell deaths. Our data shown in Fig 3B and Fig3C indicated both 3-MA and z-DEVD-fmk inhibited cell death respectively, which indicated berberine-induced cell death included both autophagic cell death and apoptosis.Section 3 The molecular mechanism on cell autophagy and apoptosis induced by berberinePreviously study indicated that CD147 inhibited starvation-induced autophagic cell death in SMMC7721 cells. Our results indicated that berberine significantly decreased the expression of CD147 at protein level, and the expression of CD147 was negatively associated with levels of berberine-induced tumor cell death. Moreover, we found overexpression of CD147 protein markedly inversed berberine-induced cell death, suggesting that CD147 may be involved in the cell death induced by berberine. Our study for the first time linked CD147 to the anti-tumor activities of berberine and indicated that CD147 down-regulation by berberine played an important role in berberine-induced cell death.This research will provide new potential method for cancer treatment. Meanwhile, it will also be an ideal example for using pharmacological methods to study Chinese traditional medicine.