The isoquinoline plant alkaloid berberine has anti-tumor effects on a variety of carcinoma cells, mainly through inhibition of cell proliferation, apoptosis induction and cell cycle arrest. However, the mechanisms underlying its role in tumor progression are unknown. In the present study, we investigated the molecular mechanisms involved in berberine-induced cell death in 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. (Cancer Sci 2011; 102: 1287–1292)
Berberine is an isoquinoline alkaloid found in a number of important medicinal plant species such as Berberis aristata and Berberis aquifolium, and has antibacterial,(1) anti-hypertensive,(2) anti-inflammatory,(3) anti-diabetic(4) and anti-hyperlipidemic effects.(5) 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.(6,7) These findings suggest that berberine is a promising candidate for clinical use in cancer chemotherapy.
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 1 million cases of HCC occur in the world each year.(8) Hepatocellular carcinoma is highly resistant to conventional systemic therapies and the prognosis for patients with advanced HCC remains poor. Although a lot of 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.(9,10) Thus, the development of novel systemic agents from natural products with low toxicity and few side-effects is being actively pursued.(11–13)
Previous studies confirm the anti-tumor effects of berberine on HCC.(14–16) 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.(17) However, the exact mechanisms underlying the anti-tumor effects of berberine are still unknown.
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,(18–20) inhibits apoptosis(21) and anoikis,(22) promotes tumor angiogenesis,(23,24) and confers resistance to some chemotherapeutic drugs.(22) These findings indicate that CD147 might be a chemotherapeutic target for treating hepatoma. Our previous study shows that CD147 might play an important role in the inhibitory regulation of autophagy and autophagic cell death in HCC cells;(25) however, whether CD147 also plays a role in mediating the anticancer effects of berberine remains unclear.
In the present study, we investigated the effects of berberine on human hepatoma cell lines HepG2 and SMMC7721 and examined the functional role of CD147 in berberine-induced cell death. To the best of our knowledge, this is the first study to explore the relationship between berberine-induced cell death and CD147 expression.
Materials and Methods
Materials. Berberine was purchased from Sigma (St. Louis, MO, USA) and dissolved in RPMI 1640 medium at a concentration of 1 m as a stock solution. The autophagy inhibitor, 3-methyladenine (3-MA), was obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA). The caspase-3 inhibitor, z-DEVD-fmk (10 mm stock solution), was obtained from BD Biosciences (Franklin Lakes, NJ, USA). The mouse anti-human CD147 and mouse anti-human β-actin monoclonal antibodies (mAb) were purchased from BioVision, Inc. (Palo Alto, CA, USA), and the horseradish peroxidase (HRP)-labeled goat anti-mouse IgG was obtained from Invitrogen (Carlsbad, CA, USA). 3-[4, 5-Dimethylthiazol-2-yl]-2, 5-diphenyltetrazolium bromide (MTT) was purchased from Sigma Chemical Co. and the pEGFP-LC3 plasmid was kindly provided by Addgene Inc. (Cambridge, MA, USA). pcDNA3.1 and pcDNA3.1-CD147 were a gift from Dr Chenggong Liao (Cell Biology Department, The Fourth Military Medical University, Xi’an, China).
Cell culture. Human hepatoma cell lines SMMC7721 and HepG2 were provided by the Institute of Cell and Biochemistry, Chinese Academy of Sciences (Shanghai, China) and grown in RPMI 1640 medium supplemented with 10% fetal bovine serum (FBS) at 37°C under a mixture of 95% room air and 5% CO2. To study berberine-induced cell death, SMMC7721 and HepG2 cells were cultured either with or without berberine at 37°C under a mixture of 95% room air and 5% CO2 for 24 h.
MTT assay. Cell viability was assessed after treatment with various concentrations of berberine for 24 h using the MTT assay as previously described.(26) In brief, SMMC7721 and HepG2 cells were plated at a density of 5 × 103 cells/well in 96-well culture plates in RPMI 1640 medium supplemented with 10% FBS. After overnight incubation, the cells were treated with various concentrations of berberine for 24 h, or with 125 μm berberine for different times (0, 24, 48, 72 or 96 h). Then, 10 μL of 5 mg/mL MTT was added to each well for an additional 4 h and the resulting formazan crystals dissolved in 100 μL DMSO. The absorbance was read at 570 nm using an automatic microplate reader (Immuno Mini NJ-2300; Inter Med, Tokyo, Japan).
Western blot analysis. To detect CD147 expression in SMMC7721 cells, samples were lysed with a radioimmunoprecipitation assay (RIPA) buffer (Beyotime Inc., NanTong, China) containing 100 μm phenylmethylsulfonyl fluoride and the protein concentrations were determined using a BCA kit (Pierce, Rockford, IL, USA). Equal amounts of total protein were separated on 12% SDS-PAGE gels and then transferred to PVDF membranes (Millipore, Bedford, MA, USA). The membranes were immunoblotted with the appropriate primary antibody diluted in tris buffered saline (TBS) buffer containing 0.05% Tween-20 and 5% non-fat dry milk at room temperature for 2 h. The following primary antibodies were used: anti-CD147 mAb (1:3000) and anti-β-actin mAb (1:2000). After extensive washing, the membranes were incubated with an HRP-labeled goat anti-mouse (1:5000) antibody.
Autophagy assay with LC3 dots. SMMC7721 cells were transfected with pEGFP-LC3 for 24 h and cultured with different concentrations of berberine. Exogenous EGFP (enhanced GFP)-fused microtubule-associated protein light chain 3 (EGFP-LC3) is a specific autophagy marker widely used in autophagy research.(27) When autophagy is stimulated, the distribution pattern of GFP-LC3 changes from a diffuse cytoplasmic pattern to a punctate pattern that labels pre-autophagosomal and autophagosomal membranes.(28) Autophagy was analyzed using an Olympus BX60 fluorescence microscope (Olympus, Tokyo, Japan). The percentage of cells with EGFP-LC3 punctate dots was determined as previously described.(25) Briefly, a minimum of 100 cells from each sample was counted in three independent experiments. The percentage of cells showing EGFP-LC3 punctate dots was calculated by dividing the number of cells showing punctate dots by the number of cells counted.
Transmission electron microscope (TEM) analysis. A TEM analysis was performed as previously described.(25) Briefly, after culture in the absence or presence of 500 μm berberine for 24 h, SMMC7721 and HepG2 cells were fixed with 3% glutaraldehyde in 0.2 m phosphate buffer (pH 7.3) for 4 h at 4°C, then post-fixed with 1% osmium tetroxide and 0.5% tannic acid for 1 h at 4°C followed by washing three times with 0.1 m phosphate buffer (pH 7.3). Next, cells were dehydrated and embedded in Epon812 (Electron Microscopy Sciences, Fort Washington, PA, USA). Finally, sections were counterstained with uranyl acetate and lead citrate, and examined using a JEM-2000EX transmission Electron Microscope (Jeol Ltd, Tokyo, Japan).
Trypan blue exclusion assay. Cell death was evaluated using a trypan blue exclusion assay as previously described.(25) In brief, SMMC7721 and HepG2 cells were cultured for 24 h in 0, 31.25, 62.5, 125, 250 or 500 μm berberine in the absence or presence of the autophagy inhibitor 3-MA (10 mm) and/or the caspase-3 inhibitor z-DEVD-fmk (100 μm). Then, both adherent and non-adherent cells were harvested, washed three times with PBS, and resuspended in 100 μL PBS. After mixing with 100 μL of 0.8% Trypan blue, the cells were counted using a hemocytometer. The number of dead cells with disrupted membranes (blue cells) per 200 cells was counted in three replicates. Cell death was expressed as the mean percentage of blue cells/total cells.
Overexpression of CD147 in SMMC7721 cells. To investigate the effects of CD147 on berberine-induced cell death, SMMC7721 cells were transfected with pcDNA3.1-CD147 or pcDNA3.1 for 24 h and then cultured with 0, 31.25, 62.5, 125, 250 or 500 μm berberine for 24 h. CD147 expression was evaluated by western blotting and cell death calculated using a Trypan blue exclusion assay.
Statistical analysis. All statistical analyses were performed using the SPSS 13.0 statistical software package (SPSS Inc., Chicago, IL, USA). Statistical significance was determined using a Student’s t-test. All statistical tests were two-sided and P <0.05 was considered statistically significant.
Berberine inhibits hepatoma cell viability. To verify the effects of berberine on hepatoma cell proliferation, SMMC7721 and HepG2 cells were treated with varying concentrations of berberine for 24 h and cell viability analyzed using a MTT assay. As shown in Figure 1(A,B), cell viability was significantly reduced by berberine in a dose-dependent manner. We also investigated whether the berberine-mediated reduction in cell viability was time dependent. Figure 1(C,D) shows that treatment of SMMC7721 and HerpG2 cells with 62.5 μm berberine inhibited cell viability in a time-dependent manner. These results show that berberine inhibits hepatoma cell growth in a dose- and time-dependent manner.
Berberine induces autophagy. To determine whether berberine induced autophagy, SMMC7721 cells were transfected with pEGFP-LC3 and exposed to different concentrations of berberine for 24 h. The formation of EGFP-LC3 punctate dots is a marker for autophagosomes, which can be observed under a fluorescence microscope.(28) As shown in Figure 2, EGFP-LC3 punctate dots were observed in SMMC7721 cells at berberine doses >31.25 μm, indicating that autophagy was induced. A diffuse EGFP-LC3 pattern was observed in the control (0 mm berberine) cells. The percentage of autophagic cells was 13.4%, 38.9%, 62.0%, 90.4% and 96.7% at 31.25, 62.5, 125, 250 and 500 μm berberine, respectively, indicating that berberine-induced autophagy was dose dependent.
Berberine induces both autophagic cell death and apoptosis. To further confirm the effect of berberine on autophagy, we evaluated the level of berberine-induced autophagy in SMMC7721 and HepG2 cells using TEM, which is currently the standard method for monitoring autophagy. Figure 3(A) shows that autophagic vacuoles (white arrows) were observed in berberine-treated SMMC7721 cells. The results suggest that a significantly higher level of autophagy occurred in SMMC7721 and HepG2 cells treated with 500 μm berberine compared with that in the control (0 μm). Moreover, we observed that cell nuclei had collapsed and disintegrated (white arrowheads) in Figure 3(A), which indicated berberine also induced apoptosis. To determine the effect of autophagy and apoptosis on cell viability, we next investigated berberine-induced cell death in SMMC7721 and HepG2 cells using a Trypan blue exclusion assay. 3-Methyladenine (3-MA), a common autophagy inhibitor, was used to inhibit autophagy and prevent autophagic cell death induced alone. It does not inhibit cell death induced by other mechanisms. z-DEVD-fmk, an apoptosis inhibitor, inhibits apoptosis only and no other types of cell death. The results showed that cell death was inhibited by both 3-MA and z-DEVD-fmk (Fig. 3B,C), which further suggests that berberine-induced cell death involves both autophagy and apoptosis. However, when 3-MA and z-DEVD-fmk were used together, cell death was not completely inhibited, indicating that berberine-induced cell death might also be induced via other mechanisms, such as necrosis.
Berberine-mediated downregulation of CD147. Previous studies have suggested that CD147 inhibits starvation-induced autophagic cell death in SMMC7721 cells.(25) Therefore, we investigated CD147 expression in SMMC7721 cells treated with different concentrations of berberine. The results showed that CD147 expression was markedly reduced in a dose-dependent manner at protein levels (Fig. 4).
Overexpression of CD147 inhibits berberine-induced cell death. To explore the role of CD147 in berberine-induced cell death, SMMC7721 cells were transfected with pcDNA3.1-CD147 or pcDNA3.1. After 24 h, the cells were cultured with 0, 31.25, 62.5, 125, 250 or 500 μm berberine for 24 h. CD147 expression was then detected by western blotting and cell death was assessed using the Trypan blue exclusion assay. As shown in Figure 5(A,B), there was no notable reduction on CD147 expression by SMMC7721 cells transfected with pcDNA3.1-CD147 (Fig. 5B) compared with those transfected with pcDNA3.1 (Fig. 5A). Furthermore, SMMC7721 cells transfected with pcDNA3.1-CD147 showed significantly lower levels of berberine-induced cell death than those transfected with pcDNA3.1 (Fig. 5C). These results suggest that overexpression of CD147 inhibits berberine-induced cell death.
Previous studies have suggested that berberine inhibits the proliferation and development of cancer cells by inhibiting Class I PI3K-Akt signaling pathways.(29,30) This is one of the most important signaling pathways involved in the negative regulation of autophagy.(31) Therefore, it is possible to speculate that berberine might induce autophagy in tumor cells. In the present study, we investigated whether berberine induced autophagic cell death in hepatoma cells. Our results showed that berberine induced both apoptosis and autophagic cell death. Next, we investigated the mechanisms underlying berberine-induced cell death in hepatoma cells. Because our previous data(25) indicated CD147 inhibits starvation-induced autophagy in SMMC7721 cells, we analyzed the expression of CD147 in SMMC7721 cells co-cultured with differing concentrations of berberine. The results showed that CD147 expression was markedly reduced by berberine. Furthermore, we demonstrated that overexpression of CD147 significantly inhibited berberine-induced cell death.
Autophagic cell death, also referred to as PCD II (program cell death II; apoptosis is referred to as PCD I), was identified relatively recently(32) and plays an important role alongside apoptosis in cancer cell death. Although autophagic cell death has been characterized in many contexts, it has not yet been extensively studied with respect to berberine-induced cancer cell death. Our present results provide compelling evidence that berberine-induced cell death in HCC cells is, at least in part, due to both autophagic and apoptotic mechanisms. This suggests that induction of autophagy by berberine might represent a novel mechanism by which berberine kills tumor cells and modulates tumor progression. These results shed further light on the anti-neoplastic activity of berberine and are in agreement with other recent reports.(16,33)
It is interesting that co-incubation of cells with both 3-MA and z-DEVD-fmk did not completely prevent berberine-induced cell death. This suggests that berberine induces not only autophagic cell death and apoptosis, but also some other types of death, such as necrosis. Although unexpected, this was in agreement with a previous report showing that berberine induces cell lysis/necrosis in murine melanoma B16 cells.(34)
We also investigated the signaling molecules that might be involved in berberine-induced HCC cell death. Our results showed that berberine significantly decreased the expression of CD147 at the protein level. Moreover, cell death was markedly reduced by overexpression of CD147, suggesting that CD147 plays a role in the mechanism of berberine-induced cell death. Our study is the first to link CD147 expression to the anti-tumor activity of berberine, and indicates that berberine-mediated downregulation of CD147 plays an important role in berberine-induced cell death. This inhibition of CD147 expression by HCC cells might further explain the anti-tumor activity of berberine.
CD147 is highly expressed on the surface of various malignant tumor cells where its main function is that of a cellular adhesion molecule that induces the secretion of matrix metalloproteinases (MMP; mainly MMP-1, MMP-2 and MMP-9), thus promoting invasion and metastasis.(20,35) It is well known that inhibition of CD147 suppresses tumor progression.(36–38) A previous study suggested that CD147 activates the Class I PI3K-Akt pathway in tumor cells,(39) which is one of the most important signaling pathways involved in the negative regulation of autophagy.(31) We recently confirmed that knockdown of the CD147 gene enhances autophagic cell death in HCC cells, probably by inhibiting the Class I PI3K-Akt signaling pathway.(25) The present study showed that berberine induces autophagic cell death in HCC cells via downregulation of CD147. Based on the findings outlined above, it is reasonable to hypothesize that berberine induces autophagic cell death, at least in part, through downregulating CD147 and the subsequent inhibition of the Akt signaling pathway, which increases the level of autophagy. A recent study confirmed that berberine suppresses the activity of Akt in HCC cells,(16) which supports this hypothesis.
Although a variety of studies have confirmed that berberine exerts its anticancer effects by inducing tumor cell apoptosis, the molecular mechanisms involved are not fully understood. Recent studies have shown that inhibition of CD147 has an anti-tumor effect through enhancing the susceptibility of cancer cells to apoptosis,(21,40) indirectly suggesting that CD147 might play an inhibitory role in cancer cell apoptosis. Based on our present findings, we can hypothesize that berberine induces apoptosis in HCC cells by downregulating CD147. Although CD147 seems to be an important molecule involved in both apoptosis and autophagic cell death induced by berberine, further experiments are needed to verify the mechanisms by which berberine downregulates expression of the CD147 gene and the exact function of CD147 in the induction of cell death.
Given its importance in cancer development and progress, CD147 might be a promising therapeutic target for cancer treatment. A series of preclinical studies confirmed that inhibition of CD147 decreases tumor cell proliferation and invasion and increases chemosensitivity to therapeutic agents.(21,36–38) However, treatments aimed at reducing CD147 expression, such as anti-CD147 monoclonal antibodies or gene silencing by RNA interference, are limited and expensive. The fact that berberine appears to downregulate CD147 directly suggests it might be a promising agent for studying the function of CD147, and for the treatment of various cancers. Berberine is a protoberberine alkaloid widely found in medicinal plants used in traditional Chinese medicine for hundreds of years. Its chloride salt has been used for several decades to treat gastroenteritis and secretory diarrhea in China. The potential use of berberine as an anti-neoplastic agent has triggered many new studies. To date, researchers have determined the definitive structure of nine metabolites of berberine in humans. These metabolites are very polar and easily excreted in urine.(41) A recent study has shown that orally administered berberine is detectable in the lung tissues of mice and inhibits in vivo tumorigenesis and growth of xenografted lung cell tumors. This indicates that viable concentrations of berberine are achievable through oral administration.(42) The relatively low toxicity of berberine at therapeutic levels also supports further development.
In conclusion, our results show for the first time that berberine induces cell death in HepG2 and SMMC7721 cells, at least in part, through a pathway involving downregulation of CD147. These results provide new insights into the function of CD147 during tumor progression and suggest a novel mechanism underlying the pharmacological effects of berberine.
This work was supported by the Science and Technology Project Foundation of Xi’an (Grant SF1028) and Scientific Research Program Funded by Shaanxi Provincial Education Department (Program No. 2010JK801,804).
The authors have no conflicts of interest to declare.