Berberine induces apoptosis through a mitochondria/caspases pathway in human hepatoma cells
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Berberine, a main component of Coptidis Rhizoma, is a plant alkaloid with a long history of medicinal use in Chinese medicine. Berberine has indicated significant antimicrobial activity against a variety of organisms including bacteria, viruses, fungi. The mechanism by which berberine initiates apoptosis remains poorly understood. In the present study, we demonstrated that berberine exhibited significant cytotoxicity in hepatoma HepG2 cells but is ineffective in Chang liver cells. Herein we investigated cytotoxicity mechanism of berberine in HepG2 cells. The results showed that HepG2 cells underwent internucleosomal DNA fragmentation after 24-h treatment with berberine (50 μM). Moreover, berberine induced the activation of caspase-8 and −3, and caused the cleavage of poly ADP-ribose polymerase (PARP) and the cytochrome c release, whereas the expression of Bid and anti-apoptosis factor Bcl-XL were decreased markedly. The loss of mitochondrial membrane potential (Δ ψm) at 24 h and activation of Fas at 12 h were also seen in the berberine-treated HepG2 cells. These findings supported the fact that the inhibitors of caspases, DEVD-FMK, IETD-FMK and VAD-FMK, prevented apoptosis and restored the expression of Bcl-XL, Bcl-2 and Bid. These results indicated that the potential of anti-hepatoma activity of berberine may be mediated through a caspases-mitochondria-dependent pathway.
KeywordsBerberine Hepatoma Apoptosis
Berberine, an alkaloid purified from Berberis species, has been extensively studied and known to exhibit multiple pharmacological activities, such as anti-protozoal, anti-hypertensive (Bova et al. 1992), anti-bacterial (Amin et al. 1969), anti-inflammation (Akhter et al. 1977), anti-cholinergic (Tsai and Ochillo 1991) and anti-arrhythmic (Wang and Zheng 1997). Moreover, an anti-HIV (Vlietinck et al. 1998) and anti-oxidative activity (Hwang et al. 2002; Yokozawa et al. 2004) has recently been reported. Berberine, the major ingredient of these herbs, has many pharmacological effects including: inhibition of DNA and protein synthesis, arrests cell cycle progress, and possesses anti-cancer effect (Kuo et al. 1995; Yang et al. 1996; Miura et al. 1997; Lin et al. 1998; Wu et al. 1999; Jantova et al. 2003; Nishida et al. 2003). Berberine was also shown to inhibit the in vitro growth of a number of human cancer cell lines. However, the molecular mechanisms underlying berberine-induced apoptosis are not yet well defined.
Members of the Bcl-2 family of proteins have been demonstrated to be associated with the mitochondrial membrane and regulate its integrity (Nomura et al. 1999). In addition, the Bcl-2 family of proteins (e.g. anti-apoptotic Bcl-2 and Bcl-XL; proapoptotic Bcl-XS and Bax) has been suggested to play a role in apoptosis (Kuwana and Newmeyer 2003; Kirkin et al. 2004; Sharpe et al. 2004). In this mitochondrial death pathway, the ratio of expression of the proapoptotic Bax protein and the antiapoptotic Bcl-2 or Bcl-XL proteins ultimately determines cell death or survival (Liu et al. 1996; Kluck et al. 1997; Lorenzo et al. 2002). Overexpression of Bcl-XL or Bcl-2 can protect some types of cells against chemotherapy agent-mediated apoptosis, suggesting that the mitochondrial pathway predominates in these types of cells. During the process of induced apoptosis, activation of the initiator caspase-8 can transmit death signals either through direct activation of the effector caspase-9 or −3, or by means of the proapoptotic Bcl-2 family member Bid, through a mitochondrial pathway (Daniel et al. 2001). In this mitochondrial death pathway, the ratio of expression of the proapoptotic Bax protein and the antiapoptotic Bcl-2 or Bcl-XL proteins ultimately determines cell death or survival (Liu et al. 1996; Kluck et al. 1997).The involvement of mitochondria in mediating apoptotic death is supported by recent studies showing a decrease in mitochondrial membrane potential (Δψm) (Bahar et al. 2000). In another study (Fulda et al. 2002), overexpression of Bcl-2 or Bcl-XL conferred protection against TRAIL in neuroblastoma, glioblastoma, breast and hepatoma cancer cell lines (Watanabe et al. 2002, 2004), but reduced Fas-induced caspase-8 cleavage, suggesting that caspase-8 was activated both upstream and downstream of the mitochondria in these cells upon treatment with TRAIL. As apoptosis induced by chemotherapy acts mainly through the mitochondrial pathway, downregulation of Bcl-2 or Bcl-XL might restore sensitivity not only to chemotherapy but also to TRAIL in some types of cancer. In particular, caspase-3 activation can be induced through a caspase-8-dependent, mitochondria-independent pathway (Korsmeyer et al. 2000), or through a caspase-9-dependent, mitochondria-dependent mechanism (Poulaki et al. 2001; Abou El Hassan et al. 2004; Wang et al. 2004).
We have recently focused on human hepatocellular carcinoma (HCC), one of the global incidence of tumor that has increased extensively and has become one of the most frequent malignant neoplasms (Kensler et al. 2004; Saffroy et al. 2004). In this study, we investigated this molecular mechanism in which berberine induces apoptosis in human HepG2 cells. We show that berberine can cause cell cytotoxicity through a mitochondria-caspases-dependent pathway. The activation of caspases lead to a fall in the contents of Bcl-2, Bcl- XL and Bid, providing a new mechanism for berberine-induced apoptosis.
Materials and methods
The HepG2 and Chang liver cell lines were originally obtained from the American Tissue Culture Collection (ATCC, USA). HepG2 and Chang liver cells were grown in Dulbecco’s minimum essential medium (Gibco) supplemented with 10% fetal calf serum (Gibco), 2 mM Glutamine, 1% non essential amino acids (NEAA) and 1% antibiotics (100 U/ml of penicillin and 100 μg/ml of streptomycin). Incubation was carried out at 37°C in a humidified atmosphere of 5% CO2 and 95% air. All experiments were performed in plastic tissue culture flasks, dish or in microplates (Nunc, Naperville, Denmark). Incubations were performed with HepG2 cells and Chang liver cells seeded on 24-well plates or 100-mm culture dishes. After plating, cells were allowed to adhere overnight and were then treated with chemical or vehicle only (control samples).
Chemical reagents and antibodies
Berberine was obtained from Sigma (St. Louis, MO, USA). Synthetic peptide inhibitors irreversibly inhibit the activity of each of the caspase-family proteases. DEVD-FMK, benzyloxy carbonyl-Asp-Glu-Val-Asp-fluoromethylketone; IETD-FMK, benzyloxy carbonyl-z-Ile-Glu-Thr-Asp-fluoromethylketone; and VAD-FMK, benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone were supplied by Chemicon. 5′,6,6′-Tetrachloro-1,1′,3,3′-tetraethyl benzimidazolo-carbocyanine iodide (JC-1), anti-β-actin were from Sigma. Anti-Bid, anti-Bcl-XL, anti-caspase 8, anti-caspase 9 and anti-poly (ADP-ribose) polymerase (PARP) antibodies, and horseradish peroxidase-linked anti-rabbit or mouse IgG were from Cell Signaling Technology, Inc. (Beverly, MA, USA). Monoclonal mouse anti-human CPP32 to detect caspase 3 and anti-Fas were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA).
Assessment of cell viability and growth
Cell viability was determined, as previously reported, by the MTT quantitative colorimetric assay, capable of detecting viable cells (Kim et al. 1993). The cells were seeded at 2×104 cells/ml density and incubated with Berberine at various concentration (0, 10, 25, 50, and 100 μM) for 24 h and 48 h. Thereafter the medium was changed and incubated with MTT (0.5 mg/ml) for 4 h. The viable cell number is directly proportional to the production of formazan which, following solubilization with isopropanol, can be measured spectrophotometrically at 563 nm. Cell growth was determined by counting cells at indicated periods of time using a Coulter counter and measured by trypan blue (0.2%) exclusion assay.
Determination of DNA fragmentation
Both detached and attached cells were harvested by scraping and centrifugation, washed in PBS (with 1 mM ZnCl2), resuspended in 0.5 ml lysis buffer (0.5% Triton X-100, 20 mM EDTA, and 5 mM Tris; pH 8.0) for 45 min. Fragmented DNA in the supernatant fraction after centrifugation at 14,000 rpm was extracted twice with phenol:chloroform:isoamyl alcohol (25:24:1, v/v/v) and once with chloroform and then precipitated with ethanol and 5 M NaCl overnight at −20°C. The DNA pellet was washed once with 70% ethanol and resuspended in Tris–EDTA buffer (pH 8.0) with 100 μg/ml RNase A incubated at 56°C for 2 h. After quantitative analysis of DNA content by spectrophotometry (260 nm), an equal amount of DNA was electrophoresed in horizontal agarose gel (1.8%) performing at 1.5 V/cm for 3 h. DNA in gel was visualized under UV light after staining with ethidium bromide (0.5 mg/ml).
Determination of mitochondrial membrane potential
The mitochondrial membrane potential was assessed by using JC-1, a lipophilic cation that can selectively enter into mitochondria (Reers et al. 1991). JC-1 was dissolved in dimethylsulfoxide to give a 1 mg/ml solution. This was further diluted to 20 μg/ml in a FACS buffer containing 5% FCS and 0.1% NaN3 in phosphate-buffered saline, and filtered using 0.45-μm filter. After the required treatments of cells (1×105), both adherent and detached cells were collected as described above and resuspended in 125 μl of the FACS buffer. The cell suspension was incubated for 20 min at room temperature with 250 μl of the filtered working solution of JC-1. Both red and green fluorescence emissions were analyzed with a flow cytometer (FACScan, Becton Dickinson, Sunnyvale, CA, USA). A minimum of 10,000 cells per sample was acquired in list mode and analyzed using Winmdi software. The decrease in mitochondrial membrane potential was determined by a decrease in the ratio of red to green fluorescence intensities.
Preparation of total cell extracts and immunoblots analysis
Cells were plated onto 15 cm2 dishes at a density of 2×105 cells/ml with or without Berberine (0, 10, 25, 50, and 100 μM, 12 and 24 h) and harvested. To prepare the whole-cell extract, cells were washed with PBS plus zinc ion (1 mM) and suspended in a lysis buffer (50 mM Tris, 5 mM EDTA, 150 mM NaCl, 1% NP 40, 0.5% deoxycholic acid, 1 mM sodium orthovanadate, 81 μg/ml aprotinine, 170 μg/ml leupeptin, 100 μg/ml PMSF; pH 7.5). After 30 min rocking at 4°C, the mixtures were centrifuged (10,000 g) for 20 min, and the supernatants were collected as the whole-cell extracts. The protein content was determined with Bio-Rad protein assay reagent using bovine serum albumin as a standard. The ECL western blotting was performed as follows. An equal gram of total cell lysate from control and Berberine-treated samples was resolved on 10–15% SDS-PAGE gels along with pre-stained protein molecular weight standard (Bio-Rad). Protein was then blotted onto NC membranes (Sartorious), membranes were reacted with primary antibodies. The secondary antibody was a peroxidase-conjugated goat anti-mouse antibody. After binding, the bands were revealed by enhanced chemiluminescence using the ECL commercial kit.
Release of cytochrome c
Cells (2×106) were harvested, washed once with ice-cold phosphate-buffered saline and gently lysed for 2 min in 80-μl ice-cold lysis buffer (250 mM sucrose, 1 mM EDTA, 20 mM Tris–HCl, pH 7.2, 1 mM dithiothreitol, 10 mM KCl, 1.5 mM MgCl2, 5 μg/ml pepstatin A, 10 μg/ml leupeptin, 2 μg/ml aprotinin). Lysates were centrifuged at 12,000 g at 4°C for 10 min to obtain the supernatants (cytosolic extracts free of mitochondria) and the pellets (fraction that contains mitochondria). The protein concentration was determined by Bio-rad protein assay kit and 25 μg of each fraction was loaded onto a 15% SDS-PAGE. Protein was then blotted onto NC membranes for detecting cytochrome c.
To quantify the percentage of cells undergoing apoptosis, we used annexin V–FITC (Biosource international, USA) as previously described. Briefly, Hep G2 cells were incubated for 24 h with or without 50 μM Berberine. Then the cells were washed twice with cold PBS and resuspended in annexin-V binding buffer (10 nM HEPES [N-2-hydroxyethylpiperazine-N′-2-ethanesulfonic acid], 140 nM NaCl, 5 nM CaCl2, pH 7.4) at a concentration of 1×106 cells/ml. After incubation, 100 μl of the solution was transferred to a 5-ml culture tube, and 5 μl annexin V–FITC and 10 μl PI (5 μg/ml, Santa Cruz, CA, USA) were added. The tube was gently vortexed and incubated for 15 min at room temperature in the dark. At the end of incubation, 1 ml of binding buffer was added, and the cells were analyzed immediately by flow cytometry. Flow cytometric analysis was performed with a FACS Caliber using the CellQuest software. Data were analyzed by CellQuest and WinMDI software.
Data were reported as mean ± standard deviation of three independent experiments and evaluated by one-way ANOVA. Significant differences were established at P<0.05.
Cytotoxic effect of berberine on HepG2 cells
Berberine-induced apoptotic death
The activation of caspases in berberine-treated cells
Effects of berberine on expression of Fas and Bcl-2 family of proteins
To further evaluate the effect of chemotherapy agents on mitochondrial apoptosis signaling pathway, we also examined whether berberine induces cell death by modulating the expression of Fas and Bcl-2 family members, which ultimately determine the cellular response to apoptotic stimuli. In the mitochondrial pathway, the death signals lead to the release of proapoptotic factors including cytochrome c, which results in the activation of caspase-9 and inactivation of IAPs, particularly XIAP, respectively (Liu et al. 1996; Kroemer and Reed 2000). The treatment of HepG2 cells for 12 or 24 h with concentrations of berberine that are sufficient to induce apoptosis does significantly alter the expression and cleavage of several key proteins related to the mitochondrial death pathway, which was examined by western blot analysis. These experiments demonstrated that treatment of berberine significantly induces release of cytochrome c from the mitochondria into the cytoplasm for 24 h (Fig. 3c). The data also indicated that the expression level of Fas protein modulates apoptosis induced by berberine in HepG2 cells. In addition, the cleavage of Bid, a substrate of caspase-8, was generated after exposure to various concentrations of berberine for 12–24 h, suggesting that the death pathway from caspase to mitochondria was activated (Fig. 3c). The expression levels of Bcl-XL, an anti-apoptosis protein, was decreased. These results indicate that the treatment of berberine leads to a shift from anti-apoptosis to pro-apoptosis by altering the function of the proteins in the Bcl-2 family, which results in the release of cytochrome c from mitochondria.
Berberine-induced reduction of mitochondrial membrane potential in hepatoma cells
Modulation of caspases in berberine-induced apoptosis in HepG2 cells
Modulation of caspases in berberine-induced loss of mitochondrial membrane potential (Δ ψm) in HepG2 cells
Modulation of Bcl-2 protein families in berberine-induced apoptosis in HepG2 cells
Several population-based studies suggested that people in Southeast Asian countries have a much lower risk of getting colon, gastrointestinal, prostate, breast cancers when compared to their Western counterparts (Dorai and Aggarwal 2004). It is very likely that their diet may influence tumorigenesis. These dietary have been recognized as chemopreventive agents and believed to be pharmacologically harmless. These chemopreventive agents have been found to suppress cancer cell proliferation, the expression of anti-apoptotic proteins, and induce apoptosis.
Berberine is one of the major components of Coptis chinesis, which is frequently utilized in proprietary Chinese herbal drugs to have wide range of pharmacological effects. In addition, berberine may possess anti-tumor promoting properties as evidenced by the inhibition of cyclooxygenase-2 transcription and N-acetyltransferase activity in colon and bladder cancer cell lines in vitro (Fukuda et al. 1999). In the current study, we demonstrated that berberine had cytotoxic effects in HepG2 cells including a typical ladder pattern of internucleosomal fragmentation, mitochondrial membrane damage, annexin V binding, and activation of caspases indicated by decreased procaspase-8 and increased cleavage of caspase-3, but had no influence on Chang liver cells.
As discussed previously, Bid, which upon cleavage by Caspase-8 and myristoylation migrates to mitochondria where it is attracted by the cardiolipin-rich contact sites between the outer and inner mitochondrial membranes (Zha et al. 2000). Bid might also inactivate anti-apoptotic Bcl-2 family members but, in addition, it seems to transduce cell death signals for their cytochrome c releasing function (Grinberg et al. 2002). The Bcl-XL, a membrane of Bcl-2 family was cleaved from 30 kDa into 16-kDa fragment in a caspase-dependent fashion, which has been shown to involve acceleration of cell death (Clem et al. 1998). The current data suggested that berberine-induced caspases activation promotes the loss of mitochondria potential from the cell in response to hitherto undefined process that was involved in berberine-induced apoptosis (Figs. 5, 6).
Fas is important in the induction of apoptosis in hepatocytes, and it plays a significant role in the pathogenesis of hepatic disease, including liver injury, hepatitis, and HCC (Galle et al. 1995). The Fas/Fas ligand (Fas-L) system plays a key part in chemotherapeutic agent that elicited apoptosis in tumors (Muller et al. 1997, 1998).Then, the failure of Fas-mediated apoptosis marked by an impairment of Fas expression or by a defect in the Fas-activated signaling pathway may be involved in hepatocarcinogenesis (Ito et al. 1998). According to our study, berberine-induced apoptosis in HepG2 cells are associated with interactions with activated Fas and caspase-8.
Apoptosis induced by different stimuli, such as death ligands, chemotherapeutic drugs, chemopreventive agents or ionizing irradiation, leads to the activation of caspases. Recent results showed that during taxol treatment in BJAB Burkitt-like lymphoma cells, both caspases-3 and −8 are part of a mitochondrial feedback amplification loop of apoptosis (von Haefen et al. 2003). These results indicate that taxol uses different apoptosis signaling pathways in BJAB Burkitt-like lymphoma cells. In our report we demonstrated that caspases-3 and −8 were activated during apoptosis induced by berberine in HepG2 cells. The berberine-induced apoptosis could be almost completely inhibited by the addition of a general caspase inhibitor, Z-VAD-FMK, a caspase-3 inhibitor, DEVD-CHO, or a caspase-8 inhibitor, IETD-CHO. Bid is cleaved downstream of the point of Bcl-2 action, catalyzed by caspase-3, upstream of caspase-8 activation, and seems to act as a potential feedback loop for the amplification of apoptosis-associated release of cytochrome c from the mitochondria (Fig. 7). The mechanism was consistent with the findings of taxol in BJAB cells (von Haefen et al. 2003).
The data demonstrated that cytotoxicity of berberine is due to the induction of apoptosis which activates pro-caspases and DNA fragmentation. Furthermore, the activation of caspases lead to a fall in the contents of Bcl-XL, Bcl-2 and Bid. These results suggest that berberine appears to possess anticancer potential in human hepatoma.
The experiments comply with the current laws of Taiwan. This work was supported by the Chung Shan Medical University Research Fund and Liver Disease Prevention & Treatment Research Foundation Grant.
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