Archives of Toxicology

, Volume 80, Issue 2, pp 62–73 | Cite as

Berberine induces apoptosis through a mitochondria/caspases pathway in human hepatoma cells

  • J. -M. Hwang
  • H. -C. Kuo
  • T. -H. Tseng
  • J. -Y. Liu
  • C. -Y. Chu
Molecular Toxicology

Abstract

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.

Keywords

Berberine Hepatoma Apoptosis 

Introduction

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

Cell culture

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.

Apoptosis assays

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.

Statistical analysis

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.

Result

Cytotoxic effect of berberine on HepG2 cells

To determine the ability of berberine to induce cell toxicity in hepatoma cell line, in comparison with Chang liver cells, an immortalized non-tumor cell line, HepG2 (2×104 per ml) and Chang liver cells were seeded in 24-well plates and incubated in the absence or presence of increasing concentrations of berberine for 24 or 48 h. For most of the anti-cancer treatment, cytotoxicity is measured by a standard MTT assay following a brief drug exposure. As shown in Fig. 1a, the survival curve shows that berberine had a dose-dependent effect on the cytotoxicity of HepG2 cells. Within 24 h of the addition of 50 μM of berberine, viability was reduced by 52% (P<0.01). After treatment with 100 μM of berberine for 24 and 48 h, 47 and 28% of the HepG2 cells survived in culture, respectively. In contrast, treatment with the same concentrations of berberine for 24 or 48 h did not elicit marked cytotoxic effects in Chang liver cells (Fig. 1b).
Fig. 1

Effect of berberine on the cell viability of HepG2 and Chang liver cells. a HepG2 and b Chang liver cells were treated with either 0.1% DMSO (as control) or berberine (10–100 μM) for 24 or 48 h, and the proportion of surviving cells was measured by the MTT assay as described in “Materials and methods”. The experiments were performed in triplicate. Data presented as means ± SD of three independent experiments. *P<0.05, **P<0.01, when compared with control group

Berberine-induced apoptotic death

To determine the ability of berberine to inhibit cell proliferation in HepG2 cells, cells were incubated in the absence or presence of increasing concentrations of berberine for 12–36 h. As shown in Fig. 2a, we observed an inhibition of growth in berberine-treated HepG2 cell lines as compared with vehicle controls. Growth inhibition was decreased (35% and 28%) after treatment with 50 or 100 μM for 36 h. To confirm the induction of apoptosis, HepG2 cells were treated with various concentrations of berberine, and DNA was isolated and analyzed by agarose gel electrophoresis. These experiments demonstrated a typical ladder pattern of internucleosomal DNA fragmentation in the treatment of berberine (Fig. 2b).
Fig. 2

Berberine-induced apoptosis in HepG2 cells. a Growth assay of berberine-treated HepG2 cells were determined by counting cells for 0–36 h. b Agarose gel electrophoresis of DNA from berberine-treated HepG2 cells. Cells were treated with indicated concentration of berberine at 24 h and assessed for DNA fragmentation assay as described in “Materials and methods” (Fig. 3). Effect of berberine on the lipid peroxidation induced by t-BHP (1.5 mM, for 30 min) in the primary cultured rat hepatocytes. Lipid peroxidation was evaluated by malondialdehyde (MDA) formation. Each column represents the mean and the SD of three independent experiments. *P<0.05, **P<0.01, compared to the control with t-BHP treatment alone

The activation of caspases in berberine-treated cells

Caspases are activated during apoptosis by proteolytic processing at specific aspartate cleavage sites (Thornberry and Lazebnik 1998). Recent studies have identified that caspases were important mediators of apoptosis caused by various apoptotic stimuli (Desagher et al. 1999). To determine whether the induction of apoptosis by berberine is through caspase pathway, the activation of three key caspases was examined by western blot analysis. Treatment of HepG2 cells with 10, 25, 50 or 100 μM berberine did induce strong cleavage of caspases-8 (fold 0.9–0.2) at 12–24 h, as compared to the control (Fig. 3a). Activation of caspase-3 was seen as the detection of its active p17 cleavage (fold 1.3–2.7) (Fig. 3a). The activity of caspases-3 had obviously increased at 24 h after 25, 50, or 100 μM berberine treatments of HepG2. The enhanced activation of caspases is indicated by decreased procaspase-8 and increased cleavage of caspase-3. On the contrary, treatment of Chang liver cells with the same concentration of berberine (10, 25, 50 or 100 μM) did not induce cleavage of caspases-8, and −3 (Fig. 3b). The cleavage of poly ADP-ribose polymerase (PARP), a substrate for caspase-3, was also produced by the berberine treatment at 24 h. Similar inactivation of caspases and intact PARP were also observed in the treatment of Chang liver cells (data not shown).
Fig. 3

Effect of berberine on caspase 8, caspase 3, and caspase 9. a Berberine-induced HepG2 activation of caspases. Equal amounts of protein from total fraction of cells, which has been treated with berberine for 12 or 24 h, were analyzed by 12% SDS-PAGE and, subsequently, immunoblotting with antibody against caspase-8, caspase-3, caspase-9, and β-actin. b Berberine produced only a marginal effect of caspase activation in Chang liver cells. Cells that were treated with berberine for the indicated times were analyzed by 12% SDS-PAGE and subsequently with antibody against caspase 8, caspase 3 and β-actin, which served as internal control. c Effect of berberine on PARP, cytochrome c, Fas and Bcl-2 family. Equal protein of total or cytosolic cell lystes of HepG2 cells treated with berberine for the indicated times were analyzed by 10% SDS-PAGE for PARP and Fas or 15% for Bcl-2 family and subsequently with antibody against cytochrome c, Bid, Bcl-XL and β-actin, which served as internal control

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

Many apoptosis stimuli including the death-inducing ligands and chemotherapy agents can activate the mitochondrial apoptosis pathway. A decline of the mitochondrial membrane potential (Δ ψm) may be an early event in the process of cell death. The loss of the mitochondrial membrane potential and the release of cytochrome c are the markers for the activation of mitochondrial pathway, which are regulated by the pro- and anti-apoptotic proteins of the Bcl-2 family. Therefore, we determined Δ ψm by FACS analyses at various times following berberine treatment in HepG2 cells. We first examined the alteration of mitochondrial membrane potential using a fluorescent dye, JC-1, which forms monomers (FL-1) at a low membrane potential or J-aggregates (FL-2) at a higher membrane potential. As shown in Fig. 4, significant decrease in the ratio of FL-2 to FL-1 is evident as early as 24 h after exposure to indicated concentration of berberine, exhibiting a loss of Δ ψm. These results suggested that initiation of growth inhibition and apoptosis of HepG2 cells by berberine is associated with changes in mitochondrial membrane potential, while the alteration was not observed in Chang liver cells (not shown).
Fig. 4

Induction of loss of mitochondrial membrane potential (Δψm) induced by berberine. Human HepG2 cells treated with indicated concentration of berberine at 24 h were analyzed. Alteration in mitochondrial membrane potential was measured by flow cytometry using JC-1 staining as described under “Materials and methods”. One of three independent experiments is shown. Data presented as means ± SD of three independent experiments. *P<0.05, **P<0.01, when compared with control group

Modulation of caspases in berberine-induced apoptosis in HepG2 cells

To address the significance of caspases activation in berberine-induced apoptosis, we used a general and potent inhibitor of caspases, Ac-DEVD-FMK (a caspase-3 inhibitor), z-IETD-FMK (a caspase-8 inhibitor) and z-VAD-FMK (caspases inhibitor). The berberine-induced cell death is significantly determined by annexin V–FITC/PI dye. The extent of apoptosis was quantified as percentage of annexin V-positive cells. After 24 h of the addition of 25 or 50 μM of berberine, the extent of apoptosis was 35% or 62%, but 2-μM Ac-DEVD-FMK, z-IETD-FMK and z-VAD-FMK pre-treatment abolishes 50 μM of berberine-induced apoptosis (Figure. 5). The extent of apoptosis was decreased (16%, 12% and 28%), as compared to untreated-control.
Fig. 5

Moderation of berberine-induced HepG2 apoptosis. HepG2 cells were pretreated with 50 μM BSA (empty control), 2 μM DEVD-FMK, 2 μM IETD-FMK, 2 μM VAD-FMK for 1 h; then the cells were treated with 50 μM berberine for 24 h or alone. The fraction of cells undergoing apoptotic cell death was detected by annexin V–FITC. The percentages presented in each frame depict the dose-dependent increase in the apoptotic cell fraction as described under “Materials and methods”. One of three independent experiments is shown. Data presented as means ± SD of three independent experiments. *P<0.05, **P<0.01, when compared with control group

Modulation of caspases in berberine-induced loss of mitochondrial membrane potential (Δ ψm) in HepG2 cells

In order to ascertain the role of mitochondrial membrane potential in berberine-induced apoptosis through caspases pathway, we tested the effects of 2-μM Ac-DEVD-FMK, z-IETD-FMK and z-VAD-FMK on berberine-induced damage. The data showed that the addition of potent inhibitor of caspases had only a mild influence in the berberine-treated groups (Fig. 6), suggesting that the decreased mitochondrial membrane potential by berberine might lead to increased susceptibility of hepatoma cells to caspase-8 or −3 mediated apoptosis.
Fig. 6

Moderation of berberine-induced loss of mitochondrial membrane potential (Δ ψm). HepG2 cells were pre-treated with 50 μM BSA, 2 μM DEVD-FMK, 2 μM IETD-FMK, 2 μM VAD-FMK for 1 h; then the cells were treated with 50 μM berberine for 24 h or alone. Alteration in mitochondrial membrane potential was measured by flow cytometry using JC-1 staining

Modulation of Bcl-2 protein families in berberine-induced apoptosis in HepG2 cells

We were intent on determining the role of Bcl-2 family of protein in berberine-induced apoptosis. The treatment of HepG2 cells for 24 h with concentrations of berberine that are sufficient to induce apoptosis does significantly cause the cleavage of caspases-8, −3 and PARP. The activation of caspases-8, −3 and cleavage PAPR induced by berberine were suppressed by pre-treatment of potent inhibitor of caspases, Ac-DEVD-FMK, z-IETD-FMK and z-VAD-FMK (Fig. 7a). To determine whether inhibition of caspases were associated with the altered expression of Bcl-2 family proteins in berberine-induced apoptosis, we determined the expression levels of Bcl-2, Bcl-XL and Bid proteins in HepG2 cells after exposure to various concentrations of berberine. As shown in Fig. 7b, treatment with 25- or 50-μM berberine for 24 h results in decreased levels of Bcl-2, Bcl-XL and Bid, but were interfered with pre-treatment of potent inhibitor of caspases. The cleavage of Bid, a substrate of caspase-8, was recovered by the addition of z-IETD-FMK (a caspase-8 inhibitor) with berberine (Fig. 7b), suggesting that the death pathway from caspase to mitochondria was activated. The Bcl-XL, an antiapoptosis protein, was cleaved in a caspase-dependent fashion, which was blocked by z-VAD-FMK (Fig. 7b). These results indicate that the elevated caspases activity in berberine-treated HepG2 cells is correlated with downregulation of Bcl-2, Bcl-XL and Bid proteins.
Fig. 7

Involvement of caspase-8, caspase-3 in berberine-induced degradation of Bcl-2 family protein. a HepG2 cells were pretreated with 2 μM DEVD-FMK, 2 μM IETD-FMK, 2 μM VAD-FMK for 1 h; then the cells were treated with 50 μM berberine for 24 h or alone. Both detached and attached cells were harvested and analyzed by 10% SDS-PAGE and subsequently immunoblotted with antisera against caspase-8, caspase-3, PARP and β-actin. b HepG2 cells were pretreated with 2 μM DEVD-FMK, 2 μM IETD-FMK, 2 μM VAD-FMK for 1 h; then the cells were treated with 50 μM berberine for 24 h or alone. Immunoblotting of Bcl-2, Bcl-XL and Bid in HepG2 were determined

Discussion

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.

Notes

Acknowledgements

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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Copyright information

© Springer-Verlag 2005

Authors and Affiliations

  • J. -M. Hwang
    • 1
  • H. -C. Kuo
    • 2
  • T. -H. Tseng
    • 1
  • J. -Y. Liu
    • 2
  • C. -Y. Chu
    • 1
  1. 1.School of Applied Chemistry, Care and Management CollegeChung Shan Medical UniversityTaichungTaiwan
  2. 2.Institute of Biochemistry and Biotechnology, Medical CollegeChung Shan Medical UniversityTaichungTaiwan

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