Introduction
Liver cancer is the fifth most common cancer worldwide and the third most common cause of cancer mortality (1,2). Hepatocellular carcinoma (HCC), which accounts for 80–90% of primary liver tumors, is characterized by a poor prognosis and high mortality (1,2). Despite therapeutic advances, the overall survival of patients with HCC has not significantly improved in the last two decades. Surgical resection, radiofrequency ablation (RFA) and percutaneous ethanol injection (PEI) are accompanied by high recurrence rates (3–5). Transarterial chemoembolization (TACE), the most commonly used non-surgical alternative, is usually reserved for palliative treatment (6), and chemotherapy and radiotherapy generally offer unsatisfactory response rates. Thus, new therapeutic strategies are urgently required for the treatment of HCC.
Vitexins are a group of complex polyphenolic antioxidants found in plants. Vitexin inhibits α-glucosidase, restrains rotavirus infection and has significant protective effects against CCl4-induced hepatotoxicity (7–9). Several vitexins have been shown to induce apoptosis of human leukemia and colon cancer cells (10,11). In previous studies, we demonstrated that a mixture of vitexin lignans, known as EVn-50, exhibits obvious in vitro and in vivo anticancer activity against hormone-dependent cancer and non-hormone dependent cancer such as breast, prostate, ovarian, gastric cancer and choriocarcinoma via a possible mechanism of promoting apoptosis of cancer cells (12). The purified vitexin compound 1 (VB1, a neolignan compound), which is the most abundant Vitexin compound in EVn-50, has potent cytotoxic effects in several cancer cell lines, including human HCC cells (13,14). The broad-ranging antitumor effects and cytotoxicity of VB1 may be mediated by alternation of Bax/Bcl-2 ratio in favor of Bax, by activation of caspases, and by suppression of the mTOR pathway (13,14). These studies strongly suggest that VB1 can be developed as a cancer preventive agent. However, to date, no studies address the effects of VB1 on the induction of apoptosis in HCC cells.
The PI3K/AKT and MEK/ERK pathways have profound effects on proliferative, apoptotic and differentiation pathways. Dysregulation of components of these cascades can contribute to resistance to other pathway inhibitors, chemotherapeutic drug resistance as well as other diseases (15). Overexpression of AKT and ERK have been reported in several types of human cancer, including HCC, and cells expressing elevated levels of AKT and ERK are less sensitive to apoptosis stimuli (16–18). Other well known risk factors for HCC such as HBV and HCV infection also seem to utilize the AKT and ERK pathways for the control of hepatocyte survival and viral replication (19). Furthermore, activation of AKT and ERK signaling pathways predicts poor prognosis in HCC (17), supporting the participation of activated PI3K/AKT and MEK/ERK axes in this disease.
FOXO3a is a member of the forkhead/winged helix box class O (FOXO) transcription factors that participates in a variety of cellular processes such as cell cycle progression, stress, detoxification, DNA damage repair, glucose metabolism and differentiation (20). During tumor development, inhibition of FOXO3a-induced transcriptional activity promotes cell transformation, tumor progression and angiogenesis (21–23). A number of kinases have been shown to suppress FOXO activity. These include phosphoinositide AKT (24) and extracellular signal-regulated kinases 1 and 2 (ERK1/2) (25). However, whether VB1 induces apoptosis in HCC cells by regulating FOXO3a transcription factor through inhibition of AKT and ERK1/2 kinases has yet to be confirmed.
In the present study, we demonstrated that the selective cytotoxicity of VB1 in HCC cells was accompanied by inhibition of AKT and ERK1/2 kinase phosphorylation, activation of FOXO3a transcription factor and apoptotic death in HepG2 cells. Furthermore, knockdown of AKT by siRNA or PD98059 enhanced VB1-induced FOXO3a transcriptional activity. These data suggest that VB1 induces apoptosis by activating FOXO3a transcription factor and that this is most likely induced by inhibition of AKT and ERK1/2 phosphorylation.
Materials and methods
Reagents
VB1 [6-hydroxy-4-(4-hydroxy-3-methoxyphenyl)-3-hydro-methyl-7-methoxy-3,4-dihydro-2-naphthaldehyde] was purified from EVn-50 (a mixture of lignan compounds obtained from Vitex negundo seed) as previously described (13). 3-(4, 5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) was purchased from Sigma Chemical Co. (St. Louis, MO, USA). Fluorouracil (5-FU) was purchased from Sigma Chemical Co. The cell apoptosis enzyme-linked immunosorbent assay (ELISA) detection kit was obtained from Roche Applied Science, Mannheim, Germany. The Apoptotic DNA Ladder Detection kit was obtained from the Bodataike Company, Beijing. The Caspase-3 Activity Detection kit, Caspase-8 Colorimetric Activity Assay kit 25, and Caspase-9 Colorimetric Activity Assay kit were purchased from Millipore, Billerica, MA, USA. Caspase inhibitors, such as zVADfmk, zDEVD-fmk, zIETD-fmk and zLEHD-fmk were purchased from R&D Systems (Minneapolis, MN, USA). Propidium iodide (PI) was purchased from Sigma Chemical Co. Antibodies against phospho-AKT, AKT, phospho-ERK1/2, ERK1/2, Bim, TRAIL, DR5, DR4, phosphor-FOXO3a, FOXO3a and β-actin were purchased from Cell Signaling Technology, Inc. (Danvers, MA, USA). Enhanced chemiluminescence (ECL) western blotting detection reagents were provided by Amersham Life Sciences Inc. (Arlington Heights, IL, USA).
Cell lines and cell culture techniques
Human HCC cell lines Hep3B, Huh-7 and HepG2 and human embryo liver L-02 cells were purchased from the China Centre for Type Culture Collection (CCTCC, Wuhan, China). The cells were maintained in Dulbecco's modified Eagle's medium (DMEM; Life Technologies, Grand Island, NY, USA) supplemented with 10% fetal bovine serum (FBS) (Invitrogen), 100 U/ml penicillin and 100 U/ml streptomycin, and cultured in a humidified atmosphere at 37°C with 5% CO2.
MTT assay
Cells were seeded in a 96-well plate at a density of 0.5×104 cells/well and maintained in serum-free medium for 24 h. This was followed by exposure to various concentrations of experimental agents that were added to each well and cultured for 24 h, prior to incubation with media containing 5 mg/ml MTT for 4 h. The cell suspension was then centrifuged and 100 μl DMSO was added to the resulting supernatant. Absorbance at the 570 nm wavelength (A570) was measured using an enzyme-labeling instrument (ELX-800 type; BioTek, Shanghai, China). Relative cell viability inhibition rate was calculated as: (average A570 of the experimental group/average A570 of the control group) × 100%.
DNA analysis
DNA content was analyzed by flow cytometry. Cancer cells with or without prior exposure to VB1 were collected by centrifugation and adjusted to 3×106 cells/ml. Pre-chilled absolute methanol was added to 0.5 ml of cell suspension and incubated overnight at 4°C. The methanol was removed by centrifugation. DNA was stained with PI staining solution (100 μg/ml PI, 0.1% Triton X, 1 mM EDTA in PBS) in the presence of an equal volume of DNase-free RNase (200 μg/ml). DNA was analyzed immediately using a Partec CyFlow ML cytometer (FACS 420; Becton-Dickinson, USA). Debris and clumps were excluded by monitoring DNA area (FL3-A) and peaks (i.e. signal height, FL3-W). Fluorescence signals from 10,000 gated cells were collected.
Histone/DNA ELISA detection of apoptosis
An ELISA kit was used to detect apoptosis in cells treated with VB1 according to the manufacturer's protocol. Briefly, cells were seeded in a 96-well plate at a density of 1×104 cells/well for 24 h. The testing agents were then added to culture medium containing 10% FBS. After 24 h, the cytoplasm of the control and experimental groups was transferred to a 96-well plate pre-coated with streptavidin that had been previously incubated with the biotinylated histone antibody and peroxidase-tagged mouse anti-human DNA for 2 h at room temperature. Absorbance was measured at 405 nm, using an EXL-800-type enzyme-linked immunosorbent apparatus (BioTek).
DNA agarose gel electrophoresis
The cells maintained in serum-free medium for 24 h were exposed to media containing various concentrations of experimental agents for 48 h. The cells were washed twice with PBS, and DNA was extracted using an apoptotic DNA ladder detection kit according to the manufacturer's instructions. The extracted DNA was maintained at 4°C overnight. The DNA (8.5 μl) was then mixed with 1.5 μl of 6X buffer solution and electrophoresed on a 20 g/l agarose gel containing ethidium bromide at 40 V. The results were observed using a DBT-08 gel image analysis system.
Analysis of caspase-3, −8 and −9 activities
The activity of caspase-3, −8 and −9 was evaluated using a Caspase-3 Activity Detection kit, Caspase-8 Colorimetric Activity Assay kit 25 and Caspase-9 Colorimetric Activity Assay kit, respectively. Briefly, cell lysates were prepared after exposure to experimental agents. The assays were performed in 96-well plates by incubating 20 μg of cell lysates in 100 μl of reaction buffer [1% NP-40, 20 mM Tris-HCl (pH 7.5), 137 mM NaCl, 10% glycerol] containing 5 μM of the caspase-3 substrate Ac-DEVD-pNA, the caspase-8 substrate Ac-IETD-pNA or the caspase-9 substrate Ac-LEHD-pNA. Lysates were incubated at 37°C for 2 h. Thereafter, absorbance at 405 nm was measured with an enzyme-labeling instrument (ELX-800 type). In the caspase inhibitor assays, cells were exposed to caspase inhibitors (i.e. 20 μM zVAD-fmk, zDEVD-fmk, zIETD-fmk or zLEHD-fmk) for 1 h prior to the addition of the agents tested.
Western blot analysis
Western blotting was performed as previously described (1). In brief, cells were lysed in RIPA buffer containing 1X protease inhibitor cocktail, and protein concentrations were determined using the Bradford assay (Bio-Rad, Philadelphia, PA, USA). Proteins were separated using 10–12.5% SDS-PAGE and transferred to membranes (Millipore, Bedford, MA, USA) in a Tris (20 mM), glycine (150 mM) and methanol (20%) buffer at 55 V for 4 h at 4°C. After blocking in 5% nonfat dry milk in TBS, the membranes were incubated with primary antibodies at 1:1,000 dilution in TBS overnight at 4°C. They were then washed 3 times with TBS Tween-20, and incubated with secondary antibodies (conjugated with horseradish peroxidase at 1:5,000 dilution in TBS) for 1 h at room temperature. The membranes were washed again 3 times in TBS Tween-20 at room temperature. Protein bands were visualized on X-ray film using an enhanced chemiluminescence detection system.
DNA transfection
The small interfering RNA (siRNA) duplexes targeting the sequence 5′-UAAUGUGCCCGUCCU UGUCUU-3′ of the human AKT1 gene, the sequence 5′-ACUCCGGGUCCAGCUCCAC-3′ of the FOXO3A gene and control siRNA oligonucleotides were purchased from Dharmacon Research, Inc. (Lafayette, CO, USA). To silence Akt or FOXO3a, HepG2 cells were transfected with double stranded siRNA of Akt or FOXO3a using Signal Silence siRNA kit from Cell Signaling Technology, Inc. (Beverly, MA, USA). Briefly, 106 cancer cells were plated in a 60-mm petri dish for 24 h in 3 ml of transfection medium containing 20 μg Lipofectamine and 100 nM siRNA for 24 h. Gene silencing in transfected cells was confirmed by western blotting.
Statistical analysis
Statistical analysis was performed using PRISM statistical analysis software (GrafPad Software, Inc., San Diego, CA, USA). Data are presented as means and standard deviations ( ± SD). Differences between groups were analyzed by one- or two-way analysis of variance (ANOVA), followed by Bonferoni's multiple comparison tests. Values of P<0.05 were considered to indicate statistically significant differences.
Results
Effects of VB1 on the proliferation of human HCC cells
Since VB1 significantly inhibited the growth of several cancer cell lines, including HCC cells (13), we first examined the effects of VB1 on cell viability in 3 HCC cell lines HepG2, Hep3B and Huh-7, using the MTT assay. Fig. 1 shows that VB1 inhibited cell viability in a concentration-dependent manner. The HepG2 cell line was the most sensitive, the Hep3B cell line was moderately sensitive and the Huh-7 cell line was the least sensitive. VB1 had little effect on the human embryo liver L-02 cell line. These data suggest that VB1 is able to inhibit HCC cell growth.
Effects of VB1 on apoptosis of HepG2 cells
VB1 has been reported to induce apoptosis in breast cancer cell lines (13). We, therefore, investigated apoptosis using flow cytometric analysis to detect increases in hypodiploid cell populations. Fig. 2A shows that VB1 increased the percentage of the sub-G1 population of HepG2 cells in a concentration-dependent manner (P<0.05). We also showed that the histone/DNA fragment of HepG2 cells increased (P<0.05) in a dose-dependent manner following exposure to VB1 (Fig. 2B). DNA fragmentation analysis by agarose gel electrophoresis showed a typical ladder pattern of inter-nucleosomal DNA fragments in HepG2 cells exposed to VB1 (5 or 10 μmol/l) for 24 h (Fig. 2C). These results suggest that VB1 inhibits HCC cell growth, in part through a mechanism involving the induction of apoptosis.
Effects of VB1 on caspase activities of HepG2 cells
Most chemopreventive agents induce apoptosis through the mitochondrial pathway. To determine the effectors involved in VB1-induced apoptotic pathways, we examined whether caspases were activated during VB1-induced apoptotic death of HepG2 cells. Fig. 3A,C and E show that exposure of HepG2 cells to VB1 increased the levels of active caspase-3, −8 and −9 in a concentration-dependent manner (P<0.05).
We next examined the effects of pan-caspase inhibitor zVAD-fmk, the caspase-3 inhibitor zDEVD-fmk, the caspase-8 inhibitor zIETD-fmk and the caspase-9 inhibitor zLEHD-fmk on VB1-induced apoptosis. The results in Fig. 3B,D and F show that zVAD-fmk abrogated apoptosis induced by VB1, and that zDEVD-fmk zIETD-fmk and zLEHD-fmk attenuated VB1-induced apoptosis. These data indicate that VB1-induced apoptosis was dependent on the activation of caspase-3, −8 and −9.
Effects of VB1 on the phosphorylated levels of AKT and ERK1/2 proteins in the HCC cell line HepG2
AKT is constitutively active in most cancer cells and promotes cell survival and apoptosis resistance (24). We, therefore, examined the effects of VB1 on AKT phosphorylation. HepG2 cells were exposed to VB1, and phosphorylation of AKT was examined by western blot analysis. VB1 significantly inhibited AKT phosphorylation in HepG2 cells, but had no effect on total expression of AKT (Fig. 4A). These data suggest that VB1 can inhibit the phosphorylation of PI3K/AKT proteins, which may play a major role in mediating its anti-apoptotic effects.
ERK1/2 kinase regulates cellular activities ranging from gene expression to mitosis, movement, metabolism and apoptosis (25). Hence, we examined the effects of VB1 on the activation of ERK1/2 kinases. Fig. 4B shows that exposure of HepG2 cells to VB1 resulted in a decrease in ERK phosphorylated protein. These results suggest that VB1 may be involved in the regulation of ERK1/2 kinase activity.
Inhibition of AKT and ERK1/2 kinases enhances VB1-induced apoptosis
We used a siRNA that specifically silences AKT to investigate the ability of AKT to induce apoptosis by VB1. Expression of siRNAs has previously been shown to silence gene expression resulting in functional inactivation of the targeted gene (26,27). Western blot analysis showed that AKT was downregulated after transfection with specific siRNA targeting AKT in HepG2 cells (Fig. 5A). The results in Fig. 5B show that inhibition of AKT by siRNA increased VB1-induced apoptosis in HepG2 cells. These results suggest that VB1-induced AKT inhibition may contribute to apoptosis of HepG2 cells.
We next examined whether VB1 induces apoptosis through inhibition of ERK1/2. HepG2 cells were exposed to 5 μM VB1 for 24 h in the presence or absence of 20 μM PD98059. The MEK1/2 inhibitor (PD98059) inhibited ERK1/2 phosphorylation and enhanced VB1-induced apoptosis in HepG2 cells (Fig. 5C and D). These data suggest that VB1-induced apoptosis may be mediated by the MEK/ERK pathway, and that inhibition of the MEK/ERK pathway enhances VB1-induced apoptosis.
Inhibition of AKT and ERK1/2 kinases enhances VB1-induced FOXO3a transcriptional activity
We next examined whether inhibition of AKT and ERK1/2 kinases regulates FOXO3a activity in the presence or absence of VB1. Knockdown of AKT by siRNA and MEK1/2 inhibitor (PD98059) inhibited the phosphorylation of FOXO3a in the presence or absence of VB1. Furthermore, knockdown of AKT by siRNA or PD98059 enhanced VB1-induced FOXO3a transcriptional activity (Fig. 6A and B). These data suggest that inhibition of AKT or ERK1/2 kinases acts synergistically with VB1 to induce FOXO3a transcriptional activity in the HCC cell line HepG2.
We next sought to examine the effects of FOXO3a transcription factor on the regulation of AKT and ERK1/2 kinases. We used a specific siRNA targeting FOXO3a in HepG2 cells. Downregulation of FOXO3a by siRNA has no effect on the expression of p-AKT, AKT, p-ERK1/2 and ERK1/2. Effects of VB1 on the phosphorylated levels of AKT and ERK1/2 proteins in the HCC cell line HepG2 were not reversed by the inhibition of FOXO3a transcriptional activity (Fig. 6C). These data suggest that the FOXO3a protein is a downstream mediator of AKT and ERK1/2 kinases.
Effects of FOXO3a transcription factor on the regulation of pro-apoptotic effects of VB1
AKT and ERK1/2 kinase have been shown to regulate the phosphorylation of FOXO3a protein (24,25). We measured the phosphorylation of FOXO3a protein using western blot analysis. As shown in Fig. 6A, VB1 inhibited the phosphorylation of FOXO3a protein. However, VB1 had no effect on the expression of total FOXO3a protein (Fig. 7A).
Western blot analysis showed that FOXO3a was downregulated after transfection with specific siRNA targeting FOXO3a in HepG2 cells (Fig. 7B). The results also indicated that inhibition of FOXO3a expression by siRNA inhibited VB1-induced apoptosis (Fig. 7C). These data suggest that VB1-induced apoptosis may be mediated by regulation of FOXO3a, and that inhibition of FOXO3a inhibits VB1-induced apoptosis.
Effects of VB1 on the expression of FOXO3a downstream apoptosis-associated target genes
It has been reported that activation of FOXO3a results in upregulation of the apoptosis-associated target gene products Bim, TRAIL, DR4 and DR5 proteins (20). We, therefore, examined the effect of VB1 on the expression of Bim, TRAIL, DR4 and DR5 proteins. These genes are direct targets of FOXO3a transcription factor. The results indicated that VB1 induced the expression of Bim, TRAIL, DR4 and DR5 proteins (Fig. 8A–D). These data further suggest that VB1-induced apoptosis in HepG2 cells is mediated through mechanisms that involve the activation of FOXO3a transcription factor.
Discussion
In the present study, we demonstrated that VB1-induced apoptosis in HCC cancer cells appears to be mediated through inhibition of AKT and ERK1/2 kinases and activation of FOXO3a transcription factor. Our results showed that inhibition of AKT and ERK1/2 kinases increased VB1-induced apoptosis, and that inhibition of FOXO3a transcription factor by siRNA blocked VB1-induced apoptosis. We also showed that VB1 upregulated Bim, TRAIL, DR4 and DR5. These findings indicate that VB1 induces apoptosis by inhibiting AKT and ERK1/2 kinases which results in activation of FOXO3a transcription factor in HCC cells. Apoptotic cells share a number of common characteristics. Caspase-3 activation, an enhanced sub-G1 cell population, histone/DNA fragmentation and DNA ladders are regarded as specific indicators of apoptosis. In the present study, we demonstrated activation of caspase-3, an increase in the sub-G1 population, histone/DNA fragments and the presence of a DNA ladder in VB1-treated HepG2 cells, thus demonstrating that VB1 induces HCC cell apoptosis. Overall, these properties of VB1 strongly suggest that it may be used as a cancer chemopreventive agent.
Caspase activation is known to play an important role in apoptosis triggered by various pro-apoptotic signals (28,29). It is generally recognized that there are two major apoptotic pathways: one involves the transduction of death signals through death receptors, and the other involves mitochondrial signaling (28,30). Both pathways require ordered activation of a set of caspases, which cleave cellular substrates resulting in the morphological and biochemical changes that characterize apoptosis. Activation of caspase-8 and −9, have respectively been shown to play a central role in mediating apoptosis signaled by death receptors and by mitochondria. In the present study, we demonstrated that VB1 was able to activate caspase-3 −8 and −9 activity. The presence of the caspase inhibitor attenuated apoptosis induced by VB1, indicating that VB1-induced apoptosis was essentially dependent on the activation of caspase-3, −8 and −9.
Forkhead O transcription factors (FOXO) factors have been shown to be dysregulated in a variety of tumor types, including HCC, owing to the constitutive activation of AKT and ERK1/2 kinases (31,32). FOXOs are regulated by synthesis, phosphorylation, acetylation and ubiquitination at three different levels involving transcriptional activity, subcellular localization and stability (33). AKT and ERK kinases are thought to phosphorylate FOXO3a at different sites in response to growth factor and insulin stimulation. Phosphorylation of FOXO3a by these oncogenic kinases results in its translocation from the nucleus to cytoplasm where it is subsequently degraded (34,35). Previous studies have shown that FOXO3a is at least one of the missing links connecting the two pathways. Inhibition of the PI3K/Akt/mTOR and MEK/ERK pathways has been shown to result in loss of phosphorylation at Akt and ERK phosphorylation sites, accompanied by nuclear accumulation and increased transcriptional activity of FOXO3a (35). In the present study, we demonstrated that inhibition of AKT and ERK1/2 kinases acts synergistically to regulate apoptotic effects of VB1 through activation of FOXO3a transcription factor. VB1 inhibited the accumulation of phosphorylated FOXO3a protein.
FOXO factors are also critical for the regulation of cell cycle arrest, cell death and DNA damage repair. Consequently, inactivation of FOXO proteins has been shown to be associated with tumorigenesis in breast and prostate cancer, glioblastoma, leukemia and hepatocellular carcinoma (20,36). The FOXO subfamily contains 4 members (FOXO1, FOXO3, FOXO4 and FOXO6), which activate or repress multiple genes such as Bim, TRAIL, DR4, DR5 and survivin that are involved in apoptosis (37–41). Our findings demonstrate that VB1 regulates the expression of FOXO3a apoptosis-associated target genes. We also showed that inhibition of FOXO3a by siRNA blocked the apoptotic effects of VB1. These results suggest that FOXO3a transcription factor mediated the anti-proliferative and pro-apoptotic effects of VB1.
Previous studies indicated that the effects of anticancer agents may be regulated through activation of FOXO transcription factors (41–43). In addition to their effects on cell cycle, apoptosis, differentiation and reactive oxygen species, FOXO proteins have also been implicated in the negative regulation of angiogenesis. For example, resveratrol and ECGG inhibit angiogenesis through activation of FOXO (44,45). Collectively, these findings demonstrate that dephosphorylation and activation of FOXO by inhibition of AKT and ERK kinases may have significant implications for the treatment and prevention of, among others, diabetic complications, cardiovascular diseases and malignant neoplastic disease.
In summary, our results suggest that VB1 induces apoptosis through regulation of FOXO3a transcription factor in human HCC cells. Pharmacological and genetic inhibition of AKT and ERK1/2 kinases may act synergistically with VB1 to induce FOXO3a transcriptional activity which are mediated by dephosphorylation. Although a variety of clinical and in vivo studies are required to explore the therapeutic potential of VB1, our present study, together with our previous studies, indicates that VB1 may be a potential agent for further development in the prevention and treatment of HCC as well as other types of cancer.