Abstract
OBJECTIVE To study the anti-tumor effect of arsenic trioxide on the HepG2 human hepatocellular carcinoma cell line, and to explore its mechanism of action.
METHODS The MTT assay was used to determine the inhibitory effect of As2O3 on HepG2 cells at various As2O3 concentrations. The expression of p-JNK, caspase-3 and PARP was detected by Western blots.
RESULTS As2O3 markedly inhibited the growth of the HepG2 cells and induced apoptosis. The results of Western blot analysis showed that the As2O3-induced apoptosis was accompanied by caspase-3 and PARP activation. p-JNK was detected at 10 min following As2O3 treatment, and preceded to peak at 20 min, and decreased by 30 min. The total protein content did not obviously change. The activation of JNK occurred prior to cell apoptosis. SP600125, a JNK inhibitor, suppressed the As2O3-induced activation of caspase-3 and PARP cleavage.
CONCLUSION As2O3 inhibits the proliferation of human HepG2 hepatocellular carcinoma cells by inducing apoptosis in vitro. As2O3-induced apoptosis is accessed through the caspase-3 pathway. The JNK signal-transduction pathway and caspase-3 are involved upstream in the As2O3-induced HepG2 apoptotic response.
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Introduction
Arsentic trioxide (As2O3) is the major active component of the traditional Chinese medicine, arsenicum sublimatum. Chinese researchers have achieved outstanding clinical results in treating acute promyelocytic leukemia (ALP) using As2O3[1,2]. Recent studies have shown that As2O3 has a significant inhibitory effect when used to treat solid tumors such as esophageal carcinoma, gastric cancer, colon carcinoma and especially hepatoma[3,4]. Several reports have indicated that As2O3 can induce hepatic cell apoptosis[5,6]. However further research is needed to elucidate the mechanism of action of As2O3, especially related to the signal-transduction pathway. In our study we used the HepG2 human hepatic cell line in an in vitro model to examine the effect of As2O3 on protein expression of c-Jun amino-terminal kinase (JNK), caspase-3 and poly (ADP-ribose) polymerase (PARP) in relation to possible mechanisms of As2O3-induced apoptosis.
Materials and Methods
Reagents
RPMI 1640 was purchased from the Huamei Biotechnology Co. BSA and MTT were obtained from the Gibco Co., and dimethyl sulfoxide (DMSO) was purchased from Sigma. Rabbit anti-phospho-SAPK/JNK antibody, rabbit anti-PARP polyclonal antibody, rabbit anti-cleaved caspase-3 antibody and SP600125 were acquired from the Jingmei Biotechnology Co., and injectable As2O3 was purchased from the Yida Medicine Co., Harbin, China.
Cell line and cell culture
The HepG2 human hepatoma cell line was provided by the First Affiliated Hospital of CMU (Shenyang, China). The cells were cultured with RPMI 1640 medium supplemented with 100 ml/L calf serum, 1×105 U/L penicillin and streptomycin at 37°C in a humidified atmosphere containing 5% CO2 in air.
MTT assay
The HepG2 cells were seeded in 96-well plates at a density of 1×104/ml and incubated for 0~72 h with different concentrations of As2O3, followed by incubation with 5 mg/mL of MTT for 4 h. Then the su-pernatant was removed following centrifugation, and 100 μl of DMSO was added. Absorbance at 570 nm (A570) was measured with an enzyme-labeling instrument. The relative cell-proliferative inhibition rate (IR)= (1-average A570 of the experimental group/average A570 of the control group)×100%.
Western blot analysis
As previously described[7], cells were treated by low-serum starvation, after which they were collected, washed 3 times with PBS, lysed in cell lysate and then centrifuged at 12,000 r/min for 20 min at 4°C. Following determination of the protein in the supernatant by the Bradford method, protein samples were electrophoresed on 100 g/L or 60 g/L SDS-PAGE at 100 mA for 3 h, and finally transferred onto PVDF membranes. The PVDF membranes were treated with BSA (10 g/L), followed by incubation with the first antibody (1:1000 dilution) for 2 h. The corresponding secondary antibody (1:3,000 dilution) was added and incubated at room temperature for 1 h. After washing the membrane, the results were observed by ECL chemiluminescence with β-actin as an internal reference.
JNK-signal transduction was blocked by pre-incubating the HepG2 cells with 20 μmol/L SP600125 for 1h, then As2O3 was added. Western blots were used to detect the expression of all of the proteins as described above.
Statistical analysis
Analysis of the data was performed with a SPSS 12.0 software package. The differences among the groups were compared by one way ANOVA and Student’s t test. The level of significance was set at P≤0.05.
Results
Inhibition of HepG2 proliferation by As2O3
The results from the MTT assay showed that various levels of As2O3 markedly inhibited HepG2 cellular proliferation (Fig.1A), in a concentration and time-dependent manner. The inhibitory ratio showed a linear correlation with the incubation time (P<0.01). With 10 μmol/L of As2O3, the inhibitory ratios at 24, 48 and 72 h were 29.6, 71.2 and 81.4%, respectively. This concentration of As2O3 caused the greatest inhibition of HepG2 cellular proliferation (Fig.1B). Therefore, we used 10 μmol/L of As2O3 in subsequent experiments.
As2O3-induced activation of caspase-3
Western blot results showed that the caspase-3 pre-cursor and its substrate PARP were activated after 24 h of treatment with As2O3. A cleaved caspase-3 band (17kDa) and cleaved PARP (85kDa) band were detected at 24 h. The cleavage increased with the time of incubation (Fig.2).
As2O3 activation of JNK
Western blots showed that JNK was activated by 10 min of As2O3 treatment, and preceded to peak at 20 min followed by a decrease at 30 min during induced apoptosis. The total protein content did not obviously change (Fig.3). The activation of JNK occurred prior to cell apoptosis.
The influence of SP600125 on JNK
The result of Western blots showed that pre-incubating HepG2 cells for 1 h with 20 μmol/L SP600125 followed by addition of As2O3 for 48 h, inhibited the expression of p-JNK. Meanwhile the activation of caspase-3 and PARP were also markedly inhibited, suggesting that the JNK-signal pathway participated in the As2O3-induced HepG2 apoptosis (Fig.4A). The results of the MTT assay showed that pre-incubation with SP600125 weakened the effect of As2O3-induced HepG2 apoptosis (Fig.4B).
Discussion
The first anti-tumor effects of As2O3 were discovered in the treatment of APL. Since then, studies have focused on the treatment of solid tumors, especially inhibition of hepatoma cells[8], and it has been shown that As2O3 can inhibit hepatoma cellular proliferation[9]. We used the MTT assay to examine the influence of different concentrations of As2O3 on the HepG2 hepatoma cells. The results showed that the inhibition by As2O3 was both time and concentration dependent, displaying an in vitro anti-tumor effect[10].
Caspase-3 is an important member of the ICE/CED-3 family, and plays a significant role in the regulation of apoptosis. It exists as a cytoplasmic non-active precursor, and when activated, promotes apoptosis along with other members of the ICE family[11,12]. Since PARP is the substrate for caspase-3, it is known as a “death substrate” for caspase-3 as its activation leads to apoptosis and cell death[13]. Therefore, activation of caspase-3 is a useful marker for initiation of apoptosis. Our result showed that addition of As2O3 to the cultured HepG2 cells resulted in activation of caspase-3 and PARP at, and after 24 h. Their activation increased with time to at least 72 h resulting in cellular apoptotic inhibition of proliferation[14]. Caspase-3, as the primary apoptotic factor is regulated by a variety of upstream signal-transduction pathways. We found that JNK was detected at 10 min following As2O3 addition, preceded to peak at 20 min and decreased from 30 min. The total protein expression did not obviously change. Thus, As2O3 activated the JNK protein[15] prior to cell apoptosis. By pre-incubating the HepG2 cells with SP600125, a specific JNK antagonist, the activation of caspase-3 and PARP was markedly suppressed, suggesting that As2O3 may activate caspase-3 to start apoptosis and proliferative inhibition through a JNK signal-transduction pathway. However, even though SP600125 can completely block the JNK pathway, it can not completely block activation of caspase-3 and the inhibitory effect of As2O3 on HepG2 cell proliferation. There may be some other pathways involved in As2O3-induced HepG2 apoptosis and inhibition of proliferation.
In summary, our study showed that As2O3 inhibited the proliferation of the HepG2 hepatoma cells by inducing apoptosis in vitro via activation of caspase-3 through a JNK signal-transduction pathway.
Footnotes
This work was supported by a grant from the National Natural Science Foundation of China (No.30572114).
- Received October 28, 2007.
- Accepted December 29, 2007.
- Copyright © 2008 by Tianjin Medical University Cancer Institute & Hospital and Springer