Contributed equally
Gallic acid (3,4,5-trihydroxybenzoic acid; GA) is a naturally occurring plant polyphenol, isolated from water caltrop, which has been reported to exert anticancer effects. The present study investigated the antiproliferative effects of GA on the HepG2 and SMMC-7721 human hepatocellular carcinoma (HCC) cell lines using MTT and colony formation assays. In particular, the underlying mechanism of GA-induced apoptosis in SMMC-7721 cells was studied
Hepatocellular carcinoma (HCC) is a primary malignancy of the liver, which is associated with high morbidity and mortality rates (
The screening of safe and effective anti-tumor compounds from Traditional Chinese Medicine is currently a hotspot of research, and a number of effective components isolated from plants have been revealed to possess significant cytotoxicity against certain cancer cells (
In the present study, the anti-proliferative effects of GA on HepG2 and SMMC-7721 human HCC cell lines were investigated.
GA (purity, 99.5%; melting point, 251°C), MTT, dimethyl sulfoxide (DMSO), rhodamine (RH)-123 and 2,7-dichlorfluorescein-diacetate were obtained from Sigma-Aldrich (St. Louis, MO, USA). Dulbecco's modified Eagle medium (DMEM), RPMI-1640 and fetal bovine serum (FBS) were purchased from Gibco Life Technologies (Carlsbad, CA, USA). Penicillin and streptomycin sulfate were purchased from GE Healthcare Life Sciences (Logan, UT, USA). Mouse anti-B-cell lymphoma (Bcl)-2, apoptosis regulator Bcl-2-like protein 4 (Bax) and cytochrome
The HepG2 and SMMC-7721 human HCC cell lines were obtained from the Cell Bank of the Chinese Academy of Science (Beijing, China). Cells were cultured in DMEM medium containing 10% FBS, 100 U/ml penicillin and 100 µg/ml streptomycin sulfate. The HL-7702 human normal hepatocyte cell line was obtained from the Chinese Academy of Science Type Culture Collection (Shanghai, China) and incubated in RPMI-1640 medium supplemented with 10% FBS. All cells were incubated at 37°C in a humidified incubator (Thermo Fisher Scientific, Waltham, MA, USA) in an atmosphere of 5% CO2. Cells were detached from the monolayer using 0.25% trypsin (Sigma-Aldrich) for 5 min once cells had grown to near confluence.
Cell viability was measured using MTT assays (
To investigate the survival of cells treated with GA, colony formation assays were performed as previously described (
Apoptotic morphology was detected using an Annexin V binding apoptosis kit, according to the manufacturer's instructions (Nanjing KeyGen Biotech Co., Ltd.). In brief, 5×105 SMMC-7721 cells/well were seeded into a 6-well plate containing a cover slip, and cultured overnight at 37°C in a humidified incubator.. Cells were treated with 0, 6.25, 12.5 and 25.0 µg/ml GA for 48 h. The treated cells were washed with phosphate-buffered saline (PBS; Beijing Dingguo Changsheng Biotech Co., Ltd.), fixed in 10% paraformaldehyde (Beijing Dingguo Changsheng Biotech Co., Ltd.) for 10 min and stained with reagent I (Giemsa) from the kit. Typical apoptotic changes to the nuclei were observed using light microscopy (CHK-213; Olympus Corp., Tokyo, Japan), and apoptotic cells were identified according to criteria from a previous study (
Apoptotic morphology was detected using acridine orange staining, according to the manufacturer's instructions. Briefly, 5×105 SMMC-7721 cells/well were seeded into a 6-well plate and cultured overnight. Cells were treated with 0, 25.0 and 50.0 µg/ml GA for 48 h. Treated cells were subsequently harvested and stained with acridine orange (100 µg/ml) for 15 min in the dark. Any morphological changes to the nuclei were observed using fluorescence microscopy (Eclipse TS100, Nikon Corp., Tokyo, Japan).
In order to evaluate oligonucleosomal fragmentation, genomic DNA was extracted and examined using agarose gel electrophoresis as previously described (
The proportion of apoptotic cells was measured using Annexin V-FITC/PI double staining, according to the manufacturer's instructions. SMMC-7721 cells (5×105 cells/well) were seeded into a 6-well plate and cultured overnight. Cells were treated with GA (0, 6.25, 12.5 and 25.0 µg/ml) for 48 h, subsequently harvested and washed twice with ice-cold PBS, and treated according to the manufacturer's instructions. Following harvesting and washing, cells were immediately analyzed using flow cytometry (FCM; COULTER® EPICS® XL™ Flow Cytometer; Beckman Coulter, Inc., Brea, CA, USA).
The levels of caspase-3, −8 and −9 activity were measured using the caspase activity assay kit according to the manufacturer's instructions. Briefly, following GA treatment (0, 6.25, 12.5, 25.0, and 50.0 µg/ml) for 48 h, ~3×105 SMMC-7721 cells were harvested and washed twice with PBS, and subsequently treated with the caspase activity assay kit according to the manufacturer's instructions. Data were obtained in triplicate and expressed as the mean ± standard deviation. The activity of caspase was normalized and expressed as ODtest / ODcontrol × 100%.
The change in MMP in SMMC-7721 cells was analyzed by flow cytometry using RH-123 staining (
The production of ROS in SMMC-7721 cells was detected by FCM using dichloro-dihydro-fluorescein diacetate (DCFH-DA; Sigma-Aldrich) as a probe according to previous methodology (
The expression levels of Bax, Bcl-2 and cytochrome
All experimental data are expressed as the mean ± standard deviation, and reproducibility was confirmed in at least three separate experiments. Statistical analysis was performed using SPSS version 13.0 (SPSS, Inc., Chicago, IL, USA).
To determine the mode of cell death observed, morphological changes in the nucleus of SMMC-7721 cells following GA treatment were analyzed using Giemsa and acridine orange staining. As demonstrated in
The present study also investigated the induction of apoptosis in SMMC-7721 cells via the occurrence of DNA fragmentation. It was observed that DNA extracted from untreated SMMC-7721 cells exhibited no fragmentation, while DNA from GA treated cells demonstrated DNA laddering, which occurred as a result of endonuclease action at sites between nucleosomes (
To assess whether the GA-induced apoptosis of SMMC-7721 cells was caspase-dependent, the catalytic activity of caspase-3, −8 and −9 was examined. The results (
Caspase activation is primarily triggered by a change in MMP, and apoptosis is also associated with loss of the MMP in a number of cases. Therefore, the present study measured the MMP of SMMC-7721 cells using RH-123 dye as marker, and flow cytometric analysis revealed depletion of the MMP. Untreated SMMC-7721 cells possessed intact plasma and a normal MMP, and the cell RH-123 fluorescence was 95.7±0.4%. However, in SMMC-7721 cells treated with 6.25, 12.5 and 25.0 µg/ml GA for 48 h, the levels of RH-123 fluorescence were 86.5±1.4, 77.1±2.2 and 67.7±2.8%, respectively; this fluorescence was statistically different from the untreated group (P<0.05;
ROS generation may correspond with a reduction in the MMP, and ROS have been observed to be involved in the induction of apoptosis in a number of systems. In order to determine whether ROS production was involved in GA-induced apoptosis of SMMC-7721 cells, the present study detected the ROS levels in GA-treated SMMC-7721 cells using DCFH-DA. Flow cytometric analysis revealed that untreated SMMC-7721 cells possessed low levels of endogenous ROS (2.6±0.2%), whereas treatment with GA significantly enhanced intracellular ROS levels. The levels of ROS generation reached 11.3±0.8, 20.1±2.3 and 47.4±2.8%, when SMMC-7721 cells were treated with 6.25, 12.5 and 25.0 µg/ml GA, respectively, for 2 h; this ROS generation was significantly different from the untreated group (P<0.05;
Mitochondria possess a critical role in caspase-dependent cell apoptosis, via the release of cytochrome
In Traditional Chinese Medicine, herbal plants and plant-derived medicines are frequently used (
Anticancer drugs are often capable of inducing apoptosis, therefore eliminating cells that harbor genetic damage or exhibit inappropriate division (
There are two primary, linked apoptotic pathways: The death receptor (extrinsic) pathway and the mitochondrial-mediated (intrinsic) pathway (
In the mitochondrial-mediated apoptotic pathway, the release of cytochrome
ROS, including oxygen ions and peroxides, are chemically reactive molecules, which may arise during periods of environmental stress (
Proteins of the Bcl-2 family have been suggested to be the primary regulators of apoptosis in the mitochondrial-mediated pathway (
Taken together, the results of the present study demonstrate that GA induced apoptosis in SMMC-7721 cells through the mitochondrial-mediated pathway. It was therefore hypothesized that apoptosis was induced as follows: ROS were generated at the induction of apoptosis. Subsequently, electron transport was disrupted and the MMP was decreased in SMMC-7721 cells. Bax protein expression was upregulated and Bax was inserted into the outer mitochondrial membrane to form channels, facilitating the release of cytochrome
In conclusion, GA is capable of inhibiting the growth of HepG2 and SMMC-7721 HCC cells, as well as inducing apoptosis in SMMC-7721 cells
The present study was supported by the Wu Jieping Medical Foundation (grant no. LDWMF-P5-2011B004). Equipment support for FCM was provided by Changchun Institute of Applied Chemistry (Chinese Academy of Sciences, Changchun, China). The authors would also like to thank Dr Zuojia Liu for significant advice on this study.
Chemical structure of gallic acid.
GA selectively inhibits the proliferation of hepatocellular carcinoma cells in a dose-dependent manner. Effect of GA treatment on the viability of (A) HepG2 cells (B) SMMC-7721 cells and (C) HL-7702 cells. (D) Effect of GA on the colony formation of SMMC-7721 and HepG2 cells. Data are expressed as the mean ± standard deviation of three separate experiments. Significant differences from untreated cells are indicated, *P<0.05, **P<0.01 vs. control group. GA, gallic acid.
Gallic acid induces development of apoptotic morphology of SMMC-7721 cells (magnification, ×200). Apoptotic morphological changes of SMMC-7721 cells were observed under light (left panels) or fluorescence (right panels) microscopy.
DNA fragment analysis of SMMC-7721 cells following treatment with various concentrations of gallic acid. DNA isolated from SMMC-7721 cells treated with 6.25, 12.5 and 25.0 µ/ml GA for 48 h, and then analyzed by agarose gel electrophoresis. GA induced DNA fragmentation.
Flow cytometric analysis of SMMC-7721 cells following treatment with gallic acid. The extent of apoptosis induced in SMMC-7721 cells treated by 6.25, 12.5 and 25.0 µg/ml GA for 48 h was measured by flow cytometry. Data from three independent experiments are shown, and values are presented as the mean ± standard deviation. FITC, fluorescein isothiocyanate; PI, propidium iodide.
Detection of the activity of (A) caspase-3, (B) caspase-9 and (C) caspase-8 in SMMC-7721 cells following treatment with various concentrations of gallic acid for 48 h. Data are expressed as the mean ± standard deviation of three independent experiments. *P<0.05, **P<0.01, vs. control group.
Flow cytometric analysis of SMMC-7721 cells following treatment with various concentrations of GA. (A) Mitochondrial membrane potential and (B) the generation of ROS in SMMC-7721 cells following treatment with various concentrations of GA. Data are expressed as the mean ± standard deviation. RH-123, rhodamine 123; DCF, 2,7-dichlorfluorescein; ROS, reactive oxygen species; GA, gallic acid.
Western blot analysis of apoptosis associated proteins Bax, Bcl-2 and cytochrome