Methyl gallate, gallic acid-derived compound, inhibit cell proliferation through increasing ROS production and apoptosis in hepatocellular carcinoma cells

Abstract

Hepatocellular carcinoma (HCC) is a global health problem. Currently, there is no effective therapeutic strategy for HCC. Methyl gallate (MG), from plant-derived phenolic gallic acid, has exhibited antitumor efficacy. However, the effect of MG on HCC is unclear. In vitro growth activity was detected by a sulforhodamine assay. A zebrafish xenotransplantation was applied to evaluate the inhibitory effect of MG. Reactive oxygen species (ROS) production, autophagy, and lysosome formation were detected by specific dyes. Finally, apoptosis was examined using annexin V-FITC/PI staining and western blot was performed to determine the molecular mechanism. It was demonstrated that MG treatment inhibited the proliferation of Hep3B, Mahlavu, and HepJ5 cells. Xenotransplantation also showed that MG inhibited the growth of Hep3B and HepJ5 cells. MG treatment increased cellular levels of superoxide and oxidative stress. Increases in autophagy and lysosome formation were found after MG treatment. The western blot analysis showed that MG activated cleavage of caspase-3 and poly (SDP ribose) polymerase (PARP), modulated levels of the Bcl2, Bax, and Bad ligands, and induced apoptosis. MG induced autophagy with notable activation of beclin-1, autophagy related 5+12 (ATG5+12), and conversion of light chain 3-I (LC3-I) to II. Our study showed that MG exposure inhibited HCC proliferation both in vitro and in vivo. And blocking autophagy enhanced MG-induced cytotoxicity in HCC cells. These findings suggested MG might serve as a powerful therapeutic supplement for human HCC patients.

Fig 1. Methyl Gallate (MG) treatment decreases hepatocellular carcinoma (HCC) cell survival.

After treating Hep3B, Mahlavu, and HepJ5 cells with different doses of MG (0~40 μg/ml) for 48h, the relative cell survival rate was determined using an SRB assay. Survival of vehicle-treated cells was defined as 100%. MG treatment showed reduction of the cell survival rate in HCC cells in a dose-dependent manner. Data are expressed as the mean±SD of three independent experiments in triplicate (* p<0.05, ** p<0.01).

Fig 2. MG-suppressed cell proliferation in a xenotransplantation model.

Zebrafish was used as the animal model for the xenotransplantation assay to determine the efficacy of MG treatment in hepatocellular carcinoma (HCC). Fluorescence labeled Hep3B and HepJ5 cells were implanted into an embryo yolk of the zebrafish, and then embryos were exposed to 40 μg/ml MG or dH₂O as a vehicle control. Proliferative activities of the HCC cell lines in the embryos (n = 20 for each group) were compared by monitoring the fluorescence intensity on days 1 and 3 post-injection (1 and 3 dpi) of MG. (a and b) MG treatment reduced the increase in cell numbers in the embryo population (from 80% for the vehicle to 33% embryos respectively) in HepJ5 cells. A decrease in the fluorescence intensity was shown after 3 days in Hep3B cells with 40 μg/ml MG treatment (c and d). In the Hep3B cell line, the increase in cell numbers in the embryo population decreased from 100% (vehicle) to 39% (40 μg/ml) within 20 embryos. Treatment with 40 μg/ml MG dramatically decreased the fluorescence intensity in HCC cells compared to the vehicle. Scale bare was 1 mm.

Fig 3. Methyl Gallate (MG) increases Reactive Oxygen Species (ROS) and superoxide levels in HepJ5 and Mahlavu cells.

Both of HepJ5 and Mahlavu cells were treated with 40 μg/ml MG for 24 h. ROS and superoxide levels were detected using specific dyes. MG treatment significantly increased ROS and superoxide levels compared to the vehicle. Data are presented as the mean±SD of three independent experiments in triplicate (* p<0.05, ** p<0.01).

Fig 4. Pretreatment with aminoguanidine hemisulfate (AGH; an antioxidant) abolishes the Methyl Gallate (MG)-induced reactive oxygen species pretreatment with aminoguanidine hemisulfate (AGH; an antioxidant) abolishes the Methyl Gallate (MG)-induced Reactive Oxygen Species (ROS) and superoxide levels.

HepJ5 and Mahlavu cells were treated with AGH and then exposed to MG. Levels of ROS and superoxide were detected using specific fluorescence dyes. (a) Significant increase of the ROS level was found after MG treatment. The MG-induced ROS level was abolished in AGH pretreated HepJ5 and Mahlavu cells. (b) The superoxide level increased after exposure to MG and was abolished with AGH pretreatment. Data are presented as the mean±SD of three independent experiments in triplicate (** p<0.01).

Fig 5. Methyl Gallate (MG) increases autophagosomes and lysosomes formation.

HepJ5 and Mahlavu cells were exposed to 40 μg/ml MG for 24 h. (a) Autophagosomes and (b) lysosomes were detected using specific dyes. The formation of autophagosomes and lysosomes increased after MG treatment compared to the vehicle. Data are presented as the mean±SD of three independent experiments in triplicate (** p<0.01).

Fig 6. Methyl Gallate (MG) treatment causes changes in autophagy- and apoptosis-related proteins.

HepJ5 cells were treated with 40 μg/ml MG or vehicle for 48h. a. Levels of autophagy-related proteins (ATG5, ATG12, Beclin-1, and LC3) were checked by western blot. Amounts of ATG5, ATG5+12, and Beclin-1 were similar between MG-treated and vehicle-treated samples. The ratio of LC3-II/LC I was high in the MG-treated sample. b. Levels of cell apoptosis-related proteins (Bcl-2, Bax, Bad, c-caspase 3 and c-PARP) were checked. MG-treated cells showed decreased Bcl-2 (antiapoptotic), increased Bax and Bad proteins (proapoptotic), and increased cleavage of caspase 3 (c-caspase3) and PARP (c-PARP). All experiments were repeated at least three times independently (** p<0.01).

Fig 7. Blocking autophagy enhances MG-induced cytotoxicity in HepJ5 cells.

The cell viability in cells treated with MG for 24 h in the presence and absence of CQ for 16 h was determined using SRB assay (a). Apoptotic cells were quantified using Annexin V-FITC/PI staining and FACS analysis after 40 μg/ml MG treatment for 48h in the presence and absence of CQ for 16 h (b). The blockage of autophagy flux was confirmed by detecting the accumulation of LC3-II using western blot (c). Data are presented as the mean±SD of three independent experiments in triplicate, ** p<0.01).