Autophagy inhibition contributes to the synergistic interaction between EGCG and doxorubicin to kill the hepatoma Hep3B cells

Abstract

(-)-Epigallocatechin-3-O-gallate(EGCG), the highest catechins from green tea, has promisingly been found to sensitize the efficacy of several chemotherapy agents like doxorubicin (DOX) in hepatocellular carcinoma (HCC) treatment. However, the detailed mechanisms by which EGCG augments the chemotherapeutic efficacy remain unclear. Herein, this study was designed to determine the synergistic impacts of EGCG and DOX on hepatoma cells and particularly to reveal whether the autophagic flux is involved in this combination strategy for the HCC. Electron microscopy and fluorescent microscopy confirmed that DOX significantly increased autophagic vesicles in hepatoma Hep3B cells. Western blot and trypan blue assay showed that the increasing autophagy flux by DOX impaired about 45% of DOX-induced cell death in these cells. Conversely, both qRT-PCR and western blotting showed that EGCG played dose-dependently inhibitory role in autophagy signaling, and that markedly promoted cellular growth inhibition. Amazingly, the combined treatment caused a synergistic effect with 40 to 60% increment on cell death and about 45% augmentation on apoptosis versus monotherapy pattern. The DOX-induced autophagy was abolished by this combination therapy. Rapamycin, an autophagic agonist, substantially impaired the anticancer effect of either DOX or combination with EGCG treatment. On the other hand, using small interference RNA targeting chloroquine autophagy-related gene Atg5 and beclin1 to inhibit autophagy signal, hepatoma cell death was dramatically enhanced. Furthermore, in the established subcutaneous Hep3B cells xenograft tumor model, about 25% reduction in tumor growth as well as 50% increment of apoptotic cells were found in combination therapy compared with DOX alone. In addition, immunohistochemistry analysis indicated that the suppressed tendency of autophagic hallmark microtubule-associated protein light chain 3 (LC3) expressions was consistent with thus combined usage in vitro. Taken together, the current study suggested that EGCG emerges as a chemotherapeutic augmenter and synergistically enhances DOX anticancer effects involving autophagy inhibition in HCC.

Figure 1. DOX treatment was found to increase autophagic activity in Hep3B cells.

(A) Electron microscopic technology showed up-regulated numbers of autophagosomes in Hep3B cells treated with 2.5 µM DOX versus control. Arrows indicate autophagic structures. Scale bars represent 500 nm. Magnification, ×40000. (B) Immunoblots showed increased expression levels of Atg5 and beclin1 in Hep3B cells treated with DOX of 1, 2.5 and 5 µM for 24 h compared with control. Protein ratios normalized to GAPDH were used to quantify fold change relative to control and are shown below each blot. Data are from a representative study (n = 3). (C) Immunofluorescence analysis indicated that elevated LC3 fluorescent signals were visualized in cells administrated with 2.5 µM DOX.

Figure 2. Autophagy suppression enhanced DOX-induced growth inhibition and cell death of Hep3B cells.

Hep3B cells were treated with vehicle (control), 2 mM 3MA, 2.5 µM DOX, or both 3MA and DOX for 24 h. Light microscopic images recorded the morphology (A) and trypan blue assay determined the cell death (B); Columns, percentage of trypan blue-positive cells; bars, SE. Data was from a representative of three independent studies. Bars = 200 µm. *p<0.05 vs. control, ^#p<0.01 vs. control.

Figure 3. Dose-dependent inhibited effect of EGCG on the autophagy and proliferation in Hep3B cells.

(A) EGCG (40 µg/ml) was found to reduce the autophagosome number in Hep3B cells. Arrows indicate autophagic structures. (B) Exposed to EGCG of 10, 20, 40 µg/ml for 24 h, the expression levels of Atg5 and beclin1 in Hep3B cells were determined at the RNA levels by qRT-PCR. (C) Cell lysates following treatment with varies concentrations of EGCG were subjected to western blotting. Protein ratios normalized to GAPDH were used to quantify fold change relative to control. Results shown are representative of three independent experiments and error bars indicate SE. (D) EGCG exerted inhibition effects on proliferation of Hep3B cells in dose- and time-dependent manner after EGCG treatment with the indicated concentrations. Bars = 500 nm. Magnification, ×40000. *p<0.05 vs. control, ^#p<0.01 vs. control.

Figure 4. Combination of EGCG and DOX promoted cell death and apoptosis in Hep3B cells.

(A) Trypan blue assay characterized the cell death of Hep3B cells treated with EGCG (10 µg/ml, 20 µg/ml) in the presence or absence of DOX (2.5 µM) for 24 h and 48 h. (B) Flow cytometry analyzed the apoptosis of Hep3B cells after addition of EGCG (10 µg/ml) in the presence or absence of DOX (2.5 µM) for 24 h. The lower panel is the summarized data. Results are representative of three independent experiments and error bars indicate SE. ^#p<0.01 vs. control.

Figure 5. Combination effects of EGCG and DOX on Hep3B cells involved autophagic modulation.

Genetic inhibition of autophagy in Hep3B was conducted with siRNAs targeting at Atg5 and beclin1. Effective knockdown of autophagy gene or protein expression levels with each siRNA was confirmed by qRT-PCR (A) and western blotting (B). By trypan blue staining, it was showed that (C) rapamycin (Rapa,100 nM), an agonist, substantially impaired the cell death in Hep3B treated with EGCG(10 µg/ml) and DOX(2.5 µM) and (D)blocking autophagy by siRNAs targeting at Atg5 and beclin1 enhanced cell death of these cells in the presence of EGCG and DOX for 48 h. Cumulative results from three independent experiments were shown as mean ± SE. C, control. ^#p<0.01 vs. control.

Figure 6. Contribution of autophagy and apoptosis to anti-tumor effects of EGCG and DOX in HCC model.

Nude mice were subcutaneously injected with Hep3B cells. When the volume of tumor reached 100³, the mice were divided into corresponding treatment groups based on both volume and weight. Tumor volume was recorded every 4 days (A) and the tumor weight was recorded (B) when the tumors were excised after 15 days treatment. The data represents means and standard deviations and error bars indicate SE. (C) LC3 protein expression in each tumor tissue section was measured by immunochemistry. Magnification, 400×. (D) Apoptosis in each tumor tissue section was measured by TUNEL staining. Positive cells were determined in three independent experiments. Three random fields representing 200 tumor cells were counted. Magnification, 200×. Cumulative results were shown as mean ± SE and error bars indicate SE. Bars = 20 µm. *p<0.05 vs. control, ^#p<0.01 vs. control, n = 6.