Effects of ginkgol C17:1 on cisplatin-induced autophagy and apoptosis in HepG2 cells

Abstract

Previous studies have demonstrated that ginkgol C17:1 significantly inhibits human liver cancer cells and enhances the anticancer activity of cisplatin in vivo and in vitro. However, the mechanism and biological function of ginkgol C17:1 on cells undergoing chemotherapy remain unclear. The aim of the present study was to determine the antitumor activity and mechanism of ginkgol C17:1 in combination with cisplatin in human hepatoblastoma HepG2 cells. The green fluorescent protein (GFP)-light chain 3 (LC3) adenovirus was transfected into HepG2 cells and autophagic flux was determined using fluorescence microscopy. Western blot analysis was also conducted to measure the expression of proteins associated with apoptosis, autophagy and their associated signaling pathways. Compared with the control group, autophagic flux and nucleus aberration rates were significantly increased (P<0.05), and the expression of proteins associated with autophagy and apoptosis were increased in the groups treated with cisplatin or ginkgol C17:1, respectively. However, following co-treatment with ginkgol C17:1 and cisplatin, the autophagic flux and the expression of autophagy proteins decreased; however, the nucleus aberration rate and apoptosis protein expression significantly increased (P<0.05) compared with the group treated with cisplatin alone. Additionally, the signaling pathways of autophagy and apoptosis were also activated following treatment with cisplatin, alone and in combination with ginkgol C17:1. Taken together, these results indicate that ginkgol C17:1 inhibits cisplatin-induced autophagy via AMP-activated protein kinase/ULK1signaling and increases cisplatin-induced apoptosis in HepG2 cells via the phosphoinositide 3-kinase/Akt/mechanistic target of rapamycin pathway.

Keywords: HepG2 cells; apoptosis; autophagy; cisplatin; ginkgol C17:1.

Figure 1.

Ginkgol C17:1 boosts autophagy and apoptosis in HepG2 cells. (A) Following treatment of HepG2 cells with the indicated concentrations of ginkgol C17:1 (0, 10, 20, 40, 80 and 160 µg/ml) for 24 h, cell viability was detected by an MTT assay. (B) The autophagy protein LC3 was examined by an immunofluorescence assay (magnification, ×200) following infection with GFP-RFP-LC3 adenovirus for 36 h and treatment with ginkgol C17:1 (40 µg/ml) for 24 h; (C) the relative rate of GFP-LC3 puncta was then quantified. (D) Following treatment of HepG2 cells with ginkgol C17:1 (40 µg/ml) with or without NH₄Cl (0.535 mg/ml) for 24 h, the expression of Beclin-1, LC3I/II and p62 were analyzed by western blotting. Following treatment with ginkgol C17:1 (40 µg/ml) for 24 h, the morphology of HepG2 nuclei was observed by (E) immunofluorescence microscopy (magnification, ×200) and they were stained with Hoechst 33342. (F) The nucleus aberration rate was then analyzed and (G) western blot analysis was performed to determine the expression of cleaved caspase-3, Bax and Bcl-2. All values are presented as the mean ± standard deviation from three independent experiments (n=5). *P<0.05 and **P<0.01 vs. control. GFP, green fluorescent protein; RFP, red fluorescent protein; LC3, light chain 3; Bcl-2, B-cell lymphoma 2; Bax, Bcl-2-associated X protein.

Figure 2.

Effects of ginkgol C17:1 on cisplatin-induced autophagy and apoptosis in HepG2 cells. (A) Following treatment with cisplatin (0, 1, 2, 4, 8 and 16 µg/ml) for 24 h, HepG2 cell viability was determined via an MTT assay. (B) Cell viability was detected by MTT assay following treatment with cisplatin (2 µg/ml) and ginkgol C17:1 (0, 20, 40 and 80 µg/ml) for 24 h. Following co-treatment with cisplatin and ginkgol C17:1, LC3 autophagosomes were detected by an (C) immunofluorescence assay (magnification, ×200) and the expression of Beclin-1, LC3I/II and p62 were analyzed by (D) a western blot assay. Under the same conditions, the morphology of HepG2 nuclei was observed by (E) immunofluorescence microscopy (magnification, ×200) staining with Hoechst 33342 and the expression of cleaved caspase-3, Bax and Bcl-2 were analyzed by (F) western blot analysis. Data are presented as the mean ± standard deviation from three independent experiments. *P<0.05 and **P<0.01 vs. control group, and ^##P<0.01. LC3, light chain 3; Bcl-2, B-cell lymphoma 2; Bax, Bcl-2-associated X protein; GFP, green fluorescent protein.

Figure 3.

Ginkgol C17:1 enhances apoptosis and inhibits the autophagy of cells, similar to 3MA. Following infection of HepG2 cells with the GFP-LC3 virus and treatment with or without cisplatin and 3-MA for 24 h, (A) LC3 autophagosomes were detected by an immunofluorescence assay and the average rate of LC3 puncta was valued (magnification, ×200). (B) Western blot analysis measuring the expression of Beclin-1 and p62 was performed. Following treatment with or without cisplatin and 3MA for 24 h, (C) nuclei morphology were observed by immunofluorescence microscopy (magnification, ×200) following staining with Hoechst 33342 and (D) the expression of Beclin-1, LC3 and p62 were measured by western blotting. Following treatment of HepG2 with cisplatin with or without ginkgol C17:1 and 3MA for 24 h, western blot analysis investigating the expression of (E) Beclin-1 and p62 and (F) Bax and Bcl-2 was performed. Data are presented as the mean ± standard deviation from five independent experiments. *P<0.05 and ^##P<0.01. Cisplatin, 2 µg/ml; 3MA, 0.75 mg/ml and ginkgol C17:1, 40 µg/ml. GFP, green fluorescent protein; LC3, light chain 3; Bcl-2, B-cell lymphoma 2; Bax, Bcl-2-associated X protein; 3-MA, 3-methyladenine.

Figure 4.

Effect of ginkgol C17:1 on the AMPK/ULK1 pathway. (A) Following co-treatment of HepG2 cells with cisplatin (2 µg/ml) and ginkgol C17:1 (0, 20, 40 and 80 µg/ml) for 24 h, western blot analysis was performed to determine the expression of AMPK, p-AMPK (Thr172), ULK1 and p-ULK1 (Ser555). (B) Following treatment of cells with cisplatin (2 µg/ml) with or without ginkgol C17:1 (40 µg/ml) and compound C (8 µg/ml) for 24 h, western blot analysis was performed to measure the expression of p-AMPK (Thr172), p-ULK1 (Ser555), Beclin-1, LC3I/II, Bax and Bcl-2. AMPK, AMP-activated protein kinase; p-, phosphorylated; LC3, light chain 3; Bcl-2, B-cell lymphoma 2; Bax, Bcl-2-associated X protein.

Figure 5.

Influence of ginkgol C17:1 on the PI3K/Akt/mTOR pathway. (A) Following co-treatment of HepG2 cells with cisplatin (2 µg/ml) and ginkgol C17:1 (0, 20, 40 and 80 µg/ml) for 24 h, western blot analysis was performed to measure the expression of proteins in the upstream pathway: PI3K and p-PI3K (Tyr458), Akt and p-Akt (Tyr315), mTOR and p-mTOR (Ser2448), JNK and p-JNK (Tyr185). (B) Following treatment of cells were with cisplatin (2 µg/ml), with or without ginkgol C17:1 (40 µg/ml) and rapamycin (100 ng/ml) for 24 h, western blotting was performed to measure the expression of p-mTOR (Ser2448), Beclin-1, LC3I/II, Bax and Bcl-2. p-, phosphorylated; PI3K, phosphoinositide 3-kinase; mTOR, mechanistic target of rapamycin; LC3, light chain 3; Bcl-2, B-cell lymphoma 2; Bax, Bcl-2-associated X protein.