Ginkgolic acid suppresses the development of pancreatic cancer by inhibiting pathways driving lipogenesis

Abstract

Ginkgolic acid (GA) is a botanical drug extracted from the seed coat of Ginkgo biloba L. with a wide range of bioactive properties, including anti-tumor effect. However, whether GA has antitumor effect on pancreatic cancer cells and the underlying mechanisms have yet to be investigated. In this study, we show that GA suppressed the viability of cancer cells but has little toxicity on normal cells, e.g, HUVEC cells. Furthermore, treatment of GA resulted in impaired colony formation, migration, and invasion ability and increased apoptosis of cancer cells. In addition, GA inhibited the de novo lipogenesis of cancer cells through inducing activation of AMP-activated protein kinase (AMPK) signaling and downregulated the expression of key enzymes (e.g. acetyl-CoA carboxylase [ACC], fatty acid synthase [FASN]) involved in lipogenesis. Moreover, the in vivo experiment showed that GA reduced the expression of the key enzymes involved in lipogenesis and restrained the tumor growth. Taken together, our results suggest that GA may serve as a new candidate against tumor growth of pancreatic cancer partially through targeting pathway driving lipogenesis.

Keywords: AMP-activated protein kinase (AMPK); cancer metabolism; ginkgolic acid (GA); lipogenesis; pancreatic cancer.

Figure 1. GA treatment suppresses the viability of cancer cells with no cytotoxic effect on non-cancer cells

Cancer cells (Panc-1, BxPC-3 and HepG2) and two normal cells (HL-7702 and HUVEC) were treated with various concentrations (0, 1, 2, 5, 10, 20, 50, and 100μM) of GA. At the indicated time points (12, 24, 36, and 48h), cell viability in each group was assessed by MTT assay.

Figure 2. GA treatment inhibits the clone formation and induces apoptosis of cancer cells

(A) The effects of GA on the colony forming ability of Panc-1, BxPC-3, and HepG2 cells. Images are representative of three independent experiments. (B) The effects of GA on cancer cells apoptosis was detected by flow cytometry. *P < 0.05.

Figure 3. GA treatment inhibits the migration and invasion ability of cancer cells

(A) Wound-scratch assay were performed in Panc-1, BxPC-3, and HepG2 cells pretreated with 20μM GA or not. Images were visualized at 0 h and 24h at a magnification of 100×. (B) The effects of GA on the invasion ability of cancer cells were assessed by Matrigel-invasion assay. Images are representative of three independent experiments. *P < 0.05.

Figure 4. GA prevents lipogenesis of cancer cells

(A) Oil Red O staining was used to visualized the lipid droplets changes in Panc-1, BxPC-3, and HepG2 cells pretreated with 20μM GA or not. (B) The effects of GA on the mRNA expression of lipogenic genes (ACC, FASN, SREBF-1 and SREBF-2) were examined by real-time PCR with β-actin as the normalized reference gene. *P < 0.05. (C) The effects of GA on the protein expression of lipogenic genes were examined by Western blotting analysis using β-actin as an internal loading control.

Figure 5. GA inhibits the expression of lipogenic genes via activating AMPK signaling

(A) The effects of GA on the activity of AMPK in Panc-1, BxPC-3, and HepG2 cells were measured by Western blotting analysis. (B) The efficiency of siRNAs targeting AMPK in BxPC-3 cells was evaluated by Western blotting. (C) Immunoblotting results revealed that knocking down AMPK expression restored GA-prevented lipogenic genes expression in cancer cells. β-actin was used as an internal loading control.

Figure 6. GA prevents the subcutaneous xenograft tumor growth and the expression of lipogenic enzyme in vivo

(A) The tumors volume was calculated every 3 days throughout the experiment. The tumor growth was dramatically retarded by GA administration. (B) Representative photograph of subcutaneous xenograft tumors derived from mice in GA group and Control group. (C) The average tumor weights in GA group and Control group were measured at the end of the experiment. *P < 0.001. (D) Representative image of H&E staining and immunohistochemical staining for PCNA and FASN in GA group and Control group.