Ginkgolic acid induces interplay between apoptosis and autophagy regulated by ROS generation in colon cancer

Biochemical and Biophysical Research Communications Volume 498, Issue 1, 25 March 2018, Pages 246-253 Biochemical and Biophysical Research Communications Author links open overlay panelYuxiaLiuaBinYangbLirongZhangcXianlingCongcZhenLiudYuHucJingZhangeHaixiaHuf a Medical Records Room, China-Japan Union Hospital of Jilin University, 126 Xiantai Road, Jilin 130033, China b Department of Breast Surgery, China-Japan Union Hospital of Jilin University, 126 Xiantai Road, Jilin 130033, China c Department of Pathology, China-Japan Union Hospital of Jilin University, 126 Xiantai Road, Jilin 130033, China d Department of TCM Pediatrics, Traditional Chinese Medicine Hospital of Luhe District, Nanjing 223300, China e Department of Nephrology, China-Japan Union Hospital of Jilin University, 126 Xiantai Road, Jilin 130033, China f Colo-rectal Surgery, China-Japan Union Hospital of Jilin University, 126 Xiantai Road, Jilin 130033, China Received 9 January 2018, Accepted 13 January 2018, Available online 17 January 2018. crossmark-logo https://doi.org/10.1016/j.bbrc.2018.01.091 Get rights and content Highlights • Ginkgolic acid suppresses the proliferation and induces cell cycle arrest. • Ginkgolic acid induces apoptosis and autophagy in human colon cancer cells. • Ginkgolic acid promotes ROS generation, induces cytotoxicity in human colon cancer cells. • Ginkgolic acid inhibits tumor growth of colon cancer in vivo. Abstract Presently, developing effective anti-colon cancer drugs still remains to be important. Ginkgolic acids (GA), as a botanical drug extracted from the seed coat of Ginkgo biloba L., possess various bioactive properties. Our findings, for the first time, indicated that GA suppressed colon cancer cell proliferation, migration and invasion. GA led to cell death through G0/G1 phase arrest. In addition, apoptosis was significantly induced by GA treatment. The intrinsic apoptosis pathway was included, proved by the release of cytochrome c (Cyto-c) from the mitochondria into the cytosol. GA-induced autophagy was supported by the dose-dependent increase of LC3BII, autophagy-related gene-5 (ATG-5) and Beclin-1. Notably, silencing ATG-5 further reduced the cell viability and enhanced apoptosis in GA-treated colon cancer cells, indicating that GA-induced apoptosis rather than autophagy contributes to colon cancer cell death. And mammalian target of rapamycin complex 1 (mTORC1) was dose-dependently reduced by GA, evidenced by the reduction of p-mTOR, p-p70 ribosomal S6 kinase (p70s6k) and p-pras40. Moreover, GA markedly resulted in reactive oxygen species (ROS) generation, along with increased H2O2 and O2−. However, blocking ROS generation using its scavenger, NAC, significantly recovered GA-induced cells death, supported by the increase of cell viability, and the decrease of apoptosis. The expressions of autophagy- and cell cycle arrest-related molecules, as well as mTORC1 were also reversed by N-acetyl-l-cysteine (NAC) in GA-treated cells. In vivo, GA reduced tumor growth without toxicity to animals. In conclusion, our study illustrated that GA caused G0/G1 phase arrest and triggered intrinsic apoptosis and autophagy modulated by ROS generation in human colon cancer, elucidating that GA might be considered as a potential agent for colon cancer therapy. Previous article Next article Keywords Colon cancer Ginkgolic acids Cell cycle arrest Apoptosis and autophagy ROS 1. Introduction Colon cancer is one of the leading causes of cancer mortality in the world [1]. And chemotherapy is widely used to prevent colon cancer. For example, cisplatin (DDP) is well-known as an effective anti-tumor drug. But it is limited due to the side effects to bodies, which highlights the requirement for novel effective and little toxic therapeutic strategies [2]. Ginkgolic acid (GA, Fig. 1A), also known as anacardic acids, is a botanical drug isolated from the seed coat of Ginkgo biloba L. [3]. GA has a wide range of bioactive properties, including anti-bacterium, anti-HIV and molluscicidal activities [4]. Recently, GA is reported to suppress human larynx cancer and tongue squamous carcinoma in the absence of toxic effects [3]. Moreover, GA blocked the growth of pancreatic cancer cells [5]. However, if GA has anti-cancer effects on human colon cancer and the underlying molecular mechanism remains little to be known. Fig. 1 Download high-res image (1MB)Download full-size image Fig. 1. Ginkgolic acid suppresses the proliferation and induces cell cycle arrest in SW-480 cells. (A) Chemical structure of ginkgolic acid (GA). (B) Colon cancer cell lines as indicated were treated with GA or DDP for 24 h, and then the IC50 values were measured using MTT analysis. (C) SW-480 cells were incubated with the described concentrations of GA for 24, 48 or 72 h, followed by MTT analysis. SW-480 cells were treated with the described doses of GA for 24 h, and then, the following researches were performed. (D) The formed colonies were stained. (E,F) The number of migrated and invaded cancer cells were evaluated, respectively. (G) The levels of ADAM12, SNAI1, Vimentin and E-cadherin were detected by western blot analysis. (H) Cell cycle analysis determined the relative cell numbers in each cell-cycle phase. And the percentages of cells in each phase were quantified. (I) RT-qPCR analysis of indicated genes after the described treatment. Each value shown represents mean ± SEM. ∗p < .05, ∗∗p < .01, ∗∗∗p < .001 versus the Con group. Cell proliferation needs a complete cell cycle, modulated by cyclin-dependent kinases (CDKs) and CDK inhibitor proteins [6]. But normal cell cycle control is not observed in cancer cells. During the progression of carcinogenesis, the cell cycle deregulation plays an essential role [7]. Apoptosis and autophagy are two important programmed cell deaths. Apoptosis is considered as an active form of chemotherapy-caused cells death, characterized by the chromatic condensation, nucleus fragmentation and caspases activity [8]. Autophagy, however, plays a dual role in preventing tumor. Autophagy could results in cell death through collaborating with apoptosis [9]. Therefore, targeting apoptosis and autophagy attract researchers' attention to find novel and effective anti-cancer drugs. ROS, including hydrogen peroxide, nitric oxide, hydroxyl radical, superoxide anion radical, and lipid peroxides, could modulate the fate of tumor cells through mediating different pathways, such as apoptosis, autophagy or ferroptosis-induced cell death [10,11]. In the present studied, we verified the role of GA in the progression of human colon cancer both in vitro and in vivo, as well as revealed the underlying molecular mechanism.