醋酸棉酚调控细胞自噬发挥抗肿瘤作用的机制研究

	醋酸棉酚调控细胞自噬发挥抗肿瘤作用的机制研究; Study on the antitumor mechanism of gossypol acetate by regulating autophagy
	蔡碧宬
导师	周宏宇
摘要	It is an promising pathway for the development of anti-tumor drugs by screening and discovering active compounds from natural products. Due to that the regulation of autophagy can be used as a potential strategy for cancer treatment in different types of cancers, this study focuses on screening compounds which can promote autophagosome formation by using stable expression GFP-LC3 293T cell line from our laboratory. It is aimed to discover natural small molecules which can regulate autophagy and exhibit potent antitumor activity. Furthermore, molecular biological techniques such as western blotting and gene silencing will be used to elucidate the mechanism of autophagy regulation by the test compound. And the role of autophagy in the antitumor activity of the test compound will be investigated as well. The screening results showed that the compound Gossypol acetate (GAA) could increase the aggregation of fluorescence puncta in GFP-LC3-293T cells, indicating that GAA induced the formation of autophagosomes. Mechanism studies found that different concentrations of GAA have different effects on autophagy regulation. Low concentration of GAA induced autophagy manifested as increased LC3-Ⅱ expression and decreased p62 expression. The induction of autophagy by GAA is attributed to the activation of AMPK and inhibition of its downstream mTORC1 complex, resulting in the activation ULK1, the initiation factor of autophagy. However, high concentration of GAA inhibited late-stage of autophagy, which was manifested as blocked degradation of LC3 and p62. The inhibition of autophagy is related to the decrease of intracellular ATP level and the reduction of the number of intracellular active lysosomes by GAA. The addition of exogenous ATP can reverse the inhibition of autophagy by high concentration GAA. Finally, in order to explore the role of autophagy regulation by GAA in its antitumor effect, siRNA-LC3 was used to knock down LC3 which is necessary to form the autophagosomes, and the effect of GAA on cell viability was further determined when LC3 was knocked down. Results showed that the knockdown of LC3 partially reversed the cell death caused by GAA, indicating that accumulation of LC3 induced by GAA played an important role in promoting cell death by GAA. In summary, this paper found that GAA is a natural small molecule with antitumor and autophagy regulating effects. Our study elucidated the mechanism involved in the regulation of autophagy by GAA and reveal the role of autophagy regulation in the antitumor activity of GAA. Our study provided important pharmacological foundation for the possible application of GAA as an antitumor drug in the future.
	2020-09
文献类型	学位论文
条目标识符	http://ir.kib.ac.cn/handle/151853/74105
专题	昆明植物所硕博研究生毕业学位论文
推荐引用方式 GB/T 7714	蔡碧宬. 醋酸棉酚调控细胞自噬发挥抗肿瘤作用的机制研究, Study on the antitumor mechanism of gossypol acetate by regulating autophagy[D],2020.

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