Wu Ming-Yue, Wang Sheng-Fang, Cai Cui-Zan, Tan Jie-Qiong, Li Min, Lu Jin-Jian, Chen Xiu-Ping, Wang Yi-Tao, Zheng Wei, Lu Jia-Hong
State Key Laboratory of Quality Research in Chinese Medicine, Institute of Chinese Medical Sciences, University of Macau, Macau SAR, China.
State Key Laboratory of Medical Genetics, Xiangya Medical School, Central South University, Changsha, Hunan, China.
Oncotarget. 2017 Sep 8;8(44):77673-77684. doi: 10.18632/oncotarget.20767. eCollection 2017 Sep 29.
Autophagy is a cellular bulk degradation pathway implicated in various diseases. Inhibition of autophagy has been regarded as a new therapeutic strategy for cancer treatment, especially in combination with chemotherapy. In our study, we identified two natural compounds, dauricine (DAC) and daurisoline (DAS), as two potent autophagy blockers through a high-content screening. DAC and DAS are alkaloids isolated from traditional Chinese medicine . We systematically examined the effects of DAC and DAS on autophagy function in HeLa cells and found that DAC and DAS induced massive formation of autophagic vacuoles and lipidation of LC3. The accumulation of autophagic vacuoles and LC3 lipidation are due to blockage of autophagosome maturation as evidenced by interrupted colocalization of autophagsosome and lysosome, increased GFP-LC3/RFP-LC3 ratio and accumulation of autophagic substrate p62. Moreover, DAC and DAS impaired lysosomal function, as indicated by reduced lysosomal protease activity and increased lysosomal pH values. Importantly, we showed that DAC and DAS strongly inhibited the lysosome V-type ATPase activity. For the therapeutic potential, we found that DAC and DAS blocked the campothecin (CPT)-induced protective autophagy in HeLa cells, and dramatically sensitized the multiple cancer cells to CPT-induced cell death. In conclusion, our result shows that DAC and DAS are autophagy inhibitors which inhibit the lysosomal degradation of auophagic vacuoles, and sensitize the CPT-induced cancer cell death. The study implies the therapeutic potential of DAC and DAS in the treatment of cancers in combination of chemotherapy by inhibiting autophagy.
自噬是一种涉及多种疾病的细胞大量降解途径。抑制自噬已被视为癌症治疗的一种新的治疗策略，尤其是与化疗联合使用时。在我们的研究中，我们通过高内涵筛选鉴定出两种天然化合物，蝙蝠葛碱（DAC）和蝙蝠葛苏林碱（DAS），它们是两种有效的自噬阻断剂。DAC和DAS是从中药中分离出的生物碱。我们系统地研究了DAC和DAS对HeLa细胞自噬功能的影响，发现DAC和DAS诱导自噬空泡大量形成以及LC3的脂化。自噬空泡的积累和LC3脂化是由于自噬体成熟受阻，这可通过自噬体与溶酶体的共定位中断、GFP-LC3/RFP-LC3比率增加以及自噬底物p62的积累来证明。此外，DAC和DAS损害了溶酶体功能，表现为溶酶体蛋白酶活性降低和溶酶体pH值升高。重要的是，我们发现DAC和DAS强烈抑制溶酶体V型ATP酶活性。对于治疗潜力，我们发现DAC和DAS阻断了喜树碱（CPT）诱导的HeLa细胞保护性自噬，并显著增强了多种癌细胞对CPT诱导的细胞死亡的敏感性。总之，我们的结果表明DAC和DAS是自噬抑制剂，它们抑制自噬空泡的溶酶体降解，并增强CPT诱导的癌细胞死亡。该研究表明DAC和DAS在通过抑制自噬联合化疗治疗癌症方面具有治疗潜力。
