Hispidin induces autophagic and necrotic death in SGC-7901 gastric cancer cells through lysosomal membrane permeabilization by inhibiting tubulin polymerization

// Long-Xian Lv 1, 2, * , Zhen-Xing Zhou 1, * , Zhan Zhou 1 , Li-Jiang Zhang 1 , Ren Yan 2 , Zhao Zhao 1 , Li-Ya Yang 2 , Xiao-Yuan Bian 2 , Hui-Yong Jiang 2 , Yu-Dong Li 1 , Yi-Sheng Sun 1 , Qin-Qin Xu 1 , Gui-Li Hu 3 , Wen-Jun Guan 1 , Yong-Quan Li 1 1 Institute of Pharmaceutical Biotechnology and College of Pharmaceutical Sciences, Zhejiang University, 310058 Hangzhou, China 2 State Key Laboratory for Diagnosis and Treatment of Infectious Diseases, Collaborative Innovation Center for Diagnosis and Treatment of Infectious Diseases, The First Affiliated Hospital, College of Medicine, Zhejiang University, 310003 Hangzhou, China 3 Department of Basic Medicine, College of Medicine, Zhejiang University, 310058 Hangzhou, China * These authors contributed equally to this work Correspondence to: Yong-Quan Li, email: lyq@zju.edu.cn Wen-Jun Guan, email: guanwj@zju.edu.cn Keywords: hispidin, autophagy, necrotic cell death, lysosomal membrane permeabilization, microtubule depolymerization Received: February 03, 2016 Accepted: February 20, 2017 Published: March 06, 2017 ABSTRACT Hispidin and its derivatives are widely distributed in edible mushrooms. Hispidin is more cytotoxic to A549, SCL-1, Bel7402 and Capan-1 cancer cells than to MRC5 normal cells; by contrast, hispidin protects H9c2 cardiomyoblast cells from hydrogen peroxide-induced or doxorubicin-induced apoptosis. Consequently, further research on how hispidin affects normal and cancer cells may help treat cancer and reduce chemotherapy-induced side effects. This study showed that hispidin caused caspase-independent death in SGC-7901 cancer cells but not in GES-1 normal cells. Hispidin-induced increases in LC3-II occurred in SGC-7901 cells in a time independent manner. Cell death can be partially inhibited by treatment with ATG5 siRNA but not by autophagy or necroptosis inhibitors. Ultrastructural evidence indicated that hispidin-induced necrotic cell death involved autophagy. Hispidin-induced lysosomal membrane permeabilization (LMP) related to complex cell death occurred more drastically in SGC-7901 cells than in GES-1 cells. Ca 2+ rather than cathepsins from LMP contributed more to cell death. Hispidin induced microtubule depolymerization, which can cause LMP, more drastically in SGC-7901 cells than in GES-1 cells. At 4.1 μM, hispidin promoted cell-free tubulin polymerization but at concentrations higher than 41 μM, hispidin inhibited polymerization. Hispidin did not bind to tubulin. Alterations in microtubule regulatory proteins, such as stathmin phosphorylation at Ser 16 , contributed to hispidin-induced SGC-7901 cell death. In conclusion, hispidin at concentrations higher than 41 μM may inhibit tubulin polymerization by modulating microtubule regulatory proteins, such as stathmin, causing LMP and complex SGC-7901 cell death. This mechanism suggests a promising novel treatment for human cancer.