Natural lignan justicidin A-induced mitophagy as a targetable niche in bladder cancer

Abstract

Accumulated dysfunctional mitochondria are involved in tumorigenesis, and it is conceivable that mitophagy, a selective form of autophagic degradation of mitochondria, plays a tumor-suppressive role. Our bioinformatics analysis identified lignan justicidin A (JA) as a potential mitophagy inducer. In HRAS-mutant human bladder cancer T24 cells, JA reduced population cell growth, changed mitochondrial membrane potential, and induced autophagy. JA-induced mitophagy was demonstrated by a reduction of mitochondrial puncta by confocal microscopy and co-localization of autophagy marker LC3 and mitochondrial matrix protein HSP60 in the autophagic vesicles by electron microscopy. These phenomena were associated with altered mitochondrial dynamics, increased expressions of HIF-1α and its target gene BNIP3, and induced co-immunoprecipitation of LC3 with BNIP3 homo-dimer. Confocal microscopy further observed co-localizations among puncta of LC3, BNIP3, and HSP60. JA raised BNIP3 expression in T24 but not E7 (HRAS wild-type) and induced stronger autophagy in T24 than in E7 cells, indicating JA preferentially caused BNIP3-mediated mitophagy in urinary tract cells with oncogenic HRAS. Furthermore, JA enhanced cytotoxicity of T24 cells to anti-cancer drugs cisplatin combined with gemcitabine. Analyses of patients’ data further showed that, in contrast to other major cancer types, lowered mitophagy in bladder urothelial carcinoma compared with normal tissues and reduced expression of mitochondrial genes in cisplatin-responsive bladder cancer cells compared with non-responsive cells suggest mitophagy acts as a tumor suppressor to avoid cisplatin resistance in bladder cancer. Overall, our data suggest the role of BNIP3 and mitophagy in anti-cancer mechanism of human bladder cancer with HRAS mutation in response to JA.