Cepharanthine-induced mitophagy through regulation of FBXL4-BNIP3 drives ferroptosis leading to robust anti-lung cancer efficacy.

Cepharanthine (Cep), a natural alkaloid from Stephania (Menispermaceae), exhibits broad-spectrum anti-cancer activity. In the present study, Cep was found to induce ferroptosis and mitophagy, for which the relationship and upstream targets remain unelucidated. Herein, the role of Cep in the induction of mitophagy was deeply investigated. Cep showed robust anti-lung cancer effects, as confirmed by decreased cell viability, elevated apoptosis, suppressed colony formation and inhibited growth of tumor grafts in Lewis cell-bearing mice. RNA-sequencing analysis revealed that Cep treatment significantly enriched differentially expressed genes (DEGs) in mitophagy and ferroptosis pathways, which were demonstrated in in vitro and in vivo experiments as well. In-depth investigations showed that inhibition of autophagy abolished Cep-mediated ferroptosis, but not vice versa. Moreover, genetic knockdown of BNIP3 dampened the mitophagy and ferroptosis of lung cancer cells induced by Cep. Additional data confirmed that Cep could bind to and thereby inhibit FBXL4, which attenuated the ubiquitination of BNIP3. FBXL4-mediated BNIP3 activation promoted the recruitment of LC3 to mitochondria and autophagic flux in the presence of Cep. Collectively, Our study elucidates a complete mechanistic pathway wherein Cep activates BNIP3-mediated mitophagy by inhibiting FBXL4, ultimately driving ferroptosis and offering a new therapeutic avenue for lung cancer.