Arctigenin induces necroptosis through mitochondrial dysfunction with CCN1 upregulation in prostate cancer cells under lactic acidosis

Abstract

Arctigenin, a mitochondrial complex I inhibitor, has been identified as a potential anti-tumor agent, but the involved mechanism still remains elusive. Herein, we studied the underlying mechanism(s) of action of arctigenin on acidity-tolerant prostate cancer PC-3AcT cells in the lactic acid-containing medium. At concentration showing no toxicity on normal prostate epithelial RWPE-1 and HPrEC cells, arctigenin alone or in combination with docetaxel induced significant cytotoxicity in PC-3AcT cells compared to parental PC-3 cells. With arctigenin treatment, reactive oxygen species (ROS) levels, annexin V-PE positive fractions, sub-G(0)/G(1) peak in cell cycle analysis, mitochondrial membrane depolarization, and cell communication network factor 1 (CCN1) levels were increased, while cellular ATP content and phospho (p)-Akt level were decreased. Pretreatment with ROS scavenger N-acetylcysteine effectively reversed the series of phenomena caused by arctigenin, suggesting that ROS served as upstream molecules of arctigenin-driven cytotoxicity. Meanwhile, arctigenin increased the levels of p-receptor-interacting serine/threonine-protein kinase 3 (p-RIP3) and p-mixed lineage kinase domain-like pseudokinase (p-MLKL) as necroptosis mediators, and pretreatment with necroptosis inhibitor necrostatin-1 restored their levels and cell viability. Treatment of spheroids with arctigenin resulted in necroptotic cell death, which was prevented by N-acetylcysteine. The siRNA-based knockdown of CCN1 suppressed the levels of MLKL, B-cell lymphoma 2 (Bcl-2), and induced myeloid leukemia cell differentiation (Mcl-1) with increased cleavage of Bcl-2-associated X (Bax) and caspase-3. Collectively, these results provide new insights into the molecular mechanisms underlying arctigenin-induced cytotoxicity, and support arctigenin as a potential therapeutic agent for targeting non-Warburg phenotype through induction of necroptosis via ROS-mediated mitochondrial damage and CCN1 upregulation.