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-G0/G1 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.

中文翻译：

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在乳酸性酸中毒下，Arctigenin通过线粒体功能障碍诱导前列腺坏死，CCN1上调。

线粒体复合物I抑制剂Arctigenin已被确认为潜在的抗肿瘤药物，但所涉及的机制仍然难以捉摸。在本文中，我们研究了Arctigenin在含乳酸的培养基中对耐酸性的前列腺癌PC-3AcT细胞起作用的潜在机制。在浓度对正常前列腺上皮RWPE-1和HPrEC细胞无毒性的浓度下，与亲代PC-3细胞相比，单独或与多西紫杉醇联合使用Arctigenin诱导的PC-3AcT细胞具有明显的细胞毒性。使用arctigenin处理后，细胞周期分析，线粒体膜去极化和细胞通讯网络因子1（CCN1）的水平升高，活性氧（ROS）水平，膜联蛋白V-PE阳性分数，sub-G0 / G1峰升高，而细胞ATP升高含量和磷酸（p）-Akt水平降低。ROS清除剂N-乙酰半胱氨酸的预处理有效逆转了由Arctigenin引起的一系列现象，这表明ROS充当了Arctigenin驱动的细胞毒性的上游分子。同时，Arctigenin可增加p受体相互作用的丝氨酸/苏氨酸蛋白激酶3（p-RIP3）和p-混合谱系激酶结构域样假激酶（p-MLKL）作为坏死病介体的水平，并用坏死抑制剂necrostatin-进行预处理1恢复其水平和细胞活力。用Arctigenin处理球体会导致坏死性细胞死亡，这可通过N-乙酰半胱氨酸来预防。基于siRNA的CCN1敲低抑制了MLKL，B细胞淋巴瘤2（Bcl-2）的水平，并诱导了髓样白血病细胞分化（Mcl-1），同时增加了Bcl-2相关X（Bax）和胱天蛋白酶的裂解-3。总的来说，