Muniraj Nethaji, Siddharth Sumit, Nagalingam Arumugam, Walker Alyssa, Woo Juhyung, Győrffy Balázs, Gabrielson Ed, Saxena Neeraj K, Sharma Dipali
Department of Oncology and the Sidney Kimmel Comprehensive Cancer Center, Johns Hopkins University School of Medicine, Baltimore, MD, USA.
MTA TTK Momentum Cancer Biomarker Research Group, Budapest, Hungary.
Carcinogenesis. 2019 Sep 18;40(9):1110-1120. doi: 10.1093/carcin/bgz015.
Withaferin A (WFA), a steroidal lactone, negatively regulates breast cancer growth however, its mechanisms of action remain largely elusive. We found that WFA blocks autophagy flux and lysosomal proteolytic activity in breast cancer cells. WFA increases accumulation of autophagosomes, LC3B-II conversion, expression of autophagy-related proteins and autophagosome/lysosome fusion. Autolysosomes display the characteristics of acidic compartments in WFA-treated cells; however, the protein degradation activity of lysosomes is inhibited. Blockade of autophagic flux reduces the recycling of cellular fuels leading to insufficient substrates for tricarboxylic acid (TCA) cycle and impaired oxidative phosphorylation. WFA decreases expression and phosphorylation of lactate dehydrogenase, the key enzyme that catalyzes pyruvate-to-lactate conversion, reduces adenosine triphosphate levels and increases AMP-activated protein kinase (AMPK) activation. AMPK inhibition abrogates while AMPK activation potentiates WFA's effect. WFA and 2-deoxy-d-glucose combination elicits synergistic inhibition of breast cancer cells. Genetic knockout of BECN1 and ATG7 fails to rescue cells from WFA treatment; in contrast, addition of methyl pyruvate to supplement TCA cycle protects WFA-treated cells. Together, these results implicate that WFA is a potent lysosomal inhibitor; energetic impairment is required for WFA-induced apoptosis and growth inhibition and combining WFA and 2-DG is a promising therapeutic strategy for breast cancer.
Withaferin A（WFA）是一种甾体内酯，对乳腺癌生长具有负向调节作用，但其作用机制仍 largely 难以捉摸。我们发现 WFA 阻断乳腺癌细胞中的自噬流和溶酶体蛋白水解活性。WFA 增加自噬体的积累、LC3B-II 转化、自噬相关蛋白的表达以及自噬体/溶酶体融合。在 WFA 处理的细胞中，自噬溶酶体显示出酸性区室的特征；然而，溶酶体的蛋白降解活性受到抑制。自噬流的阻断减少了细胞燃料的循环利用，导致三羧酸（TCA）循环的底物不足以及氧化磷酸化受损。WFA 降低催化丙酮酸向乳酸转化的关键酶乳酸脱氢酶的表达和磷酸化，降低三磷酸腺苷水平并增加 AMP 激活的蛋白激酶（AMPK）的激活。AMPK 抑制可消除而 AMPK 激活可增强 WFA 的作用。WFA 和 2-脱氧-D-葡萄糖联合对乳腺癌细胞产生协同抑制作用。BECN1 和 ATG7 的基因敲除不能使细胞从 WFA 处理中恢复；相反，添加丙酮酸甲酯以补充 TCA 循环可保护 WFA 处理的细胞。总之，这些结果表明 WFA 是一种有效的溶酶体抑制剂；WFA 诱导的细胞凋亡和生长抑制需要能量损伤，并且联合使用 WFA 和 2-DG 是一种有前景的乳腺癌治疗策略。
