College of Basic Medicine, Hebei University, Baoding, Hebei, 071000, China.
Key Laboratory of Pathogenesis Mechanism and Control of Inflammatory-autoimmune Diseases in Hebei Province, Hebei University, Baoding, Hebei, 071000, China.
Curr Cancer Drug Targets. 2024;24(5):534-545. doi: 10.2174/0115680096262915231026050602.
The energy supply of certain cancer cells depends on aerobic glycolysis rather than oxidative phosphorylation. Our previous studies have shown that withaferin A (WA), a lactone compound derived from , suppresses skin carcinogenesis at least partially by stabilizing IDH1 and promoting oxidative phosphorylation. Here, we have extended our studies to evaluate the anti-tumor effect of WA in liver cancer.
Differential expression of glycolysis-related genes between liver cancer tissues and normal tissues and prognosis were verified using an online database. Glycolysis-related protein expression was detected using western blot after overexpression and knockdown of IDH1 and mitochondrial membrane potential assay based on JC-1, and mitochondrial complex I activity was also detected. The inhibitory effect of WA on the biological functions of HepG2 cells was detected along with cell viability using MTT assay, scratch assay, clone formation assay, glucose consumption and lactate production assay. Western blot and qRT-PCR were used to detect the expression of proteins and genes related to IDH1, p53 and HIF1α signaling pathways.
We first identified that IDH1 expression was downregulated in human liver cancer cells compared to normal liver cells. Next, we found that treatment of HepG2 cells with WA resulted in significantly increased protein levels of IDH1, accompanied by decreased levels of several glycolytic enzymes. Furthermore, we found that WA stabilized IDH1 proteins by inhibiting the degradation by the proteasome. The tumor suppressor p53 was also upregulated by WA treatment, which played a critical role in the upregulation of IDH1 and downregulation of the glycolysis-related genes. Under hypoxic conditions, glycolysis-related genes were induced, which was suppressed by WA treatment, and IDH1 expression was still maintained at higher levels under hypoxia.
Taken together, our results indicated that WA suppresses liver cancer tumorigenesis by p53-mediated IDH1 upregulation, which promotes mitochondrial respiration, thereby inhibiting the HIF-1α pathway and blocking aerobic glycolysis.
某些癌细胞的能量供应依赖于有氧糖酵解而非氧化磷酸化。我们之前的研究表明，从印度大麻中提取的内酯化合物醉茄素 A（WA）通过稳定 IDH1 和促进氧化磷酸化，至少部分抑制皮肤癌的发生。在此，我们将研究扩展到评估 WA 在肝癌中的抗肿瘤作用。
使用在线数据库验证肝癌组织与正常组织中糖酵解相关基因的差异表达及其与预后的关系。通过 IDH1 的过表达和敲低后检测糖酵解相关蛋白的表达，使用 JC-1 进行线粒体膜电位测定，还检测了线粒体复合物 I 的活性。通过 MTT 测定、划痕实验、克隆形成实验、葡萄糖消耗和乳酸生成实验检测 WA 对 HepG2 细胞生物学功能的抑制作用。Western blot 和 qRT-PCR 用于检测与 IDH1、p53 和 HIF1α 信号通路相关的蛋白和基因的表达。
我们首先发现与正常肝细胞相比，IDH1 在人肝癌细胞中的表达下调。接下来，我们发现 WA 处理 HepG2 细胞导致 IDH1 蛋白水平显著升高，同时几种糖酵解酶的水平降低。此外，我们发现 WA 通过抑制蛋白酶体降解来稳定 IDH1 蛋白。WA 处理还上调了肿瘤抑制因子 p53，这对于 IDH1 的上调和糖酵解相关基因的下调起着关键作用。在缺氧条件下，诱导糖酵解相关基因，WA 处理可抑制该诱导作用，同时 IDH1 表达在缺氧条件下仍维持在较高水平。
综上所述，我们的研究结果表明，WA 通过 p53 介导的 IDH1 上调抑制肝癌肿瘤发生，从而促进线粒体呼吸，抑制 HIF-1α 通路并阻断有氧糖酵解。
