Aims

In tumor cells, shikonin treatment has been reported to inhibit glycolysis by suppressing the activity of pyruvate kinase M2 (PKM2) and to induce apoptosis by increasing reactive oxygen species (ROS) production. However, hepatocellular carcinoma (HCC) shows variable sensitivity to shikonin treatment, and the mechanism for these differences remains unclear. We evaluated the effects of shikonin on metabolic and oxidative pathways in sensitive and refractory HCC cell lines to identify mechanisms of differential sensitivity.

Main methods

Cell viability and apoptosis were evaluated by MTT assay, PI/Annexin V and JC-1 staining. Mitochondrial function was further evaluated by measurements of ROS and mitochondrial mass. Oxygen consumption rates, NAD⁺/NADH, ATP and lactate were measured as indicators of energy metabolism and glycolysis. Protein expression associated with glycolysis and apoptosis was evaluated by western blotting, RT-qPCR and immunofluorescence staining.

Key findings

The sensitivity to shikonin treatment was significantly higher for HepG2 cells than for HCCLM3 cells, with less dramatic effects in HCCLM3 cells on apoptosis, ROS, and oxidative phosphorylation. Shikonin up-regulated mitochondrial biogenesis to increase mitochondrial oxidative phosphorylation in HepG2 cells, but displayed the opposite trend in HCCLM3 cells. Mechanistically, shikonin promoted nuclear expression of PKM2 and HIF1α in HCCLM3 cells, with upregulation of glycolysis-related gene transcription and glycolysis.

Significance

These results suggest that PKM2 rewires glucose metabolism, which explains the differential sensitivity to shikonin-induced apoptosis in HCC cells. Our findings elucidate mechanisms for differential responses to shikonin, provide potential biomarkers, and indicate a theoretical basis for targeting glycolytic enzymes in refractory HCC.

中文翻译：

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紫草素通过 PKM2 和 HIF1α 差异调节葡萄糖代谢，以克服难治性 HCC 细胞系中的细胞凋亡

 目标

据报道，在肿瘤细胞中，紫草素治疗可通过抑制丙酮酸激酶 M2 (PKM2) 的活性来抑制糖酵解，并通过增加活性氧 (ROS) 的产生来诱导细胞凋亡。然而，肝细胞癌（HCC）对紫草素治疗表现出不同的敏感性，并且这些差异的机制仍不清楚。我们评估了紫草素对敏感和难治性 HCC 细胞系代谢和氧化途径的影响，以确定差异敏感性的机制。

 主要方法

通过MTT测定、PI/Annexin V和JC-1染色评估细胞活力和凋亡。通过测量 ROS 和线粒体质量进一步评估线粒体功能。测量耗氧率、NAD ⁺ /NADH、ATP和乳酸作为能量代谢和糖酵解的指标。通过蛋白质印迹、RT-qPCR 和免疫荧光染色评估与糖酵解和细胞凋亡相关的蛋白表达。

 主要发现

HepG2 细胞对紫草素处理的敏感性显着高于 HCCLM3 细胞，而 HCCLM3 细胞对细胞凋亡、ROS 和氧化磷酸化的影响较小。紫草素在 HepG2 细胞中上调线粒体生物合成，增加线粒体氧化磷酸化，但在 HCCLM3 细胞中表现出相反的趋势。从机制上讲，紫草素促进 HCCLM3 细胞中 PKM2 和 HIF1α 的核表达，上调糖酵解相关基因的转录和糖酵解。

 意义

这些结果表明 PKM2 重新连接葡萄糖代谢，这解释了 HCC 细胞对紫草素诱导的细胞凋亡的不同敏感性。我们的研究结果阐明了紫草素差异反应的机制，提供了潜在的生物标志物，并为针对难治性 HCC 中的糖酵解酶提供了理论基础。