Shikonin inhibited glycolysis and sensitized cisplatin treatment in non-small cell lung cancer cells via the exosomal pyruvate kinase M2 pathway

The active ingredient of the traditional Chinese medicine comfrey is shikonin, a naphthoquinone compound. The focus of this study was to investigate the effect of shikonin on the proliferation, invasion, migration, and chemoresistance of non-small cell lung cancer (NSCLC) cells, and to explore its underlying molecular biological mechanisms. The results show that shikonin inhibited the viability, proliferation, invasion, and migration of NSCLC cells A549 and PC9, and induced apoptosis. As the inhibitor of pyruvate kinase M2 (PKM2), a key enzyme in glycolysis, shikonin inhibited glucose uptake and the production of lactate, the final metabolite of aerobic glycolysis. <i>In vivo</i> chemotherapeutic assay showed that shikonin reduced the tumor volume and weight in NSCLC mice model and increased the sensitivity to cisplatin chemotherapy. Histoimmunology experiments showed the combination of shikonin and cisplatin downregulated the expression of PKM2 and its transcriptionally regulated downstream gene glucose transporter 1 (Glut1) in tumor tissue. In an assessment of glucose metabolism, micro-PET/CT data showed a combination of shikonin and cisplatin inhibited the fluorodeoxy glucose (¹⁸F-FDG) uptake into tumor. Since exosomal PKM2 affected the sensitivity to cisplatin in NSCLC cells, we also demonstrated shikonin could inhibit exosome secretion and exosomal PKM2 through the administration of exosomal inhibitor GW4869. Furthermore, shikonin sensitized cisplatin treatment by reducing the extracellular secretion of exosomal PKM2. In conclusion, we suggest that shikonin not only inhibits PKM2 intracellularly but also reduces glycolytic flux and increases cisplatin sensitivity through the exosomal pathway.

中药紫草的活性成分为紫草素（shikonin），一种萘醌类化合物（naphthoquinone compound）。本研究聚焦于探讨紫草素对非小细胞肺癌（non-small cell lung cancer, NSCLC）细胞增殖、侵袭、迁移及化疗耐药性的影响，并探索其潜在的分子生物学机制。研究结果显示，紫草素可抑制A549与PC9两种非小细胞肺癌细胞的活力、增殖、侵袭与迁移，并诱导细胞凋亡。作为糖酵解（glycolysis）关键酶丙酮酸激酶M2（pyruvate kinase M2, PKM2）的抑制剂，紫草素可抑制葡萄糖摄取以及有氧糖酵解（aerobic glycolysis）的终产物乳酸的生成。体内（in vivo）化疗实验结果表明，紫草素可缩小非小细胞肺癌小鼠模型的肿瘤体积与重量，并增强其对顺铂（cisplatin）化疗的敏感性。组织免疫学实验显示，紫草素与顺铂联合使用可下调肿瘤组织中PKM2及其转录调控的下游基因葡萄糖转运蛋白1（glucose transporter 1, Glut1）的表达水平。在葡萄糖代谢评估中，微型正电子发射计算机断层扫描/计算机断层扫描（micro-PET/CT）数据显示，紫草素与顺铂联合给药可抑制肿瘤对氟代脱氧葡萄糖（fluorodeoxy glucose, ¹⁸F-FDG）的摄取。鉴于外泌体（exosome）包裹的PKM2可影响非小细胞肺癌细胞对顺铂的敏感性，我们还通过外泌体抑制剂GW4869处理证实，紫草素可抑制外泌体分泌以及外泌体中PKM2的含量。此外，紫草素可通过减少外泌体PKM2的胞外分泌增敏顺铂治疗。综上，我们认为紫草素不仅可在细胞内抑制PKM2活性，还可通过外泌体通路减少糖酵解通量并增强顺铂的化疗敏感性。