双氢青蒿素干预糖酵解关键酶组增强gefitinib治疗肺腺癌敏感性的机制研究

Lung adencarcinoma cancer has a globally high morbidity rate in Yunnan Province. The approach to treatment is often late, and outcomes are suboptimal with traditional treatment. Molecular targeted therapy can promote the prognosis of patients. Gefitinib is the main target of NSCLC, and acquired drug resistance often occurred at 12-18 months after the treatment. We found that DHA can enhance the sensitivity of lung adenocarcinoma cell lines to Gefitinib in vivo and in vitro. Combined with DHA antimalarial mechanism and bioinformatics analysis, further study found that DHA can inhibit the activity of glycolysis key enzymes PKM2 and LDHA, thus reducing the ATP and lactic acid production. The knock-down of PKM2 could enhance the sensitivity of Gefitinib to lung adenocarcinoma cells and increase the affinity between Gefitinib and TK. Therefore, we propose the potential mechanism of how DHA increase the Gefitinib sensitivity, that DHA inhibits the activities of PKM2 and LDHA in the key enzymes of glycolysis, enhances the affinity between Gefitinib and TK, and blocks EGFR downstream pathways, then the sensitivity enhanced. In this study, we are going to use methodologies like enzyme activity assays, to obtain the solid evidence that DHA increases the Gefitinib treatment sensitivity by interfering with key enzymes of glycolysis, and to provide the scientific basis for the sensitization effect of Artemisinin drugs.

云南肺腺癌全球高发，就诊时多处晚期，传统治疗获益较少，分子靶向治疗可延长患者预后。gefitinib是肺腺癌的主要靶点，多在治疗12-18月后发生获得性耐药。我们研究发现DHA能在体内外增加肺腺癌细胞系对gefitinib敏感性。结合DHA抗疟机制及生物信息学分析，进一步研究发现，DHA可抑制糖酵解关键酶PKM2和LDHA活性，降低ATP及乳酸产量。而敲减PKM2可增强肺腺癌细胞对gefitinib的敏感性，增加gefitinib和TK之间的亲和力。由此，我们提出DHA增加gefitinib敏感性可能的作用机制：DHA抑制糖酵解关键酶PKM2和LDHA活性，抑制糖酵解，阻断EGFR下游通路，增加gefitinib敏感性。本项目拟进一步采用酶活性检测等技术，旨在获得DHA干预糖酵解关键酶组增强gefitinib敏感性治疗肺腺癌的证据，为青蒿类药物增敏提供科学依据。

云南宣威地区肺腺癌全球高发，且就诊时多处于晚期，失去手术机会。晚期患者从传统的化疗和放疗中获益较少，分子靶向治疗是其主要手段。Gefitinib作为 EGFR-TKI，可显著延长 NSCLC 患者生存期，但大部分患者在治疗 12-18月后对 gefitinib 的敏感性降低，发生获得性耐药，寻找增敏剂显得尤为重要。. 前期研究中，课题组发现DHA可增强gefitinib对耐药突变细胞株 H1975（EGFR L858R+T790M）和敏感突变株 H3255（EGFR L858R）的抑制作用及克隆形成能力，分别使H1975（ EGFR L858R+T790M）对gefitinib的敏感性增加4.15倍，H3255（EGFR L858R）对gefitinib的敏感性增强6.93倍，结合既往青蒿素抗疟机制研究的最大突破（可共价结合疟原虫的丙酮酸激酶（pfPKM）和乳酸脱氢酶（pfLDH），抑制糖酵解）及我们进一步机制研究发现，双氢青蒿素干预糖酵解关键酶 PKM2和LDHA活性，并不影响其蛋白表达，抑制糖酵解，增强 gefitinib与TK之间的亲和性。裸鼠成瘤实验结果表明，DHA联合gefitinib可在体内显著抑制耐药突变细胞株 H1975（EGFR L858R+T790M）和敏感突变株 H3255（EGFR L858R）在裸鼠体内的生长及瘤体湿重。更为重要的是，IHC结果显示gefitinib耐药患者PKM2或LDHA的表达明显高于gefitinib敏感的患者，生存分析软件提示PKM2或LDHA高表达患者总生存期明显差于低表达的患者。. 通过本项目研究，初步证实DHA 可抑制肿瘤细胞糖酵解关键酶PKM2和LDHA活性，抑制糖酵解，增强 gefitinib 与TK之间的亲和性，增强肺腺癌细胞对 gefitinib 的敏感性，为青蒿素类药物增加EGFR-TKI敏感性提供可靠证据，进一步为临床推广提供充分的实验依据。通过本项目研究，发表SCI学术论文3篇，中文科技核心期刊论文2篇，云南科技进步奖三等奖1项，云南省卫生科技成果奖1项，授权实用新型专利2项，软件著作权1项。