基于缝隙连接蛋白靶点的青蒿素B对顺铂抗肺癌的增效减毒作用机制研究

Artemisinin B is one kind of anti-tumor active ingredient from Artemisia annua. Connexin (Cx) possesses tumor suppressor properties and can be a great potential target for cancer therapy. Cisplatin (DDP) is an antitumor drug in first-line therapy for cancer and can be differentially accumulated in tumor and non-tumor cells, which may be mediated by Cx. DDP has a strong cytotoxic effect on cancer cells, but severe side effects limit its clinical application. In the previous study, we found that Artemisinin B increased the cytotoxicity of DDP, up-regulated expression of Cx and enhanced the function of gap junction (GJ) in lung cancer cells, suggesting that it may have the synergism and detoxication effects in the treatment of lung cancer. To explore of the mechanism of synergism and detoxication effects of Artemisinin B in combination with DDP, the studies will be carried out as follows: (1) Evaluating synergism and detoxication effects of Artemisinin B in combination with DDP; (2) Further investigating if the mechanism involves Cx, GJ and other target proteins; (3) Exploring the signaling pathways involved in the regulation of GJ and Cx by Artemisinin B. The project aims to investigate the synergism and detoxication effects of Artemisinin B and to explore the underlying mechanism associate with Cx. The research findings can provide a scientific interpretation of the synergism and detoxication effects of Artemisinin B in combination with DDP, as well as the scientific information for the new application of artemisinins.

青蒿素B系中药黄花蒿中抗肿瘤活性成分。缝隙连接蛋白（Cx）具肿瘤抑制因子特性，能介导顺铂在肿瘤和非肿瘤细胞中的差异性积累，是极具潜力的抗肿瘤靶点。一线抗肿瘤药物顺铂对肿瘤细胞有着强大杀伤作用但严重的毒副作用成为限制其应用的瓶颈问题。我们前期研究发现：青蒿素B能协同顺铂抑制肺癌细胞的增殖且能改变肺癌细胞中Cx的表达及缝隙连接（GJ）功能，提示其对顺铂抗肺癌方面可能具有“增效减毒”作用且与Cx有关。为解决此科学问题，本项目拟在前期工作基础上，进行如下研究：（1）全面评价青蒿素B对顺铂抗肺癌的“增效减毒”作用；（2）确认青蒿素B“增效减毒”作用是否由Cx介导；（3）探究青蒿素B对Cx及GJ调控的相关信号通路。本项目系国内外首次基于Cx靶点对青蒿素B在抗肿瘤方面“增效减毒”作用机制进行研究，其结果可为青蒿素B的“增效减毒”作用给予科学阐释，并为拓展青蒿素类活性成分新用途提供科学资料。

青蒿素B（代号YA-2）系中药黄花蒿中抗肿瘤活性成分。缝隙连接蛋白（Cx）具有肿瘤抑制因子特性，是极具潜力的抗肿瘤作用靶点。一线抗肿瘤药物顺铂（DDP）对肿瘤细胞有强大杀伤作用，但严重的毒副作用和耐药性成为限制其应用的瓶颈问题。我们前期研究发现：青蒿素B能协同DDP抑制肺癌细胞增殖且能增强Cx的表达及缝隙连接（GJ）功能。本项目主要研究内容包括评价青蒿素B对顺铂抗肺癌的“增效减毒”作用并基于Cx靶点研究其作用机制。主要研究结果如下：（1）YA-2与DDP单独作用于人肺癌A549细胞72h后均呈增殖抑制活性，IC50值分别为24.06±13.74µM及2.49±1.24µM；二者联合应用具有相加及协同作用，Q值在0.85≤Q≤1.15及Q>1.15的范围之内；YA-2（6.25μM）与DDP（2.5μM）联用时比单独用药诱导细胞凋亡的作用更优；（2）YA-2作用A549细胞48h后可明显增强细胞间GJ功能，可促进A549细胞中Cx43总蛋白及膜蛋白的表达，Cx43的mRNA水平明显提高；（3）体内实验结果显示：YA-2（50mg/kg）协同DDP（3mg/kg）抑制A549细胞和顺铂耐药株A549/DDP细胞裸鼠移植瘤的生长，且高表达Cx43能够增加细胞对DDP的敏感性；（4）YA-2协同DDP抑制四株非小细胞肺癌（NSCLC）A549、HCC827、H1299和H460细胞的增殖和转移，促进凋亡，其机制与促进Cx43表达，激活MAPK（P38和JNK）/P53信号通路有关；（5）YA-2协同DDP抑制顺铂耐药株A549/DDP细胞增殖和转移，促进凋亡，其机制与促进Cx43蛋白表达，抑制MEK1/ERK信号通路，激活P53通路有关；（6）YA-2协同DDP抑制NSCLC的作用机制与激活Cx43介导的GJ通路有关，其机制为：YA-2促进Cx43的表达，使Cx43介导的GJ通路数量增加，同时促进Cx43蛋白C末端去磷酸化从而激活GJ通路；YA-2促进细胞内Fe3+转化成Fe2+，从而使细胞外Ca2+内流增加，导致GJ通路开放。本项目系国内外首次基于Cx靶点研究青蒿素B对顺铂抗肺癌的“增效减毒”作用机制，该研究结果可为青蒿素B作为创新药物的研发提供科学依据，并为拓展青蒿素类活性成分新用途提供科学资料。