双氢青蒿素抗结直肠癌及逆转5-FU耐药的分子机制

5-fluorouracil (5-FU) resistance is a major issue in treating malignant diseases. Identifying and developing novel chemotherapeutic agents with high effectiveness and low side-effects that can improve the clinical efficacy of 5-FU are still in great demand. Artemisinin, a well-known antimalarial drug, has recently been reported to possess potential anti-cancer activity. In our primary experiments, we demonstrated that dihydroartemisinin (DHA), a derivate of artemisinin, could efficiently inhibit proliferation of colorectal cancer cells, and synergistically enhanced the cytotoxic effect of 5-FU. Moreover, the 5-FU resistance in p53-deficient HCT116 cells could be reversed by DHA. On the basis of these results , further investigations will be performed in this project, including 1) take advantage of the cell lines and animal models, to elucidate the anti-colorectal cancer effect and action mechanisms of DHA; 2) evaluate the synergistic effect of DHA and 5-FU, and illuminate the mechanisms of their action, to access the suitability of DHA as a novel chemotherapeutic agent in the combined therapy; 3) analyze gene expression profiles modulated by the synergistic action of DHA and 5-FU, identify novel target genes and explore their functions, to further clarify the molecular mechanisms of 5-FU resistance. The findings of this project will shed new light on the underlying mechanism of 5-FU resistance, and will hopefully promote the development of DHA as a clinical anti-cancer drug in the combination therapy.

易产生耐药性是影响5-氟尿嘧啶(5-FU )临床总体疗效的重要因素，而筛选高效低毒的增敏剂对提高5-FU的疗效具有重要意义。青蒿素是有效的抗疟药之一，近年来还发现其具有很好的抗肿瘤作用。我们的研究发现，双氢青蒿素（DHA）能抑制结直肠癌细胞生长，与5-FU联合用药具显著的协同增效作用。在HCT116细胞上DHA能够逆转因p53基因缺失导致的5-FU耐药。在此基础上我们拟开展进一步的研究：1）从细胞和动物水平检测DHA对结直肠癌的抑瘤作用，阐明其分子机制；2）通过DHA与5-FU等联合用药，检测协同增效的作用，探讨其分子机制，为将DHA转化为化疗辅助新药提供依据；3)分析DHA逆转5-FU耐药产生过程中的基因表达变化，发现新靶点基因，研究其功能，以进一步探明5- FU耐药产生的分子机制。本研究结果不仅可以加深对5-FU耐药机制的理论认识，还具有把DHA开发成临床化疗辅助药物的应用价值。

本课题旨在探讨双氢青蒿素（DHA），一种一线抗疟药，在人类结直肠癌的抑癌作用及其机制，并探究其可逆转5-FU耐药的分子机制。我们首先在多种人类肠癌细胞株中证实了DHA的抑癌作用，并发现双氢青蒿素在多种结直肠癌中的IC50远低于另一类青蒿素衍生物青蒿琥酯。接下来，我们采用流式细胞仪对DHA作用后的肠癌细胞进行分析检测凋亡细胞及凋亡蛋白印记，证实了DHA对肠癌细胞的抑癌作用伴随着凋亡上调及细胞线粒体膜电位降低和细胞色素C的释放。这提示DHA可能通过调控线粒体的相关功能改变，从而产生对肠癌细胞的抑癌作用。下一步，我们发现在P53基因敲除的人肠癌HCT116细胞（HCT116 P53-/-）中，对比野生型HCT116细胞，传统化疗药物5-FU的抑癌作用大大降低，即对5-FU耐药。但在5-FU与DHA共同作用后，原本对5-FU耐药的HCT116 P53-/-的增殖再次被有效抑制。针对这个现象，我们在HCT116 P53-/-细胞中通过对活性氧簇（ROS）、凋亡细胞、BCL-2家族蛋白等的检测，初步阐明了DHA是通过调节BCL-2与BAX蛋白的表达，调节上调了ROS介导的细胞凋亡，从而提高了5-FU在HCT116 P53-/-细胞中的抑癌效果。最后，我们通过对转录组测序，对比了在HCT116 P53-/-细胞分别在5-FU单药与DHA、5-FU联用后的转录组表达改变，进行了生物信息学分析后，获得了差异表达谱，并筛选了新的靶点基因。结果表明，DHA通过促进ATF4的表达，引起ROS表达增加从而促进HCT116 P53 - / - 细胞凋亡，这可能是DHA与5FU联用逆转5FU耐药的作用机制。本研究证实了双氢青蒿素在人结直肠癌细胞中的逆转耐药作用，揭示了DHA作为化疗增敏剂的理论基础，为结直肠癌的联合化疗提供了新思路。