噻唑烷类化合物抗肿瘤作用及其作用机制研究

In recent years, cancer is the leading category of cancer threatening the health of people in many countries. The emergence of drug resistance is a major challenge to the success of cancer chemotherapy. Although the drug resistance can be caused by various mechanisms, the ultimate failure of chemotherapy often results from multidrug resistance (MDR), a phenotype of cross-resistance to multiple drugs with diverse chemical structures. .The principle and technique of pharmacology, molecular cell biology and chemical genetics are used in this study. The overall objective of this proposal is to investigate the anti-cancer effects of new agents, thiazolidinone compounds (MMPT, YFZ-11, YFZ-22, YFZ-38 and YFZ-69), and the possible mechanism in inhibiting tumor cells growth. Firstly, the in vitro effects of thiazolidinone compounds (MMPT, YFZ-11, YFZ-22, YFZ-38 and YFZ-69), on cell viability and apoptosis of various cancer cells, including drug resistant cancer cells overexpressing P-glycoprotein, and wild-p53, mutant-p53 and null-p53 cancer cells, will be determined by sulforhodamine B (SRB) assay, H33258 stained assay, caspases activity assay, western blotting and so on. Together with these results, the antitumor activity and selectivity of thiazolidinone compounds (MMPT, YFZ-11, YFZ-22, YFZ-38 and YFZ-69) will be optimized by exploring the structure-activity relationship (SAR) in cancer cells, including chemoresistant cells. The compound, which is effective against cancer cells and sensitive inducing apoptosis in cancer cells, including chemoresistant cells but not in normal cells, will be screened and decided as lead compound, which antitumor effect is also P-glycoprotein and p53 status independent. Secondly, to test the in vivo effect of lead compound, we established subcutaneous A549 tumors in nude mice. Finally, the mechanism of lead compound is further discussed by using the technique of Real-time quantitative PCR, bioinformatics and RNA interference and so on. The goal of this proposal is to develop anticancer therapeutics that effectively treat refractory tumors but are largely nontoxic to normal cells. The hypothesis to be tested is that agents, which can induce cytotoxic effects in cancer cells, including resistant cancer cells, but not in normal cells, and whose antitumor activity does not depend on P-glycoprotein and p53 status, will be a new valuable way for developing safe, tumor-selective anticancer therapeutics.

近年来，癌症严重威胁人类的身体健康。多药耐药性是造成肿瘤化疗失败的主要原因。本研究运用药理学、分子细胞生物学和化学遗传学的原理技术方法，对（MMPT、YFZ-11、YFZ-22、YFZ-38及YFZ-69）5种噻唑烷类化合物的抗肿瘤作用和可能的作用机制进行研究。首先通过体外抑瘤检测(SRB检测)与凋亡诱导检测，结合结构-活性分析把抑瘤效果好且能有效诱导肿瘤细胞凋亡（不依赖P-gp和p53表型）的化合物确认为先导化合物。再建立裸鼠移植肿瘤模型检测先导化合物的体内抗肿瘤活性。最后通过荧光实时定量PCR、生物信息学和RNA干扰等技术进一步探讨先导化合物的作用机制。项目期望筛选出对耐药细胞株有效而对正常细胞株低毒的噻唑烷类化合物，其抗肿瘤活性不依赖P-gp和p53表型，为抗肿瘤药物筛选提供一个新思路。

肺癌是威胁人类健康的一大疾病，在全世界癌症死因中高居榜首。目前所用化疗药物在化疗初期效果明显，可有效诱导肺癌细胞凋亡，但毒副作用严重且易引起耐药，从而导致化疗失败。因此寻找高效、毒副作用小、能克服多药耐药且专门作用于肺腺癌细胞的化疗药物迫在眉睫。本研究运用药理学、分子细胞生物学和化学遗传学的原理技术方法，通过一系列体内外实验，对5种噻唑烷类化合物（YFZ-11、YFZ-22、YFZ-22A、 YFZ-38、 YFZ-69）的抗肿瘤作用和可能的作用机制进行研究。一方面，通过体外抑瘤检测(SRB 检测)与凋亡诱导检测，结合结构-活性分析，把抑瘤效果好且能有效诱导肿瘤细胞凋亡（不依赖 P-gp 和 p53 表型）的化合物确认为先导化合物。通过SRB法检测5种噻唑烷类化合物类型的对非小细胞肺癌细胞系（A549、H460、 H1792、 H226、A549/TaxR、H460/TaxR）以及正常的Wi-38、HELF细胞的生长抑制作用。发现5种噻唑烷化合物能够抑制多种肺癌细胞的生长，呈现了明显的时间剂量依赖性，且对正常细胞毒性比较小。通过多种凋亡检测方法，研究发现5种噻唑烷类化合物都能有效诱导细胞凋亡，并且多种凋亡途径共同参与、相互协调来完成，这将有效克服耐药问题。结合结构-活性分析，我们把抑瘤效果好且能有效诱导肿瘤细胞凋亡（不依赖 P-gp 和 p53 表型）的YFZ-22A确认为先导化合物。建立裸鼠移植肿瘤模型检测先导化合物的体内抗肿瘤活性。另一方面，探究ZYF-22A诱导细胞凋亡的机制。通过转录组测序、生物信息学和 RNA 干扰等技术进一步探讨先导化合物诱导细胞凋亡的作用机制。结果表明，多种Caspase参与了ZYF-22A诱导的细胞凋亡，由此推知，线粒体途径、膜受体途径与内质网途径共同参与了YFZ-22A诱导的A549细胞凋亡。项目筛选出的对耐药细胞株有效而对正常细胞株低毒的噻唑烷类化合物（YFZ-22A），其抗肿瘤活性不依赖 P-gp 和 p53 表型，为抗肿瘤药物筛选提供一个新思路，也可为化学遗传学、细胞生物学和药理学研究积累研究资料，推动这些学科的发展。