以降低AKR1C1表达为新靶标的抗小细胞肺癌候选药物分子机制研究

1， Small cell Lung cancer is mainly treated with chemotherapy since its high malignant degree which is hard to be treated with surgery operations. However, the medicine for chemotherapy is quite limited. Consequently, there is a great clinical significance to develop drugs with totally new mechanism. Wentilactone A is a new compound which is extracted from marine microorganism. it induces growth inhibition of small cell lung cancer cells in vivo and in vitro. Moreover, its effects is equal to cisplatin and even better than cyclophosphamide while the side effects is much less than cisplatin. Pre-clinical trials have all proved that it can be the anti-tumor drug candidate for small cell lung cancer. The result of gene chip suggests that the AKR1C1 mRNA expression of NCI-H446 cells could be decreased by 122 times after Wentilactone A treatment. Moreover the protein expression of AKR1C1 is also dcreased as follow, and overwxpression of AKR1C1 could reverse the effect of Wentilactone A. AKR1C1 is a Phase Ⅱ enzyme. The expression of AKR1C1 gene always maintains at high level in many cancers, which may related on drug resistance of these cancers. And recent reports suggested that high level of AKR1C1 decreased the expression of AIF, and promoted cell growth and migration. However, there is no dugs that can effectively decrease the expression of AKR1C1. This study will further approve that Wentilactone A could decrease the expression of AKR1C1 mainly through the Nrf2/ARE signalling pathway, thereby causing the cell apoptosis, cell growth inhibition and migration inhibitory of small cell lung cancer cells. This research will provide a new target for the chemotherapy of small cell lung cancer.

小细胞肺癌由于恶性程度高，难以手术治疗，主要以化疗为主，但目前化疗药物少且毒副作用大，开发毒副作用小的新型靶向性药物具有重大的临床意义。WA是从海洋微生物中分离的新化合物，前期研究证明其有针对性的抗小细胞肺癌作用，疗效与顺铂相当，但毒副作用远小于顺铂，初步完成了临床前研究，成药前景良好。全基因组表达芯片结果显示WA能使NCI-H446细胞的AKR1C1基因表达下降122倍，细胞中AKR1C1蛋白表达也明显下调，过表达该基因能逆转WA的作用。AKR1C1为Ⅱ相解毒酶基因，在多种肿瘤中高表达，不仅与肿瘤耐药有关，近年来有研究表明其高表达能抑制凋亡诱导因子生成，促进肿瘤细胞的生长和转移。目前临床尚无以降低AKR1C1表达为靶标的药物。本课题拟从分子水平探讨WA主要通过Nrf2/ARE途径靶向作用AKR1C1基因，从而引起细胞凋亡，抑制增殖和迁移，旨在为小细胞肺癌的化疗提供新的作用靶点和作用药物。

肺癌是我国死亡率最高的恶性肿瘤。小细胞肺癌（small cell lung cancer， SCLC）约占15%，其特点为癌细胞生长快，扩散转移早，恶性程度高。SCLC患者死亡率和复发率高，5年生存率不足10%，因此开发疗效好、副作用小的药物具有十分重大的意义。Wentilactone A (WA)是一种从海洋微生物次级代谢产物中分离得到的去甲二萜类小分子化合物。本课题通过检测WA对NCI-H1688、LTEP-sm等SCLC细胞株增殖及细胞凋亡的影响，并利用人全基因组表达谱芯片筛选等实验揭示WA抑制SCLC增殖的具体分子机制。体内体外实验结果显示WA显著抑制SCLC细胞增殖和迁移，诱导SCLC细胞凋亡，抑制SCLC细胞裸鼠移植瘤生长。生物信息学分析结果表明：凋亡通路、PI3K-AKT等信号通路相关基因的表达情况在WA处理前后有显著差异（P<0.05），其中变化最明显的基因是AKR1C1基因，下调94.74倍。AKR1C1基因干扰后，SCLC细胞凋亡率增加，裸鼠移植瘤生长速度变慢，与WA作用后的效果一致；相反，AKR1C1基因功能获得后裸鼠移植瘤生长速度变快，同时c-FLIP表达增加（P<0.05），c-caspase-3表达降低（P<0.05），凋亡率减少（P<0.05）。此外，WA处理后SCLC细胞IGF-1R/IRS-1/PI3K/AKT/Nrf2信号通路磷酸化水平受到抑制，同时AKR1C1表达量降低。IGF-1具有逆转WA诱导SCLC细胞株凋亡的作用。上述研究结果表明WA抑制IGF-1R/IRS-1/PI3K/AKT/Nrf2信号通路磷酸化，进而抑制AKR1C1基因的表达，诱导SCLC细胞株凋亡。本研究为小细胞肺癌药物开发提供了一个很有前景的前体化合物。项目资助发表核心论文3篇，SCI论文2篇。培养硕士研究生2名，博士研究生1名。项目投入经费65万，支出58.5万，各项支出基本与预算相符。剩余经费6.5万元。