姜黄素通过抑制MMP-2 酶切TRAIL促进DcR1阳性肺腺癌凋亡

TRAIL choosed to induce specific and strong apoptosis in tumor cells. Although some tumors，including adenocarcinoma keep resistance to TRAIL-induced apoptosis，the benefits of its safety for normal cells droved people making effect to overcome the resistance. Functional death inducing signaling complex (DISC) would be assembled in lipid raft among cell membrane following TRAIL bounding with DR4 or DR5.But the decoy receptors DcR1 and DcR2 would attenuated cell death by occupying TRAIL and blocking DISC formation. An adenocarcinoma cells,A549 (with a phenotype of DR4+DR5+DcR1+ DcR2- ) keeps strong resistance to rhTRAIL or TRAIL gene transfection. Except DcR1, shedding mTRAIL from cell membrane by MMP-2 could also be responsible for this phenomenon. We found that lower concentration of curcumin could up-regulated TRAIL loading on cell membrane and promoted apoptosis induced by TRAIL gene transfection. Based on these, membrane-bound TRAIL was determined by flow cytometry. TRAIL and its receptors localized in lipid rafts would be analyzed by immunoprecipitation and laser scanning confocalmicroscope.The efficacy of curcumin to improve TRAIL-induced apoptosis and tumor extinction could also be investigated in animal experiment.The proposal has a solid preliminary studies,innovative ideas and reasonable design and methods. This study will provided a well theoretical foundation for develop new drug to promoted sensitivity of adenocarcinoma to TRAIL.

转染TRAIL基因可选择性、高效地诱导肿瘤细胞凋亡,但肺腺癌严重耐药。TRAIL高效、低毒的优势吸引我们不断探索增敏策略。TRAIL与功能性受体DR4、DR5在细胞膜脂筏内组装为死亡诱导信号复合物(DISC)；而诱骗受体DcR1和DcR2通过竞争结合TRAIL阻断凋亡。表型为DR4+DR5+DcR1+ DcR2-肺腺癌A549对TRAIL高度耐受，除DcR1外，MMP-2可能通过酶切跨膜型TRAIL的脱落而降低DISC组装，从而参与耐药。在发现低浓度姜黄素上调TRAIL 跨膜分布，并增强A549对TRAIL诱导凋亡敏感性的基础上，采用流式细胞术、免疫共沉淀等技术，结合动物实验来阐明姜黄素新的药理作用："通过抑制MMP-2 酶切TRAIL而促进DcR1阳性肺腺癌凋亡"。本项目前期准备扎实、设计新颖、研究方案合理，将为应用低浓度姜黄素联合TRAIL 抗肿瘤奠定理论基础。

肿瘤坏死因子相关凋亡配体( tumor necrosis factor related apoptosis inducing ligand, TRAIL ) 选择性、高效地诱导多种肿瘤细胞凋亡,重组人TRAIL蛋白（recombinate human TRAIL, rhTRAIL）治疗肿瘤已经完成临床研究。肺腺癌为高发肿瘤, 80%以上肺腺癌DR4和DR5为阳性，但诱骗受体DcR1阳性率也在60%以上 。对肺腺癌TRAIL耐受机制和姜黄素增敏作用的研究，如下问题尚需要深入探讨：①与截短rhTRAIL 比较，全长rhTRAIL是否可克服在DcR1+肿瘤的耐受？②低浓度姜黄素是否可增强DcR1+肿瘤对TRAIL的敏感性？③ROS和脂筏是否可影响两者联合诱导凋亡的效应？前期研究发现低浓度的姜黄素可促进转染TRAIL基因NK细胞诱导结肠癌和白血病细胞凋亡效应。在此基础上深入研究，发现低浓度姜黄素增强表型TRAIL+DR5+DcR1+HSP70high 的肺腺癌A549细胞对TRAIL的敏感性。低浓度姜黄素也可增强DcR1低表达肺腺癌H460细胞对截短rhTRAIL的敏感性；对DcR1高表达细胞，姜黄素与全长的rhTRAIL联合诱导凋亡效应更强。低浓度姜黄素可拮抗MMP-2对A549和NK细胞mTRAIL的脱落作用；姜黄素与全长rhTRAIL联合，还可通过促发活性氧、上调DR5表达、及诱导线粒体途径凋亡而增强DcR1+ 肺腺癌对TRAIL的敏感性。研究发现，姜黄素对mTRAIL表达、DR5表达和促进凋亡效应均与浓度相关，7.5μmol/L姜黄素可上调TRAIL表达，增强脂筏依赖的细胞凋亡；但高浓度姜黄素（≥40μmol/L）无上述效应。不同类型肿瘤对TRAIL的耐受机制不同，研究中还针对脑肿瘤干细胞对TRAIL耐受机制进行探讨，为继续深入研究姜黄素的增敏作用奠定基础。初步探讨了姜黄素对极化单核巨噬细胞ROS和表型的影响，为今后模拟肺腺癌在非可控炎症微环境中对TRAIL治疗的反应建立基础。