PI3K/AKT/FOXO3a信号通路对结直肠癌细胞持续低剂量率放射敏感性的贡献及机制

The clinical benefits of image-guided radioactive implantation for recurrent rectal carcinoma had been convinced, while basic research of biological effect and associated molecular mechanisms after continuous low dose rate irradiation on colorectal cancer was absent. It was convinced that activation of PI3K/AKT/FOXO3a pathway could start cell self-protection response, decrease radiosensitivity of tumor cells and reduce treatment effect. Our early research found that continuous low dose rate irradiation could decrease the expression level of AKT and downstream proteins regulating the process of DNA repair and apoptosis. It is premised that continuous low dose rate irradiation might impact radiosensitivity of colorectal cancer cell via PI3K/AKT/FOXO3a pathway. Our purpose is to observe the expression level of key proteins of PI3K/AKT/FOXO3a pathway which might impact the procession of cell proliferation and survival, after treatment of PI3K agonist IGF-1, PI3K inhibitor LY294002, RNAi down-regulate FOXO3a and continuous low dose rate irradiation. The target proteins include PI3K, FOXO3a, p27, p21, CyclinB1 and CyclinD1. Our research want to clarify the role of PI3K/AKT pathway in continuous low dose rate irradiation, find out key point of the pathway, and do some exploration for tumor cell regulation network after continuous low dose rate irradiation, and provide necessary basic research evidence for clinical use of 125I radioactive seed.

放射性粒子植入治疗局部复发直肠癌临床应用前景良好。我们前期研究发现持续低剂量率照射对结直肠癌细胞具有较强的杀伤作用，伴有AKT表达下降。我们推测PI3K/AKT/FOXO3a通路可能参与调控结直肠癌细胞对持续低剂量率照射的敏感性。本项目拟在此基础上观察持续低剂量率照射后PI3K/AKT/FOXO3a通路中PI3K、FOXO3a、p27、p21、CyclinB1和CyclinD1等影响细胞增殖和凋亡蛋白表达量的改变。观察特异性激动、抑制PI3K表达，以及RNAi干涉技术敲低FOXO3a后，结直肠癌CL187细胞放射敏感性的变化，进行深入、系统的分子机制研究，论证该通路对结直肠癌细胞持续低剂量率照射敏感性的贡献。此研究有助于阐明PI3K/AKT/FOXO3a通路在持续低剂量率照射中的地位，为筛选和确定持续低剂量率照射治疗局部复发直肠癌患者的潜在获益人群提供必要的基础实验依据。

放射性粒子植入治疗局部复发直肠癌临床应用前景良好。我们前期研究发现持续低剂量率照射对结直肠癌细胞具有较强的杀伤作用，伴有AKT表达下降。我们推测PI3K/AKT/FOXO3a通路可能参与调控结直肠癌细胞对持续低剂量率照射的敏感性。本研究探索125I粒子持续低剂量率照射诱导肿瘤细胞新的死亡方式有利于明确125I粒子持续性低剂量率照射发挥抗癌活性的分子机制。研究发现125I粒子持续性低剂量率照射诱导保护性自噬和类凋亡样死亡。碘125粒子射线照射后LC3-II/ LC3-I比例随照射剂量增加而升高，加入氯喹比率进一步增高。照射后，LC3在胞浆中呈点状分布，且随照射剂量增高点状分布增多；LC3与线粒体蛋白Tim23共定位，氯喹处理和碘125粒子射线照射后LC3与线粒体蛋白Tim23共定位增多，出现大量线粒体自噬囊泡。照射后线粒体膜电位下降47%，线粒体ATP生成降低36%，线粒体活性氧水平上调123%。深入研究结果提示125I粒子持续性低剂量率照射损伤HCT116细胞线粒体引起线粒体ROS增高，高水平的线粒体活性氧上调HIF-1α及下游靶基因BINP3和NIX的表达，并触发保护性线粒体自噬保护HCT116细胞免于凋亡。125I粒子持续性低剂量率照射后部分HCT116细胞胞浆中出现大量圆形的空泡，免疫荧光检测发现这些空泡来自于肿胀的内质网。25I粒子持续性低剂量率照射活化PI3K/AKT信号通路，引起AIP1蛋白表达下降，放线菌酮抑制蛋白的合成后125I粒子持续性低剂量率照射诱导的胞浆空泡化减少。深入研究表明Ly294002抑制PI3K/AKT信号通路的活化后125I粒子持续性低剂量率照射诱导的类凋亡样细胞死亡下降。这些研究结果提示PI3K/AKT信号通路在125I粒子持续性低剂量率照射诱导HCT116细胞类凋亡中起重要作用。