内质网应激介导刺芒柄花素逆转结肠癌耐药机制的研究

In the past 30 years, the chemotherapy in colorectal cancer have been developed rapidly, however, five year survival rate of patients with ⅢC、IV stage is only 44.3% and 8.1％ respectively. One of the main reasons for the chemotherapy failure is multidrug resistance to chemotherapeutic drugs after chemotherapy. Therefore, It is meaningful to Look for safe and effective drug to minus chemotherapy poison and enhance chemotherapy sensitivity. The research group previously found that formononetin can reverse 5-FU resistance, and endoplasmic reticulum is in a state of stress when these two drugs used together. CHOP plays an important role in endoplasmic reticulum stress reactive apoptosis, while PERK-eIF2α- ATF4 pathway plays a key role in raising CHOP protein expression. This study focus on formononetin regulating expression of colon cancer 5-FU-resistance cell CHOP, pointing at PERK mediated signalling pathways, constructing recombinant CHOP Lentivirus-mediated RNA interference, transfecting colon cancer 5-FU-resistant cell,in order to verify the dependence of formononetin in reversing the drug- resistant endoplasmic reticulum stress.

过去30年结肠癌化疗方案经历了不断的发展，但ⅢC、IV期结肠癌五年生存率分别仅为44.3%和8.1％。化疗后肿瘤对化疗药物产生多药耐药是导致化疗失败的主要原因之一。因此，寻找安全有效的药物对现有的化疗药物减毒增敏或逆转多药耐药具有重要意义。本课题组前期发现，刺芒柄花素能逆转结肠癌5-氟尿嘧啶（5-FU）耐药，两药联用时内质网处于应激状态，CHOP在内质网应激反应性凋亡中具有重要作用，PERK-eIF2α- ATF4通路在上调CHOP蛋白表达中起关键作用。本研究将围绕刺芒柄花素对结肠癌5-FU耐药细胞CHOP表达的调控，针对PERK介导的信号通路，构建重组CHOP RNAi慢病毒，转染结肠癌5-FU耐药细胞，验证刺芒柄花素逆转耐药内质网应激的依赖性。

目的：阐明刺芒柄花素逆转肿瘤耐药的作用机制，尤其是内质网应激在对肿瘤耐药逆转中的作用，明确其逆转细胞耐药的作用靶点和途径。方法：建立结肠癌耐药株，通过CCK-8检测刺芒柄花素与5-FU协同效应，流式细胞术检测刺芒柄花素干预后耐药细胞的凋亡情况，WB分析刺芒柄花素干预后耐药细胞株凋亡途径PERK-ATF4-CHOP通路各蛋白表达水平；ER Tracker染色观察药物干预前后细胞内质网功能变化；蛋白芯片高通量分析胞内Ser/Thr和Tyr激酶的磷酸化；通过建立CHOP-RNAi Lentivirus的结肠癌耐药细胞，重复验证。结果：CCK8结果：刺芒柄花素协同5-FU可显著抑制耐药细胞、5979、NC耐药细胞的生长；流式细胞术结果：刺芒柄花素协同5-FU可促进耐药细胞的凋亡；Western Blot结果：耐药细胞经药物干预24h后，协同组elf2α、P-elf2α、ATF4、CHOP、Caspase3、PERK蛋白表达量均有明显增加，MRP蛋白表达量明显减少；5979耐药细胞协同组PERK、P-PERK、eif2α、P-eif2α、ATF4、Caspase3蛋白表达量均有明显增加，MAP蛋白表达量有明显降低，除P-eif2α、Caspase3外（P<0.05），NC细胞协同组PERK、P-PERK、eif2α、P-eif2α、ATF4、CHOP、Caspase3蛋白表达量均有明显增加，MAP蛋白表达量稍有增加，除eif2α、Caspase3、MRP外（P<0.05）；体内实验显示，协同组elf2α、P-elf2α、ATF4、CHOP、Caspase3、PERK蛋白表达量均有明显增加；ER Tracker染色结果：经刺芒柄花素干预后，ER Tracker标记的内质网在耐药细胞、5979细胞、NC细胞内呈现出高亮度的红色斑块；蛋白芯片结果：刺芒柄花素逆转结肠癌耐药可能通过MAPK、VEGF、ErbB及TCR信号通路发挥作用。结论：刺芒柄花素协同5-FU能显著逆转人结肠癌耐药细胞的耐药性；刺芒柄花素逆转人结肠癌耐药的作用可能是通过诱导细胞凋亡实现；PERK-elf2α-ATF4通路的活化可能是介导刺芒柄花素逆转耐药过程的重要分子机制。