贝伐单抗联合SN-38抑制结肠癌细胞增殖的机制

Combination with SN-38 on human colon cancer LoVo cells

目的探讨贝伐单抗联合伊立替康（CPT-11）的活性代谢产物SN-38对结肠癌细胞增殖的抑制作用及其机制。方法在缺氧条件下，将贝伐单抗和sN-38作用于人结肠腺癌细胞LoVo，应用四甲基偶氮唑蓝（MTT）法，检测药物对细胞增殖的抑制作用。分别采用逆转录聚合酶链反应（RT—PCR）、Western blot和酶联免疫吸附试验（ELISA），分析药物作用后缺氧诱导因子1α（HIF-1α）及血管内皮生长因子（VEGF）在基因和蛋白表达水平的变化；通过药物对丝裂原激活蛋白激酶（MAPK）和磷脂酰三磷酸肌醇-丝苏氨酸蛋白激酶（P13K-AKT）通路的影响，分析贝伐单抗和sN-38的联合作用机制。结果贝伐单抗联合sN-38序贯给药方案较同时给药对细胞增殖的抑制更强，且以先sN-38后贝伐单抗的给药顺序作用最强，IC50值为（0．057±0．009）μmol／L，较相反给药顺序强3．8倍，较同时给药强8．9倍。HIF—1α的表达水平随着缺氧时间的延长而升高，贝伐单抗和sN-38同时作用可显著抑制其表达；VEGF的表达也随着缺氧时间的增加逐渐增加，SN-38作用12h以上可使VEGF表达下降59．4％，200μg/ml的贝伐单抗作用8h几乎可完全抑制VEGF表达，此时加入SN-38仍可使VEGF表达进一步降低。MAPK通路的p-ERK表达随缺氧时间的增加而升高，sN-38和贝伐单抗同时作用可使p-ERK的表达完全阻断，这与两药对HIF-1α的抑制是一致的。ERK的选择性抑制剂PD98059能够增强贝伐单抗对p-ERK的抑制，同时也增加了贝伐单抗对HIF-1α的抑制。p-AKT的表达虽然随着缺氧时间的延长有所升高，但贝伐单抗和sN-38对其影响并不显著。结论VEGF靶向药物联合化疗药物对细胞增殖的抑制是方案依赖性的，贝伐单抗和sN-38通过对HIF-1α和MAPK通路的调控发挥协同作用。

Objective To observe the anti-proliferation effect of bevacizumab and SN-38 （active metabolite of irinotecan） , and investigate the possible mechanisms of these two agents. Methods Human colon cancer LoVo cells were cultured under hypoxic conditions. Inhibition of cell proliferation was evaluated by MTT assay. The drug modulation on HIF-1α, VEGF, ERK and AKT were assessed by the following assays. The mRNA expression of HIF-1α and VEGF were measured by RT-PCR. The protein expression of HIF-1α, ERK and AKT were evaluated by Western blot analysis, and VEGF by ELISA assay. Results Among different combination schedules, Bevacizumab given after SN-38 show most synergistic anti-proliferation effect. Under hypoxic conditions, the expression of HIF-1α and VEGF increased as time accumulated, Bevaeizumab combined with SN-38 almost completely inhibited the expression of HIF-1α and VEGF. Moreover, the MAP kinase pathway was involved in the drug modulation of HIF-1α and VEGF. Conclusion These findings suggest the anti-proliferation effect of bevacizumab and SN-38 was schedule-dependent, and the synergistic effect of Bevacizumab and SN-38 was related to drug modulation of the HIF-1α and MAP kinase pathway.