巨噬细胞在结肠癌CT-26细胞获得性5-FU耐药中的作用

Role of macrophage in secondary resistance to 5-FU in colorectal cancer cell line CT-26

目的探讨巨噬细胞在结肠癌CT26细胞对5-FU产生获得性耐药过程中的作用及其机制。方法分别用正常培养基(NM)、巨噬细胞培养上清(CM)及预先用低剂量5-FU处理过的巨噬细胞培养上清[CM(5-FU)]处理CT-26细胞,并给予10μmol/L 5-FU处理,用CCK-8检测各组细胞增殖情况,PI/Annexin-V流式细胞术检测各组细胞凋亡情况及Western blot检测各组细胞凋亡信号通路。建立CT26小鼠皮下移植瘤模型,应用上述条件培养基处理联合5-FU化疗,动态观察移植瘤生长情况。结果检测细胞凋亡情况发现,5-FU处理组CT26细胞的凋亡细胞百分比显著高于对照组(P<0.05);Western blot检测结果显示5-FU处理组凋亡相关蛋白cleaved caspase-3及P-JNK的表达较对照组显著上调(P<0.05);Western blot检测结果及结肠癌CT26细胞小鼠移植瘤模型发现P-JNK抑制剂可显著抑制由5-FU诱导的CT26细胞Caspase-3凋亡信号通路的激活;CCK-8检测结果及小鼠移植瘤模型中均发现CM(5-FU)能够显著降低CT26细胞对5-FU的化疗敏感性;细胞凋亡检测及Western blot检测结果发现CM(5-FU)可抑制由5-FU诱导的P-JNK/Caspase-3凋亡信号通路的激活。结论 5-FU可激活结肠癌CT-26细胞的P-JNK依赖的Caspase-3凋亡信号通路以发挥抗癌作用;5-FU诱导的巨噬细胞可导致结肠癌CT26细胞产生5-FU获得性耐药,其机制可能是5-FU诱导的巨噬细胞上清可拮抗CT-26细胞中5-FU激活的P-JNK/Caspase-3凋亡信号。

Objective To determine whether macrophage participates in secondary chemoresistance to 5-fluorouracil （5-FU） in colorectal cancer cell line CT26 and explore the underlying mechanism. Methods CT26 cells were cultured in normal medium （NM）, macrophage-conditioned medium （CM） or 5-FU-treated macrophage-conditioned medium [CM（5-FU）], followed by 10 μmol/L 5-FU treatment. CCK-8 assay was used to detect cell proliferation, PI/Annexin-V assay was used to detect cell apoptosis, and Western blotting was used to detect the expression of apoptosis related proteins. Furthermore, subcutaneous tumor-bearing mouse models were established. Growth of xenografts was observed after 5-FU treatment in different culture media. Results Compared with the non-treatment control group, 5-FU treatment induced apoptosis in CT26 cells （P〈0.05） and up-regulated expression of p-JNK and cleaved caspase-3 （P〈0.05）. P-JNK inhibitor could significantly inhibit the activation of caspase-3 signaling pathway induced by 5-FU treatment in the xenografts. P-JNK-dependent caspase-3 signaling pathway in the cells cultured with CM（5-FU） was significantly inhibited, and the resistance of CT26 cells to 5-FU was induced. Conclusion 5-FU inhibits the growth of CT26 cells by activating p-JNK-dependent caspase-3 pathway. Macrophage treated with 5-FU can induce secondary resistance to 5-FU in CT26 cells. The culture supernatant of 5-FU-induced macrophage can inhibit p-JNK/caspase-3 signaling activated by 5-FU in CT26 cells.