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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 investigate the role and mechanism of macrophages in acquiring acquired resistance to 5-FU in colon cancer CT26 cells. Methods CT-26 cells were treated with normal culture medium (NM), macrophage culture supernatant (CM) and macrophage culture supernatant [CM (5-FU) The cells were treated with 10μmol / L 5-FU. The proliferation of each group was detected by CCK-8. The apoptosis of each group was detected by PI / Annexin-V flow cytometry and the apoptosis signal pathways were detected by Western blot. The subcutaneously transplanted tumor model of CT26 mice was established. The above conditioned medium was used to treat combined 5-FU chemotherapy to observe the growth of the transplanted tumor. Results The percentage of apoptotic cells in CT26 cells treated with 5-FU was significantly higher than that in the control group (P <0.05). The results of Western blot showed that the apoptosis-related proteins cleaved caspase-3 and 5-FU The expression of P-JNK was significantly up-regulated compared with that of the control group (P <0.05). Western blot and CT26 cells in colon cancer model showed that P-JNK inhibitor could significantly inhibit the expression of Caspase-3 The results of CCK-8 assay and mouse xenograft model showed that CM (5-FU) could significantly reduce chemosensitivity of CT26 cells to 5-FU. The results of apoptosis assay and Western blot CM (5-FU) was found to inhibit the activation of the P-JNK / Caspase-3 apoptotic signaling pathway induced by 5-FU. Conclusions 5-FU can activate the P-JNK-dependent Caspase-3 apoptotic signaling pathway in colon cancer CT-26 cells to exert anticancer effects; 5-FU-induced macrophages can induce 5-FU production in colon cancer CT26 cells The mechanism may be that 5-FU-induced macrophage supernatant can antagonize 5-FU-activated P-JNK / Caspase-3 apoptosis signal in CT-26 cells.