姜黄素、氯丙胺对人结肠癌肿瘤样干细胞CD44、Lgr5表达的作用

Effect of curcumin and chloropropionic amine on expression of CD44 and Lgr5 in human colon cancer stem-like cells

目的:探讨姜黄素和胃泌素受体不全拮抗剂氯丙胺对体外培养的人结肠癌肿瘤样干细胞生长增殖的抑制作用及作用机制.方法:取对数生长期的结肠癌SW480细胞用10%的完全培养基继续培养48 h后,换用无血清培养基进行同步化培养24 h,然后进行分组:姜黄素组、氯丙胺组、联合用药组,每一种药物组又分为对照组、低浓度组、中浓度组、高浓度组,联合用药组的浓度梯度依次为两药浓度的对应叠加,继续培养48 h,加入FITC标记的小鼠抗人CD44单克隆抗体20μL,同时在对照组细胞中加入同型对照抗体IgG2b 20μL,4℃避光放置30 min,然后使用目筛网滤过,上流式细胞仪进行检测CD44的表达变化.采用Western blot检测Lgr5在3种不同药物浓度作用下在细胞中的蛋白表达差异.结果:姜黄素和氯丙胺均呈时间和剂量依赖性抑制SW480细胞的生长增殖,且二者具有协同抑制作用;单独应用姜黄素和氯丙胺干预时,SW480细胞增殖逐渐减慢,数量减少,细胞逐渐变形,并出现崩解及死亡,细胞中CD44蛋白质的表达较对照组有所降低,Lgr5蛋白质的表达均低于对照组,单独用药组与对照组比较具有统计学意义(P<0.05);二者联合应用时,CD44、Lgr5蛋白质的表达均显著降低,而且与对照组和各单一用药组比较差异具有统计学意义(P<0.05).结论:(1)姜黄素和氯丙胺呈剂量依赖性地抑制体外培养的人结肠癌肿瘤样干细胞的生长增殖,且二者具有协同抑制作用;(2)姜黄素和氯丙胺可能是通过下调CD44和Lgr5分子的表达来抑制肿瘤干细胞增殖.

AIM： To investigate the synergistic effect of curcumin and gastrin receptor antagonist chloropropionic amine on the proliferation of human colon cancer stem-like cells in vitro and the possible mechanisms.METHODS： Colon cancer SW480 cells in logarithmic growth phase were cultured in complete medium containing 10% serum for 48 h, synchronized by culturing in serum-free medium for 24 h, and then divided into three groups： a curcumin group, a chloropropionic amine group, and a combination group. Each group was further divided into a control group, low-, medium-, and high-concentration groups. After treatment, cells were cultured for another 48 h, and FITC labeled mouse anti-human CD44 monoclonal antibody was added, while the isotype control antibody IgG2 b was used in the control group. After incubation at 4 ℃ for 30 min, CD44 expression was detected by flow cytometry. Western blot was used to detect the expression of Lgr5.RESULTS： Curcumin and chloropropionicamine, alone or in combination, could inhibit SW480 cell growth and proliferation time- and dose-dependently, and they had a synergistic effect. Curcumin and chloropropionic amine alone significantly decreased CD44 and Lgr5 protein expression compared with the normal control group（P 0.05）. Chloropropionic amine combined with curcumin also significantly CD44 and Lgr5 protein expression compared with the normal control group, and the decrease was more significant in the combination group compared with either of them alone（P 0.05）.CONCLUSION： Both curcumin and chloropropionic amine inhibit the growth and proliferation of SW480 cells time- and dose-dependently. Curcumin and chloropropionic amine may inhibit CD44 and Lgr5 expression to prevent the growth and proliferation of colon cancer stem cells.