mu阿片受体活化促进胰岛素样生长因子Ⅰ致乳腺癌细胞增殖

mu opioid receptor activation promotes insulin-like growth factor Ⅰ-stimulated breast cancer cell proliferation

目的观察mu阿片受体(MOR)在胰岛素样生长因子Ⅰ(IGFⅠ)致人乳腺癌细胞增殖行为中的作用。方法选取表达MOR的人乳腺癌细胞株MCF-7,采用Western印迹法(n=3)检测IGFⅠ刺激对细胞内MOR蛋白表达的影响。采用细胞计数试剂盒8(CCK-8,n=6)和流式细胞术(n=3)分析外源性MOR激动剂(DAMGO)和特异性MOR拮抗剂甲基纳曲酮(MNTX)对IGFⅠ致乳腺癌细胞增殖的影响。采用Western印迹法(n=3)检测MOR不同活化状态下,IGFⅠ下游蛋白激酶B(Akt)通路的激活水平。结果Western印迹法结果显示,以20、100、1 000μg/L不同质量浓度IGFⅠ刺激24h,IGFⅠ为20和100μg/L时MOR蛋白的相对表达水平均显著高于对照组(P值均<0.01),在IGFⅠ质量浓度为100μg/L时作用最显著。以100μg/L IGFⅠ刺激MCF-7细胞15和30min,以及1、6、12和24h,刺激15 min时MOR蛋白的表达水平即开始升高,刺激30min和1、6、12、24h时均显著高于对照组(P值分别<0.05、0.01),在刺激6h时作用达到高峰。流式细胞术检测细胞周期的结果显示,采用100μg/L IGFⅠ刺激MCF-7细胞24h后,处于增殖期的细胞数量显著多于对照组(P<0.01);采用10μmol/L DAMGO预处理2h后再与IGFⅠ共同刺激24h,处于增殖期的细胞数量进一步增多,显著高于对照组和IGFⅠ单独刺激组(P值均<0.01);而DAMGO单独刺激组处于增殖期的细胞数量与对照组相比差异无统计学意义(P>0.05)。CCK-8检测细胞活力的结果与细胞周期的检测结果一致。此外,MCF-7单独用1μmol/L MNTX预处理2h,细胞活力与对照组相比差异无统计学意义(P>0.05);MNTX预处理2h后再与IGFⅠ共同刺激,细胞活力与IGFⅠ单独刺激组相比差异无统计学意义(P>0.05);但在DAMGO与MNTX同时预处理2h后再给予IGFⅠ,细胞活力显著低于IGFⅠ与DAMGO联合刺激组(P<0.01)。Western印迹法检测结果显示,采用100μg/L IGFⅠ刺激MCF-7细胞24h后,磷酸化Akt(p-Akt)的表达水平显著高于对照组(P<0.01);10μmol/L DAMGO预处理2h后再给予100μg/L IGFⅠ刺激24h,p-Akt的表达水平显著高于IGFⅠ单独刺激组(P<0.01);MCF-7细胞单独用DAMGO或MNTX预处理2h,p-Akt的表达水平与对照组相比差异均无统计学意义(P值均>0.05);DAMGO和MNTX同时预处理2h后再给予IGFⅠ,其p-Akt的表达水平显著低于IGFⅠ与DAMGO联合刺激组(P<0.05)。结论IGFⅠ能够上调人乳腺癌细胞中MOR的表达,活化高表达的MOR能够提高Akt信号通路在IGFⅠ作用下的激活水平,促进IGFⅠ致MCF-7细胞增殖的作用,该作用可以被MOR特异性拮抗剂所阻断。

Objective To investigate effects of mu opioid receptor （MOR） activation on insulin-like growth factor Ⅰ （IGF Ⅰ ）-induced proliferation in breast cancer cell line Michigan Cancer Foundation-7 （MCF-7）. Methods MCF-7 cells, which expressed MOR, were exposed to IGF I and the protein expression of MOR was tested with Western blot. Seven groups were then assigned, control group, IGFⅠ-treated group, DAMGO-treated group, methylnaltrexone （MNTX）-treated group and IGF I plus DAMGO-treated group, IGF I plus MNTX-treatedgroup, IGF I plus DAMGO and MNTX-treated group. Cell proliferation of each group was measured using flow cytemetry and cell counting kit-8 （CCK-8）. Activation of protein kinase B （Akt） was determined by Western blot. Results Both 20 μg/L and 100 μg/L IGFⅠ promoted the protein expression of MOR in MCF-7 cells （P〈0.01）, especially 100 μg/L IGF I . Then cells were stimulated by 100 μg/L IGF I for 15 min, 30 min, 1 h, 6 h, 12 h and 24 h, respectively. The protein level of MOR began to increase at 15 min, and the effect reached a peak at 6 h （P〈0.01） and lasted for 24 h （P 〈 0.05）. The results of flow cytometry showed that compared with control group, IGF I -treated group had more proliferative cells （P〈0.01） at 24 h after 100 μg/L stimulation, and the cells were further increased in IGF I plus DAMGO-treated group （P〈0.01）. However, there was no significant difference in the proliferative cell counting between DAMGO-treated group and control group （P〉0. 05）. Consistent results could be observed in cell viability detected by CCK-8. In addition, in the presence of MNTX, a highly selective MOR antagonist, the activity of MCF-7 cells in IGF I -treated group and DAMGO-treated group was notably lower than that in IGF I plus DAMGO-treated group （both P〈0.01）. But there was no significant difference in the cell viability between MNTX-treated group and control group （ P〉0.05）, between MNTX-treatedgroup and IGF I -treated group （P〉0.05）. Similarly, phosphorylated Akt （p-Akt） expression was significantly higher in IGF I plus DAMGO-treated group than control group and IGF I group （both P〈0.01）; furthermore, these effects could also be blocked by MNTX （P〈0.05）. Conclusion Expression level of MOR in MOF-7 cells is upregulated upon IGF I treatment. Activating MOR significantly increases IGF I -stimulated activation of Akt signaling and promotes cell proliferation, which can be blocked by specific MOR antagonist. （Shanghai Med J, 2018, 41： 607-611）