大蒜素通过AKT/NF-κB通路诱导大肠癌细胞凋亡的实验研究

Experimental study on allicin inducing apoptosis of colorectal cancer cells by AKT/NF-κB pathway

目的探索大蒜素通过丝苏氨酸激酶/核转录因子-κB(AKT/NF-κB)通路对大肠癌细胞凋亡的影响。方法将大肠癌Lovo细胞分为对照组、大蒜素低、中、高剂量组(大蒜素,20mg/L、40mg/L和80 mg/L)和阳性对照组(顺铂,10μmol/L)。MTT法检测大蒜素对大肠癌细胞的半抑制浓度(IC50),检测各组细胞增殖;流式细胞仪检测各组细胞周期和凋亡;Western blot检测Akt/NF-κB信号通路、Ki-67、增殖细胞核抗原(PCNA)、B细胞淋巴瘤/白血病-2(Bcl-2)、BCL2相关X蛋白(Bax)和裂解的半胱氨酸天冬氨酸蛋白酶3(cleaved Caspase-3)的表达;将培养细胞分为sh-NC组(空白质粒)、sh-Akt组(质粒转染)、大蒜素组+sh-NC组(终浓度为20 mg/L)、大蒜素组+sh-Akt组(终浓度为20 mg/L),检测转染后各组细胞的增殖、凋亡、蛋白NF-κB p65、p-NF-κB p65、cleaved Caspase-3的表达。结果大蒜素对大肠癌细胞的抑制作用呈药物浓度依赖性,IC50为(40.27±1.23)mg/L;与对照组相比,大蒜素低、中、高剂量组和阳性对照组的细胞抑制率和凋亡率均明显升高(P<0.05),有丝分裂S期细胞和G2/M期细胞所占比例明显增加(P<0.05),Ki-67、PCNA、Bcl-2/Bax、p-Akt/Akt和p-NF-κB p65/NF-κB p65的表达明显下降(P<0.05),cleaved Caspase-3的表达明显升高(P<0.05),且随着大蒜素剂量的增加,其作用逐渐增强(P<0.05);与sh-NC组相比,sh-Akt组和大蒜素+sh-NC组细胞抑制率和凋亡率明显升高(P<0.05),p-NF-κB p65/NF-κB p65表达明显下降(P<0.05),cleaved Caspase-3表达明显升高(P<0.05);与sh-Akt组相比,大蒜素+sh-Akt组细胞抑制率和凋亡率明显升高(P<0.05),p-NF-κB p65/NF-κB p65表达明显下降(P<0.05),cleaved Caspase-3表达明显升高(P<0.05)。结论大蒜素可能通过抑制Akt/NF-кB信号通路,抑制大肠癌细胞增殖,诱导细胞凋亡。

Objective To explore effects of allicin on apoptosis of colorectal cancer cells by serine/threonine protein kinase/nuclear transcription factor-κB(AKT/NF-κB)pathway.Methods Colorectal cancer Lovo cells were divided into control group,lowdose,medium-dose and high-dose allicin group(allicin,20 mg/L,40 mg/L and 80 mg/L),and positive control group(cisplatin,10μmol/L).The semi-inhibition concentration(IC50)of allicin on colorectal cancer cells was detected by MTT assay.The cell proliferation in each group was detected.The cell cycle and apoptosis in each group were detected by flow cytometry.The expression of Akt/NF-κB signaling pathway,Ki-67,proliferating cell nuclear antigen(PCNA),B cell lymphoma/leukemia-2(Bcl-2),BCL2-related X protein(Bax)and cleaved cysteinyl aspartate specific proteinase 3(cleaved Caspase-3)was detected by Western blot.The cultured cells were divided into sh-NC group(blank plasmid),sh-Akt group(plasmid transfection),allicin+sh-NC group(final concentration of 20 mg/L),allicin+sh-Akt group(final concentration of 20 mg/L),and the proliferation,apoptosis and expression of protein NF-κB p65,p-NF-κB p65,cleaved caspase-3 were detected after transfection.Results The inhibition effect of allicin on colorectal cancer cells showed drug concentration-dependence,and IC50 was(40.27±1.23)mg/L.Compared with control group,inhibition rate and apoptosis rate of cells were significantly increased in low-dose,medium-dose and high-dose allicin group,and positive control group(P<0.05),and proportion of cells in mitotic S phase and G2/M phase was significantly increased(P<0.05),expression of Ki-67,PCNA,Bcl-2/Bax,p-Akt/Akt and p-NF-κB p65/NF-κB p65 was significantly decreased(P<0.05),and expression of cleaved Caspase-3 was significantly increased(P<0.05).And its effect was gradually enhanced with increase of allicin dose(P<0.05).Compared with the sh-NC group,the inhibition and apoptosis rates of sh-Akt group and allicin+sh-NC group were significantly increased(P<0.05),the expression of p-NF-κB p65/NF-κB p65 was significantly decreased(P<0.05),and the expression of cleaved caspase-3 was significantly increased(P<0.05).Compared with the sh-Akt group,the inhibition rate and apoptosis rate of allicin+sh-Akt group were significantly increased(P<0.05),p-NF-κB p65/NF-κB p65 expression was significantly decreased(P<0.05),cleaved caspase-3 expression was significantly increased(P<0.05).Conclusion Allicin may inhibit proliferation of colorectal cancer cells and induce apoptosis by inhibiting Akt/NF-кB signaling pathway.