吗啡对人鼻咽癌CNE-2细胞增殖和凋亡的影响

目的 探讨吗啡对人鼻咽癌CNE-2细胞增殖与凋亡的影响及可能的机制.方法 以含0.1、1、10、100、1000mg/L吗啡的培养液作用于体外培养的鼻咽癌CNE-2细胞,等体积培养基作为对照组.MTT法检测吗啡作用24、48和72 h的细胞增殖抑制率,并在不同浓度吗啡作用细胞48 h后,用Hoechst33258荧光染色、流式细胞仪检测细胞凋亡,Western免疫印迹检测Bc1-2、Bax和caspase-3、cleaved-caspase-3蛋白表达.结果 相同作用时间点0.1～100 mg/L吗啡并不影响CNE-2细胞的增殖.而在1000 mg/L吗啡作用下细胞增殖受到明显的抑制,在24、48、72 h CNE-2细胞增殖抑制率随作用时间延长而增加,分别达到(15.0±4.4)%、(30.7±4.9)%和(50.0±4.4)%.48 h后,荧光显微镜下对照组CNE-2细胞未见明显凋亡,0.1～100mg/L吗啡组仅见少量凋亡,而在1000mg/L吗啡组,可见到大量细胞出现典型的凋亡形态学改变.流式细胞仪检测结果显示0.1～100 mg/L吗啡组细胞凋亡率与对照组差异并无统计学意义(P>0.05),而1000mg/L吗啡组细胞凋亡率则明显增加[(39.33±6.03)%比(9.36±1.57)%,P<0.05].Western免疫印迹检测显示在1000 mg/L吗啡作用CNE-2细胞48 h后,Bcl-2蛋白的表达量较对照组明显下降,Bax表达增多,caspase-3活性升高,cleaved-caspase-3表达增多.而其他浓度作用下CNE-2细胞Bcl-2、Bax和caspase-3、cleaved-caspase-3蛋白表达未见明显变化.结论 大剂量吗啡可诱导鼻咽癌CNE-2细胞的凋亡,其机制可能与吗啡上调CNE-2细胞Bax蛋白表达,下调其Bcl-2蛋白表达,引起caspase-3活化有关.

Effect of morphine on proliferation and apoptosis of human nasopharyngeal carcinoma cell line CNE-2

Objective To study the effect of morphine on proliferation and apoptosis of human nasopharyngeal carcinoma cell line CNE-2 and the possible mechanism. Methods CNE-2 cells were treated with culture medium containing different concentrations of morphine(0.1,1,10,100 and 1000 mg/L) in study groups or with the same volume culture medium in control group. The inhibition rate of cell proliferation was measured by MTT assay after the CNE-2 cells were incubated with morphine for 24,48 h and 72 h. At 48hours after treatment with different concentrations of morphine,apoptosis of CNE-2 cells was assessed by Hoechst33258 fluorescence stained and flow cytometry (FCM),the protein levels of Bcl-2,Bax,caspase-3and cleaved-caspase-3 were measured by Western blotting. Results At all the time-spots,treatment with morphine at concentrations ranging from 0.1 to 100 mg/L was not shown to interfere with proliferation of CNE-2 cells. However,growth inhibition was observed with 1000 mg/L morphine,which appeared more intense along with time[(15.0±4.4)% at 24 h,(30.7±4.9)% at 48 h and(50.0±4.4) at 72 h]. At 48 hours,fluorescence microscopy showed that apoptosis of CNE-2 was not present in the control group,modest in 0.1-100 mg/L morphine treated groups,and typical in a great percentage of cells in 1000 mg/L morphine treated group. FCM showed that the apoptosis rate in 0.1-100 mg/L morphine treated groups did not differ significantly from the control group,but was remarkably increased in the 1000 mg/L morphine treated group as compared with the control group[(39.33±6.03)% vs (9.36± 1.57)%,P<0.05]. By Western blotting,treatment with 1000 mg/L morphine was shown to result in inhibited Bcl-2 expression,increased Bax expression,induced caspase-3 activation and enhanced cleaved-caspase-3 production,whereas these were not observed with 0.1,1,10,100 mg/L morphine,as compared with the control group. Conclusion Highdose morphine may induce apoptosis of human nasopharyngeal carcinoma cell line CNE-2 by the downregulation of Bcl-2,up-regulation of Bax that together leads to activation of caspase-3.