| Objective:To study the effect of sanguinarine(SAN)on autophagy and proliferation of nasopharyngeal carcinoma 5-8F cells,and to preliminarily explore the relationship between autophagy and proliferation of 5-8F cells.Methods:1.MTT and RTCA were used to detect the effect of different concentrations of SAN on 5-8F cells proliferation;2.MDC staining was used to observe the effect of SAN on autophagy;transmission electron microscope was used to observe the formation of intracellular autophagosomes after SAN intervened;3.Western Blot was used to detect the expression levels of proliferation-related proteins PCNA,autophagy-related proteins p62 、 Beclin1 、 LC3Ⅱ/Ⅰ,AMPK/mTOR signaling pathway key proteins AMPK,p-AMPK,mTOR.Results:1.SAN inhibits the proliferation of 5-8F cells: MTT and RTCA results show that compared with the control group,different concentrations of SAN treated 5-8F cells for 24 h,36h,48 h can inhibit the proliferation of 5-8F cells(P<0.01).5-8F cells were treated with SAN(2.5,5μmol/L)for 24 h for subsequent experiments.Western Blot results showed that the expression of PCNA could be reduced after 24 hours of SAN treatment(P<0.01).2.SAN induced autophagy in 5-8F cells: MDC results showed that compared with the Control group,SAN treatment enhanced the autophagy of 5-8F cells(P<0.01);Transmission electron microscopy results showed that,compared with the Control group,the effect of SAN After 5-8F cells,the intracellular autophagosomes increased significantly;Western Blot results showed that,compared with the Control group,the expression of Beclin1 and LC3Ⅱ/Ⅰ protein in the SAN group was increased(P<0.01),and the expression of p62 protein was down-regulated(P<0.01);3.The role of autophagy in the inhibition of 5-8F cell proliferation by SAN: MDC results showed that compared with SAN group,SAN combined with 3-MA treatment reduced the effect of autophagy;Transmission electron microscopy results showed that compared with SAN group,SAN combined with 3-MA group decreased intracellular autophagosomes;Western Blot results showed that compared with SAN group,SAN combined 3-MA group had down-regulated Beclin1 and LC3Ⅱ/Ⅰ protein expression(P<0.01),and p62 protein expression increased(P<0.01);The results of MTT and RTCA results showed that SAN acting on 5-8F cells alone or 3-MA combined with SAN can inhibit the proliferation of 5-8F cells,and the inhibitory effects of SAN group were more significant(P<0.01);Western Blot results showed that compared with the SAN group,the PCNA expression level in the combination of 3-MA and SAN was increased(P<0.01).That is,the inhibition of autophagy weakens the effect of SAN in inhibiting the proliferation of 5-8F.4.SAN activates the AMPK/mTOR signaling pathway: Western Blot results show that after 24 hours of SAN treatment of 5-8F cells,the expression levels of AMPK and p-AMPK increased,while the expression levels of mTOR decreased.5.SAN induces 5-8F cell autophagy and inhibits cell proliferation through AMPK/mTOR signaling pathway: After adding AMPK signaling pathway inhibitor Compound C,the results of MDC staining showed that the autophagy effect of SAN combined with Compound C group was reduced compared with SAN group(P<0.01).RTCA results showed that compared with SAN group,the inhibitory effect of SAN combined with Compound C group on 5-8F cells was reduced.Western Blot results showed that,compared with the control group,the expression of AMPK and p-AMPK decreased,and the expression level of mTOR increased.Compared with the SAN group,the proliferation-related protein PCNA and autophagy-related protein p62 in the SAN combined with Compound C group increased,and the expression of Beclin1 and LC3Ⅱ/Ⅰ decreased.That is,inhibition of AMPK/mTOR signaling pathway weakens the effect of SAN in inducing autophagy and inhibiting cell proliferation.Conclusion:1.SAN inhibits the proliferation of 5-8F cells and induce autophagy;2.SAN inhibits cell proliferation by inducing autophagy,and its mechanism may be related to the activation of AMPK/mTOR signaling pathway. |
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