Study On The Inhibition Of Nasopharyngeal Carcinoma Cell Proliferation By Sanguinarine Through MAPK/ERK Signaling Pathway

Objective:This paper investigates whether sanguinarine inhibits the proliferation of nasopharyngeal carcinoma CNE2 cells by regulating the MAPK/ERK signaling pathway,and further explores the possible mechanism of action of SAN against nasopharyngeal carcinoma.Methods:1.The proliferation of CNE2 cells was monitored by a real-time label-free cell function analyser(RTCA)at different concentrations(0.3125,0.625,1.25,2.5,5,10 ?M)of SAN,cisplatin(Cisplatin 4?g·mL-1),MAPK/ERK signalling pathway activator(ISO)and inhibitor PD98059.2.The MTT method was used to detect the proliferation of CNE2 cells at different concentrations(0.3125,0.625,1.25,2.5,5,10 ?M)of SAN,cisplatin(Cisplatin 4 ?g·mL-1)acting on CNE2 cells at 24,36 and 48 h.The IC50 values of SAN at different time points were calculated(half inhibition rate concentration).3.The CytationTM 5 cell imaging multifunctional assay system was used to monitor and detect the effects of different concentrations of SAN(1.25?M,2.5?M),cisplatin(Cisplatin4 ?g mL-1)and control group(Control group)on CNE2 cells for 12,24,36,48 and 60 hours,and the changes in CNE2 cell morphology.Cell morphological changes.4.The CNE2 cell cycle distribution was measured by flow cytometry at different concentrations of SAN(1.25?M,2.5?M),cisplatin(Cisplatin4 ?g·mL-1)and control group(Control group)for 24 hours.5.Western Blot method was used to detect the effects of different concentrations of SAN(1.25?M,2.5 ?M),cisplatin(Cisplatin 4?g·mL-1)and control(Control group)on CNE2 cells,MAPK/ERK signalling pathway key proteins p-c-Raf,p-MEK,p-ERK1/2 and proliferation-associated protein PCNA expression.The expression of key proteins p-c-Raf,p-MEK,p-ERK1/2 and proliferation-associated protein PCNA was measured in the control group(Control group),ISO 20?M group,ISO+SAN group,SAN 2.5?M group and PD980592 ?M group after the effect on CNE2 cells.Results:1.Effect of SAN on the proliferation of CNE2 cellsThe results of RTCA and MTT method experiments showed that SAN could significantly inhibit the proliferation of CNE2 cells compared with Control group(P<0.01),and the inhibition rate of CNE2 cell proliferation became more obvious with the extension of time and the increase of drug concentration.2.CytationTM5 Cell Imaging Multifunctional Assay System monitors the morphology of CNE2 cells by SANCNE2 cells showed strong inhibition of CNE2 cell growth after 12h treatment with different concentrations of SAN.After 60h treatment with SAN,CNE2 cell morphology changed significantly as the concentration of SAN increased,CNE2 cells showed a decrease in the number of adherent cells,an increase in the cell gap,irregular cell morphology and a ruptured cell membrane.3.Effect of SAN on CNE2 cell cycleFluorescence double-staining flow cytometry showed that SAN acted on CNE2 cells for 24h,decreasing the G0/G1 ratio and increasing the S-phase ratio,blocking the CNE2 cell cycle in S-phase(P<0.01).4.Effect of SAN on the expression of proliferative proteins and key proteins of MAPK/ERK signaling pathway in CNE2 cells Western blot showed that the expression of MAPK/ERK signaling pathway key proteins p-c-Raf,p-MEK,p-ERK1/2 and proliferation-related protein PCNA were significantly decreased in CNE2 cells after treatment with different concentrations of SAN compared with Control group(P<0.05).5.Role of MAPK/ERK signaling pathway in the inhibition of CNE2 cell proliferation by SANRTCA results showed that CNE2 cells proliferated faster in the 2.5?M SAN group than in the 2.5 ?M SAN group after the addition of activator ISO,while Western blot showed that the expression levels of MAPK/ERK signaling pathway key proteins p-c-Raf,p-MEK,p-ERK1/2 and p-ERK1/2 increased in the ISO 20 ?M group compared with the Control group.The expression levels of p-c-Raf,p-MEK,p-ERK1/2 protein and proliferation-related protein PCNA increased in the ISO+SAN group compared with the SAN 2.5 ?M group,and decreased in the rest of the groups(P<0.05);the expression of p-c-Raf,p-MEK,p-ERK1/2 protein and proliferation-related protein PCNA decreased more significantly in the SAN group compared with the ISO+SAN 2.5 ?M group(P<0.01).It is more likely that SAN can down-regulate MAPK/ERK signaling pathway related proteins,thus inhibiting CNE2 cell proliferation.Conclusion:The ability of SAN to inhibit the proliferation of CNE2 cells in nasopharyngeal carcinoma may be related to the fact that SAN inhibits the expression of p-c-Raf,p-MEK,p-ERK1/2 and PCNA,key proteins of the MAPK/ERK signaling pathway,and subsequently downregulates MAPK/ERK signaling pathway activity.