| Objective: Nasopharyngeal carcinoma(NPC)is a common malignant tumor of the head and neck,and China is one of the regions with a high incidence of NPC,which has become a major health problem of social concern.Berbamine,(BBM)is a bibenzylisovaline alkaloid extracted from the Chinese herb Berberis genus in China,which has antiinflammatory,whitening,anti-hypertensive and anti-arrhythmic effects.Some studies have shown that BBM has some growth-inhibiting effects on leukemia,osteosarcoma,melanoma and other tumors.As a natural source of herbal monomer,BBM also has the advantages of low cost and few adverse effects.However,the function of BBM in the development of nasopharyngeal carcinoma has not been studied,and the potential mechanism by which it exerts its antitumor effects remains unclear.Therefore,in this study,we will carry out the first study on the effects of BBM on NPC cell proliferation,apoptosis,invasion,migration and explore the signaling pathways of its effects.In addition,we further validate the above in vitro experimental results in an in vivo tumorigenic assay in nude mice,aiming to provide new ideas for the clinical drug treatment of NPC.Methods: In this study,NPC cell lines 6-10 B and CNE-2 were selected as the subjects,and the cells were treated with BBM at 0,2,4,8,16,32,64 and 128 μg/ml.The changes of cell viability under different concentrations of treatment were detected by the cell counting kit-8(CCK-8)and the appropriate treatment concentrations were screened.We evaluated the cell proliferation ability of the selected cells at different concentrations by Ed U imaging kit.And we used Annexin V-FITC/PI apoptosis kit and flow cytometry to detect the effect of BBM on apoptosis,scratch assay to explore the effect of BBM on cell migration,transwell invasion assay to detect the effect of BBM on cell invasion ability,propidium iodide(PI)staining by flow cytometry to explore the effect of BBM on cell invasion ability,and propidium iodide(PI)staining by flow cytometry to explore the effect of BBM on cell cycle.Subsequently,we used Western Blot,(WB)to detect the effector indicators of relevant cellular functions(Cyclin D,CDK2,E-cadherin,N-cadherin,BAX,Bcl-2,etc.)and MDM-2,p53 protein expression.Then,we selected 6-10 B cells inoculated in the axilla of Bal B/c nude mice for the preparation of a nude mouse tumor model.The experimental group was injected with BBM 30 mg/kg and 60 mg/kg intraperitoneally,and the control group was injected with the same amount of saline,and the body weight and tumor size were measured regularly.The mice were executed and the tumors were weighed,and the tumor specimens were collected for WB and immunohistochemistry(IHC)staining to detect the expression of MDM-2 and p53 proteins.Results: In the CCK-8 assay we found that NPC cell viability was inhibited with increasing BBM concentration,and this change showed some concentration and time dependence.In the Ed U proliferation assay,we found that the cell proliferation ability gradually decreased with increasing BBM concentration,which was consistent with the results of CCK-8.The results of Annexin V-FITC/PI apoptosis assay showed that the apoptosis ability increased with increasing BBM concentration.The results of wound healing assay showed that the wound area of both cell lines in the drug-added group was larger than that of the control group.Transwell invasion assay showed that the number of cells crossing the transwell in the treated group was significantly lower than that in the control group,and the higher the drug concentration,the lower the number of cells.Notably,in the cell cycle assay,we found that the proportion of cells in G0/G1 phase increased significantly in both NPC cell lines after BBM intervention,suggesting that BBM may cause cell cycle arrest.As we expected,in WB experiments,the expression of various effect indicators representing cell proliferation,apoptosis,migration and invasion also conformed to the above phenotypic experimental results.In addition,WB of in vivo experiments,WB and IHC of in vitro experiments showed a decrease in MDM-2 expression and a significant increase in p53 expression in the dosing-treated group.Conclusion: BBM significantly inhibited the proliferation,migration,and invasion ability of NPC cells,and this inhibition was concentration-dependent;in contrast,BBM could arrest the NPC cell cycle in the G0/G1 phase,which in turn caused apoptosis;in addition,WB experiments verified that BBM could inhibit MDM-2 and then activate the P53-P21 pathway,and P21 protein could act on the cell cycle from G0/ G1 to S-phase cell cycle transition,causing cell cycle arrest and thus promoting apoptosis... |
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