Anti-tumor Effects Of Proteasome Inhibitor MG132 For Human Adenoid Cystic Carcinoma Cells Through NRF2/KEAP1 Signaling Pathway

Objective:Adenoid cystic carcinoma(ACC)is one of the most common malignant salivary gland tumors.Its strongly peripheral invasion makes the boundary with tissues unclear.Due to the hematogenous infection,adenoid cystic carcinoma has a high rate of tumor metastasis.Its unique biological characteristics and complex structures contribute to its poor survival rates.Over the years,a large amount of researches are seeking new treatments for adenoid cystic carcinoma.The proteasome inhibitor(PI)MG132 has a satisfactory therapeutic effect in the treatment of certain solid tumors,however there is little relevant literature to demonstrate how the proteasome inhibitor can treat adenoid cystic carcinoma.While we found that the high expression of nuclear factor erythroid-2-related factor 2(NRF2)was increased in pathological sections of patients who had suffered from adenoid cystic carcinoma.In this study,different concentrations of proteasome inhibitors were added to adenoid cystic carcinoma cells cultured in vitro to observe the effects on the proliferation and apoptosis of adenoid cystic carcinoma cells,further more to detect the expression of NRF2/KEAP1 pathway after the stimulation of proteasome inhibitors.In order to investigate the effect of proteasome inhibition on adenoid cystic carcinoma and its mechanism.The aim of this study is to provide some experimental basis for the future clinical treatment and drug development of adenoid cystic carcinoma.Methods:After the culture of adenoid cystic carcinoma cells in vitro,cell-counting kit 8(CCK8)was performed to determine the effects of different concentrations of proteasome inhibitors on the proliferation of adenoid cystic carcinoma cells.Three appropriate concentrations were chosen for the following experiment to make research observations.Flow cytometry analysis was used to detect the effect of proteasome inhibitors on apoptosis.At the same time,we applied cellular immunofluorescence to determine the effect of proteasome inhibitors on the NRF2/KEAP1 pathway of adenoid cystic carcinoma cells.Quantitative real-time polymerase chain reaction(RT-PCR)to explore the NRF2/KEAP1 pathway and P62 expression.NRF2 inhibitor(ML385)was utilized to evaluate the role of NRF2/KEAP1 pathway in proteasome inhibitor-induced apoptosis.All analyses in this experiment were performed using graphpad pyramid 8.0 software.with a two-tailed student’s test,data are expressed as mean±standard error.In all analyses,P<0.05 was considered statistically significant.Results:1.We discovered that MG-132 significantly suppressed the growth of ACC-83 cells and induced apoptosis through The results of cell viability test.Compared with the blank control group containing no proteasome inhibitor medium,the experimental group at a concentration of 10μmol/L,40p.mol/L,70μmol/L significantly inhibited the proliferation of ACC-83 cells(P<0.05).Cell flow cytometry results showed that at concentrations of lO μmol/L,40μmol/L,and 7 μmol/L,proteasome inhibitors induced different degrees of apoptosis in adenoid cystic carcinoma cells(P<0.05).2.Compared with the control group,the immunofluorescence results showed that after culturing with the proteasome inhibitor,the experimental group NRF2 dramatically increased,and KEAP1 decreased compared with the control group.The results of western blotting proved that NRF2 protein was up-regulated and KEAP1 protein was down-regulated,as was the expression of P62 protein.In addition,the results of RT-PCR showed that the expression of NRF2 increased,and KEAP1 and P62 had a similar decrease trend(P<0.05).Inhibition of NRF2 reduced the therapeutic effect of MG-132 on ACC.3.After adding the NRF2 inhibitor ML385,it can be seen from the results of cell viability detection and cell flow cytometry that the effect of proteasome inhibition on adenoid cystic carcinoma cells’inhibition of proliferation and induction of apoptosis is reduced,and as Western blot verifies that the interaction of MG132 and ML385 at the same time compared with the addition of only MG132 shows that the expression of NRF2 protein is reduced.Conclusion:As the data of this experiment shown,it was confirmed that the proteasome inhibitor MG 132 inhibited cell proliferation and induced apoptosis in vitro on adenoid cystic carcinoma cells.Its role in inhibiting cell proliferation and promoting apoptosis may be related to the regulation of the NRF2/KEAP1 pathway.Proteasome inhibitor therapy will be a new strategy and new direction for adenoid cystic carcinoma,which will be beneficial for patients with adenoid cystic carcinoma.But specific applications should be clinically tested.