Effects Of Epirubicin And Berberine On Proliferation,Apoptosis,and Autophagy Of Salivary Adenoid Cystic Carcinoma (ACC2) Cells

Background:Salivary adenoid cystic carcinoma occurs in the parotid gland,submandibular gland,sublingual gland and small salivary gland.It is the most common malignant tumor of salivary glands.The current treatment of salivary adenoid cystic carcinoma is surgical excis ion,combined with postoperative radiotherapy and chemotherapy.However,it has a high recurrence rate.Salivary adenoid cystic carcinomas often affect the secretion of saliva which results in dry mouth,difficulty eating and obvious pain.Besides,it also occurs in the maxillofac ial region,often affecting the patient's beauty,greatly reducing the quality of life of patients.Therefore,in order to reduce the recurrence rate of salivary adenoid cystic carcinoma,we need new and effective chemotherapeutic drugs.Epirubic in is a chemotherapeutic drug that has been widely used in the treatment of various cancers such as bladder cancer,liver cancer and breast cancer,and the curative effect is remarkable.However,its effect on salivary adenoid cystic carcinoma is not yet clear.Berberine is a kind of traditional Chinese medic ine which has been used for a long time in our country.It was initially used in the treatment of antipyretic and analgesic,antibacterial and gastrointestinal diseases.In recent years,researchers have found that it also has anti-tumor effect.Therefore,our experiments evaluated the effects of epirubicin and berberine on the proliferation,apoptosis and autophagy in the salivary adenoid cystic carcinoma ACC2 cell line.Objective:1.To evaluate the effects of epirubic in and berberine on proliferation,apoptosis and autophagy in ACC2 cell line.2.Study the mechanism of epirubicin-induced ACC2 cell apoptosis.3.To assess the relationship between autophagy and cell resistance.Methods:1.Though CCK8 assay,study the effects of epirubic in(0 ?g/ml,0.5?g /ml,1.0?g /ml,2.0 ?g /ml)and berberine(0 ?g/ml,3.5 ?g/ml,7.0 ?g/ml,14 ?g/ml)at different concentrations in ACC2 cell line.And study the effects of epirubicin(1.0?g /ml)and berberine(7.0 ?g/ml)at different points(24h,48 h,72h)in ACC2 cell line.2.ACC2 cells were treated with epirubicin(1.0 ?g / ml)and berberin(7.0?g/ml)respectively,then stained with Annexin V-FITC / PI and detected by flow cytometry to study the ability of epirubic in and berberine to induce apoptosis.3.To investigate the effects of epirubicin and berberine on autophagy in ACC2 cells,western blot was used to detect the expression of LC3 and P62 protein in ACC2 cells treated with berberine(7.0 ?g / ml)and epirubicin(1.0 ?g / ml),and the number of autophagy spots was detected by immunofluorescence.4.To study the signal pathway that epirubicin induces apoptosis,western blot and immunofluorescence were used to detect protein expression of Bax,Bcl-2 and cytochrome C in ACC2 cells treated with epirubicin(1.0?g / ml).And RT-q PCR was used to detect the relative mRNA level of Bax and Bcl-2.5.Autophagy inhibitor chloroquine was added to epirubicin-treated cells to inhibit autophagy,then we use CCK8 assay and Annexin V / PI staining to evaluate the relationship between autophagy and drug resistance.Results:1.Under the same action time,cell viability decreased with the increasing of drug concentration.And at the same concentration,the longer the drug action time,the lower the cell viability.2.When the concentration of epirubicin was 1 ?g / ml,about 10.4% of the cells showed apoptosis.And when the concentration of berberine was 7.0?g /ml,approximately 14.2% of the cells showed apoptosis.3.The expression of LC3 II protein increased in the epirubicintreated cells,and the expression of LC3 I did not change significantly,while the expression of P62 protein decreased.In berberine-treated cells,the expression of LC3 I,LC3II and P62 protein showed no significant change.Immunofluorescence indicated that there were obvious autophagy spots in ACC2 cells treated with epirubicin.4.Western blot and immunofluorescence showed that the protein expression of Bax and cytochrome C were significantly increased,while the expression of Bcl-2 protein was not significantly changed.RT-qPCR showed that the relative mRNA level of Bax was significantly increased,while the relative mRNA level of Bcl-2 had no obvious change.5.CCK8 assay showed that the cell viability in the group treated with both epirubicin and chloroquine was obviously lower than that in the group treated with epirubicin alone.Annexin V / PI staining assay showed that the apoptosis ratio in the group treated with epirubicin and chloroquine was significantly higher than the group treated with epirubicin alone.Conclusion:1.Epirubic in and berberine reduced ACC2 cell viability in a concentration-and time-dependent manner.And epirubic in has a stronger effect than berberine.2.Both epirubicin and berberine could induce ACC2 cell apoptosis.3.Epirubicin could induce autophagy in ACC2 cells.Berberine had no significant effect on autophagy in ACC2 cells.4.Epirubic in promoted the release of cytochrome C by increasing Bax expression,which ultimately caused ACC2 cell apoptosis.5.Autophagy increased ACC2 cell resistance to epirubicin.