Inhibition Of Autophagy Promotes The Anti-tumor Effect Of Metformin In Oral Squamous Cell Carcinoma

ObjectiveOral Squamous Cell Carcinoma(OSCC)is a common malignant tumor in the head and neck,accounting for more than 90% of oral cancer.OSCC mainly occurs in the tongue,gingiva,and buccal mucosa.Major risk factors of OSCC include smoking,alcohol abuse,betel nut chewing,and Human Papilloma Virus(HPV)infection.OSCC is highly aggressive,malignant,prone to metastasis,and has a poor prognosis.At present,the main treatment of OSCC is surgical radical resection,supplemented by radiotherapy,chemotherapy,immunotherapy,gene therapy,and targeted therapy.Great progress has been made in the treatment of OSCC in recent years,but due to tumor recurrence,distant metastasis,and the emergence of drug resistance,the treatment of OSCC cannot achieve the expected effect,with a five-year survival rate being only 50%-60%.In recent years,a large number of preclinical studies have found that metformin,as a first-line drug for diabetes,can inhibit the growth of liver cancer,breast cancer,prostate cancer,etc.The researchers also found that metformin can also restrain the progress of OSCC,but the specific mechanism is not fully clear.Therefore,our topic will focus on the anti-OSCC mechanism of metformin.In addition,the antitumor effect of metformin requires greater drug concentration,which may cause unexpected side effects.Therefore,our study also focuses on how to reduce the drug concentration of metformin when it is used for anti-OSCC.The researchers found metformin induces protective autophagy of tumor cells,which will prevent the tumor cells from apoptosis.It provides us with a new thought on how to improve the anti-OSCC effect of metformin.Whether we can use autophagy inhibitors such as chloroquine(CQ)and Hydroxychloroquine(HCQ)to inhibit protective autophagy so as to improve the apoptosis level of OSCC induced by metformin and promote the anti-OSCC effect of metformin.Methods1.OSCC cell lines were treated with different concentrations of metformin,and the anti-proliferation effect of metformin on OSCC cells was detected by CCK-8 assay and colony formation assay.The effects of different concentrations of metformin on the migration and invasion of OSCC cells were detected by cell scratch assay and cell invasion assay.Cell cycle assay and Western Blot(WB)assay were used to detect the effect of metformin on the cell cycle distribution of OSCC.2.OSCC cell lines were treated with different concentrations of metformin,and the effects of metformin on apoptosis and ROS level of OSCC cells were detected by apoptosis assay,Reactive Oxygen Species(ROS)assay,and WB assay.The m RFP-GFP-LC3 adenovirus transfection assay,Transmission Electron Microscopy(TEM),and WB assay were used to detect the effect of metformin on the level of autophagy in OSCC cells.3.OSCC cells were treated with different concentrations of metformin,with or without Hydroxychloroquine(HCQ).CCK-8 assay,apoptosis assay,and WB assay were used to detect the effects of the combination of metformin and HCQ on the proliferation and apoptosis of OSCC cells.Tumor xenograft assay,Immunohistochemistry(IHC),and Hematoxylin-eosin(HE)staining were used to detect the anti-OSCC effect of metformin in vivo and the safety of metformin.Results1.The result of the CCK-8 assay showed that with the increasement of metformin concentration(0,3,6,12,24,48 m M),the viability of OSCC cells decreased significantly.Colony formation assay showed that the clone number of OSCC cells decreased significantly with the increasement of metformin concentration(0,12,24 m M).Cell migration assay showed that the scratch area of CAL27 cells increased with the increasement of metformin concentration(0,12,24 m M).Cell invasion assay showed that with the increasement of metformin concentration(0,12,24 m M),the number of CAL27 cells crossing the Transwell chamber decreased.Cell cycle assay showed that with the increasement of metformin concentration(0,12,24 m M),the number of OSCC cells arrested in the G1 phase increased significantly.Moreover,the WB assay showed that metformin also increased the expression level of P21 protein and decreased the expression level of Cyclin d1 protein in CAL27 cells.2.Apoptosis assay showed that with the increasement of metformin concentration(0,12,24 mm),the apoptosis level of OSCC cells was increased,and WB assay showed that metformin could increase the expression level of Cleaved-caspase3 protein and Bax protein,and reduce the expression level of Bcl-2.ROS assay showed that with the increasement of metformin concentration(0,12,24 m M),the ROS level in CAL27 cells decreased.CAL27 cells were transfected with m RFP-GFP-LC3 adenovirus and treated with different concentrations of metformin(0,12,24 m M)at the same time.With the increasement of metformin concentration,the amount of yellow fluorescence and red fluorescence in CAL27 cells increased.The result of TEM showed that the number of autophagosomes and autophagosomes in CAL27 cells treated with metformin was increased.WB assay showed that metformin could increase the expression level of LC3II/LC3 I and Beclin 1,and reduce the expression level of P62 in CAL27 cells.3.CCK-8 assay showed that the combination of metformin and HCQ(20μM)significantly decreased the viability of OSCC cells compared with metformin alone.Compared with metformin alone,the combination of metformin and HCQ(20μM)significantly increased the apoptosis level of OSCC cells.Tumor xenograft assay and IHC assay in nude mice showed that metformin with or without HCQ could both inhibit the growth of OSCC in vivo,and the effect of the combination of metformin and HCQ was more significant.HE staining of vital organs showed that metformin alone or the combination of metformin and HCQ had no obvious toxicity and side effects in nude mice.ConclusionsThis study found that metformin inhibited the proliferation,migration,and invasion of OSCC cells in a concentration-dependent manner,and could arrest the OSCC cell cycle in the G1 phase.Metformin promoted OSCC cell apoptosis in a ROS-independent manner.Metformin could also induce protective autophagy in OSCC cells.Inhibition of autophagy with HCQ could promote apoptosis in OSCC cells induced by HCQ.Finally,this study verified that the combination of metformin and HCQ is a safe and effective treatment for OSCC in vivo.