Anti-cancer Effect And Molecular Mechanism Of Tanshinone ⅡA-induced Autophagy In Oral Squamous Cell Carcinoma Cells

Oral squamous cell carcinoma(OSCC)is the most common primary tumor in the oral cavity.At present,the main treatment methods of oral cancer include surgery and radiotherapy,but the traditional treatment methods are inadequate,and a considerable proportion of patients with oral cancer may have metastasis and recurrence.In order to improve the therapeutic efficiency of oral squamous cell carcinoma(OSCC),a large number of studies have been used to explore new drug targets.Autophagy is considered as one of the possible targets.Autophagy is a ubiquitous pathway of material degradation in eukaryotic cells,which plays an important role in maintaining intracellular homeostasis and organelle renewal.In the process of tumorigenesis and development,autophagy runs through the whole process.Autophagy maintains homeostasis and has dual attributes of inhibiting tumors and promoting tumors.Natural anti-tumor small molecule compounds play an important role in chemotherapeutic drugs.Nowadays,searching for new anti-cancer drugs from natural drugs has become a research hotspot.Salvia miltiorrhiza Bge.,a labiate plant,is the dry root and rhizome of Salvia miltiorrhiza Bge.Tanshinone II A is a diterpenoid quinone compound isolated from Salvia miltiorrhiza,which is considered as the main active ingredient of Salvia miltiorrhiza.It has been reported that tanshinone II A has broad-spectrum anti-tumor activity and can sensitize oral squamous cell carcinoma cells to radiotherapy by inducing autophagy.Therefore,we speculate that tanshinone II A may also have anti-tumor effect on oral squamous cell carcinoma cells by regulating autophagy.In this study,we focused on whether tanshinone ⅡA can inhibit oral squamous cell carcinoma cells,whether tanshinone ⅡA can induce autophagy of oral squamous cell carcinoma cells,what is the molecular mechanism of tanshinone ⅡA-induced autophagy of oral squamous cell carcinoma,and the relationship between the inhibition and autophagy of tanshinone ⅡA on oral squamous cell carcinoma cells,so as to explore the potential therapeutic value of tanshinone ⅡA and autophagy of oral squamous cell carcinoma.The process may play a role in the treatment of oral squamous cell carcinoma.In this study,human oral squamous cell carcinoma cell line SCC-9 and SCC-9 cell line knocking down beclin1 expression were used for in vitro experiments,and OSCC nude mice model was used for in vivo experiments.After treating SCC-9 cells with different concentrations of tanshinone ⅡA,the inhibitory effect of tanshinone ⅡA on the proliferation of SCC-9 cells was detected by CCK8 assay,the apoptotic level of SCC-9 cells was detected by flow cytometry,the autophagy level of SCC-9 cells was detected by transmission electron microscopy and immunofluorescence assay,and BECLIN1,ATG7,ATG12-ATG5,PI3 K,AKT,mTOR C1 and so on were determined by Western blotting test.Protein expression level.In the follow-up experiment,cells were divided into A blank group: adding fresh medium;B TAN group: adjusting the concentration of tanshinone ⅡA in culture medium to 18μM;C siBECLIN1 group: knocking down the expression of beclin1 gene in SCC-9 cells,the same as blank group;D TAN+siBECLIN1 group: knocking down the expression of beclin1 gene in SCC-9 cells,adjusting the concentration of tanshinone ⅡA in culture medium to 18μM;CQ group Chloroquine,an autophagy inhibitor,was added to the culture medium.The levels of apoptosis,autophagy and expression of autophagy-related proteins were detected.In vivo experiments,male BALB/c-nu mice were randomly divided into three experimental groups,blank group: nude mice were subcutaneously injected with SCC-9 cells;TAN group: nude mice were subcutaneously injected with SCC-9 cells,then local injection of tanshinone ⅡA;Besiclin 1+TAN: SCC-9 cells with the expression of beclin1 gene knocked down were injected with the tumorigenesis method of TAN group;mice were anesthetized and sacrificed four weeks later.The growth curve of the tumors was depicted by recording the volume of the tumors regularly;the structure of the tumors was observed by HE staining;the expression of autophagy-related protein was detected by immunohistochemical staining and Western blotting.Cell experiments in vitro confirmed the inhibitory effect of tanshinone ⅡA on the proliferation of SCC-9 cells.Tanshinone ⅡA induced caspase-3-dependent apoptosis in SCC-9 cells.It was also found that tanshinone ⅡA promoted autophagy of SCC-9 cells by inducing BECLIN1/ATG7/ATG12-ATG5 and inhibiting PI3K/AKT/mTOR.The autophagy induction of tanshinone ⅡA on SCC-9 cells mainly depends on the activity of BECLIN1.Autophagy promotes the anti-tumor effect of tanshinone ⅡA.The results of OSCC nude mice model further validated the results of cell experiment in vitro.Tanshinone ⅡA can inhibit the growth of SCC-9 tumors in BALB/c-nu mice,and knocking down BECLIN1 SCC-9 tumors can resist the anti-tumor effect of tanshinone ⅡA;giving tanshinone ⅡA can induce autophagy of SCC-9 tumors;the autophagy level of SCC-9 tumors knocking down BECLIN1 is lower than that of SCC-9 tumors under the same treatment conditions.Based on the results,we infer that the inhibitory effect of tanshinone ⅡA on OSCC cells is partly dependent on the mechanism of BECLIN1: the knockdown of beclin1 gene blocks the effect of tanshinone ⅡA on SCC-9 cells in vivo and in vitro.This study preliminarily explained the mechanism of tanshinone ⅡA: through inducing BECLIN1/ATG7/ATG12-ATG5 pathway and inhibiting PI3K/AKT/mTOR pathway,OSCC autophagy was induced,thus inhibiting the growth of tumors.This study confirmed the anti-tumor effect of tanshinone ⅡA on OSCC cells,suggesting that the regulation of autophagy may become a new target for the treatment of OSCC.