Effect And Mechanism Of Timosaponin AⅢ Combined With Cisplatin In The Treatment Of Head And Neck Squamous Cell Carcinoma

Background and Objective:Head and neck squamous cell carcinoma(HNSCC)is the sixth most common malignant and aggressive cancer worldwide,and long-term smoking,alcohol consumption and high-risk human papillomavirus infection are considered to be the main carcinogenic factors.At present,surgical treatment,chemotherapy,radiotherapy and other treatments are commonly used in clinical practice,and the prognosis is generally poor and recurrence is easy.In recent years,the first-line chemotherapeutic drug cisplatin(CDDP)has been widely used in clinical treatment of head and neck tumors with remarkable curative effect.therefore,the treatment of cisplatin combined with other drugs has become a research hotspot.Timosaponin AIII(TAIII)is a natural steroidal saponin,isolated from the Asian Chinese herb Anemarrhena asphodeloides,and some related studies have demonstrated that it has pharmacological effects against a variety of tumors,but the anti-tumor mechanism is not clear,and its antitumor effect on human head and neck squamous cell carcinoma and its molecular mechanism have not been studied.Therefore,the purpose of this study was to verify that timosaponin AⅢ cooperates with cisplatin to inhibit the proliferation,induction of mitochondrial dysfunction,oxidative stress and apoptosis of human head and neck squamous cell carcinoma cells,and to explore the molecular mechanism of PI3K/Akt/FOXO3 a signaling pathway in anti-tumor.The drug combination therapy and molecular targeted therapy of human head and neck squamous cell carcinoma provide experimental basis,and provide new research ideas for the prevention and treatment of human head and neck squamous cell carcinoma.Methods:1.In vitro experiments,human head and neck squamous cell carcinoma cell lines Fa Du and CAL-27 were cultured,and the cells were treated with different concentration gradients of timosaponin AⅢ and cisplatin,and the CCK-8 assay to detect the proliferation inhibition of human head and neck squamous cell carcinoma Fa Du and CAL-27 by the drug group.2.Detection of apoptosis levels in human head and neck squamous cell carcinoma Fa Du and CAL-27 cells by flow cytometry.3.Detection of oxidative stress levels in human head and neck squamous cell carcinoma Fa Du and CAL-27 cells by flow cytometry and fluorescence microscopy.4.The difference of mitochondrial membrane potential of human head and neck squamous cell carcinoma Fa Du and CAL-27 cells was detected by fluorescence microscopy.5.Western blotting experiments were used to detect the protein expression levels of Bax,Bcl-x L,Cleaved Caspase-3,T-PI3 K,P-PI3 K,T-Akt,P-Akt,T-FOXO3 a and P-FOXO3 a.Immunofluorescence(IF)assay was used to detect the expression level of P-AKT protein.6.In vivo experiments,male BALB/c-Nude mice were randomly divided into 4 experimental groups,namely control group,TAⅢ group,CDDP group and TAⅢ+CDDP group.Nude mice were injected with Fa Du cells under the armpit to bear the tumor,and the drug was injected intraperitoneally.The weight change and tumor volume of the mice were recorded regularly.After the mice were sacrificed,the tumors were taken,and the weight and volume of the tumor were recorded to make tumor slices.7.Observe the cell structure of tumor sections after HE staining.8.TUNEL detects the level of apoptosis in tumor sections.9.Immunohistochemistry(IHC)detection of P-Akt protein expression changes in tumor sections.Results:1.Timosaponin AⅢ combined with cisplatin can significantly inhibit the proliferation of Fa Du and CAL-27 cells,and the inhibition results are drug concentration-dependent.2.The apoptosis of Fa Du and CAL-27 cells was detected by FITC/PI double staining by flow cytometry,and the apoptosis rate of the drug group was significantly higher than that of the control group.3.Using DCFH-DA fluorescent probe,flow cytometry and fluorescence microscope were used to detect the oxidative stress level of Fa Du and CAL-27 cells.Compared with the control group,the drug group promoted the oxidative stress of tumor cells,TAⅢ+CDDP The group had the highest level of oxidative stress.4.JC-1 fluorescent probe was used to detect the changes of mitochondrial membrane potential of Fa Du and CAL-27 cells by fluorescence microscope.The mitochondrial membrane potential of the control group was at a normal high potential,and the mitochondrial membrane potential of the TAⅢ,CDDP and TAⅢ+CDDP groups showed significant downward trend.5.The protein expression results showed that compared with the control group,the expression levels of pro-apoptotic proteins Bax and Cleaved Caspase-3 in the drug group gradually increased,and the expression level of the anti-apoptotic protein Bcl-x L gradually decreased;the signaling pathway protein T-PI3 K,T-Akt,T-FOXO3 a expression levels gradually increased,P-PI3 K,P-Akt,P-FOXO3 a expression levels gradually decreased.6.HNSCC tumor-bearing nude mice model proved that TAⅢ,CDDP and TAⅢ+CDDP group could significantly inhibit the growth of human head and neck squamous cell carcinoma xenografts,and TAⅢ+CDDP group had the most obvious inhibitory effect.7.The results of HE staining showed that the cell structure of the drug group was significantly damaged.8.The results of TUNEL experiment showed that the apoptosis rate of the drug group was significantly higher than that of the control group under in vivo conditions.9.Immunohistochemistry(IHC)results showed that the expression of P-Akt in the drug group decreased,indicating that Akt-related molecular signaling pathways are involved in the anti-cancer process.Conclusion and Research significance:1.This study is the first to explore the specific induction of cell proliferation inhibition,oxidative stress and apoptosis by timosaponin AⅢon human head and neck squamous cell carcinoma.2.Combining timosaponin AⅢ with the clinical frontier chemotherapy drug cisplatin,it is the first time that timosaponin AⅢ and cisplatin can induce the death of human head and neck squamous cell carcinoma both in vivo and in vitro,by inhibiting proliferation and inducing mitochondrial dysfunction,oxidative stress and apoptosis to explain the anti-tumor mechanism.3.The antitumor effect of timosaponin AⅢ and cisplatin on human head and neck squamous cell carcinoma may be through regulating the PI3K/Akt/FOXO3 a signaling pathway to generate excessive ROS and mitochondrial dysfunction,thereby promoting the apoptosis of human head and neck squamous cell carcinoma.