Study On The Clinical Efficacy Of Sodium Cantharidate Injection Combined With Chemotherapy On Esophageal Squamous Carcinoma And Its Mechanism Of Action

Esophageal Carcinoma is one of the common clinical tumors of the digestive tract in China,and its pathological types mainly include esophageal squamous carcinoma and esophageal adenocarcinoma,and the pathological types of esophageal cancer in China are mainly esophageal squamous cell carcinoma.Due to the lack of plasma membrane layer in esophagus,tumor cells will invade and migrate in the early stage of development,which leads to poor prognosis of patients.Sodium Cantharidate(SC)is a semi-synthetic derivative of the hydrolyzed and extracted extract of Chinese traditional medicine zanthoxanthin,which has a wide range of anti-tumor activity,and clinical experiments have shown that it is effective against lung cancer,gastric cancer,colorectal cancer,cervical cancer and other malignant tumors,and its use with radiotherapy drugs has the effect of enhancing the efficacy and reducing the adverse effects of patients.In this study,we firstly observed the clinical effect of sodium cantharidateacid injection combined with chemotherapeutic drugs in the treatment of esophageal squamous carcinoma,then explored and analyzed the network interaction between sodium cantharidateacid and esophageal carcinoma using network pharmacology and molecular docking techniques,predicted the potential action targets of sodium cantharidateacid against esophageal carcinoma,and finally preliminarily investigated the molecular mechanism of sodium cantharidateacid in the treatment of esophageal carcinoma by in vitro cellular assay.Part I Clinical efficacy of sodium cantharidate injection combined with chemotherapy on esophageal squamous carcinomaObjective To observe the clinical efficacy of sodium cantharidate injection combined with chemotherapeutic drugs in the treatment of esophageal cancer.Methods A total of 60 patients with squamous esophageal cancer were included in this study,and the patients were randomly divided into a control group and a treatment group of 30 patients each according to the random number table method.The chemotherapy alone group was treated with TP chemotherapy regimen,and the combined treatment group was treated with TP chemotherapy regimen combined with Sodium Cantharidate injection regimen,with a 2-week cycle and 4 cycles of chemotherapy.The recent efficacy and the differences in TCM evidence score,KPS functional status score,serum tumor markers(CEA,CA19-9,SCCA)and toxic side effects of chemotherapy before and after treatment were observed.Results In terms of TCM symptoms scores,both groups were effectively improved after treatment,but the overall effective rate of the combined treatment group was higher than that of The chemotherapy alone group,and the difference was statistically significant(P<0.05),indicating that the combined treatment group was significantly better than The chemotherapy alone group in improving the TCM symptoms.In terms of KPS scores,there was a statistically significant difference between the two groups(P<0.05),indicating that the combined treatment group was significantly better than The chemotherapy alone group in improving patients’ physical strength.In terms of tumor markers,tumor markers CEA,CA19-9 and SCCA were reduced after treatment in both groups,but there was no significant difference between the two groups(P>0.05).In terms of toxic side effects blood system,gastrointestinal reactions,liver and kidney function,there was no statistical difference between The chemotherapy alone group and the combined treatment group in terms of blood system and liver and kidney function(P>0.05),and there was a statistical difference in terms of improving patients’ gastrointestinal symptoms such as nausea and vomiting and diarrhea(P<0.05),and the study results showed that the combined treatment group was superior to the chemotherapy alone group in improving the gastrointestinal symptoms of patients with nausea and vomiting and diarrhea group.Conclusion Sodium Cantharidate combined with chemotherapy for esophageal squamous carcinoma can significantly improve the TCM symptoms,improve the clinical symptoms of patients and reduce the toxic side effects of chemotherapy drugs.Part Ⅱ Study on the mechanism of esophageal cancer inhibition by Sodium Cantharidate based on network pharmacologyObjective To predict and analyze the anti-Esophageal cancer mechanism of Sodium Cantharidate by network pharmacology.Methods The potential gene targets of Cantharidin were predicted from the Swiss Target Prediction database and PharmMapper database,and the genes related to Esophageal cancer were collected from GeneCards database,discrete database and OMIM database,the intersection genes of Cantharidin and Esophageal cancer were obtained by using genny intersection tool,and the intersection genes were introduced into STRING database to construct protein-protein interaction network,and were analyzed by Cytoscape3.9.0,and to obtain the key target protein of Sodium Cantharidate on Esophageal carcinoma.The core target was molecularly docked with the Sodium Cantharidate molecule using AutoDockTools-1.5.6 software to further validate the potential for interaction between the two.Go Enrichment Analysis and Kegg enrichment pathway analysis were performed on key proteins from David database to predict the possible molecular mechanism of action of Sodium Cantharidate against Esophageal cancer.Results A total of 5819 Esophageal disease genes were obtained,167 potential gene targets and 134 main pathways were identified,and PI3K-Akt signal pathway was predicted to be the key signal pathway of anti-Esophageal cancer effect of Cantharidin.Conclusion Cantharidin may play an anti-Esophageal cancer role by affecting the key targets of Aktl,Src,Casp3,HSP90AA1,MMP9 and regulating the PI3K-Akt pathway.Part Ⅲ Inhibition of proliferation,invasion and migration of human esophageal cancer KYSE-30 cells by Sodium Cantharidateacid and its mechanismObjective To investigate the effect of sodium cantharidateacid on the proliferation,invasion and migration ability of human esophageal squamous carcinoma KYSE-30 cells and to explore its mechanism of action to further validate the results of network pharmacological analysis..Methods Human esophageal squamous carcinoma KYSE-30 cells KYSE-30 cells were treated with different concentrations of sodium cantharidateacid(0,0.625,1.25,2.5,5,10 and 20 mg/L)for 24h and 48h,respectively,to observe the changes of drug effects on human esophageal squamous carcinoma KYSE-30 cells,and to analyze the effects of sodium cantharidateacid on human esophageal squamous carcinoma KYSE-30 cells using the CCK-8 method.The effect of sodium cantharidateacid on the proliferation ability of human esophageal squamous carcinoma KYSE-30 cells was analyzed by CCK-8 method.In order to reduce the effect of cytotoxicity on the experiment,the concentration of sodium zebranate was set to three concentration gradients,including low concentration 0.4mg/L,medium concentration 0.8mg/L and high concentration 1.2mg/L.The effects of sodium zebularine on the migration and invasion ability of human esophageal squamous carcinoma KYSE-30 cells were observed in the cell clone formation assay,the scratch assay and the invasion migration assay,respectively,and the effects of sodium zebularine on the migration and invasion ability of human esophageal squamous carcinoma KYSE-30 cells were detected by Western Blot assay.The expression of EMT-related markers and core proteins of PI3K-Akt signaling pathway in human esophageal squamous carcinoma KYSE-30 cells was examined by Western Blot.Results The results of proliferation assay and clone formation assay showed that sodium cantharidateacid significantly inhibited the proliferation of human esophageal squamous carcinoma KYSE-30 cells.The results of cell scoring and invasion migration assays showed that sodium cantharidateacid inhibited the migration invasion of human esophageal squamous carcinoma KYSE-30 cells in a concentration-dependent manner(*P<0.05,**P<0.01,***P<0.001).The expression levels of proteins such as PI3K and p-Akt,N-cadherin and Vimentin were significantly decreased,and E-cadherin protein expression was increased(*P<0.05,**P<0.01,***P<0.001).Conclusion Sodium Cantharidateacid can effectively inhibit the proliferation,invasion and metastasis of esophageal cancer,and its mechanism of action may be related to the regulation of PI3K-Akt signaling pathway.