A Multicenter,Double-blind,Randomized,Controlled Trial Of Caffeic Acid Efficacy On Advanced/recurrent Esophageal Squamous Cell Carcinoma Patients With Low Expression Of KDM4C In China

Part one Caffeic acid inhibits proliferation,migration and invasion of esophageal squamous carcinoma cellsObjective: Our group first screened the ESCC tumor stem cell subpopulation of ALDHbri+ and found that the KDM4 C is highly expressed in it.After knocking down KDM4 C,Several biological functions of stem cells such as malignant proliferation,self-renewal,tumor formation in vivo were significantly lower than before,suggesting that KDM4 C is a potential molecular therapeutic target for ESCC.A number of studies have shown that caffeic acid not only has anti-inflammatory,immune regulation,white blood cells,platelets and other functions,but also has anti-tumor effects that inhibit various tumor proliferation,invasion and metastasis,which has not been reported in esophageal squamous cell carcinoma.This study investigated the inhibitory effects of caffeic acid on malignant biological behaviors such as proliferation,migration and invasion of esophageal squamous carcinoma cells.Methods:A luciferase-labeled ESCC cell KDM4 C KYSE30 stably overexpressing KDM4 C and the ESCC cell shKDM4 C KYSE150 knocked down KDM4 C stably was established by lentiviral infection.RT-PCR and Western blot were used to detect the expression of KDM4 C in stable overexpressed/knockdown cells,and the overexpression and knockdown efficiency were detected.shCon KYSE150,Con KYSE30,shKDM4 C KYSE150 cells were treated with different concentrations of caffeic acid,and cell proliferation was detected by CCK 8 kit,and IC50 was calculated.shCon KYSE150,Con KYSE30,and shKDM4 C KYSE150 cells were treated with 20uM/L caffeic acid,and the cell migration and invasion abilities were detected by two kinds of Transwell chambers containing no Matrigel and Matrigel.Nude mice were divided into control group and caffeic acid treatment group.The luciferase-labeled shCon KYSE150,shKDM4 C KYSE150,KDM4 C KYSE30,and Con KYSE30 cells were inoculated subcutaneously in the shoulders of nude mice respectively.100 ul of caffeic acid(100 mg/Kg dissolving in 3g/L sodium carboxymethylcellulose)were injected intraperitoneally to caffeic acid treatment group,and the control group were injected intraperitoneally with the same volume of 3g/L sodium carboxymethylcellulose.The longest and shortest diameter of the tumor weremeasured twice a week.On the 35 th day,the small animal was imaged to detect the growth of the subcutaneous tumor,and then the mice were sacrificed by cervical dislocation.The tumor was removed and weighed.We calculated the tumor volume and drawn the tumor growth curve.Then we used independent sample t test to analyze the deference of subcutaneous tumor volume,weight and fluorescence intensity.Results:1.The shKDM4 C KYSE150 and KDM4 C KYSE30 stable cell lines were established;KDM4C mRNA expression in KDM4 C KYSE30 was increased by5.8-fold compared with the control Con KYSE30 cells.Compared with the control shCon KYSE150 cells,the expression of KDM4 C m RNA was decreased in shKDM4 C KYSE150.0.74 times;2.The expression of KDM4 C protein in KDM4 C KYSE30 was increased by 1.1times compared with the control group of Con KYSE30 cells.Compared with the control shCon KYSE150 cells,the expression of KDM4 C protein in shKDM4 C KYSE150 was decreased by 0.7 times.The proliferation of shCon KYSE150,Con KYSE30 and shKDM4 C KYSE150cells in vitro was significantly decreased in different doses of caffeic acid,and the IC50 was 23.31 uM/L,32.51 uM/L and 38.21 uM/L,respectively.4.After knockdown of KDM4 C,shKDM4C KYSE150 cells migrated and invaded to the basal plane of the chamber were significantly reduced(p <0.05);5.After 20 uM/L caffeic acid treatment,the inhibition rates of cell migration were:shCon KYSE150=65.88%(p <0.0001);shKDM4C KYSE150=36.51%(p=0.0009);Con KYSE30=35.38%(p=0.0085)The cell invasion inhibition rates were: shCon KYSE150=67.65%(p <0.0001);shKDM4C KYSE150=36.99%(p <0.0001);Con KYSE30=37.66%(p <0.0001),both statistically significant;6.The subcutaneous tumor formation of caffeic acid in nude mice showed that the subcutaneous tumor growth of shKDM4 C KYSE150 was significantly decreased after knockdown of KDM4 C.The volume,weight and fluorescence intensity of subcutaneous tumors were significantly decreased at the end of the experiment(p=0.0014);The volume,mass and fluorescence intensity of subcutaneous tumors were significantly reduced compared with the control groups(P<0.05).Further calculation of tumor mass inhibition rate showed that the inhibition rate of shCon KYSE150 with high expression of KDM4C(74.80%)was greater than that of knockdown shKDM4CKYSE150(39.7%),and the inhibition rate of caffeic acid to overexpressed KDM4 C KDM4C KYSE30(61.53%)was higher than that of low The inhibition rate of Con KYSE30 expressing KDM4C(30.39%).Conclusion: Based on the above basic experimental results,we conclude that caffeic acid can inhibit the malignant biological behaviors such as proliferation,migration and invasion of ESCC cells,and the inhibition of caffeic acid on KDM4C-expressing ESCC cells is greater than that of KDM4C-expressing ESCC cells.The results suggest that coffee Acid is a potential new ESCC inhibitory drug,which provids an experimental basis for the clinical application of caffeic acid in the treatment of esophageal squamous cell carcinoma.Part twoA multicenter,double-blind,randomized,controlled trial of caffeic acid efficacy on advanced/recurrent esophageal squamous cell carcinoma patients with low expression of KDM4C in ChinaObjective: Based on the results of previous basic experiments,we demonstrated that caffeic acid can significantly inhibit the proliferation,invasion,migration and growth of subendothelial tumors in vitro.To further evaluate the efficacy and safety of caffeic acid in patients with ESCC,we applied for a registration named "A multicenter,double-blind,randomized,controlled trial of caffeic acid efficacy on advanced/recurrent esophageal squamous cell carcinoma patients in China".This topic is one of its sub-subjects to evaluate the efficacy,safety,and quality of life on advanced/recurrent esophageal squamous cell carcinoma patients with low expression of KDM4 C in ChinaMethods: Pathologically confirmed patients with stage IV esophageal squamous cell carcinoma or esophageal squamous cell carcinoma who were eligible for inclusion in each study center were enrolled in this clinical trial after signing an informed consent form.Before the caffeic acid treatment,the blood samples of the patients and the biopsy/surgical resection of the specimens were collected.The expression of KDM4 C in ESCC tissues was detected by immunohistochemistry at the Molecular Biology Laboratory of the First Affiliated Hospital of Henan University of Science and Technology.Our center's joint drug manufacturer randomized the caffeic acid tablets and placebo tablets.The patients were enrolled according to the drug number(theingredients were caffeic acid tablets or placebo tablets): 0.3g(3 tablets)at a time,3 per day Second,oral,long-term medication,until the patient has intolerable side effects or death.During the follow-up period,the blood routine was reviewed every 2 weeks,and liver function,liver enzymes,blood lipids and other indicators were checked every 8weeks to detect drug side effects,adverse events were recorded at any time,esophageal imaging was reviewed every 8 weeks,and a quality of life questionnaire was filled to evaluate the efficacy and Quality of Life.Statistical analysis was performed using SPSS 24.0 software.The measurement data was expressed as x±SD.The t-test or rank sum test was used for the count data.The chi-square test and Fisher's exact test were used for the measurement data.The test level was ? =0.05,P<0.05 was statistical.significance.Results: The clinical trial registration was completed in June 2017,and the clinical trial number was NCT03070262.In October 2018,the clinical trial was successfully launched in Luoyang,Anyang,Nanyang,Kaifeng,Zhengzhou and other centers.As of March 12,2019,25 patients were enrolled in the study centers,7patients died,11 patients had high KDM4 C expression,and 14 patients had low expression.Because the study has not been completed,it has not been unmasked.It is impossible to further analyze the data of caffeic acid group and placebo group,and evaluate the drug efficacy,drug-related adverse reactions and differences in quality of life.The current data showed that the incidence of 1-2 cases of symptomatic adverse events in KDM4 C low expression group was low,1 case of hypotension,1 case of night sweats,mild hematologic adverse reactions,the incidence was less than 50%,and the grade 1-2 was poor.Reaction,no 3-4 grade adverse events occurred,no fatal adverse events occurred.The quality of life of patients with KDM4 C low expression group before and after treatment was in the two areas of social function and total health status.The four symptom areas of fatigue,nausea and vomiting,shortness of breath and diarrhea improved,and the trend of fatigue and fatigue improved significantly(P=0.066).There were no significant changes in the emotional function,cognitive function,pain,insomnia,and constipation.The physical function,role function,loss of appetite,and economic difficulties were worse than before,but there was no statistical difference(P> 0.05).Conclusion: At present,the clinical trials have not been uncovered,and the evaluation of caffeic acid efficacy and comparative analysis of quality of life arecurrently unevaluable.Based on current data,we can conclude that caffeic acid/placebo is safe for patients with advanced/recurrent esophageal squamous cell carcinoma with low KDM4 C expression and does not add additional adverse effects.