Research Of Fu-Zheng-Jie-Du Formula On Reversing Drug Resistance Induced By Cancer Cell Stemness Induced By CGAS/Wnt16b/β-catenin Axis Mediated In Cancer Associated Fibroblasts

Backgroud:Gastric cancer(GC)is one of the most common malignant tumor in China a majority of patients were diagnosed in advanced stage,hence chemotherapy is a fundamental therapeutic option.The relapse and metastasis of tumor is one of the leading cause responsible for the death of GC patients,drug resistance is the critical reason.Extended research revealed that by the mean time chemotherapy drugs kill cancer cells,they also affect the other cells in the tumor microenvironment(TME),such as cancer associated fibroblasts(CAFs),faciliate the development of drug resistance.CAFs became senescent upon DNA damaged by chemotherapy drugs,which caused the secretion of senescence associated secretory phenotype(SASP),is an important mechanism of CAFs mediated drug resistance.Cyclic GMP-AMP synthase(cGAS)is an important DNA sensor in which exist in cytoplasm,when detected endogenous DNA in cytoplasm,cGAS can activate NF-κB according to the downstream signal transduction to promoting a variety of cytokines expression including Wnt16b.Wnt16b is a ligand of Wnt/β-catenin signal pathway and overexpressed in CAFs which undergo chemotherapy.Wnt16b can promote the stemness of cancer cells by activating β-catenin,and cancer stem cells(CSCs)play key role in chemorsistance.Hence it is important in mediated the cGAS/Wnt16b/β-catenin axis in promoting the therapeutic effect of chemotherapy.Fu-Zheng-Jie-Du formula(FZJD)is an effective formula which used in Guang’anmen Hospital of China Academy of Chinese Medical Science in treating tumor relapse and metastasis.Our previous researches proved that FZJD can promote the therapeutic effect of chemotherapy in tumor-bearing mice model by modulating the subtype proportion of tumor associated macrophages(TAMs),which known as an other important cel lpopulation in TME.FZJD can also inhibiting tumor angiogenesis and impro’ve the immunosuppression of tumor.The present study aimed to further investigate wether the chemotherapy synergistic effect of FZJD is associated with modulating the function of CAFs.Purpose:To establishment the DNA damaged CAFs model caused by chemotherapy and CAFs/cancer cells hybrid tumor-bearing mice model in order to observe the drug resistance-promoting effect of CAFs in vitro and vivo.Then investigate wether FZJD is effective in mediating the cGAS/Wnt16b/β-catenin axis to overcome drug resistance.Method:The basic experimental research is divided into three parts:1.Establish and evaluate the DNA damaged CAFs model;2.Observe the affect of FJZD in cGAS/Wnt16b//β-catenin axis mediated drug resistance;3.Establish 5-FU resistant tumor-bearing mice model and observe the affect of FZJD in drug resistance and investigate the relative mechanisms.1.Establish and evaluate of the DNA damaged CAFs model① MRC-5 and 5-FU was respectively choosen as CAFs and inducer,CCK8 assay was performed to screen suitable intervention conditions.② Immnuofluorescence(IF)assay was performed to detect the expression ofγH2AX in DNA damaged MRC-5(dMRC-5).③ Annexin V/PI double staining assay was performed to detect the apoptosis of dMRC-5.④ qRT-PCR assay was performed detect the cGAS gene expression in dMRC-5.2.Observe the affect of FJZD in cGAS/Wnt16b//β-catenin axis mediated drug resistance① Preparation of FZJD contained serum:20 SD rat were divided into contol group and FZJD group,then administration pure water of FZJD solution(0.33g/ml)respectively by oral gavage(OG)for consecutively five times,then blood was gathered form abdominal aorta.② Western-blot was performed to detected the cGAS expression in MRC-5 and dMRC-5 which undergo different interventions.③IF was to detected the Wnt16b expression in MRC-5 and dMRC-5 which undergo different interventions.④Conditioned medium(CM)was collected from MRC-5 and MRC-5 which undergo different interventions to culture MKN45,then CCK8 was performed to detect IC50.⑤Western-blot was performed to detected the β-catenin expression in MKN45 which was cultured by different CM.3.Establish 5-FU resistant tumor-bearing mice model and observe the affect of FZJD in drug resistance and investigate the relative mechanisms.① NOD-SCID mice was used to implant MKN45 or MKN45/dMRC-5.Control and 5-FU group was setted in MKN45 bearing mice.Control,5-FU,FZJD and FZJD+5-FU group was setted in MKN45/dMRC-5 hybird bearing mice.Results:1.Over 90%yH2AX positive cell were detected after 5-FU(0.8mM)treatment for 72h,over 65%cell still alive,cGAS gene expression in 5-FU group was significantly higher than control group(P<0.0001),dMRC-5 modelwas established.2.The cGAS expression in dMRC-5 was significantly higher than MRC-5(P<0.0001).The cGAS level in dMRC-5 upon different interventions was:Control group>BS group>FS group>G150 group>Blank group.3.The Wnt16b expression in dMRC-5 was significantly higher than MRC-5(P<0.001).The Wnt16b level in dMRC-5 upon different interventions was:Control group>BS group>G150 group>FS group>Blank group.4.The IC50 level in MKN45 which cultured withMKN45 CM or different groups of dMKN45 CM was upregulated,ranked as:Control group>BS group>Normal group>G150 group>FS group>Blank group.5.The β-catenin expression in MKN45 which culturedwith MKN45 CM or different groups of dMKN45 CM was significantly higher than MKN45 in the Blank group(P<0.0001).The β-catenin level in dMRC-5 upon different interventions was:Control group>BS group>G150 group>FS group>Normal group>Blank group.6.Average tumor weight in control group in MKN45/dMRC-5 hybrid bearing mice was higer than their compartment in MKN45 bearing mice,Tumor inhibition rate in 5-FU group in MKN45/dMRC-5 hybrid bearing mice was lower than their compartment in MKN45 bearing mice.5-FU resistance tumor-bearing mice model was establishmented.7.Average tumor weight in MKN45/dMRC-5 hybrid bearing mice was ranked as:Control group>5-FU group>FZJD group>FZJD+5-FU group.The data in FZJD+5-FU group was significantly lower than Control group(P<0.01).8.Tumor inhibition rate in different groups in MKN45/dMRC-5 hybrid bearing mice was ranked as:FZJD+5-FU group>FZJD group>5-FU group>Control group.9.The tumor tissue CD44 expression in 5-FU group in MKN45/dMRC-5 hybrid bearing mice and MKN45 bearing mice was significantly higher than their compartment(P<0.01).CD44 expression in different groups in MKN45/dMRC-5 hybrid bearing mice was ranked as:FZJD group>Control group>FZJD+5-FU group>Control group.10.β-catenin expression in 5-FU group was higher than control group in MKN45 bearing mice.The level of β-catenin in MKN45/dMRC-5 hybird mice ranked as:5-FU group>Control group>FZJD+5-FU group>FZJD group.Conclusion:1.The cGAS and Wnt16b was overexpressed in MRC-5 unpon 5-FU treatment.2.FZJD is effective in overcome drug resistance by inhibiting cancer cell stemness mediated by dMRC-5.The mechanism relied on mediating cGAS/Wntl6b/β-catenin axis.3.dMRC-5 facilitated tumor growth and resistant to chemotherapy.The mechanism of FJZD improve the therapeutic effect of 5-FU is involved in suppress the β-catenin expression and cancer cell stemness.