The Mechanism Of EGCG Reversing The Primary Drug Resistance Of Colon Cancer By Inhibiting The MMP/Syndecan-1/EGFR/Autophagy Pathway

Object: The drug resistance of colon cancer is difficult to solve,fluorouracil combined with other chemotherapy drugs is the main chemotherapy regimen,but more than 50%of patients are not sensitive to chemotherapy.Although KRAS NRAS BRAF wild-type patients may benefit from anti-EGFR mab(Cetuximab/Erbitux),more than 20% of KRAS NRAS BRAF wild-type patients are not sensitive to Cetuximab/Erbitux combined with chemotherapy.Previous studies of our group found that matrix metalloproteinase(MMP)increased in colon cancer patients.As a transmembrane protein,heparin sulfate proteoglycan syndecan-1(Sdc-1)can be cleaved by MMP to form soluble Sdc-1.Soluble Sdc-1 can activate EGFR through HB-EGF and induce drug resistance of colon cancer,but the specific mechanism is not yet known.EGCG is a natural inhibitor of MMP.Therefore,it is speculated that EGCG may inhibit Sdc-1shedding and EGFR activation by inhibiting MMP,thus reverse drug resistance.In this project,it is intended to further study the specific mechanism of Sdc-1 inducing drug resistance of colon cancer to fluorouracil and Cetuximab/Erbitux through EGFR,and to study the regulatory effect of EGCG on MMP/ Sdc-1 /EGFR axis and its influence on primary drug resistance of colon cancer,so as to provide theoretical basis and therapeutic means for reversing primary drug resistance.Methods: This study was divided into three parts.The first part screened colon cancer cells resistant to fluorouracil and Cetuximab/Erbitux and explored the mechanism of drug resistance.In the second part,the effect and mechanism of EGCG on primary drug resistance of colon cancer were explored in vitro.The third part clarify the effect and mechanism of EGCG on primary drug resistance of colon cancer in vivo.Results:The first part of the study showed that primary drug-resistant SW48 cells and Caco-2 cells were successfully screened out,and KRAS,NRAS and BRAF were wildtype.There were many vesicles in the primary drug-resistant cells,and the vesicles contained cytoplasm under electron microscope.Transcriptome sequencing revealed significant changes in MMP and autophagy related molecules in the primary drugresistant cells.The co-localization of Sdc-1 and EGFR on the cell membrane was observed by confocal laser to facilitate the interaction between Sdc-1 and EGFR.The expression of MMP7 and MMP9 was significantly increased in drug-resistant cells,Sdc-1 and HB-EGF on cell membrane were proteolysed by MMP7 and MMP9,resulting in the increase of soluble Sdc-1 and HB-EGF proteins in cell supernatant,while Sdc-1 and HB-EGF gene expression had no significant change.Soluble Sdc-1and HB-EGF activate RAS/RAF/MEK/ERK pathways downstream of EGFR,thereby promoting NOXA expression.NOXA and Beclin-1 competitively bind MCL1,the increased NOXA result in the release of Beclin-1 from the Beclin-1/ MCL1 complex,which ultimately activated the autophagy pathway and induced drug resistance.The second part and the third part of the study showed that EGCG could inhibit the expression of MMP7 and MMP9,reduce the proteolysis of MMP on Sdc-1 and HBEGF,reduce the level of soluble Sdc-1 and HB-EGF,thus inhibit the EGFR pathway and autophagy flow,and enhance the sensitivity of drug-resistant cells to fluorouracil and Cetuximab/Erbitux in vitro and in vivo.Conclusions: KRAS,NRAS and BRAF wild-type colon cancer cells were successfully screened and resistant to fluorouracil and Cetuximab/Erbitux,and the mechanism of drug resistance was the Sdc-1/HB-EGF/EGFR/ autophagy pathway activated by MMP.EGCG can reverse primary resistance by inhibiting MMP/ Sdc-1/HB-EGF/EGFR/autophagy pathway.