Study On The Mechanism Of Natural Compound EGCG Enhancing The Anti-colorectal Cancer Effect Of Irinotecan

Background Colorectal cancer(CRC)is one of the top three malignant tumors in terms of incidence and mortality in the world.The most common Colorectal carcinoma is adenocarcinoma,including Colorectal adenocarcinoma(COAD)and Rectum adenocarcinoma(READ),which makes up 95% to98% of all cases.CRC screening is one of effective strategies for early diagnosis and prevention of disease-related death,which can be treated surgically or even cured,while advanced and/or metastatic CRC requires additional chemotherapy and radiation and usually gets a poor prognosis.For stage III and IV colorectal cancer patients,chemotherapy is an integral part of treatment.Irinotecan,a common chemotherapy drug for CRC,can be used alone or in combination with Fluorouracil and other drugs,but its significant side effects are the main problem in the clinical application.Epidemiological investigation showed that green tea can reduce the risk of many chronic diseases,such as diabetes and cardiovascular diseases.In vitro and in vivo experiments also suggested that catechins,especially epigallocatechin-3-gallate(EGCG),which are the main active components of green tea,have the functions of inhibiting oxidative stress and inflammation,reducing blood lipid and blood sugar,regulating gene expression and molecular signal pathway,etc.In recent years,the anti-tumor activity of natural active products such as EGCG had been widely concerned by people,especially in the prevention and treatment of gastrointestinal tumors such as gastric cancer and colorectal cancer.EGCG and other polyphenols are often used as adjuvant anti-cancer drugs because of their negligible side effects and abroad effectiveness.The purpose of this study is to explore the inhibitory effect of EGCG combined with irinotecan on colorectal cancer.Methods1.In vitro study on the molecular mechanism of EGCG combined with irinotecan in enhancing the anti-colorectal cancer effect(1)CCK8 assay,wound healing assay and Transwell assay were employed to determine the effects of EGCG and/or irinotecan on the proliferation,migration,and invasion of RKO and HCT116 colorectal cancer cells.(2)Cell cycle,apoptosis,autophagy vesicles,ROS,and mitochondrial membrane potential were quantified by flow cytometry.(3)Immunofluorescence was applied to observe the intracellular GRP78 and γ-H2 AX protein expression.Changes of protein levels were examined by Western blot and m RNA levels by q PCR.(4)Constructed plasmid GRP78 and si RNA were transfected into cells for over expressing and silencing the GRP78 gene.(5)GRP78 protein on the cell surface was detected by ELISA.2.In vivo study on the anticancer effect of irinotecan combined with EGCG in mice(1)After the subcutaneous tumor model of human colorectal cancer cell line HCT116 was established,the nude mice were randomized 4 groups to administrate drugs: control,irinotecan,EGCG,irinotecan plus EGCG.(2)The weight and tumor volume of nude mice was monitored.After 3 weeks,blood samples were collected from anesthetized mice and serum was prepared.VEGF content was detected by ELISA.The tumor tissue was collected from the mice and the tumor mass was measured.(3)The immunohistochemical staining of the tumor tissue was used to detect the expression of VEGF,Ki67,γ-H2 AX,Cleaved-Caspase3,Beclin-1,and GRP78 proteins in the tissue sample,and Western blot was used to detect the expression of VEGF,γ-H2 AX,BAX,Bcl-2,GRP78,and LC3 proteins.3.Bioinformatics analysis of GRP78 and its expression difference in colorectal cancer patients(1)Through the HPA and GEPIA databases,the differences of GRP78 expression in colorectal cancer and normal tissues as well as the relationships between GRP78 expression and five-year survival,overall survival and disease-free survival in colonic adenocarcinoma and rectal adenocarcinoma were analyzed using bioinformatics methods.(2)The m RNA and protein expression of GRP78 in tumor and peri-tumor was determined by q PCR and IHC.Results1.In vitro study on the molecular mechanism of EGCG combined with irinotecan in enhancing the anti-colorectal cancer effect CCK8 assays showed that irinotecan at the concentration of 0.5 μM and above,EGCG 20 μM or more,significantly suppressed the proliferation of both RKO and HCT116 cells.When 0.5 μM irinotecan was combined with 20 μM and 50 μM EGCG,the synergistic effect was significant.The wound healing and Transwell experiments also confirmed that the combination of the two drugs had a greater inhibitory effect on cell migration and invasion than either drug alone.EGCG could aggravate DNA damage of RKO and HCT116 cells by inhibiting topoisomerase I,promote the protein expression of γ-H2 AX,and cleavaged ATM.DNA damage resulted in S and G2 cell cycle arrest of RKO and HCT116 cells,and the down-regulation of p-Rb,CDK4,cyclin D1,and cyclin B1.EGCG could also eliminate ROS in colorectal cancer cells and destroy the redox state of tumor cells.JC-1 and annexin V-FITC&PI double staining data showed that the mitochondrial depolarization induced by EGCG might be the leading cause of apoptosis.EGCG significantly increased the number of autophagic vesicles with MDC staining and LC3BⅡ/I ratio.When 3-MA was chosen to inhibit autophagy,cell apoptosis was also inhibited.On the contrary,rapamycin promoted cell apoptosis,suggesting that EGCG may induce cell apoptosis by inducing autophagy.GRP78 is a molecular chaperone that mediates the folding of new peptides in the endoplasmic reticulum(ER).EGCG promoted the expression of GRP78 in colorectal cancer cells,indicating that the cells were at the risk of endoplasmic reticulum stress(ERS)and UPR pathway activation.When transfected with plasmid or si RNA to overexpress or knockdown GRP78,the corresponding apoptosis was either promoted or inhibited,suggesting that EGCG could increase the apoptosis induced by GRP78 mediated ERS.2.In vivo study on the anti cancer effect of irinotecan combined with EGCG in mice After implementing the HCT116 colorectal cancer cell line into the right-back of BALB/C nude mice for 2 weeks,the average tumor volume exceeded 100 mm3.The mice were randomized to administrate drugs for 3 weeks.Except for the control group,the mice did not lose weight significantly.Compared with the initial stage,the tumor volume of the control group increased significantly and slightly in the irinotecan group or EGCG group,while decreased in the combination group.The results acquired from IHC and ELISA showed that there was no significant difference in the expression of VEGF in each group.EGCG promoted the expression of GRP78,cleaved-caspase3,γ-H2 AX,and Beclin-1 in tumor tissues while inhibited Ki67,which was consistent with the results of cell experiments in vitro.3.Bioinformatics analysis of GRP78 and its expression difference in colorectal cancer patients GEPIA and HPA database analysis showed that the GRP78 in colorectal cancer was significantly higher than that in peri-tumor tissues,and no remarkable relevance between the 5-year survival rate and GRP78 expression level in CRC was found.Compared with patients with low GRP78 expression,the higher the GRP78 expression contributed to worse overall survival and disease-free survival in COAD,but there was no significant difference.While the situation was contrary to READ.The results obtained from IHC and q PCR indicated that the expression of GRP78 in READ was remarkably higher than that in peri-tumor tissues.Conclusion Our study found that EGCG could enhance the anti-tumor effect of irinotecan.The results of in vitro cell experiments showed that EGCG and irinotecan could inhibit Topoisomerase I in colorectal cancer RKO and HCT116 cells,resulting in more extensive DNA damage,thus inducing cell cycle arrest and inhibiting cell proliferation,migration and invasion.EGCG promoted apoptosis,which might be related to lowering mitochondrial membrane potential and depolarization,and also to converting autophagy signal into apoptosis signal.EGCG could eliminate ROS in cells,which could be related to cell death by reducing the drug resistance of cells to irinotecan and increasing drug sensitivity.At the same time,EGCG promoted the expression of GRP78,and made the constitutive UPR of cells transform into endoplasmic reticulum stress,which was also the possible reason for the increase of apoptosis.In vitro animal experiments showed that compared with irinotecan or EGCG used alone,the combination of the two drugs had stronger inhibitory effect on subcutaneous colorectal cancer in mice.Compared with the control group,EGCG and/or irinotecan only slightly inhibited the expression of VEGF in tumor tissues,but the serum VEGF content was slightly increased,suggesting that EGCG could inhibit tumor angiogenesis without inhibiting physiological VEGF expression or secretion.EGCG alone or in combination with irinotecan inhibited Ki67 in tumor tissue.Consistent with the results of cell experiments in vitro,EGCG also promoted the expression of γ-H2 AX,Cleaved-Caspase3,Beclin-1,and GRP78 in tumors,which was further confirmed by Western blot experiment.Through bioinformatics analysis of the GEPIA and HPA databases,we found that the expression level of GRP78 in tumors was significantly higher than that in normal tissues,which was consistent with the IHC and q PCR results of GRP78 in rectal adenocarcinoma specimens.However,the expression level of GRP78 was not closely related to the five-year survival rate,the overall survival rate and the disease-free survival rate.