The Inhibitory Effects Of Metformin Combined With 5-FU On Proliferation And Metastasis Of Colorectal Cancer

Background and objective:Colorectal cancer(CRC)is one of the most common malignant tumors.According to the 2020 Global Cancer Statistics,colorectal cancer is the third most common malignancy in the world and has the second highest mortality rate.At present,with the early systematic screening and continuous improvement of treatment programs,colorectal cancer mortality in developed countries is on the decline.In many developing countries,however,mortality is still rapidly rising.According to Chinese statistics in 2020,the incidence of colorectal cancer ranks the second among all malignant tumors,and the mortality rate ranks the fifth.The incidence of colon cancer has increased significantly,and some diagnosed patients are in the middle and advanced stages.It has seriously threatened the physical and mental health and even life safety of residents,and caused a great medical economic burden on families and society.Metastasis,especially distant metastasis,is a major cause of prognosis and death in patients with colorectal cancer.Nearly all colorectal cancer patients are at risk for metastasis,and about 90%of cancer-related deaths in the clinic are caused by metastasis.Currently,surgery is the main treatment for colorectal cancer,and chemotherapy,as one of the major postoperative treatments,has undoubted benefits for colorectal cancer,but its drug resistance and side effects can not be ignored.Therefore,a new drug and strategy is urgently needed to inhibit the metastasis of colorectal cancer and reduce the adverse reactions of chemotherapy.Metformin is an oral antidiabetic agent of the biguanide family,which is recommended as the first-line antidiabetic agent for Type 2 diabetes mellitus(T2DM)in many diabetes management guidelines due to its precise efficacy,low cost and relatively good tolerability.Metformin reduces blood glucose mainly by reducing liver gluconeogenesis and improving insulin sensitivity,without causinghypoglycemia.Because of the significant correlation between type 2 diabetes mellitus and cancer risk,the relationship between diabetes drugs and cancer prevention has attracted much attention in recent years.Studies have shown that metformin hascertain inhibitory effects on a variety of tumors.Metformin can inhibit not only the proliferation,invasion and migration of tumor cell lines in vitro,but also solid tumor metastasis in vivo and in animal models.At present,the role of metformin in colorectal cancer is still controversial.Metformin has been found to reduce the risk and mortality of colorectal cancer in patients with type 2 diabetes.In contrast,metformin has been found to have no protective effect on the incidence or survival of colorectal cancer.In addition,most studies have been conducted in patients with type 2 diabetes and colorectal cancer,and the effects of metformin on colorectal cancer in non-diabetic patients has not been widely explored.Based on the antitumor effect of metformin,researchers have extensively studied the effects of metformin combined with different chemotherapy drugs on tumors.Studies have shown that metformin could significantly enhance the sensitivity of different tumor cells to chemotherapy drugs such as cisplatin,carboplatin,adriamycin and paclitaxel,and the anti-tumor effect was significantly enhanced when metformin combined with different chemotherapy drugs.5-fluorouracil(5-FU)is one of the basic chemotherapy agents for colorectal cancer,widely used in clinical stage ?,? and ? patients,and is the only chemotherapy agent that can improve the 12-month survival rate of colorectal cancer patients at present.However,the overall response rate of patients with advanced colorectal cancer treated with 5-FU monotherapy was about 10-15%,and the survival rate was not significantly improved,and the response rate of 5-FU combined with other chemotherapy drugs was less than 50%.The innate and acquired drug resistance of tumor cells to 5-FU seriously affects its chemotherapy effect,and some patients treated with 5-FU will have serious toxic and side effects.Some studies have found that metformin could significantly improve the anti-proliferation ability of 5-FU on colorectal cancer cell lines,but the effect of metformin combined with 5-FU on the invasion and metastasis of colorectal cancer has not been reported in current studies.Therefore,in order to explore the effects of metformin combined with 5-FU on the proliferation and metastasis of colorectal cancer and its possible mechanism,the effects of metformin combined with 5-FU on the proliferation and invasion of colorectal cancer cell lines HCT116 and SW1463 were examined in vitro cytological experiments.In vivo,the effects of metformin combined with 5-FU on the growth and metastasis of colorectal cancer were observed by using an orthotopic cecal implant model in nude mice.In addition,Tandem Mass Tag(TMT)-labeled quantitative proteomics and bioinformatics analysis were used to further explore the possible mechanism of metformin alone and combined with 5-FU in inhibiting the proliferation and metastasis of colorectal cancer.The specific content of this study includes the following three parts:Part ? The effects of metformin combined with 5-FU on proliferation and invasion of colorectal cancer cell lines HCT116 and SW1463 in vitroObjective:To investigate the effects of metformin combined with 5-FU on the proliferation and invasion of colorectal cancer cell lines HCT116 and SW1463 in vitro.Methods:1.Human colon cancer cell line HCT116 and human rectal cancer cell line SW1463 were used as experimental objects.2.CCK-8 assay was used to detect the cell viability of metformin(0,1mM,5mM,10mM,20mM and 40mM)or 5-FU(0,5?M,50?M,100?M,200?M and 300?M)treated for 24h.Then,the Inhibition Concentration(IC)of metformin and 5-FU on the two cell lines was calculated respectively.The appropriate concentrations of metformin and 5-FU were selected for subsequent experiments according to the use concentration ?half maximal inhibitory concentration(IC50)and the parallelism(comparability)between groups.CCK8 assay was used to detect the cell viability of the two cell lines after treated with metformin combined with different concentrations of 5-FU for 24h.3.The effects of metformin and/or 5-FU on the clonal formation ability of two cell lines were determined by plate clonal formation experiment.4.The effects of metformin and/or 5-FU on the invasion ability of the two cell lines were determined by Transwell invasion assay.Results:1.CCK8 assay showed that different concentrations of metformin(0,1 mM,5 mM,10 mM,20 mM and 40 mM)or 5-FU(0,5 ?M,50 ?M,100?M,200?M and 300?M)could inhibit the proliferation of HCT116 and SW1463 cells.in a dose-dependent manner.And with the increase of drug concentration,its inhibitory effect on proliferation increased gradually.The IC50 of metformin in HCT116 and SW1463 cell lines were 4.6mM and 13.6mM,respectively,and the IC50 of 5-FU was 4.8?M and 1269.0?M,respectively.Both 5mM metformin and 5?M5-FU were close to IC50 of HCT116 cells and IC25 of SW1463 cells.According to the use concentration ?IC50 and the parallelism between groups,5mM metformin 5?M 5-FU were selected in subsequent experiments.Compared with 5-FU alone,metformin combined with 5-FU could inhibit the proliferation of HCT116 cells and SW1463 cells significantly(P<0.05).2.The plate clonal formation experiment showed that:Compared with the control group(41.3±4.0),the number of HCT116 cells clones in the metformin group(23.3±3.5),the 5-FU group(24.7±5.5)and the metformin combined with 5-FU group(5.7±1.5)was significantly decreased(all P<0.05).Compared with the 5-FU group(24.7±5.5),the number of HCT116 cells clones in the metformin combined with 5-FU group(5.7±1.5)was significantly decreased(P<0.05).Compared with the control group(132.3±31.6),the number of SW1463 cells clones in the metformin group(69.7±11.2),the 5-FU group(75.0±13.2)and the metformin combined with 5-FU group(37.7±16.3)was significantly decreased(all P<0.05).Compared with the 5-FU group(75.0±13.2),the number of SW1463 cells clones in the metformin combined with 5-FU group(37.7±16.3)was significantly decreased(P<0.05).3.Transwell invasion test showed that:Compared with the control group(69.8±11.2),the number of HCT116 invasive cells in the metformin group(38.6±9.7)and the metformin combined with 5-FU group(23.0±7.1)was decreased significantly(all P<0.05),and the number of HCT116 invasive cells in the 5-FU group(51.0±16.3)showed no significant difference(P>0.05).Compared with the 5-FU group(51.0±16.3),the number of HCTI 16 invasive cells in the metformin combined with 5-FU group(23.0±7.1)was significantly decreased(P<0.05).Compared with the control group(87.8±9.5),the number of SW1463 invasive cells in the metformin group(50.2±9.8)and the metformin combined with 5-FU group(37.8±9.5)was significantly decreased(all P<0.05),and the number of SW1463 invasive cells in the 5-FU group(76.6±13.0)showed no significant difference(P>0.05).Cbmpared with the 5-FU group(76.6±13.0),the number of SW1463 invasive cells in the metformin combined with 5-FU group(37.8±9.5)was significantly decreased(P<0.05).Conclusion:Metformin can inhibit the proliferation and invasion of colorectal cancer cell lines HCT116 and SW1463.Metformin combined with 5-FU has a synergistic effect,which can significantly increase the inhibition of colorectal cancer cell proliferation and invasion.Part ? The effects of metformin combined with 5-FU on the growth and metastasis of colorectal cancer in nude miceObjective:To investigate the effects of metformin combined with 5-FU on the growth and metastasis of colorectal cancer by constructing an orthotopic cecal implant model in nude mice.Methods:1.Establishment of subcutaneous implant tumor model in nude mice:The HCT116 cell suspension was injected subcutaneously in 5-week-old male nude mice.After 4-5 weeks after inoculation,the tumor grew to 1.5cm in diameter,and the tumor was removed and cut into tissue blocks about 1mm3 size for use.2.Construction of cecal orthotopic implantation model for colorectal cancer in nude mice:20 male nude mice with good growth at 5 weeks of age were selected.Mice were anesthetized by intraperitoneal injection of 10%chloral hydrate(0.3 ml/100g)and then the abdomen was sterilized with 75%alcohol.An incision(0.5cm)was made in the left lower abdomen to pull out the cecum.The serosa at the site of implantation was removed.The tumor tissues(1mm3)were sutured on the wall of the cecum by surgical suture.Subsequently,the cecum was refolded to the abdominal cavity,followed by skin suturing.3.Drug intervention in nude mice model of colorectal cancer:One week after transplantation,the mice were randomly divided into four groups:(a)control group(n=5),received intraperitoneal injection of 0.9%sodium chloride;(b)metformin group(n=5),received oral lavage of metformin(250 mg/kg)per day;(c)5-FU group(n=5),received intraperitoneal injection of 5-FU(25mg/kg)once a week;(d)co-treatment group(n=5),received oral lavage of metformin(250 mg/kg)per day combined with 5-FU(25mg/kg)via intraperitoneal injection once a week.Four weeks after treatment,all mice were sacrificed after cervical dislocation followed by measuring the tumor growth and observation of metastasis.The carcinoma in situ and suspected metastatic foci were removed,their volume and weight were measured,and then partially were cut into small pieces and frozen in liquid nitrogen,partially were fixed in the neutral formalin for paraffin embedding.4.Immunohistochemical Ki-67 staining was used to detect the proliferation index of in situ implantable carcinoma.Results:1.Volume of implanted carcinoma in situ:Compared with the control group(1097.9±603.6 mm3),the volume of carcinoma in situ decreased significantly in the 5-FU group(237.6±131.3 mm3)and the co-treatment group(224.5±79.3 mm3)(all P<0.05).Compared with the control group(1097.9±603.6 mm3),there was no significant difference in the volume of carcinoma.in situ in metformin group(768.3±224.0 mm3)(P>0.05).Compared with the 5-FU group(237.6±131.3 mm3),there was no significant difference in the volume of carcinoma in situ in the co-treatment group(224.5±79,3 mm3)(P>0.05).2.Weight of implanted carcinoma in situ:Compared with the control group(1.00±0.35 g),the weight of carcinoma in situ was significantly reduced in the 5-FU group(0.48±0.34 g)and the co-treatment group(0.42±0.24 g)(all P<0.05).Compared with the control group(1.00±0.35 g),there was no significant difference in the weight of carcinoma in situ in metformin group(0.88±0.20 g)(P>0.05).Compared with the 5-FU group(0.48±0.34 g),there was no significant difference in the weight of carcinoma in situ in the co-treatment group(0.42±0.24 g)(P>0.05).3.Visual and microscopic examinations revealed that there were multiple distant metastases in all control mice(5/5)and 80%of the mice in 5-FU group(4/5).Metastatic sites/organs included pancreas,liver,intestine,omentum and renal capsule.Mice in the metformin group(1/5,20%)and co-treatment group(1/5,20%)only showed liver metastasis.Compared with control group(5/5,100%),the distant metastatic rate showed significant decline in metformin group(1/5,20%)and co-treatment group(1/5,20%)(all P<0.05).No significant differences were noticed in the distant metastasis rate between the co-treatment group(1/5,20%)and the 5-FU group(4/5,80%)(P>0.05).4.Ki-67 proliferative index of implanted carcinoma in situ:Compared with the control group(93.6%±3.5%),the Ki-67 proliferative index of metformin group(74.0%±6.5%),5-FU group(70.0%±7.9%)and co-treatment group(56.0%±6.5%)was significantly decreased(all P<0.05).Compared with the 5-FU group(70.0%±7.9%),the Ki-67 proliferative index of co-treatment group(56.0%±6.5%)was significantly decreased(P<0.05).Conclusion:Metformin can inhibit the proliferation and distant metastasis of colorectal cancer in nude mice,and the combination of metformin and 5-FU has a synergistic effect,which can significantly increase the inhibition of colorectal cancer proliferation and distant metastasis.Part ? The study on mechanism of metformin alone or combined with 5-FU in inhibiting the proliferation and metastasis of colorectal cancerObjective:Tandem Mass Tag(TMT)labeled quantitative proteomics and bioinformatics were employed to investigate the possible mechanism of metformin alone and combined with 5-FU in inhibiting the proliferation and metastasis of colorectal cancer.Methods:1.A total of 6 samples were randomly selected,including 3 samples in the control group and 3 samples in the metformin group.2.Tandem Mass Tag(TMT)labeled quantitative proteomics were employed to screen out differentially expressed proteins in the control group and metformin group.3.The bioinformatics analysis,including clustering analysis,GO functional annotations and enrichment analysis,KEGG pathway annotations and enrichment analysis were used to analyze the molecular function,biological process,cell metabolism and signal transduction pathway of differentially expressed proteins,so as to screen out the key regulatory proteins which were closely related to cell proliferation and metastasis of colorectal cancer in this study.4.Western Blot was used to verify the expression of relevant differential proteins in implanted carcinoma in situ.5.Immunohistochemical staining technique was used to verify the expression of relevant differential proteins in implanted carcinoma in situ.Results:1.Compared to the control group,70 differentially expressed proteins in metformin group including 17 up-regulated differentially expressed proteins and 53 down-regulated differentially expressed proteins were screened out by TMT proteomics analysis.2.GO functional annotation and enrichment analysis showed that three proteins including CDKN1A,GDF15 and CRIP1 were closely related to cell proliferation and metastasis.CDKN1A protein is mainly related to the functions of cell cycle,negative regulation of epithelial cell proliferation,programmed cell death and cyclin serine/threonine kinase inhibitors.GDF15 protein is mainly related to cell growth factor activity,negative regulation of epithelial cell proliferation,negative regulation of cell migration,cell adhesion,negative regulation of vascular development and other functions.CRIP1 protein is mainly related to immune response,cell response to organic matter and antibiotics,actin filaments depolymerization and cleavage,DNA double-strand fracture repair,cytoskeleton formation and other functions.Protein quantification and difference analysis showed that CDKN1A protein expression was significantly up-regulated in metformin group,which was 1.254 times higher than that in control group.The protein expression of GDF15 was significantly up-regulated,which was 1.859 times that of the control group.The expression of CRIP1 protein was significantly down-regulated,which was 0.733 times that of the control group.KEGG pathway annotation and enrichment analysis showed that there were 117 metabolic and signal transduction pathways significantly affected by 70 differential proteins,among which CDKN1A was involved in P53,PI3K-Akt,ErbB and other signaling pathways,and GDF15 was involved in cytokine-cytokine receptor interaction.3.Western Blot was used to verify the expression of CDKN1A,GDF15 and CRIP1 in implanted carcinoma in situ.Compared with the control group(1.0±0.0),the expression of CDKN1A protein was significantly increased in the metformin group(4.1±1.3),5-FU group(6.3±1.3)and co-treatment group(9.6±2.7)(all P<0.05).Compared with 5-FU group(6.3±1.3),the expression of CDKN1A protein in co-treatment group(9.6±2.7)was significantly increased(P<0.05).Compared with the control group(1.0±0.0),the expression of GDF15 protein in the metformin group(3.5±1.5)and co-treatment group(5.4±2.2)was significantly increased(all P<0.05),but there was no significant difference in the expression of GDF15 protein in the 5-FU group(1.4±0.4)(P>0.05).Compared with 5-FU group(1.4±0.4),the expression of GDF15 protein in co-treatment group(5.4±2.2)was significantly increased(P<0.05).Compared with the control group(1.0±0.0),the expression of CRIP1 protein was significantly decreased in metformin group(0.6±0.1)and co-treatment group(0.3±0.1)(all P<0.05),while there was no significant difference in the expression of CRIP1 protein in 5-FU group(0.8±0.1)(P>0.05).Compared with 5-FU group(0.8±0.1),the expression of CRIP 1 protein in co-treatment group(0.3±0.1)was significantly decreased(P<0.05).4.Immunohistochemical staining technique was used to verify the expression of CDKN1 A,GDF15 and CRIP 1 in implanted carcinoma in situ.Compared with the control group(1.4±1.3),the staining scores of CDKN1A protein were significantly increased in the metformin group(3.4±1.1),5-FU group(4.6±0.9)and co-treatment group(6.0±0.7)(all P<0.05).Compared with 5-FU group(4.6±0.9),the staining scores of CDKN1A protein in co-treatment group(6.0±0.7)were significantly increased(P<0.05).Compared with the control group(0.8±1.1),the staining scores of GDF 15 protein in the metformin group(4.6±0.5)and co-treatment group(5.6±0.5)were significantly increased(all P<0.05),but there was no significant difference in the staining scores of GDF 15 protein in the 5-FU group(1.4±1.3)(P>0.05).Compared with 5-FU group(1.4±1.3),the staining scores of GDF 15 protein in co-treatment group(5.6±0.5)was significantly increased(P<0.05).Compared with the control group(6.2±0.8),the staining scores of CRIP1 protein were significantly decreased in metformin group(4.4±0.5)and co-treatment group(2.8±0.8)(all P<0.05),while there was no significant difference in the staining scores of CRIP1 protein in 5-FU group(5.4±1.1)(P>0.05).Compared with 5-FU group(5.4±1.1),the staining scores of CRIP 1 protein in co-treatment group(2.8±0.8)were significantly decreased(P<0.05).Conclusion:A variety of proteins are involved in the inhibitory effects of metformin on colorectal cancer,and the mechanism may be related to the up-regulation of CDKN1A and GDF15 and down-regulation of CRIP1 protein expression.These three proteins may be key proteins in the synergistic inhibition of colorectal cancer proliferation and metastasis by metformin combined with 5-FU.