Proliferation Inhibition Of Metformin On Colorectal Cancer Cells By Regulating The Urea Cycle Via AMPK/p53

In the liver,the Urea Cycle（UC）is composed of five catalytic enzymes:carbamoyl phosphate synthase 1（CPS1）and ornithine transcarbamylase（OTC）are mitochondrial enzymes,argininosuccinate lyase（ASL）,argininosuccinate synthase（ASS1）and Arginase1（ARG1）are cytoplasmic enzymes.In recent years,the relationship between urea cycle metabolism disorder and tumor occurrence and development has attracted much attention.Increasingly research results showed that changes in urea cycle enzymes are not only related to tumorigenesis and development,also associated with poor survival in hepatocellular,breast,and colorectal cancers etc.Cytoplasmic ornithine（an intermediate product of the urea cycle）is a specific substrate for the production of putrescine by ornithine decarboxylase（ODC,also known as ODC1）.Polyamines such as putrescine,spermine and spermidine play an important role in the proliferation of tumor cells.The results of previous studies proved that increased ODC1 activity can promote cell proliferation thereby accelerate the growth of tumor cells.It is suggested that inhibition of ODC1 activity may be a potential target for tumor inhibition.Colorectal cancer is the fourth most common cancer in the world.Although the overall incidence of CRC has decreased significantly in recent years,it continues to increase in patients under 50-years-old.The prognosis of colorectal cancer varies according to cancer stage,with 5-year survival rate of about 90%for stage I disease and a dismal～10%for stage IV patients.Although 60%of patients present with a resectable disease at the time of diagnosis,nearly half of these patients stand curative surgery alone and another 20～25%who receive post-surgical adjuvant chemotherapy,experience cancer relapse,metastatic disease,and eventual death.At present,the treatment of colon cancer is still based on chemotherapy,commonly used chemotherapy drugs,such as oxaliplatin,have great toxic and side effects,moreover,drug resistance often occurs.Thus,it is very necessary to find an anti-CRC drug with less side effects and highly effective.Metformin（Met）is an oral biguanide drug,approved by the FDA in1994 as a first-line drug for the treatment of diabetes.Multiple clinical studies have shown that metformin has improved survival in diabetic patients with different types of tumors,suggesting its potential anticancer effects.Previous studies have found that as a classic drug for regulating glucose metabolism,metformin can induce autophagy,anti-aging,anti-oxidative stress,anti-endoplasmic reticulum stress,anti-inflammatory,anti-fibrosis,etc.through AMPK-dependent or independent pathways,At the same time,the activation of AMPK pathway induced by metformin can induce the activation of p53 and enhance the activity of p53.The p53 gene is the most common tumor suppressor genes in human cancers.Under normal conditions,p53 strictly controls the physiological processes of cell proliferation,senescence,DNA repair and cell death.Recent research results indicate that p53 also plays an important role in the metabolism of normal cells and cancer cells.The abnormality of its regulatory function is closely related to changes in glucose metabolism and amino acid metabolism,revealing the role of p53 in affecting tumor progression by regulating metabolism in the body.The urea cycle is an important part of amino acid metabolism,The latest research shows that it can play an anti-tumor effect by inhibiting the urea cycle.The effect of metformin on inhibiting the proliferation of colon cancer cells has been reported but the mechanism is lacking;then,whether metformin regulates urea cycle enzymes and ODC1 activity through AMPK/p53 signaling pathway to inhibit the proliferation of colon cancer cells has not been reported yet.Objective:In the first part of this study,HCT116 cells were inoculated subcutaneously in the right lower axilla of 5-week-old female BALB/c-nu mice to form tumors,the experimental group was fed with Met,the effect of Met on tumor growth,urea cycle and putrescine production were detected;the second part of the study used human colon cancer cells HCT116 and SW480 as the research object,the effect of Met on colon cancer cell proliferation,urea cycle and putrescine production were detected,combining the effects of Met in vivo and in vitro to explore its mechanism of inhibiting the proliferation of colon cancer cells,it provided a new theoretical and experimental basis for the clinical application of Met in the treatment of colon cancer.MethodPart I:HCT116 cells were inoculated subcutanically in the right lower axilla of 5-week-old female BALB/c-nu mice to form tumors.The experimental group was treat with Met with concentration of 200 mg/kg/day and the control group was fed with 200μL saline/day for 28 days.The body weight and tumor size of nude mice were measured and recorded every day.After the treatment,the nude mice were sacrificed,the tumor was taken,weighed and photographed,before the nude mice were sacrificed,mouse blood was collected,blood biochemical detection of the effect of Met treatment on blood urea and uric acid levels in nude mice;tumor tissue was collected,and the effect of Met on tumor tissue urea cycle and putrescine production were identified and quantified by LC-MS/MS;the difference in the expression of ODC1 gene and protein in normal colon tissue and colon cancer tissue were detected via bioinformatics technology;Western blot was used to detect protein expression of AMPK/p53,urea cycle enzymes and ODC1 protein in the tumor.Part II:Using human colon cancer cells HCT116 and SW480 as the experimental object,the CCK-8,colony formation method were used to detect the effect of Met on the proliferation of cells;Flow cytometry was used to test the effects of Met on cell cycle of CRC cells;the effect of Met on urea cycle and putrescine production of cells were identified and quantified by LC-MS/MS,the relationship between the expression of ARG1,TP53 and the survival curve of colon cancer patients were detected via Bioinformatics technology;Western blot was used to detect protein expression of AMPK,urea cycle proteins and ODC1 in CRC cells;pre-treatment of Nutlin-3（p53 activator）and pft-α（p53 inhibitor）before being treated with Met,changes of the proliferation in CRC cells were checked by Ed U and colony formation;the cell cycle was detected by flow cytometry and the expression of p53 and ODC1 proteins were measured by Western blot.ResultPart I:1.The qualified measurement results showed that small tumor volume and reduced tumor weight in Met-treated mice compared with the control group;the blood biochemical results show that the urea and urca level in the Met group are decreased compared with the control group,and statistically significant（P<0.001）.2.The results of LC-MS/MS showed that compared with the control group,the tumor tissues metabolite levels of the nude mice in the Met treatment group were significantly changed,and the putrescine level was significantly decreased,which was statistically significant（P<0.05）.3.Bioinformatics results showed that the expression of ODC1 in colorectal cancer was greatly higher than that of the normal colon tissues,and the staining intensity of ODC1 in normal colon tissues was either no or light brownish yellow,whereas that in colon cancer tissues showed medium to strong brownish yellow staining.4.Western blot results showed that compared with the control group,the expression levels of AMPK,p53 and p21 in the tumor tissue of the Met group mice were significantly increased,The expression level of CPS1,OTC,ARG1 and ODC1 was significantly decreased and had statistical significance（P<0.05）.Part II:1.The results of CCK-8 method showed that after Met treatment of HCT116 and SW480 cells,the proliferation of HCT116 and SW480 cells were significantly inhibited in a time and dose-dependent manner（P<0.05）.The IC₅₀ of Met treatment for 24 h on CRC cells were 40 mmol/L and 42mmol/L respectively;the IC₅₀ of Met treatment for 48 h on cells were 20mmol/L and 30 mmol/L respectively.2.Clone formation experiment:It was proved that the inhibition of cell proliferation increased with the concentration increase of Met（P<0.001）.3.The cell cycle detecting by FCM showed that Met could induce the cell cycle arrest at G1 phase,with statistical significance（P<0.001）.4.The results of LC-MS/MS showed that compared with the control group,the metabolite levels of HCT116 cells in the Met treatment group were significantly changed,and the putrescine level was significantly decreased,which was statistically significant（P<0.001）.5.Bioinformatics results showed that low expression of TP53 and high expression of ARG1 are associated with poor prognosis and survival.6.Western blot results showed that compared with the control group,the expression levels of AMPK and p-AMPK in the Met group cells were significantly elevated,and the expression levels of ARG1,OTC and ODC1were obvious reduced and had statistical significance（P<0.05）.7.After Nutlin-3 treatment,Ed U,clone formation experiment,and FCM results showed that compared with the control group,the proliferation of HCT116 cells in the Nutlin-3 and Met treatment groups was significantly inhibited,and the cell cycle was arrested at the G1 phase.Western blot results showed that compared with the control group,the expression level of p53protein in the Nutlin-3 and Met treatment groups was significantly up-regulated,while the expression level of ODC1 protein was significantly down-regulated.Pft-αdid not exhibit inhibitory effect on cell proliferation compared with control by Ed U measures;however,the cell proliferation was inhibited by treatment with metformin or pft-αcombined with metformin;Met can rescue the p53 inhibition caused by Pft-αand reduce the expression of ODC1 protein detected by Western blot.Conclusion1.Met can inhibit the growth of colon cancer cells in vivo,and inhibit the urea cycle and putrescine production.2.Met can inhibit the proliferation of colon cancer cells in vitro,induce cell cycle arrest in G1 phase,and inhibit the urea cycle and putrescine production.3.Met may play its role in inhibiting the proliferation of colon cancer cells by activating AMPK/p53 signaling pathway,inhibiting the urea cycle and putrescine production.