Inhibitory Effect Of Aripiprazole On Colorectal Cancer By Mediating MDM2

Background and Objective:As the third most common cancer and the second most lethal cancer in the world,colorectal cancer has attracted more and more attention.At present,the main treatment options include surgery,radiotherapy,chemotherapy and other ways,but the treatment effect is still not optimistic.The new use of old drugs is slowly coming into people’s sight as a low-cost alternative.In recent years,more and more reports have proved that antipsychotic drugs can inhibit the occurrence and development of tumors.Aripiprazole,the third generation antipsychotic drug,is widely used in clinic because of its good efficacy and less side effects.It has been reported that aripiprazole can cause cell cycle arrest and inhibit cell proliferation in breast cancer cells.P53 gene plays an important role in the occurrence,development and regulation of tumors,which is a hot topic in recent years.P53 plays an important role in the regulation of cell cycle during DNA damage and other stressors.The tumor suppression of p53 is closely related to the regulation of some proteins.MDM2(Mouse double minute 2),as a major negative regulator of p53,can affect the development of tumors by interacting with p53.At present,MDM2-p53 inhibitors have entered clinical trials.Nutlin-3,the first MDM2-p53 small molecule inhibitor,blocks the interaction of p53 and MDM2 by binding MDM2,which both inhibits the activity of MDM2 and activates the function of p53protein and its network.Xhibits inhibition of various tumor cell activities.Whether aripiprazole may affect the activity of colorectal cancer by inhibiting MDM2-p53interaction is worth investigating.The purpose of this study was to explore the role and molecular mechanism of MDM2 in the inhibition of colorectal cancer by aripiprazole,and to provide new ideas for the clinical treatment of colorectal cancer and the design and development of MDM2 antagonists.Methods and Results:1.Different concentrations of aripiprazole(0,5,10,20μM)were used to treat the logarithmic growth phase of cervical cancer cells Hela,liver cancer cells Hep G2,non-small cell lung cancer cells A549,colorectal cancer cells HT-29,colorectal cancer cells HCT116 and normal colon cells FHC.MTT assay was used to detect the survival rate at 24 h,48 h and 72 h after drug intervention.The results showed that aripiprazole had no obvious antitumor activity on cervical cancer cell Hela,liver cancer cell Hep G2 and non-small cell lung cancer cell A549,but significantly inhibited the proliferation of colorectal cancer cells(HCT116 and HT-29),with IC50 values of 11.53μM and 6.23μM(P<0.01)at 72 h,which were significantly lower than the IC50 value(100μM)of fluorouracil on HT-29 cells.In addition,aripiprazole has no significant toxicity to normal colonic cells.In vivo,BALB/c mice were inoculated with HT-29 colorectal cancer cells subcutaneously,and then treated with different doses of aripiprazole(0,10,30 mg/kg)by gavage twice a day for 14 consecutive days,during which the body weight was monitored;the size of the tumor was detected after administration,and the hepatotoxicity and nephrotoxicity were assessed by H&E staining.The results showed that aripiprazole also had inhibitory effect on colorectal cancer cells,and the volume of tumor-bearing tissue in mice decreased gradually with the increase of the dose of aripiprazole(P<0.01).Aripiprazole had no significant effect on body weight and no significant hepatotoxicity and nephrotoxicity.In conclusion,aripiprazole can inhibit the proliferation of colorectal cancer cells in vitro and in vivo.2.Transcriptome analysis and network pharmacology explore the role of MDM2in the inhibition of colorectal cancer by aripiprazoleThe tumor-bearing tissues of mice treated with aripiprazole were transcribed and sequenced,and the targets of aripiprazole on colorectal cancer were screened by combining Dis Ge NET and Gene Cards website data.Three groups of data were enriched to 375 overlapping targets such as MDM2,in which the transcription level of MDM2was significantly down-regulated by aripiprazole.Cell cycle pathway,Ubiquitination pathway,p53 signaling pathway and other signaling pathways also changed accordingly.The m RNA levels of MDM2 and its promoter transcription factors IRF8,SP1,EIF4E were significantly down-regulated in the tumor-bearing mice by q PCR(P<0.05).The correlation between MDM2 and p53 expression and the prognosis of colorectal cancer patients was analyzed in the clinical database GEPIA,suggesting that the survival time of colorectal cancer patients with low MDM2 expression and high p53 expression was longer.3.Involvement of MDM2 in the mechanism of cell cycle inhibition by aripiprazole in colorectal cancer cellsTo further explore the mechanism by which aripiprazole inhibits colorectal cancer,we performed flow cytometry based on the changes in transcriptome enrichment to cell cycle pathways.The results of flow cytometry showed that aripiprazole could block the cell cycle of HT-29 cells in G0/G1 phase and exert its anti-tumor activity after 24 h of aripiprazole intervention.The expression of MDM2,p21 and Cyclin D1 in vitro and in vivo was detected by Western blot,and the results showed that aripiprazole could significantly down-regulate the expression of MDM2 and Cyclin D1(P<0.05),and significantly up-regulate the expression of p21(P<0.01).The MDM2 knock-down vector was constructed and used in combination with 20μM aripiprazole to treat HCT116 cells.After MDM2 knockdown plasmid was constructed and transfected into the cells,the cell survival rate was detected by MTT assay,and the expression of MDM2 and cell cycle related proteins was detected by WB.MTT results showed that compared with Control group,the cell viability after MDM2knockdown was significantly decreased(P<0.001);compared with 20μM aripiprazole group,the cell viability of MDM2 knockdown group combined with aripiprazole group was also significantly decreased(P<0.05).WB results showed that Cyclin D1 protein level was significantly down-regulated(P<0.05)and p53 and p21 protein levels were significantly up-regulated(P<0.05)in MDM2 knockdown group compared with Control group;Compared with the 20μM aripiprazole group,p53 was significantly up-regulated in the MDM2 knockdown group and the aripiprazole combination group(P<0.05).The expression of p53 in HT-29 cells was detected by immunofluorescence.The expression of p53 in HT-29 cells was detected by Western blot.Immunofluorescence results showed that 20μM aripiprazole significantly up-regulated the expression of p53(P<0.01).WB results showed that 20μM aripiprazole up-regulated the expression of p53 protein in vitro and in vivo(P<0.05),and significantly up-regulated p53 in the nucleus(P<0.001).Pearson method was used to analyze the protein expression of p53and MDM2,and the results showed that the increase of p53 protein expression was significantly correlated with the decrease of MDM2 protein expression(R~2=0.4964,P=0.0005).Ubiquitination assay was used to detect the ubiquitination of p53 in HT-29 cells treated with aripiprazole and MDM2 inhibitor(Nutlin-3)respectively.The results showed that compared with the Control group,the ubiquitination level of p53 was decreased by(25.9±2.3)%with aripiprazole and(17.5±2.5)%with Nutlin-3.In order to test whether aripiprazole can bind MDM2 like the inhibitor Nutlin-3,we used Discovery Studio to predict the binding of aripiprazole to MDM2,and cell thermal migration assay(CETSA)to verify the binding of aripiprazole to MDM2.The results showed that there wereπ-cation,π-,π-alkyl interactions between aripiprazole and MDM2,and the molecular docking score was 103.309.The results of CETSA showed that the thermal stability of MDM2 protein was improved after treatment with 20μM aripiprazole for 30 min,suggesting that aripiprazole could bind to MDM2 in cells.In order to investigate whether 5-HT receptor,the pharmacological target of aripiprazole,could act as the upstream target of MDM2 to regulate MDM2,5-HT agonist H9523 was introduced to further explore the possible mechanism of aripiprazole down-regulating MDM2 expression.The results showed that the downregulation of AKT-MDM2 induced by aripiprazole was restored when 5-HT agonist was used in combination with aripiprazole.These results suggest that aripiprazole may inhibit the expression of MDM2 through 5-HT receptor.Conclusions:In vitro and in vivo,aripiprazole exerts its antitumor activity in colorectal cancer by binding to MDM2,inhibiting MDM2-p53 signaling pathway,increasing p53 nuclear accumulation,inhibiting p53 ubiquitination and arresting cell cycle in a concentration-dependent manner.It provides a new theoretical basis and experimental evidence for the anti-tumor research of aripiprazole and the treatment of colorectal cancer.