The Role Of BCL9 In Aripiprazole Inhibiting Colon Cancer Cells Proliferation And Its Molecular Mechanism

Background and Objective:Colon cancer is a relatively common cancer,which can easily cause great damage to the body,leading high incidence and mortality rates in modern society.To date,there is a dearth of drugs to treat colon cancer securely and potently,so we need to discover new drugs to save patients’ lives.In recent years,many antipsychotic drugs have been proven to have antitumor effects.Aripiprazole(Ari),as a third-generation antipsychotic drug,has also shown inhibitory effects on tumor proliferation,but its mechanism is still unclear.Current research has found that the most significant cause of colon cancer is overreaction of WNT-β-catenin pathway.BCL9(B-cell lymphoma 9)is a transcription factor of β-catenin,it is a critical factor in the activating the WNT-β-catenin pathway,which has an important impact on colon cancer.The purpose of this study is to explore the effects of aripiprazole on the proliferation of colon cancer cells,and to explore the key role and mechanism of BCL9.This study can provide novel ideas for the development of clinical anti-tumor drugs.Methods and Results:1.The effects of aripiprazole on proliferation,cell cycle and tumorigenesis of colon cancer cells (1)0 μM,6 μM,12 μM and 24 μM Ari were treated separately on A549 lung cancer cells,Hep G2 liver cancer cells,SK-N-SH neuroblastoma cells,FHC normal intestinal cells,HCT116 and HT29 colon cancer cells for 24 h,48 h and 72 h,then using MTT colorimetric to check.The experimental results showed that Ari had the most significant effect on HT29 colon cancer cells(P < 0.001).However,there was no significant inhibitory effect on FHC cells.(2)Animal experiments were carried out in BALB/c nude mice with xenograft tumors on scapular species,treated with 10 mg/kg and 30 mg/kg Ari for 14 days.The tumor weight significantly lower than in the control group(P < 0.01).2.BCL9 is involved in aripiprazole-induced cell cycle arrest in colon cancer cells(1)Using bioinformatics methods,BCL9,a gene closely related to WNT pathway activation,was successfully enriched from TCGA clinical database and xenograft transcriptome data.GO analysis and KEGG analysis enriched cell cycle and mitochondrial-related pathways.Meanwhile,colon cancer cell cycle was found to be blocked in G0/G1 phase after Ari intervention by flow cytometry(P < 0.05).(2)q PCR and WB experiments detected tumor tissues,BCL9 m RNA and BCL9,β-catenin,and Cyc D1 protein levels of were significantly lower than control group(P< 0.01).(3)After 0 μM,6 μM,12 μM,and 24 μM Ari were treated on HT29 cells for 24 h,PCR and WB experiments detected that BCL9 m RNA and BCL9,β-catenin,and Cyc D1 protein levels of were significantly lower than control group(P < 0.01).The mortality of colon cancer cells was increased when BCL9 was knocked down detected by MTT assay,while BCL9,β-catenin,and Cyc D1 protein expression levels were significantly lower than control group(P < 0.01).Under the combined effect of BCL9 knockdown and Ari drug treatment,BCL9,β-catenin,and Cyc D1 protein expression levels decreased even more significantly(P < 0.001).3.Mechanism of aripiprazole inhibiting colon cancer cell cycle arrest through BCL9(1)On the one hand,molecular docking showed that Ari could competitively inhibit the band of BCL9 and β-catenin by Discovery Studio.CO-IP assay confirmed that Ari weakened the binding of BCL9 and β-catenin.Furthermore,immunofluorescence experiments verified that after Ari treatment,the expression levels of β-catenin in the nucleus were decreased.These results suggest that Ari affects the binding of BCL9 and β-catenin,blocks β-catenin to entry the nuclear and inhibit the WNT pathways.(2)On the other hand,based on the transcriptome data,the expression of COX-2and COX-4,which are closely related to mitochondrial activity,were significantly reduced on Ari.Mitochondrial experiments found that after 9 hours treatment with 12μM Ari,mitochondria were damaged,and WB experiments confirmed the reduced expression levels of COX-2 and COX-4 proteins.Studies have shown that HIF-1α is closely related to mitochondria.As a transcriptional activator of BCL9,HIF-1α may affect BCL9 expression.q PCR and WB experiments were performed to show that Ari reduced the expression of HIF-1α and BCL9.Furthermore,dual-luciferase assays showed that after 24 hours treatment with 12 μM and 24 μM Ari,the binding of HIF-1α to BCL9 promoter was significantly weakened(P < 0.01).The above experimental results indicate that Ari damages mitochondria,reducing the expression of BCL9 by HIF-1α.Conclusion:This study confirmed that Ari affects cell cycle progression and mitochondrial regulation,and BCL9 is a key target for inhibiting colon cancer.The main mechanism was revealed: Ari mediated HIF-1α to reduce the expression of BCL9 by damaging mitochondria,and affected the binding of BCL9 to β-catenin,thus inhibiting the activation of WNT-β-Catenin pathway,and ultimately leading to cell cycle arrest.