Study On The Inhibitory Mechanism Of A Novel Chalcone Derivative CX258 In Colorectal Cancer

Colorectal cancer（CRC）is the third most common malignant tumor worldwide,which seriously threatens human health.At present,some small molecule drugs have been approved for the treatment of CRC,but they have problems,such as low efficacy,poor targeting and high drug resistance.Therefore,it is urgent to develop new therapeutic strategies and targets for CRC treatment.Dysregulation of Wnt/β-catenin signaling is associated with many cancers including CRC,thus Wnt signaling is a promising target for drug development.Using our natural compounds library,we screened and identified a novel family of chalcone derivatives（CXs）by detecting Wnt signaling inhibition and CRC cell proliferation.In this study,we performed further in vitro and in vivo studies of CX258 to investigate its effect and related molecular mechanisms in CRC.The results of this study provide potential candidate drugs and new ideas for the treatment of CRC.Methods and results:1.The chalcone derivative CX258 was chemically synthesized.The structure and purity of CX258 was confirmed by ¹H-NMR,¹³C-NMR,HRMS and HPLC.Cell level activity verification showed that CX258 inhibited the proliferation of CRC cells,and its IC50 value was determined in four CRC cell lines.Western blot and Wnt reporter gene assay confirmed that CX258 inhibited the expression of Wnt signaling pathway and its downstream target genes Axin2 and c-Myc at the transcriptional level.2.RNA-Seq was used to analyze the changes of differential gene expression in the transcriptome of CRC cell line LS174T before and after CX258 treatment.Based on the significance ranking,Degree Scores,pathway enrichment analysis and literature background knowledge,five hub genes were selected,which were RRM2,TOP2A,UBE2C,CDK1 and AURKB.q RT-PCR verified that the expression of five hub genes in LS174T and DLD-1 cells were decreased after CX258 treatment,which was consistent with the RNA-seq results.The TCGA database was analyzed by the GEPIA program,and the expression levels of these five genes were significantly increased in CRC.Protein-protein interaction（PPI）network analysis showed that these five genes interacted with each other.This study mainly focused on TOP2A and CDK1,which ranked first in significance and Degree Score respectively.3.Western blot showed that CX258 significantly inhibited the expression of TOP2A,and the reduction of TOP2A expression inhibited the expression of CDK1.In addition,reduction of TOP2A expression impaired the inhibitory effect of CX258 on CRC cell proliferation,cell cycle arrest,and inhibition of Wnt target genes.These results suggest that TOP2A may be a potential regulator of CX258 regulating Wnt/β-catenin signaling pathway to inhibit the proliferation and cycle progression of colorectal cancer cells.4.The xenograft model of CRC in NOD/SCID mice was successfully established,and the tumor suppressive activity of CX258 was confirmed in vivo.CX258significantly inhibited the tumor growth after administration with no significant toxicity observed within 15 days.Western blot,HE staining,and immunohistochemical staining further verified that cell cycle-related P53 and P21were significantly activated but CDK1 expression was inhibited in CX258-treated tumor tissues.TOP2A and Wnt target genes were significantly reduced in CX258-treated tumor specimens.Conclusions:Chalcone derivative CX258 inhibits the expression of CDK1 and induces G2/M phase arrest of CRC cells,thereby inhibiting the proliferation of CRC cells.Its mechanism of action may be to inhibit the expression of the downstream target genes of Wnt/β-catenin-mediated signaling pathway by inhibiting the expression of TOP2A.Therefore,CX258 is a potential novel inhibitor of TOP2A/Wnt/β-catenin signaling.TOP2A is a potential mediator of Wnt signaling and cell cycle regulation.This may lead to new therapeutic strategies and drugs for the treatment of CRC.