Study On The Mechanisms Of A Novel Retinoid WYC-209 Induced Tumor Repopulating Cells Apoptosis

Chemotherapy is one of the commonly used treatments for cancer.However,anticancer efficacy of existing chemotherapy drugs is not significant due to the drug resistance on some stemmed and tumorigenic cancer cells.Thus,developing novel therapeutic drugs which can efficiently kill such malignant tumorigenic cells is of great significance for cancer treatment.A three-dimensional soft fibrin gel culture system was developed for screening tumor repopulating cells(TRCs)with high self-renewal capacity,high tumorigenicity and high drug resistance,which could be used as a cell model for the screening of novel anticancer drugs.Based on the structure of the third-generation retinoid Tazarotene,a novel anticancer drug with high efficiency in inhibiting the growth of TRCs was finally screened and named WYC-209 after multiple molecular designs and syntheses.It was found that WYC-209 could efficiently induce apoptosis of multiple cancer cell lines in culture and inhibit lung metastasis of murine melanoma TRCs in immune-competent mice with no apparent toxicity.However,there is not a comprehensive study on how WYC-209 induced apoptosis of TRCs.Therefore,B16-F1 TRCs will be used as a cell model to explore the molecular mechanism of WYC-209-induced apoptosis of TRCs in depth and provide new ideas for the treatment of tumors,especially for malignant tumors in next step.Main research contents and results are as follows:(1)WYC-209 induced TRCs apoptosis by down-regulating cellular traction.By using elastic round microgels to measure traction force between melanoma TRCs,it was found that cellular traction had been decreased to very low levels at the early stage of WYC-209-induced apoptosis.Besides,modulating Cdc42 protein expression level could change the expression of filamentous actin(F-actin)and thus the cellular traction force.The efficiency of WYC-209-induced apoptosis in TRCs was changed when the cellular traction force changed.These results confirmed that WYC-209 decreased cellular traction force by downregulating Cdc42 protein and thereby decreasing F-actin content,which ultimately led to apoptosis of TRCs.(2)WYC-209 reduced the traction of TRCs by inducing retinoid acid receptor gamma subunit(RARγ)translocation.Immunofluorescence assays and chromatin immuneprecipitation assays revealed that WYC-209 decreased enrichment of the transcription factor RARγ in the Cdc42 promoter region by promoting RARγ translocation from the nucleus to the cytoplasm,thereby down-regulating the transcriptional expression of Cdc42 gene and inducing the reduction of cellular traction force.(3)WYC-209 activated apoptotic gene expression via inducing cellular traction reduction and chromatin decondensation.Transmission electron microscopy images showed that WYC-209 was able to open chromatin structure in melanoma TRCs,which is a prerequisite for transcriptional activation.Further studies showed that WYC-209 decreased histone 3 lysine 9 trimethylation(H3K9me3)level and elevated histone 3 lysine 9acetylation(H3K9ac)level by down-regulating Cdc42 protein expression,resulting in chromatin decondensation.The chromatin immunoprecipitation assay revealed that WYC-209 induced chromatin decondensation in the promoter regions of P21,P27,P53 andγH2AX genes,thereby activating the transcriptional expression of these genes and ultimately contributing to the apoptosis of TRCs.In addition,inducing chromatin decondensation could indeed enhance the inhibitory effect of WYC-209 on melanoma TRCs in vivo.These results elucidated the mechanism of WYC-209-induced apoptosis in TRCs,which provided a theoretical basis for further translational applications of WYC-209,as well as new ideas for the treatment of malignant tumors.