Study On The Molecular Mechanism Of 6-methoxyflavone-induced S-phase Arrest And Apoptosis In HeLa Cells

Background and objectiveIn recent years,the incidence,mortality,lymph node involvement rate,distant metastasis rate,and recurrence rate of cervical cancer are relatively high.The incidence,drug resistance rate,recurrence rate,ovarian metastasis rate,pelvic lymph node metastasis rate,and distant metastases rate of cervical adenocarcinoma are quite high.This means that it has a worse prognosis than squamous cell carcinoma.Therefore,in-depth research into low-toxicity and high-sensitivity therapeutic drugs and their mechanisms of action along with the development of more effective treatment strategies are still the top priority of current cervical cancer prevention and treatment.This study aimed to explore the molecular mechanism of S-phase arrest and apoptosis induced in He La cells by 6-methoxyflavones and these key genes needed to evaluate their efficacy.Methods1.Screening and experimental verification of small molecule compounds in traditional Chinese medicine: 13729 compounds were screened using big data screening technologies such as network pharmacology and bioinformatics;the antitumor activity of 6-methoxyflavone was verified by in vitro and in vivo experiments;transcriptome sequencing analysis was performed to evaluate biological functions and mechanisms of 6-methoxyflavones.2.6-Methoxyflavones induce S-phase arrest via the CCNA2/CDK2/p21CIP1 pathway in He La cells: Gene set enrichment analysis was used to screen the biological functions of principal components;the cell cycle stage was detected using flow cytometry;q PCR and Western blot were used to detect the m RNA and protein expression levels of genes related to the cell cycle regulatory network after6-methoxyflavone intervention;molecular docking analysis was used to assess ligand-receptor affinity;gain-of-function experiments and nude-mouse xenograft experiments were used to study the mechanism of action of CDK2,a key gene in the cell cycle pathway;the clinical characteristics thereof were analyzed using patient data from the TCGA database.3.6-Methoxyflavones induce apoptosis in He La cells via the p-PERK/p-EIF2?/ATF4/CHOP pathway: Hoechst33342 staining and flow cytometry were used to detect the apoptosis rate in He La cells;transmission electron microscopy was used to observe subcellular structure;q PCR and Western blot were used to detect the m RNA and protein expression levels of genes related to the apoptotic pathway after 6-methoxyflavone intervention;molecular docking was used to screen key genes in the apoptosis pathway;experiments performed to clarify the mechanism of action of the key gene EIF2AK3/PERK included gain-of-function and loss-of-function,phosphorylation inhibition,flow cytometry-based apoptosis-detection,co-immunoprecipitation,and nude mouse xenografts;the clinical characteristics thereof were analyzed using patient data from the TCGA database.Results1.Network pharmacology was used to obtain 13729 small molecule compounds.Through pharmacokinetic,target gene,differential expression,and functional enrichment screening,we narrowed our results down to the small-molecule compound6-methoxyflavone.Functional enrichment analysis of 178 targets in the Pharm Mapper database revealed that 6-methoxyflavones were significantly associated with cell proliferation,cell cycle,apoptosis,and protein phosphorylation processes.A CCK-8assay revealed that 6-methoxyflavone significantly inhibited He La cell proliferation.Nude mice transplanted with tumors revealed that significant inhibition of tumor growth after treatment with 6-methoxyflavone.Functional enrichment analysis after transcriptome sequencing revealed that 6-methoxyflavones were significantly associated with cell proliferation,cell cycle,apoptosis,endoplasmic reticulum stress,and protein modification processes.2.Two targets,CDK2 and CCNA2,were screened using principal component analysis combined with differential expression analysis.Flow cytometric cell cycle assays showed that 6-methoxyflavone significantly induced S-phase arrest in He La cells.q PCR and Western blot analysis showed that 6-methoxyflavone significantly downregulated the m RNA and protein expression levels of CDK2 and CCNA2 in He La cells and significantly upregulated the expression level of p21CIP1;this was consistent with the results of transcriptome sequencing.Molecular docking showed that 6-methoxyflavone had the strongest affinity for CDK2.Moreover,6-methoxyflavone interacts non-covalently with CCNA2 and CDK2 mainly via 18 hydrophobic interactions,3 salt bridges,and 3 hydrogen bonds.Western blot analysis showed that a gain-of-function of CDK2 could reverse its low expression levels as induced by 6-methoxyflavone intervention.In vivo experiments showed that6-methoxyflavone significantly down-regulated the protein expression level of CDK2.Therefore,6-methoxyflavone induces S-phase arrest in He La cells via the CCNA2/CDK2/p21CIP1 signaling pathway.In addition,clinical analysis showed that the m RNA expression levels of CCNA2,CDK2,and p21CIP1 were significantly correlated with drug sensitivity.Furthermore,the m RNA expression level of CDK2 was significantly correlated with the histological classification of cervical cancer patients.p21CIP1 m RNA expression levels were significantly correlated with the FIGO stage of cervical cancer patients.3.Hoechst33342 staining,flow cytometry,and transmission electron microscopy showed that 6-methoxyflavone significantly induced apoptosis in He La cells.q PCR and Western blot analysis showed that 6-methoxyflavone significantly upregulated the m RNA and protein expression levels of EIF2S1/EIF2?,ATF4,and DDIT3/CHOP in He La cells.The m RNA and protein expression levels of EIF2AK3/PERK were significantly downregulated while the protein expression levels of p-PERK and p-EIF2? were significantly upregulated;this was consistent with the transcriptome sequencing results.Molecular docking showed that 6-methoxyflavone has the strongest affinity for EIF2AK3/PERK.The noncovalent interactions between6-methoxyflavone and PERK,ATF4,and CHOP included 17 hydrophobic interactions,1 ?-cation interaction,1 ?-stacking,and 1 hydrogen bond.Flow cytometry and Western blot analysis showed that EIF2AK3/PERK gain-of-function experiments and GSK2656157 significantly altered both 6-methoxyflavone-induced apoptosis levels in He La cells and the expression levels of p-PERK.In vivo experiments showed that6-methoxyflavone significantly upregulated the protein expression of p-PERK and p-EIF2?.Co-immunoprecipitation experiments showed an interaction between p-PERK and p-EIF2?.Therefore,6-methoxyflavone induced apoptosis in He La cells through the p-PERK/p-EIF2?/ATF4/CHOP pathway.In addition,an analysis of clinical features showed that the m RNA expression levels of EIF2AK3/PERK and EIF2S1/EIF2? were significantly correlated with the histological classification of cervical cancer patients,and the m RNA expression level of DDIT3/CHOP was significantly correlated with the FIGO stage.Conclusions1.6-Methoxyflavones inhibits the proliferation of cervical cancer cells.2.6-Methoxyflavones induce S-phase arrest in He La cells through the CCNA2/CDK2/p21CIP1 pathway.3.6-Methoxyflavones induce apoptosis in He La cells through the p-PERK/p-EIF2?/ATF4/CHOP pathway.4.CDK2 is a key gene in the CCNA2/CDK2/p21CIP1 pathway.The gain-of-function of CDK2 significantly up-regulated the expression levels of CDK2 and CCNA2 and reversed the low expression level of CDK2 as induced by6-methoxyflavone intervention.5.p-PERK is a key factor in the p-PERK/p-EIF2?/ATF4/CHOP pathway.EIF2AK3/PERK overexpression and GSK2656157 significantly altered the phosphorylation level of PERK and 6-methoxyflavone-induced apoptosis level in He La cells.6.6-Methoxyflavones inhibit the growth of subcutaneously transplanted tumors in nude mice.