The Mechanisms Of Ellagic Acid Against Hepatocellular Carcinoma And Synergistic Effects Of Its Combinations With Two Chemotherapeutic Agents

Ellagic acid(EA),a natural polyphenolic compound,is produced in different plants.Recently,EA has gained considerable attention due to its various pharmacological potentials.At present,EA has been proven to inhibit several types of cancers in vitro and in vivo,suggesting EA is a potential anticancer drug.However,the effect of EA on hepatocellular carcinoma(HCC)was less studied and the underlying mechanisms were still unclear.Moreover,most of patients have to receive high dose of chemotherapeutic agents in the clinical treatment of HCC,but it usually leads to multiple severe side effects.Therefore,it is significantly important to develop novel drugs and treatment approaches for clinical treatment of HCC.In this study,we used HCC and normal cell lines to investigate the anticarcinogenic effects and mechanisms of EA.In addition,we evaluated the antitumor effects and toxicity of EA in combination with doxorubicin hydrochloride(DOX)and cisplatin(DDP)on HCC in both cell-based assays and a nude mouse model to provide an experimental basis for clinical application and new drug development.We obtained results listed in following:1.We proved that EA significantly inhibited the cell viability of HepG2 cells in a dose and time dependent manner by WST-1 assay.Flow cytometric analysis demonstrated that EA caused cell cycle arrest in G0/G1 phase and increased the apoptotic percentages of HepG2 cells in a dose dependent manner.Furthermore,we found that EA treatment resulted in DNA damage by comet assay.2.The Illumina HiSeq sequencing platform was used to perform high-throughput transcriptome sequencing(RNA-seq)for two groups of HepG2 cells,which were with or without EA treatment.14123 differentially expressed genes were identified by multiple bioinformatic methods,of which 9323 genes were up-regulated and 4800 genes were down-regulated.Through the KEGG analysis of differentially expressed genes,we found that DNA replication signaling pathway was most significantly regulated by EA according to p value.3.Transcriptional levels of CDKN2B,CDKN2D,CDKN1A,CDK4,CDK2,CCNE1,E2F1,TFDP1,MCM2,MCM3,MCM4,MCM5,MCM6 and MCM7 were validated by RT-qPCR.We obtained nearly identical results between RNA-seq and RT-qPCR,suggesting the data of RNA-seq is fairly credible.In addition,by combining RT-qPCR and Western Blot,we confirmed that EA could affect the expression of Gl/S checkpoint regulatory genes at transcriptional and protein levels in HepG2 cells.4.A network of genes/ptoteins co-expression was constructed through the STRING database to predict core gene.We infected sh-p21 into the HepG2 cells to reduce the endogenous expression of p21.We then found that the silencing of p21 could reduce the sensitivity of HepG2 cells to EA by WST-1 and colony formation assays.It demonstrated that EA can inhibit the proliferation of HepG2 cells by targeting p21.5.In vitro experiments,our data revealed that EA could remarkably potentiate the anticancer activities of DOX and DDP against HCC cells,and trigger mitochondria-mediated apoptosis.Meanwhile,drug co-treatments could reduce the dose of chemotherapeutic agents,thereby alleviating the cytotoxicity to normal hepatocyte.6.In vitro experiments,EA in combination with DOX could significantly inhibit HepG2-luc2 xenograft tumor growth,and did not cause adverse reactions in the mouse body.