ABSTRACT
The aim of the present study was to demonstrate that ribavirin, a known inhibitor of eIF4E and inosine 5'-phosphate dehydrogenase (IMPDH), also inhibits histone methyltransferase zeste homolog 2 (EZH2). A computational searching revealed that ribavirin has a high structural similarity to 3-deazaneplanocin A (DZNep). The growth inhibitory effects of ribavirin as well as its effects upon epigenetic enzymes were evaluated in various cancer cell lines. siRNA assays were used to downregulate eIF4E, EZH2 and IMPDH to determine the contribution of these targets to the growth inhibitory effects of ribavirin. Ribavirin decreased EZH2 expression, inhibited histone methyltransferase activity and decreased H3K27 trimethylation. Ribavirin induced variable growth inhibition in a number of cell lines and downregulation of the targets, EZH2, eIF4E and IMPDH1 and 2 by siRNA led to comparable growth inhibition while no significant further reduction in viability was observed when siRNA transfected cells were treated with ribavirin. The results showed that ribavirin inhibits these cancer targets and should thus be studied for cancer therapy.
Subject(s)
Antineoplastic Agents/pharmacology , Antiviral Agents/pharmacology , Cell Proliferation/drug effects , Eukaryotic Initiation Factor-4E/drug effects , IMP Dehydrogenase/drug effects , Neoplasms/genetics , Polycomb Repressive Complex 2/drug effects , Ribavirin/pharmacology , Apoptosis/drug effects , Cell Line, Tumor , Computer Simulation , Drug Repositioning , Enhancer of Zeste Homolog 2 Protein , Eukaryotic Initiation Factor-4E/genetics , HeLa Cells , Histone Methyltransferases , Histone-Lysine N-Methyltransferase/drug effects , Humans , IMP Dehydrogenase/genetics , MCF-7 Cells , Neoplasms/metabolism , Polycomb Repressive Complex 2/genetics , RNA, Small InterferingABSTRACT
BACKGROUND: Down regulation of genes coding for nucleoside transporters and drug metabolism responsible for uptake and metabolic activation of the nucleoside gemcitabine is related with acquired tumor resistance against this agent. Hydralazine has been shown to reverse doxorubicin resistance in a model of breast cancer. Here we wanted to investigate whether epigenetic mechanisms are responsible for acquiring resistance to gemcitabine and if hydralazine could restore gemcitabine sensitivity in cervical cancer cells. METHODOLOGY/PRINCIPAL FINDINGS: The cervical cancer cell line CaLo cell line was cultured in the presence of increasing concentrations of gemcitabine. Down-regulation of hENT1 & dCK genes was observed in the resistant cells (CaLoGR) which was not associated with promoter methylation. Treatment with hydralazine reversed gemcitabine resistance and led to hENT1 and dCK gene reactivation in a DNA promoter methylation-independent manner. No changes in HDAC total activity nor in H3 and H4 acetylation at these promoters were observed. ChIP analysis showed H3K9m2 at hENT1 and dCK gene promoters which correlated with hyper-expression of G9A histone methyltransferase at RNA and protein level in the resistant cells. Hydralazine inhibited G9A methyltransferase activity in vitro and depletion of the G9A gene by iRNA restored gemcitabine sensitivity. CONCLUSIONS/SIGNIFICANCE: Our results demonstrate that acquired gemcitabine resistance is associated with DNA promoter methylation-independent hENT1 and dCK gene down-regulation and hyper-expression of G9A methyltransferase. Hydralazine reverts gemcitabine resistance in cervical cancer cells via inhibition of G9A histone methyltransferase.
Subject(s)
Antimetabolites, Antineoplastic/metabolism , Deoxycytidine/analogs & derivatives , Drug Resistance, Neoplasm/drug effects , Epigenesis, Genetic/physiology , Gene Expression Regulation, Neoplastic/physiology , Hydralazine/pharmacology , Uterine Cervical Neoplasms/drug therapy , Antimetabolites, Antineoplastic/therapeutic use , Blotting, Western , Cell Culture Techniques , Cell Line, Tumor , Chromatin Immunoprecipitation , DNA Primers/genetics , Deoxycytidine/metabolism , Deoxycytidine/therapeutic use , Equilibrative Nucleoside Transporter 1/metabolism , Female , Histocompatibility Antigens , Histone Deacetylases/metabolism , Histone-Lysine N-Methyltransferase/antagonists & inhibitors , Humans , Reverse Transcriptase Polymerase Chain Reaction , GemcitabineABSTRACT
Aberrant DNA methylation and histone deacetylation participate in cancer development and progression; hence, their reversal by inhibitors of DNA methylation and histone deacetylases is a promising cancer therapy. Experimental data demonstrate that these inhibitors in combination do not only show synergy in antitumor effects but also in whole genome global expression. Ten pairs of pre- and post-treatment cervical tumor samples were analyzed by microarray analysis. Treatment for seven days with hydralazine and valproate (HV) in patients up-regulated 964 genes. The two pathways possessing the highest number of up-regulated genes comprised the ribosome protein and the oxidative phosphorylation pathways, followed by MAPK signaling, tight junction, adherens junction, actin cytoskeleton, cell cycle, focal adhesion, apoptosis, proteasome, Wnt signaling, and antigen processing and presentation pathways. Up-regulated genes by HV, clustered with down-regulated genes in untreated primary cervical carcinomas and were more alike as compared with up-regulated genes from untreated patients in terms of gene ontology. Increased acetylated p53 was also observed. Epigenetic therapy with HV leads to gene reactivation in primary tumors of cervical cancer patients as well as protein acetylation. A number of these reactivated genes have a definitive role as a tumor suppressors. The global expression pattern induced by HV suggests this therapy has an impact on pathways related to energy production which may promote apoptosis.
