title: DNA methylation-independent reversion of gemcitabine resistance by hydralazine in cervical cancer cells.
abstract: 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.
  
  
  RESULTS
  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
  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.
authors.affiliation: Division of Clinical Research, Instituto Nacional de Cancerologia, Mexico City, Mexico.
authors.forename: Myrna
authors.initials: M
authors.lastname: Candelaria
authors.forename: Erick
authors.initials: E
authors.lastname: de la Cruz-Hernandez
authors.forename: Lucia
authors.initials: L
authors.lastname: Taja-Chayeb
authors.forename: Enrique
authors.initials: E
authors.lastname: Perez-Cardenas
authors.forename: Catalina
authors.initials: C
authors.lastname: Trejo-Becerril
authors.forename: Aurora
authors.initials: A
authors.lastname: Gonzalez-Fierro
authors.forename: Alma
authors.initials: A
authors.lastname: Chavez-Blanco
authors.forename: Ernesto
authors.initials: E
authors.lastname: Soto-Reyes
authors.forename: Guadalupe
authors.initials: G
authors.lastname: Dominguez
authors.forename: Jaenai E
authors.initials: JE
authors.lastname: Trujillo
authors.forename: Jose
authors.initials: J
authors.lastname: Diaz-Chavez
authors.forename: Alfonso
authors.initials: A
authors.lastname: Duenas-Gonzalez
mesh_terms: D000964:Antimetabolites, Antineoplastic / Q000378:metabolism* / Q000627:therapeutic use; D015153:Blotting, Western; D018929:Cell Culture Techniques; D045744:Cell Line, Tumor; D047369:Chromatin Immunoprecipitation; D017931:DNA Primers / Q000235:genetics; D003841:Deoxycytidine / Q000031:analogs & derivatives* / Q000378:metabolism / Q000627:therapeutic use; D019008:Drug Resistance, Neoplasm / Q000187:drug effects*; D044127:Epigenesis, Genetic / Q000502:physiology*; D033721:Equilibrative Nucleoside Transporter 1 / Q000378:metabolism; D005260:Female; D015972:Gene Expression Regulation, Neoplastic / Q000502:physiology*; D006649:Histocompatibility Antigens; D006655:Histone Deacetylases / Q000378:metabolism; D011495:Histone-Lysine N-Methyltransferase / Q000037:antagonists & inhibitors; D006801:Humans; D006830:Hydralazine / Q000494:pharmacology*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D002583:Uterine Cervical Neoplasms / Q000188:drug therapy*; D000093542:Gemcitabine
chemical_list: D000964:Antimetabolites, Antineoplastic; D017931:DNA Primers; D033721:Equilibrative Nucleoside Transporter 1; D006649:Histocompatibility Antigens; C438649:SLC29A1 protein, human; D003841:Deoxycytidine; D006830:Hydralazine; C445405:EHMT2 protein, human; D011495:Histone-Lysine N-Methyltransferase; D006655:Histone Deacetylases; D000093542:Gemcitabine