title: Mitoxantrone mediates demethylation and reexpression of cyclin d2, estrogen receptor and 14.3.3sigma in breast cancer cells.
abstract: In addition to its action as a topoisomerase II poison, mitoxantrone is activated by formaldehyde to bind DNA, forming DNA-adducts specifically at 5'CpG and CpA sequences, with an enhancement of adducts at methylated CpG sites. The butyric acid prodrug, AN-9 (pivaloyloxymethyl butyrate), releases formaldehyde upon cellular hydrolysis and our previous studies have shown that mitoxantrone acts synergistically with AN-9 in cytotoxicity assays. In this paper, we investigated the impact of methylation levels in the cell on mitoxantrone-induced cytotoxicity using the colon cancer cell line HCT116 and its derived DNA methyltransferase (DNMT) 1 and DNMT 3a knockout (DKO8) cell line. We found that decreased methylation levels in the DNMT-null cells led to at least a 2-fold reduction in mitoxantrone-induced cytotoxicity. Next, we studied the impact of mitox-antrone alone, and in combination with AN-9, on hypermethylated genes and their mRNA expression in breast cancer cells. Using methylation-specific PCR and RT-PCR, we found that mitoxantrone treatment of breast cancer cell lines resulted in demethylation of the 14.3.3s, Cyclin D2 and ERa genes, followed by re-expression of their mRNA. The effect of mitoxantrone on re-expression of key genes involved in cell cycle regulation, and ensuing death of the cells may be an additional, previously undiscovered mechanism of action of mitoxantrone.
authors.affiliation: Breast Cancer Program, Sidney Kimmel Comprehensive Cancer Center at Johns Hopkins, Baltimore, Maryland 20231, USA.
authors.forename: Belinda S
authors.initials: BS
authors.lastname: Parker
authors.forename: Suzanne M
authors.initials: SM
authors.lastname: Cutts
authors.forename: Abraham
authors.initials: A
authors.lastname: Nudelman
authors.forename: Ada
authors.initials: A
authors.lastname: Rephaeli
authors.forename: Don R
authors.initials: DR
authors.lastname: Phillips
authors.forename: Saraswati
authors.initials: S
authors.lastname: Sukumar
mesh_terms: D048948:14-3-3 Proteins; D000970:Antineoplastic Agents / Q000627:therapeutic use*; D017209:Apoptosis / Q000187:drug effects; D001943:Breast Neoplasms / Q000188:drug therapy* / Q000378:metabolism / Q000473:pathology; D002087:Butyrates / Q000494:pharmacology; D002455:Cell Division / Q000187:drug effects; D003110:Colonic Neoplasms / Q000188:drug therapy / Q000378:metabolism / Q000473:pathology; D056742:Cyclin D2; D016213:Cyclins / Q000235:genetics / Q000378:metabolism*; D004248:DNA (Cytosine-5-)-Methyltransferases / Q000172:deficiency / Q000235:genetics / Q000378:metabolism; D019175:DNA Methylation; D000090205:DNA Methyltransferase 3A; D004273:DNA, Neoplasm / Q000378:metabolism; D004317:Doxorubicin / Q000494:pharmacology; D005260:Female; D015972:Gene Expression Regulation, Neoplastic; D020868:Gene Silencing; D006801:Humans; D008942:Mitoxantrone / Q000627:therapeutic use*; D012333:RNA, Messenger / Q000378:metabolism; D011960:Receptors, Estrogen / Q000235:genetics / Q000378:metabolism*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014407:Tumor Cells, Cultured; D014446:Tyrosine 3-Monooxygenase / Q000235:genetics / Q000378:metabolism*
chemical_list: D048948:14-3-3 Proteins; D000970:Antineoplastic Agents; D002087:Butyrates; C080336:CCND2 protein, human; D056742:Cyclin D2; D016213:Cyclins; D004273:DNA, Neoplasm; C000717967:DNMT3A protein, human; D012333:RNA, Messenger; D011960:Receptors, Estrogen; C070441:pivalyloxymethyl butyrate; D004317:Doxorubicin; D008942:Mitoxantrone; D014446:Tyrosine 3-Monooxygenase; D004248:DNA (Cytosine-5-)-Methyltransferases; D000090205:DNA Methyltransferase 3A