2205

Postulating that genes relevant for effective therapy of melanoma may be silenced by inappropriate methylation, the DNA demethylating agent 5-AZA-dC was assessed in human WM164 and A375 melanoma xenografts in nude mice. For design of these studies, target genes that might be modulated by methylation silencing were assessed in vitro by qRT-PCR. Using >20 custom primers (ABI) mRNA expression of genes with known CpG islands within 1Kb of transcription start sites or with reported increases in response to 5-AZA-dC were assessed after 0.2 uM of 5-AZA-dC for 96h. 5-AZA-dC resulted in minimal cellular toxicity but increased in A375 cells mRNA for RASSF1A >500x, XAF1>70x, and SHP-1>20x. In WM164 cells RASSF1A did not increase but AIM2 increased >200x and SHP-1 >80x. Methylation specific PCR confirmed reversal of RASSF1A and XAF1 methylation after 5AZA-dC treatment. To assess effects in vivo, treatment was started with 5-AZA-dC (3mg/kg i.p. 5d/week every other week for WM164 or every week for A375) 3d after inoculation of 2x10^6 (WM164) or 2d after 5x10^5 (A375) tumor cells on both flanks. Tumors were measured and animals were weighed 3 times per week during treatment. Mice were euthanized when tumors reached >1.5cm or if obvious toxicity developed. Tumors were harvested, bisected, and snap frozen or placed into TRIzol for assessment of gene expression. Corresponding to findings in vitro, RASSF1A expression increased in A375 xenografts, but did not increase in WM164 xenografts. 5-AZA-dC treatment reduced tumor burden by 56% in WM164 and by 72% in A375 xenografts respectively. Suggesting possible relevance for treatment either alone or in combination with other therapeutics, 5-AZA-dC resulted in antitumor effects in melanoma xenografts with correspondence in vitro and in vivo of expression of genes silenced by methylation and potentially important for apoptosis

98th AACR Annual Meeting-- Apr 14-18, 2007; Los Angeles, CA

American Association for Cancer Research
2007