DNA methyltransferase inhibitors influence on the DIRAS3 and STAT3 expression and in vitro migration of ovarian and breast cancer cells.

Objectives: Downregulation of DIRAS3 (DIRAS family, GTP-binding Ras-like 3) is related to ovarian and breast cancer progression. A possible mechanism that silences this gene is the promoter region DNA methylation. The potential reversibility of this epigenetic mechanism makes it more attractive candidate for new mode of cancer treatment. DIRAS3 regulates cell cycle, tumor dormancy and inhibits cancer cell growth and motility, all of which may indirectly depend on interaction with STAT3 (Signal Transducer and Activator of Transcription 3) classified as a potential oncogene. The restoration of DIRAS3 expression could inhibit cell proliferation and invasiveness.

Material And Methods: Human ovarian carcinoma cell line (A2780) and human breast cancer cell line (MCF7) were exposed to two DNA methyltransferase inhibitors (DNMTi): decitabine (5-aza-2'-deoxycytidine) [25 μM and 12.5 μM] and RG108 [150 μM and 100 μM]. In vitro migration changes of cancer cells were examined with wound healing assay. After 7 days of DNMTi treatment cells were harvested and DNA and RNA was isolated. The methylation status of the promoter sequences of DIRAS3 and STAT3 genes was determined using methylation specific PCR (MS-PCR). Level of target genes' expression was quantified using quantitative reverse transcription PCR (QRT-PCR).

Results And Conclusions: The in vitro wound healing assay showed changes in the migration rate of both adherent cell lines after DNMTi treatment compared to the untreated cells. Relative balance between methylated and unmethylated variants of DIRAS3 after MS-PCR was shifted towards unmethylated version after DNMTi treatment in A2780 cells. Statistically significant dose dependent effect of decitabine and RG108 on DIRAS3 expression in A2780 cells was observed.