Abstract 2990: The combination of Trichostatin A and 5-aza-2’-deoxycytidine achieves superior anticancer efficacy on ovarian cancer via regulation of migration, invasion, apoptosis, and cell cycle.
Authors: Brewer Molly A. , Meng Fanliang , Sun Guigin , Yu Yanhong 
Published: August 2014

Objective: To investigate the anticancer efficacy and mechanism of Trichostatin A (TSA) and 5-aza-2’-deoxycytidine (5-aza-CdR) on ovarian cancer cells.

Material and methods: The effect of TSA and 5-aza-CdR was evaluated on the human ovarian cancer cell line SKOV3 in vitro and in vivo. Optimal dose combinations of TSA and 5-aza-CdR was assessed by MTS assay, migration and invasion by transwell assay and BD invasion chamber, expression of epithelial to mesenchymal transition (EMT) markers (Twist, E-cadherin, and N-cadherin), MMP-2/9, apoptosis markers (Bcl-2 and Bcl-x/L), cell cycle markers (p21 and p27), epigenetic markers (DNMT3A/3B, HDAC1/2, LSD1, Ac-H3/H4 and H3K4me2, H3K9me2) by Western blot. Spheroid formation was evaluated in serum free medium when exposed to the combination of drugs. Tumorigenicity of cancer cells treated both before and after implantation subcutaneously and intraperitoneally was assessed in SCID mice.

Results: The effect of fixed ratio combinations of TSA and 5-aza-CdR on cell viability was assessed and 0.1 μM TSA and 5 μM 5-aza-CdR achieved the maximal synergistic effect (Combination Index =0.421). Tumorigenicity of SKOV3 cells pretreated with both drugs was significantly suppressed when compared with either drug alone or untreated control (p&lt;0.001) in SCID mice models. Migration capacity was markedly suppressed through the induction of E-cadherin and suppression of N-cadherin (MET), and invasion was suppressed at least partially via inhibition of MMP-2 and MMP-9 with the combined treatment. The combination drugs markedly inhibited both spheroid size (p&lt;0.001) and spheroid number (p=0.002), through inhibition of Twist, N-cadherin, MMP-2, MMP-9 and induction of E-cadherin. The combination of TSA (0.1 mg/kg) and 5-aza-CdR (5 mg/kg) significantly suppressed tumor implantation (p&lt;0.001), and no apparent toxicity was found (control vs. combined, mice weight, p=0.0537) and expression of Bcl-2 and Bcl-x/L was suppressed while p21 and p27 were induced in tumor nodules. Epigenetic markers DNMT3A/3B and HDAC1/2, enzymes important to tumor suppressor genes, were markedly inhibited with the combination. Acetylation of histone H3 and H4 were more markedly stimulated with the combination than with either agent alone. H3K4me2 expression was induced and H3K9me3 and LSD1 were suppressed by exposure to the combined drugs.

Conclusion: These results suggest that the combination of TSA and 5-aza-CdR significantly suppresse tumorigenicity of ovarian cancer cells by inhibiting migration and invasion via suppression of N-cadherin and MMP-2/9, induction of E-cadherin, regulation of apoptosis and cell cycle via suppression of Bcl-2 and Bcl-x/L and induction of p21 and p27, which may be epigenetically regulated by DNA methylation and histone modification.

Citation Format: Molly A. Brewer, Fanliang Meng, Guigin Sun, Yanhong Yu. The combination of Trichostatin A and 5-aza-2’-deoxycytidine achieves superior anticancer efficacy on ovarian cancer via regulation of migration, invasion, apoptosis, and cell cycle. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2990. doi:10.1158/1538-7445.AM2013-2990