The anti-angiogenic effect of histone deacetylase inhibitors interfere with DNA methyl transferase inhibitor activity in vivo in prostate cancer models Free

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The combination of Histone Deacetylase Inhibitors (HDACI), and DNA Methyl Transferase Inhibitors (DNMTI) have been shown to act synergistically in enhancing cell death and causing re-expression of silenced genes in many cancer cell lines. We tested the efficacy of valproic acid (VPA), an HDACI, and 5-Aza-2’deoxycytidine (DAC), a DNMTI, in inhibiting the growth of prostate cancer (PCa) cell lines, PC-3 and DU-145 cells, and found that synergism in growth inhibition was seen in vitro but not in vivo. METHODS and RESULTS: In Vitro: The growth inhibition in PC-3 cells after 48h treatment with single agent DAC (0.25 µM) was 52%, VPA (1mM) was 6.5%, and the combination was 76.8%. On the other hand, DU-145 cells responded differently to the same drugs. The growth inhibition of DU-145 cells after 48h treatment with single agent DAC (0.25 µM) was 8.32%, VPA (1mM) was 29.5%, and the combination was 81.9%. The same pattern of differing sensitivity of the cell lines to DAC and VPA was seen in the colony formation assay: The decrease in number of colony forming units in PC-3 cells after 48h pre-treatment with single-agent DAC (0.25 µM) was 53.2%, VPA (1mM) was 19.1%, and the combination was 86.8% while the decrease in colony forming units of DU-145 cells after 48h pre-treatment with single-agent DAC (0.25 µM) was 32.5%, VPA (1mM) was 28.4%, and the combination was 81.8%. Thus, irrespective of the differing sensitivities of the two cell lines to the single agents, the combination of VPA and DAC displayed a significant growth-inhibitory effect on DU-145, and PC-3 PCa cell lines in vitro. In Vivo: While growth of DU-145 and PC-3 PCa cell lines in vivo was inhibited by the two drugs used singly, the combination did not have any additive effect in mediating this growth inhibition. Our hypothesis is that one of the molecular events that inhibit the combination of drugs from acting effectively, at least in PC-3 cells, is the anti-angiogeneic effect of VPA. The anti-angiogenic effect was assayed by staining for the endothelial cell marker, CD31, a determinant of blood vessel density. PC-3 but not DU-145 xenografts had a significantly lower CD31 count in the VPA-treated group compared to untreated controls (Average CD31 count/40X field in untreated PC-3 xenografts = 32.97±4.57, and in 0.4% VPA treated xenografts = 17.13 ± 3.27; a 48% decrease in vessel density). Whether the same anti-angiogenic effect is seen in DAC treated mice bearing both DU-145 and PC-3 cells is under investigation. CONCLUSIONS: VPA, an HDACI, acting as an anti-angiogenic agent, may inhibit perfusion of the second agent (DAC) to the PC-3 xenograft, thus inhibiting DAC from realizing its full potential. Our hypothesis as to the differential effect in vitro versus in vivo is that at least in PC-3 cells, VPA acts as an anti-angiogenic agent.