Antitumor Effect Based On The Interaction Of DNA Methyltransferase Inhibitor And Histone DeacetylaseInhibitor

OBJECTIVE Both aberrant DNA methylation and altered histone acetylation are two key mechanisms in epigenetics, which play an important role in the development and progression of cancer. DNA methyltransferase (DNMT) inhibitor, 5-aza-cytidine (5-aza-C), was widely studied because DNA hypomethylation induced the re-activation of tumor suppressor genes that were silenced by methylation-mediated mechanisms, could inhibit proliferation and induce differentiation of tumor. Histone deacetylase (HDAC) inhibitor, sodium phenylbutyrate (SPB), has multiple effects on tumor cells that include inhibition of proliferation, induction of differentiation and induction or repression of gene expression. The aims of this research were as follows:(l)To compare the proliferation inhibition and apoptosis effect of 5-aza-C or SPB used alone or in combination.(2)To evaluate the synergistic effect of 5-aza-C and SPB.(3)To analyze the influence of 5-aza-C and SPB on the expression of DNMTl,DNMT3a and HDAC when they were used alone or in combination.(4)To evaluate the effect of 5-aza-C and SPB on re-activation of genes that are silenced by epigenetics mechanisms.METHODS (1) The antiproliferation and apoptosis effect were assayed by ordinary microscopy, transmission electron microscopy, flow cytometry and DNA fragment. (2) The synergistic effect of 5-aza-C and SPB were observedby 5-aza-C combination with SPB or 5-aza-C (or SPB) combination with nolatrexed, a thymidylate synthase inhibitor, in vivro. (3)The influence of 5-aza-C and SPB used alone or in combination on the expression of DNMT1, DNMT3a and HDAC was measured by RT-PCR. (4) Used microarray to evaluate the effect of 5-aza-C and SPB on re-activation of genes that are silenced by epigenetics mechanisms.RESULTS (1) 5-aza-C used alone or SPB used alone or both used in combination all have antiproliferation, differentiation and apoptosis effect when showed by ordinary microscopy and flow cytometry.Both 5-aza-C used alone and two drugs used in combination caused nucleus change and DNA fragment when showed by transmission electron microscopy and flow cytometry, but these did not show in case of SPB used alone. (2)Results showed both 5-aza-C and SPB have markedly synergistic effect. (3) DNMT1, DNMT3a and HDAC were inhibited 59.3%, 38.6% and 7.4% respectively when 5-aza-C used alone; they were inhibited 7.6%, 4.6% and 60.3% respectively when SPB used alone; and they were inhibited 78.9%, 75.0% and 84.4% respectively When two drugs used in combination. (4) Four genes were down-regulated when 5-aza-C used alone, while fourteen genes were activated at the same time. Two genes were down-regulated by SPB used alone while elevated fourteen genes.When two drugs combination, twelve genes were inhibited, and forty-six genes were activated.CONCLUSIONS (1) Both 5-aza-C and SPB have favorable anti antiproliferation and apoptosis effect. (2) Both 5-aza-C and SPB have markedly synergistic effect, inhibitors of DNMT and HDAC may have well foreground in drugs combination and overcome muti-drugs resistance. (3)Aberrant epigenetics inactivated many genes which suppress tumorigenesis, inhibitors of DNMT and HDAC can reverse this morbidity. (4) The combination of HDAC and DNMT inhibition is very effective (and synergistic) in inducing apoptosis, differentiation and cell growth arrest, this suggested that methylation and deacetylation are coupled with each other.