Abstract
Background: The epigenetic combinations of DNA demethylating agents and histone deacetylase (HDAC) inhibitors have demonstrated clinical benefits for non-small cell lung cancer (NSCLC) treatment, however, there are few studies uncovering the underlying molecular mechanism of the combinations. Our previous study showed that DNA demethylating agent Azacitidine (Aza) demethylated CpG sites in paired box gene 5 (pax5) promoter region, but did not induce pax5 mRNA or protein expression.
Methods: In this study we used epigenetic combination of Aza and HDAC inhibitor Vorinostat (SAHA) to treat NSCLC cells and to elucidate the underlying molecular mechanism. We treated pax5- silenced NSCLC H460 cells with Aza+SAHA combination at sub-toxic concentration and detected the re-expression of pax5 mNRA and protein. Results: The results showed demethylation of CpG sites in pax5 promoter region by Aza treatment and increased DNA accessibility for protein binding by SAHA treatment. The combination of Aza+SAHA significantly increased p53 protein binding to DNA in pax5 promoter region (p<0.01). More efficient binding of the transcription factor p53 to pax5 promoter region is likely because SAHA increased accessibility of the chromatin conformation and Aza-demethylated DNA was more permissive, allowing transcription factors to bind. Conclusion: Our study not only explained an underlying mechanism, that pax5 re-expression was induced by Aza+SAHA combination in H460 cells via p53, but also demonstrated a pattern showing that the combination of demethylating agent and HDAC inhibitor can re-activate tumor suppressor gene (TSG) which is associated with the enhancement of transcription factors binding to the promoter region of the TSG.Keywords: Epigenetics, HDAC, methylation, non-small cell lung cancer, pax5, p53.
[1]
Yoo, C.B.; Jones, P.A. Epigenetic therapy of cancer: past, present and future. Nat. Rev. Drug Discov., 2006, 5(1), 37-50.
[2]
Esteller, M. Cancer epigenomics: DNA methylomes and histone-modification maps. Nat. Rev. Genet., 2007, 8(4), 286-298.
[3]
Feinberg, A.P.; Ohlsson, R.; Henikoff, S. The epigenetic progenitor origin of human cancer. Nat. Rev. Genet., 2006, 7(1), 21-33.
[4]
Esteller, M.; Corn, P.G.; Baylin, S.B.; Herman, J.G. A gene hypermethylation profile of human cancer. Cancer Res., 2001, 61(8), 3225-3229.
[5]
Herman, J.G.; Baylin, S.B. Gene silencing in cancer in association with promoter hypermethylation. N. Engl. J. Med., 2003, 349(21), 2042-2054.
[6]
Jakopovic, M.; Thomas, A.; Balasubramaniam, S.; Schrump, D.; Giaccone, G.; Bates, S.E. Targeting the epigenome in lung cancer: expanding approaches to epigenetic therapy. Front. Oncol., 2013, 3, 261.
[7]
Stathis, A.; Hotte, S.J.; Chen, E.X.; Hirte, H.W.; Oza, A.M.; Moretto, P.; Webster, S.; Laughlin, A.; Stayner, L.A.; McGill, S. Phase I study of decitabine in combination with vorinostat in patients with advanced solid tumors and non-Hodgkin’s lym-phomas. Clin. Cancer Res., 2011, 17(6), 1582-1590.
[8]
Silverman, L.; Verma, A.; Odchimar-Reissig, R. A phase I/II study
of vorinostat, an oral histone deacetylase inhibitor, in combination
with azacitidine in patients with the myelodysplastic syndrome
(MDS) and acute myeloid leuke-mia (AML). Initial results of the
phase I trial: a New York Cancer Consortium. J. Clin. Oncol., 2008, 26(Abstract), 7000..
[9]
Juergens, R.A.; Wrangle, J.; Vendetti, F.P.; Murphy, S.C.; Zhao, M.; Coleman, B.; Sebree, R.; Rodgers, K.; Hooker, C.M.; Franco, N. Combination epigenetic therapy has efficacy in patients with refractory advanced non-small cell lung cancer. Cancer Discov., 2011, 1(7), 598-607.
[10]
Pesek, M.; Kopeckova, M.; Benesova, L.; Meszarosova, A.; Mukensnabl, P.; Bruha, F.; Minarik, M. Clinical significance of hypermethylation status in NSCLC: evaluation of a 30-gene panel in patients with advanced disease. Anticancer Res., 2011, 31(12), 4647-4652.
[11]
Kanteti, R.; Nallasura, V.; Loganathan, S.; Tretiakova, M.; Kroll, T.; Krishnaswamy, S.; Faoro, L.; Cagle, P.; Husain, A.N.; Vokes, E.E. PAX5 is expressed in small-cell lung cancer and positively regulates c-Met transcription. Lab. Invest., 2009, 89(3), 301-314.
[12]
Vidal, L.J.; Perry, J.K.; Vouyovitch, C.M.; Pandey, V.; Brunet-Dunand, S.E.; Mertani, H.C.; Liu, D.X.; Lobie, P.E. PAX5alpha enhances the epithelial behavior of human mammary carcinoma cells. Mol. Cancer Res. MCR, 2010, 8(3), 444-456.
[13]
Proulx, M.; Cayer, M.P.; Drouin, M.; Laroche, A.; Jung, D. Overexpression of PAX5 induces apoptosis in multiple myeloma cells. Int. J. Hematol., 2010, 92(3), 451-462.
[14]
Liu, W.; Li, X.; Chu, E.S.; Go, M.Y.; Xu, L.; Zhao, G.; Li, L.; Dai, N.; Si, J.; Tao, Q. Paired box gene 5 is a novel tumor suppressor in hepatocellular carcinoma through interaction with p53 signaling pathway. Hepatology, 2011, 53(3), 843-853.
[15]
Steinbach, J.P.; Kozmik, Z.; Pfeffer, P.; Aguzzi, A. Overexpression of Pax5 is not sufficient for neoplastic trans-formation of mouse neuroectoderm. Int. J. Cancer, 2001, 93(4), 459-467.
[16]
Qiu, X.; Liang, Y.; Sellers, R.S.; Perez-Soler, R.; Zou, Y. Aerosol azacytidine inhibits orthotopic lung cancers in mice through Its DNA demethylation and gene reactivation effects. PLoS One, 2014, 9(10), e109874.
[17]
Vichai, V.; Kirtikara, K. Sulforhodamine B colorimetric assay for cytotoxicity screening. Nat. Protoc., 2006, 1(3), 1112-1116.
[18]
Thompson, R.F.; Suzuki, M.; Lau, K.W.; Greally, J.M. A pipeline for the quantitative analysis of CG dinucleotide methylation using mass spectrometry. Bioinformatics, 2009, 25(17), 2164-2170.
[19]
Mahesh, S.; Saxena, A.; Qiu, X.; Perez-Soler, R.; Zou, Y. Intratracheally administered 5-azacytidine is effective against orthotopic human lung cancer xenograft models and devoid of important systemic toxicity. Clin. Lung Cancer, 2010, 11(6), 405-411.
[20]
Tsai, H.C.; Li, H.; Van Neste, L.; Cai, Y.; Robert, C.; Rassool, F.V.; Shin, J.J.; Harbom, K.M.; Beaty, R.; Pappou, E. Transient low doses of DNA-demethylating agents exert durable antitumor effects on hematological and epithelial tumor cells. Cancer Cell, 2012, 21(3), 430-446.
[21]
Christman, J.K. 5-Azacytidine and 5-aza-2′-deoxycytidine as inhibitors of DNA methylation: mechanistic studies and their implications for cancer therapy. Oncogene, 2002, 21(35), 5483-5495.
[22]
Zheng, X.; Naiditch, J.; Czurylo, M.; Jie, C.; Lautz, T.; Clark, S.; Jafari, N.; Qiu, Y.; Chu, F.; Madonna, M.B. Differential effect of long-term drug selection with doxorubicin and vorinostat on neuroblastoma cells with cancer stem cell characteristics. Cell Death Dis., 2013, 4, e740.
[23]
Gore, M.A.; Morshedi, M.M.; Reidhaar-Olson, J.F. Gene expression changes associated with cytotoxicity identified using cDNA arrays. Funct. Integr. Genomics, 2000, 1(2), 114-126.
[25]
Esteller, M. Aberrant DNA methylation as a cancer-inducing mechanism. Annu. Rev. Pharmacol. Toxicol., 2005, 45, 629-656.
[26]
Petta, V.; Gkiozos, I.; Strimpakos, A.; Syrigos, K. Histones and lung cancer: Are the histone deacetylases a promising therapeutic target? Cancer Chemother. Pharmacol., 2013, 72(5), 935-952.
[27]
Yang, X.J.; Seto, E. Collaborative spirit of histone deacetylases in regulating chromatin structure and gene expression. Curr. Opin. Genet. Dev., 2003, 13(2), 143-153.
[28]
Lu, T.Y.; Kao, C.F.; Lin, C.T.; Huang, D.Y.; Chiu, C.Y.; Huang, Y.S.; Wu, H.C. DNA methylation and histone modification regulate silencing of OPG during tumor progression. J. Cell. Biochem., 2009, 108(1), 315-325.
[29]
Yun, J.; Song, S.H.; Park, J.; Kim, H.P.; Yoon, Y.K.; Lee, K.H.; Han, S.W.; Oh, D.Y. Gene silencing of EREG mediated by DNA methylation and histone modification in human gastric cancers. Lab. Invest., 2013, 93(10), 1165.
[30]
Ezhkova, E.; Tansey, W.P. Chromatin immunoprecipitation to study protein-DNA interactions in budding yeast. Methods Mol. Biol., 2006, 313, 225-244.
[31]
Sarkar, S.; Horn, G.; Moulton, K.; Oza, A.; Byler, S.; Kokolus, S.; Longacre, M. Cancer development, progression, and therapy: an epigenetic overview. Int. J. Mol. Sci., 2013, 14(10), 21087-21113.
[32]
Sarkar, S.; Goldgar, S.; Byler, S.; Rosenthal, S.; Heerboth, S. Demethylation and re-expression of epigenetically silenced tumor suppressor genes: sensitization of cancer cells by combination therapy. Epigenomics, 2013, 5(1), 87-94.
[33]
Bhaskara, S.; Jacques, V.; Rusche, J.R.; Olson, E.N.; Cairns, B.R.; Chandrasekharan, M.B. Histone deacetylases 1 and 2 maintain S-phase chromatin and DNA replication fork progression. Epigenetics Chromatin, 2013, 6(1), 27.
[34]
Deaton, A.M.; Bird, A. CpG islands and the regulation of transcription. Genes Dev., 2011, 25(10), 1010-1022.
[35]
Stegmaier, P.; Kel, A.E.; Wingender, E. Systematic DNA-binding
domain classification of transcription factors. Genome informatics
International Conference on Genome Informatics,, 2004, 15(2), 276-286.
[36]
Sandelin, A.; Carninci, P.; Lenhard, B.; Ponjavic, J.; Hayashizaki, Y.; Hume, D.A. Mammalian RNA polymerase II core promoters: insights from genome-wide studies. Nat. Rev. Genet., 2007, 8(6), 424-436.
[37]
Beckerman, R.; Prives, C. Transcriptional regulation by p53. Cold Spring Harb. Perspect. Biol., 2010, 2(8), a000935.
[38]
Espinosa, J.M. Mechanisms of regulatory diversity within the p53 transcriptional network. Oncogene, 2008, 27(29), 4013-4023.
[39]
Lai, S.L.; Perng, R.P.; Hwang, J. p53 gene status modulates the chemosensitivity of non-small cell lung cancer cells. J. Biomed. Sci., 2000, 7(1), 64-70.
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