Abstract
Paired box gene1 (PAX1) is essential for normal chordate development and has been recently characterised to be a tumour suppressor gene which is frequently hypermethylated in different cancer types. We investigated the reactivation of PAX1 using curcumin and resveratrol in HeLa, SiHa and Caski cell lines and role of hypermethylation in 39 CpG sites of PAX1 promoter from −6 to −286 region in regulating its expression. Curcumin in HeLa and SiHa cells and resveratrol in Caski cells caused significant (P < 0.01) reactivation of PAX1 expression as shown by qRT PCR, but reversal of promoter hypermethylation was not observed across the three cell lines. Interestingly, even positive control 5-aza-2′-deoxycytidine was not found to be effective to cause demethylation of CpG sites under consideration suggesting the promoter region to be resistant towards hypomethylating effects as shown by bisulphite sequencing. However, a striking correlation between PAX1 reactivation and Ubiquitin-like with PHD and RING finger domains 1 (UHRF1) downregulation after treatment with curcumin and resveratrol in HeLa, SiHa and Caski cell lines was observed which was further confirmed after transient silencing of UHRF1 expression. PAX1 reexpression was also obtained in Caski and SiHa cell lines after treatment with sodium butyrate, a histone deacetylase inhibitor, suggesting that PAX1 reactivation by curcumin and resveratrol may be due to their effect on histone deacetylase mediated through downregulation of UHRF1 which can regulate both DNA methylation and histone acetylation.
Similar content being viewed by others
References
Guerrero-Preston R, Michailidi C, Marchionni L, et al. Key tumor suppressor genes inactivated by “greater promoter” methylation and somatic mutations in head and neck cancer. Epigenetics. 2014;9:1031–46.
Chao TK, Ke FY, Liao YP, Wang HC, Yu CP, Lai HC. Triage of cervical cytological diagnoses of atypical squamous cells by DNA methylation of paired boxed gene 1(PAX1). Diagn Cytopathol. 2011;41:41–6.
Takimoto A, Mohri H, Kokubu C, Hiraki Y, Shukunami C. Pax1 acts as a negative regulator of chondrocyte maturation. Exp Cell Res. 2013;319:3128–39.
Peters H, Wilm B, Sakai N, Imai K, Maas R, Balling R. Pax1 and pax9 synergistically regulate vertebral column development. Development. 1999;126:5399–408.
Singh V, Sharma P, Capalash N. DNA methyltransferase-1 inhibitors as epigenetic therapy for cancer. Curr Cancer Drug Targets. 2013;13:379–99.
Su HY, Lai HC, Lin YW, Chou YC, Liu CY, Yu MH. An epigenetic marker panel for screening and prognostic prediction of ovarian cancer. Int J Cancer. 2009;124:387–93.
Huang YK, Peng BY, Wu CY, Su CT, Wang HC, Lai HC. DNA methylation of PAX1 as a biomarker for oral squamous cell carcinoma. Clin Oral Investig. 2014;18:801–8.
Elaine HL, Ng SL, Li JL, et al. Cervical dysplasia: assessing methylation status (methylight) of CCNA1, DAPK1, HS3ST2, PAX1 and TFPI2 to improve diagnostic accuracy. Gynecol Oncol. 2010;119:225–31.
Vasiljević N, Scibior-Bentkowska D, Brentnall AR, Cuzick J, Lorincz AT. Credentialing of DNA methylation assays for human genes as diagnostic biomarkers of cervical intraepithelial neoplasia in high-risk HPV positive women. Gynecol Oncol. 2014;132:709–14.
Chang CC, Huang RL, Wang HC, Liao YP, Yu MH, Lai HC. High methylation rate of LMX1A, NKX6-1, PAX1, PTPRR, SOX1, and ZNF582 genes in cervical adenocarcinoma. Int J Gynecol Cancer. 2014;24:201–9.
Lai HC, Ou YC, Chen TC, et al. PAX1/SOX1 DNA methylation and cervical neoplasia detection: a Taiwanese Gynecologic Oncology Group (TGOG) study. Cancer Med. 2014;3(4):1062–74.
Greger V, Debus N, Lohmann D, Höpping W, Passarge E, Horsthemke B. Frequency and parental origin of hypermethylated RB1 alleles in retinoblastoma. Hum Genet. 1994;94:491–6.
Alhosin M, Sharif T, Mousli M, et al. Down-regulation of UHRF1, associated with re-expression of tumor suppressor genes, is a common feature of natural compounds exhibiting anti-cancer properties. J Exp Clin Cancer Res. 2011;30:41.
Bronner C, Achour M, Arima Y, Chataigneau T, Saya H, Schini-Kerth VB. The UHRF family: oncogenes that are druggable targets for cancer therapy in the near future. Pharmacol Ther. 2007;115:419–34.
Achour M, Mousli M, Alhosin M, et al. Epigallocatechin-3-gallate up-regulates tumor suppressor gene expression via a reactive oxygen species-dependent down-regulation of UHRF1. Biochem Biophys Res Commun. 2013;430:208–12.
Lai HC, Lin YW, Huang TH, et al. Identification of novel DNA methylation markers in cervical cancer. Int J Cancer. 2008;123:161–7.
Zhang Y, Chen FQ, Sun YH, Zhou SY, Li TY, Chen R. Effects of DNMT1 silencing on malignant phenotype and methylated gene expression in cervical cancer cells. J Exp Clin Cancer Res. 2011;30:98.
Mulder GB, Manley N, Maggio-Price L. Retinoic acid-induced thymic abnormalities in the mouse are associated with altered pharyngeal morphology, thymocyte maturation defects and altered expression of Hoxa3 and Pax1. Teratology. 1998;58:263–75.
Holland LZ, Holland ND. Expression of amphiHox-1 and amphiPax-1 in amphioxus embryos treated with retinoic acid: insights into evolution and patterning of the chordate nerve cord and pharynx. Development. 1996;122:1829–38.
Stefanska B, Rudnicka K, Bednarek A, Fabianowska-Majewska K. Hypomethylation and induction of retinoic acid receptor beta 2 by concurrent action of adenosine analogues and natural compounds in breast cancer cells. Eur J Pharmacol. 2010;638:47–53.
Parashar G, Parashar NC, Capalash N. Curcumin causes promoter hypomethylation and increased expression of FANCF gene in SiHa cell line. Mol Cell Biochem. 2012;365:29–35.
Bock C, Reither S, Mikeska T, Paulsen M, Walter J, Lengauer T. BiQ Analyzer: visualization and quality control for DNA methylation data from bisulfite sequencing. Bioinformatics. 2005;21:4067–8.
Parashar G, Capalash N. Expression of the TIMP2 gene is not regulated by promoter hypermethylation in the Caski cell line. Oncol Lett. 2012;3:1079–82.
Hagemann S, Heil O, Lyko F, Brueckner B. Azacytidine and decitabine induce gene-specific and non-random DNA demethylation in human cancer cell lines. PLoS One. 2011;6(3):e17388. doi:10.1371/journal.pone.0017388.
Si J, Boumber YA, Shu J, Qin T, Ahmed S, He R, Jelinek J, Issa JP. Chromatin remodeling is required for gene reactivation after decitabine-mediated DNA hypomethylation. Cancer Res. 2010;70(17):6968–77.
Bronner C, Krifa M, Mousli M. Increasing role of UHRF1 in the reading and inheritance of the epigenetic code as well as in tumorigenesis. Biochem Pharmacol. 2013;86:1643–9.
Sharif T, Auger C, Alhosin M, et al. Red wine polyphenols cause growth inhibition and apoptosis in acute lymphoblastic leukaemia cells by inducing a redoxsensitive up-regulation of p73 and down-regulation of UHRF1. Eur J Cancer. 2010;46:983–94.
Abusnina A, Keravis T, Yougbaré I, Bronner C, Lugnier C. Anti-proliferative effect of curcumin on melanoma cells is mediated by PDE1A inhibition that regulates the epigenetic integrator UHRF1. Mol Nutr Food Res. 2011;55:1677–89.
Raynal NJ-M, Si J, Taby RF, et al. DNA methylation does not stably lock gene expression but instead serves as a molecular mark for gene silencing memory. Cancer Res. 2012;72:1170–81.
Kim HJ, Bae SC. Histone deacetylase inhibitors: molecular mechanisms of action and clinical trials as anti-cancer drugs. Am J Transl Res. 2011;3:166–79.
Acknowledgments
This study was supported by Council of Scientific and Industrial Research (CSIR), New Delhi, India.
Conflict of interest
The author(s) declare that they have no competing interests.
Author information
Authors and Affiliations
Corresponding author
Electronic supplementary material
Below is the link to the electronic supplementary material.
Rights and permissions
About this article
Cite this article
Parashar, G., Capalash, N. Promoter methylation-independent reactivation of PAX1 by curcumin and resveratrol is mediated by UHRF1 . Clin Exp Med 16, 471–478 (2016). https://doi.org/10.1007/s10238-015-0366-1
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s10238-015-0366-1