Does Wnt/β-catenin pathway contribute to the stability of DNMT1 expression in urological cancer cell lines?

Abstract

DNA methylation is considered as one of the most important epigenetic mechanisms and it is catalyzed by DNA methyltransferases (DNMTs). DNMT1 abundance has been frequently seen in urogenital system tumors but the reasons for this abundance are not well understood. We aimed to look into the effects of Wnt/b-catenin signaling pathway on overexpression of DNMT1 and aberrant expression of UHRF1 and HAUSP which are responsible for stability of DNMT1 at transcriptional and protein levels in urogenital cancers. In this context, firstly, Wnt/b-catenin signaling pathway was activated by using SB216763 which is a glycogen synthase kinase-3 (GSK3) b inhibitor. Cell proliferation levels in bladder cancer cells, renal cell carcinoma, and prostate cancer cells treated with GSK3b inhibitor (SB216763) were detected by WST-1 reagent. WIF-1 gene methylation profile was determined by methylation-specific PCR (MSP); expression levels of target genes -catenin and WIF-1 by real-time PCR; and protein levels of -catenin, DNMT1, pGSK3(Ser9), HAUSP, and UHRF1 by Western Blot. Our results indicated that treatment with SB216763 caused an increased cell proliferation at low dose. mRNA levels of -catenin increased after treatment with SB216273 and protein levels of pGSK3b(Ser9), b-catenin, and DNMT1 increased in comparison to control. HAUSP and UHRF1 were either up-regulated or down-regulated at the same doses depending on the type of cancer. Also, we showed that protein levels of DNMT1, b-catenin, HAUSP, and UHRF1 decreased after re-expression of WIF-1 following treatment with DAC. In Caki-2 cells, -catenin pathway might have accounted for the stability of DNMT1 expression, whereas such relation is not valid for T24 and PC3 cells. Our findings may offer a new approach for determination of molecular effects of Wnt/b-catenin signal pathway on DNMT1. This may allow us to identify new molecular targets for the treatment of urogenital cancers.

The effect of heat shock protein 90 inhibitors on histone 4 lysine 20 methylation in bladder cancer

“…Total protein extractions have been previously described (Varol et al, 2015[ 32 ]). Briefly, cells were lysed in RIPA lysis buffer (CST, USA) containing 1 mM PMSF (Sigma-Aldrich, Germany).…”

Oncogenic roles of the cholesterol metabolite 25‑hydroxycholesterol in bladder cancer

“…The effect of 24S-hydroxycholesterol and 25-hydroxycholesterol on the proliferation of bladder cancer cells was assessed via an MTT assay (Sigma-Aldrich, Merck KGaA). The MTT analysis was performed as previously described (35,36). Cells were plated at a density of 8x10 3 cells/well in 96-well plates in DMEM supplemented with 10% FBS and incubated at 37˚C in a 5% CO 2 incubator for 12 h. Cells were treated with 24S-hydroxycholesterol (10 -6 M), 25-hydroxycholesterol (10 -6 M) and vehicle DMSO (control), for 48 h. Subsequently, MTT (5 mg/ml) was added to the wells (20 µl/well) and the plates were incubated at 37˚C for 4 h in a 5% CO 2 incubator.…”

“…Western blot analysis. Western blotting was performed as previously described (35,36). Total protein was extracted from T24 and RT4 cells using extraction buffer comprising NaCl/Pi containing 0.5% Triton X-100, 1 mM EDTA, 1 mM phenylmethyl sulfonyl fluoride and complete protease inhibitors (Roche Diagnostics).…”

Expression profiles of CD11b, galectin-1, beclin-1, and caspase-3 in nasal polyposis*

“…Western blot analysis of caspase-3 was performed as described previously in a recent study (13). Briefly, the cells were lysed in lysis buffer solution (Cell Signaling Technology, Inc., Danvers, MA, USA) containing 1 mM phenylmethanesulfonyl fluoride (Sigma-Aldrich).…”