5-aza-2′-deoxycitidine inhibits cell proliferation, extracellular matrix formation and Wnt/β-catenin pathway in human uterine leiomyomas

Abstract

Background Uterine leiomyoma is a benign tumor with unclear pathogenesis and inaccurate treatment. This tumor exhibits altered DNA methylation related to disease progression. DNMT inhibitors as 5-aza-2′-deoxycytidine (5-aza-CdR), have been suggested to treat tumors in which DNA methylation is altered. We aimed to evaluate whether DNA methylation reversion with 5-aza-CdR reduces cell proliferation and extracellular matrix (ECM) formation in uterine leiomyoma cells to provide a potential treatment option. Methods Prospective study using uterine leiomyoma and adjacent myometrium tissues and human uterine leiomyoma primary (HULP) cells (n = 16). In tissues, gene expression was analyzed by qRT-PCR and DNMT activity by ELISA. Effects of 5-aza-CdR treatment on HULP cells were assessed by CellTiter, western blot, and qRT-PCR. Results DNMT1 gene expression was higher in uterine leiomyoma vs myometrium. Similarly, DNMT activity was greater in uterine leiomyoma and HULP cells (6.5 vs 3.8 OD/h/mg; 211.3 vs 63.7 OD/h/mg, respectively). After 5-aza-CdR treatment on HULP cells, cell viability was reduced, significantly so at 10 μM (85.3%). Treatment with 10 μM 5-aza-CdR on HULP cells significantly decreased expression of proliferation marker PCNA (FC = 0.695) and of ECM proteins (COLLAGEN I FC = 0.654; PAI-1, FC = 0.654; FIBRONECTIN FC = 0.733). 5-aza-CdR treatment also decreased expression of Wnt/β-catenin pathway final targets, including WISP1 protein expression (10 μM, FC = 0.699), c-MYC gene expression (2 μM, FC = 0.745 and 10 μM, FC = 0.728), and MMP7 gene expression (5 μM, FC = 0.520 and 10 μM, FC = 0.577). Conclusions 5-aza-CdR treatment inhibits cell proliferation, ECM formation, and Wnt/β-catenin signaling pathway targets in HULP cells, suggesting that DNA methylation inhibition is a viable therapeutic target in uterine leiomyoma.

Emerging role of different DNA methyltransferases in the pathogenesis of cancer

“…The Wnt/β-catenin signaling pathway is a common class of signaling pathways in cancer and its activation endows cancer cells with sustained self-renewing growth properties and is associated with therapy resistance (Bugter et al, 2021). In an experiment of Carbajo-García MC et al, the findings have demonstrated that in human uterine leiomyoma primary (HULP) cells, aberrant overexpression of DNMT1 induced abnormal DNA methylation and subsequently activated the Wnt/β-catenin signaling pathway, contributing to cell proliferation and extracellular matrix (ECM) formation (Carbajo-Garcia et al, 2021). In addition, a significant Frontiers in Pharmacology frontiersin.org study on the mechanism of treatment for mantle cell lymphoma (MCL) indicated that the inactivation of the Wnt/β-catenin signaling pathway and downregulation of DNMT1 synergistically suppressed MCL, and these results could guide the dosing in the clinical treatment of MCL (Li et al, 2017).…”

Integrative analysis of the DNA methylome and transcriptome in uterine leiomyoma shows altered regulation of genes involved in metabolism, proliferation, extracellular matrix, and vesicles

“…Inhibition of DNA methylation upregulated the tumor suppressors EFEMP1 , HTATIP2 , FBLN2 , and ARHGAP10 , supporting the notion that epigenetic modification of DNA methylation is involved in the expression of tumor suppressor genes that trigger UL development. Because 5‐aza‐CdR inhibits cell proliferation, extracellular matrix formation, and Wnt/β‐catenin signaling pathway targets in HULP cells [31,41], our findings suggest that reversing DNA methylation of tumor suppressor genes could inhibit these processes. Extracellular matrix has been identified as a potential target for future therapeutics in UL [49], and we demonstrated that EFEMP2 and FBLN2 were associated with aberrant DNA methylation and that demethylation recovered their expression, suggesting potential therapeutic impact.…”

“…Human UL primary (HULP) cells obtained from individual patients ( n = 10) were isolated from UL samples as previously described [31]. To evaluate the effect of 5‐aza‐CdR on the selected downregulated and hypermethylated genes, HULP cells were treated with 0 μ m (control) or 10 μ m 5‐aza‐CdR (Abcam, Cambridge, UK) for 72 h, with the medium replaced every 24 h. Total RNA from HULP cells was obtained using a Qiagen RNeasy Mini Kit, and cDNA was synthesized using a PrimeScript RT Kit (Takara, Shiga, Japan).…”

Deciphering the Role of Histone Modifications in Uterine Leiomyoma: Acetylation of H3K27 Regulates the Expression of Genes Involved in Proliferation, Cell Signaling, Cell Transport, Angiogenesis and Extracellular Matrix Formation

“…To evaluate the effect of SAHA (Abcam, Cambridge, UK) on the selected downregulated and hypoacetylated genes, HULP cells were isolated from UL tissues ( n = 10) from selected women, as previously described [ 36 ] and treated with 0 μM (0.01% DMSO as a control) or 10 μM of SAHA for 48h. Then, total RNA was extracted from HULP cells using a Qiagen RNeasy Mini kit, and cDNA was synthesized using a Takara PrimeScript RT reagent kit; qRT-PCR was performed to evaluate gene expression CD40 , GIMAP8 , IL15, GPX3 and DPT in HULP cells treated with or without SAHA, as described above.…”