Effect of 5-Aza-2'-deoxycytidine on SLC22A18 in glioma U251 cells

Abstract

Abstract. SLC22A18 [solute carrier family 22 (organic cation transporter) member 18] is located within the 11p15.5 cluster, and may be a new tumor suppressor gene; evidence of SLC22A18 hypermethylation is documented in several types of human cancers. In order to determine whether SLC22A18 hypermethylation is involved in glioma, we determined the SLC22A18 gene protein expression, mRNA expression and methylation status in glioma U251 cells before and after treatment with 5-Aza-2'-deoxycytidine (5-Aza-CdR), and observed the change in growth. Glioma U251 cells treated with 5-AzaCdR were analyzed by flow cytometry to identify any change in their cell cycle profiles. Tumors induced via the injection of untreated U251 cells were measured. Immunohistochemistry, reverse transcriptase-polymerase chain reaction (RT-PCR) and PCR-based methylation assay were carried out to determine SLC22A18 gene protein expression, mRNA expression and methylation status in glioma U251 cells before and after treatment with 5-Aza-CdR. The treated cells showed an increase in their proportion in G1, from 79.2 to 83.5%, and a decrease in S phase, from 12.4 to 5.8%. The apoptotic rate increased from 6.4 to 15.8%. Tumors induced via the injection of untreated U251 cells were approximately 1.46 cm 3 in size, whereas the tumors induced by U251 cells treated with 5-Aza-CdR averaged 0.88 cm 3 in size. The expression levels of SLC22A18 protein and mRNA in U251 cells were increased following treatment with 5x10 -7 M 5-Aza-CdR. Prior to 5-Aza-CdR treatment, the SLC22A18 gene demonstrated hypermethylation and therefore could not be cleaved by HpaII and MspI. It is known that only the DNA digested with HpaII or MspI can be amplified. Following treatment with 5-Aza-CdR, the SLC22A18 gene became demethylated, and could then be cleaved by both of the enzymes, and this failed to be amplified. 5-Aza-cdR may induce glioma U251 cell division and apoptosis and enhance demethylation and protein and mRNA expression of SLC22A18. The hypermethylation of SLC22A18 may be related to the transcriptional silencing of this gene. The growth inhibitory effects of 5-Aza-CdR treatment in vivo remain recognizable.

Decitabine Treatment of Glioma-Initiating Cells Enhances Immune Recognition and Killing

“…Consistent with our findings, in a human lung carcinoma cell line, DAC was found to induce G2/M cell cycle arrest and increased the expression of p21 (a cyclin-dependent kinase inhibitor, cell cycle regulator) [38]. Furthermore, DAC cytostatic effects on the established U251 GBM cell line [39] and on one primary GBM cell line [31] were reported. Concerning the impact of DAC on cell viability, it is known that DAC effects on tumour cells are highly dose-dependent: for cultured leukaemia cells, it was reported that DAC-mediated re-expression of hypermethylated genes and changes in cell signalling pathways were achieved using DAC doses that did not induce acute cytotoxicity [40].…”

Hydroxyapatite nanoparticles inhibit the growth of human glioma cells in vitro and in vivo

“…In this experiment, U251 and SHG44 cells with early apoptotic signals, stained with annexin-V, and cells with late death signals, stained with PI, were considered and quantified, and the apoptotic cells were analyzed using CellQuest software. 11,12 Each assay was performed in triplicate.…”

Epigenetic changes in gastroenteropancreatic neuroendocrine tumours

“…64 The antiproliferative effect of decitabine has been tested in different tumour histotypes with encouraging results. [65][66][67][68][69][70][71] However, its anticancer activity in patients with refractory and metastatic solid tumours led to only limited responses because of difficulty to calibrate steady-state blood concentration and a narrow therapeutic index, resulting in dose-limiting haematologic toxicities. [72][73][74] Therefore, considering that the therapeutic potential of decitabine is hampered by its systemic instability, further studies are required to improve drug stability in solution and efficacy of delivery, in order to minimise toxicities, and to prolong epigenetic outcomes.…”