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

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-Aza-CdR 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 cm³ in size, whereas the tumors induced by U251 cells treated with 5-Aza-CdR averaged 0.88 cm³ 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.