Promoter methylation and downregulation of SLC22A18 are associated with the development and progression of human glioma

Abstract

Background: Downregulation of the putative tumor suppressor gene SLC22A18 has been reported in a number of human cancers. The aim of this study was to investigate the relationship between SLC22A18 downregulation, promoter methylation and the development and progression of human glioma.

Method: SLC22A18 expression and promoter methylation was examined in human gliomas and the adjacent normal tissues. U251 glioma cells stably overexpressing SLC22A18 were generated to investigate the effect of SLC22A18 on cell growth and adherence in vitro using the methyl thiazole tetrazolium assay. Apoptosis was quantified using flow cytometry and the growth of SLC22A18 overexpressing U251 cells was measured in an in vivo xenograft model.

Results: SLC22A18 protein expression is significantly decreased in human gliomas compared to the adjacent normal brain tissues. SLC22A18 protein expression is significantly lower in gliomas which recurred within six months after surgery than gliomas which did not recur within six months. SLC22A18 promoter methylation was detected in 50% of the gliomas, but not in the adjacent normal tissues of any patient. SLC22A18 expression was significantly decreased in gliomas with SLC22A18 promoter methylation, compared to gliomas without methylation. The SLC22A18 promoter is methylated in U251 cells and treatment with the demethylating agent 5-aza-2-deoxycytidine increased SLC22A18 expression and reduced cell proliferation. Stable overexpression of SLC22A18 inhibited growth and adherence, induced apoptosis in vitro and reduced in vivo tumor growth of U251 cells.

Conclusion: SLC22A18 downregulation via promoter methylation is associated with the development and progression of glioma, suggesting that SLC22A18 is an important tumor suppressor in glioma.

Figure 1

SLC22A18 is downregulated in glioma. Representative images of SLC22A18 immunohistochemical staining in (A) adjacent normal brain tissue and (B) glioma, × 200. (C) Semiquantitative analysis of SLC22A18 expression in glioma and the adjacent brain tissue, P value compares overall SLC22A18 expression in each group.

Figure 2

SLC22A18 expression in patients stratified by tumor grade and recurrence. (A) Semiquantitative analysis of SLC22A18 immunohistochemical staining in low grade (WHO I-II) glioma and high grade (WHO III-IV) glioma. (B) Semiquantitative analysis of SLC22A18 immunohistochemical staining in gliomas which recurred and those which did not recur within six months after surgery, P values compare overall SLC22A18 expression in each group.

Figure 3

Correlation between SLC22A18 promoter methylation and SLC22A18 mRNA and protein expression. (A) SLC22A18 promoter methylation analysis. In patients 1, 8, 15 and 30, the SLC22A18 promoter was methylated in glioma and not the adjacent brain tissue. The SLC22A18 promoter is also methylated in U251 cells. T, glioma; N, adjacent brain tissue; m, methylated; u, unmethylated. (B) SLC22A18 RT-PCR mRNA expression in patients 1, 8, 15 and 30. GAPDH was used as an internal control. (C) Western blot of SLC22A18 protein expression in patients 1, 8, 15 and 30. ß-actin was used as an internal control. Both SLC22A18 mRNA and protein expression are significantly downregulated in gliomas with promoter methylation, compared to the corresponding adjacent normal brain tissues.

Figure 4

Semiquantitative analysis of SLC22A18 protein expression in gliomas with and without SLC22A18 promoter methylation. P-value compares overall SLC22A18 expression in each group.

Figure 5

The demethylating agent 5-aza-2-deoxycytidine restores SLC22A18 expression in U251 cells and suppresses cell growth. (A) Western blotting indicating a significant increase in SLC22A18 expression after treatment of U251 cells with 2 μg/ml 5-aza-2-deoxycytidine for 7 days: 1, control cells; 2, treated cells; ß-actin was used as an internal control. (B) Growth curves demonstrating reduced cell numbers in the 5-aza-2-deoxycytidine-treated group, compared to empty plasmid transfected U251-EV cells and untransfected U251 cells.

Figure 6

PCR amplification of SLC22A18 in U251-SLC22A18 stable cell lines. M, DNA molecular mass marker; lane 1, culture medium only control; lane 2, empty plasmid transfected U251-EV cells; lane 3, untransfected U251 cells; lane 4, U251-SLC22A18 cells.

Figure 7

Western blot of SLC22A18 protein expression in U251-SLC22A18 stable cell lines. Lane 1, U251-SLC22A18 cells; lane 2, empty plasmid transfected U251-EV cells; lane 3, untransfected U251 cells. β-actin was used as loading control.

Figure 8

Cytotoxic effect of SLC22A18 expression in U251 cells. U251-SLC22A18, empty plasmid transfected U251-EV cells and untransfected U251 cells were cultured in plastic 96-well plates and quantified using the MTT assay.

Figure 9

Effects of SLC22A18 expression on U251 cell adhesion. (A) U251 cell adhesion to ECM (Fn and Matrigel). (B) U251 cell adhesion to ECV304.

Figure 10

Effects of SLC22A18 expression on tumor growth in vivo. (A) Subcutaneous tumor model. a, U251 cells group; b, U251-EV cells group; c, U251-SLC22A18 cells group. (B) Tumor growth curves of each group over 28 days.

Figure 11

SLC22A18 mRNA and protein is expressed in neurons but not astrocytes or oligodendrocytes in vitro. SLC22A18 RT-PCR (A) and Western blot (B). Lane 1, neurons; lane 2, astrocytes; lane 3, oligodendrocytes; M, standard DNA molecular mass marker.

Figure 12

SLC22A18 mRNA and protein is expressed in neurons but not astrocytes or oligodendrocytes in vivo. (A, B) SLC22A18 immunohistochemical staining in the hippocampus and (C) at high magnification in CA1. (D) Olfactory bulb including glomeruli (Gl), mitral cells (Mi), and the inner granule layer (IGL). (E) High magnification view of mitral cells. (F) Small SLC22A18-positive periglomerular cells. (G) Cerebellum showing weak staining of the internal granule layer (IGL), strong staining of Purkinje cells (P) and occasional positive cells in the molecular layer (ML). No staining of dendrites was evident in the ML. (H) High magnification of Purkinje cells. (I) High magnification of the IGL, showing cytoplasmic SLC22A18 staining. (J) White matter tracts in the cerebellum indicated by *. (K) Corpus callosum indicated by * with SLC22A18-positive amygdala (Am) observed at the right edge. (L) High magnification of a double stained astrocytic cell (SLC22A18, red brown; GFAP, blue gray) in white matter tracts of the cerebellum. All sections were counterstained with methyl green, scale bars = 20 μm.