Overexpression of N-Myc Downstream-Regulated Gene 2 (NDRG2) Regulates the Proliferation and Invasion of Bladder Cancer Cells In Vitro and In Vivo

N-Myc downstream-regulated gene 2 (NDRG2) is a candidate tumor suppressor gene, which plays an important role in controlling tumor growth. The aim of this study was to investigate the expression of NDRG2 gene in bladder cancer (BC) tissues and several bladder cancer cell lines, and to seek its clinical and pathological significance. Ninety-seven bladder carcinoma and 15 normal bladder tissue sections were analyzed retrospectively with immunohistochemistry. The human bladder cancer cell line T24 was infected with LEN-NDRG2 or LEN-LacZ. The effects of NDRG2 overexpression on T24 cells and T24 nude mouse xenografts were measured via cell growth curves, tumor growth curves, flow cytometric analysis, western blot and Transwell assay. NDRG2 was highly expressed in normal bladder tissue, but absent or rarely expressed in cacinomatous tissues (χ2=8.761, p < 0.01). The NDRG2 level was negatively correlated with tumor grade and pathologic stage(r=-0.248, p < 0.05), as well as increased c-myc level (r=-0.454, p< 0.001). The expression of NDRG2 was low in the three BC cell lines. T24 cells infected with LEN-NDRG2 showed inhibition of proliferation both in vitro and in vivo, and NDRG2 overexpression can inhibit tumor growth and invasion in vitro

<i>NDRG2</i> overexpression inhibits proliferation of human bladder cancer cells.

<p>(A and B) Colony formation assay. Upregulation of <i>NDRG2</i> inhibits colony formation. (C) The cell growth curves of T24 cells by MTT method. All the assays were repeated independently for at least three times. The results are shown as the mean ± SD (*=p<0.001).</p

The influences of <i>NDRG2</i> overexpression on T24 cells infected with lentivirus.

<p>(A) The effect of <i>NDRG2</i> overexpression on G1 cell cycle and apoptosis regulators as assayed by western blot. (B) Relative quantification of protein expression, normalized to GAPDH levels. The figure shows the tendency of each group as indicated. All the assays were repeated independently for at least three times. The results are shown as the mean ± SD (** p<0.01)..</p

FCA of cell cycle arrest and apoptosis induced by overexpression of <i>NDRG2</i>.

<p>(A) T24cells infected with LEN-<i>NDRG2</i> lentivirus were more obviously arrested in G0/G1cycle. (B) Percentages of apoptotic cells in LEN-<i>NDRG2</i> groups were greater than in the other two groups(1–3). 48 h after the infection (4–6); 72 h after the infection; (1 and 4) blank control (PBS); (2 and 5) LEN-LacZ; (3 and 6) LEN-<i>NDRG2</i>. All the assays were repeated independently for at least three times. The results are shown as the mean ± SD.</p

Overexpression of <i>NDRG2</i> suppresses human bladder cancer cell invasion, migration and expression of MMP-2 and MMP-9.

<p>(A) Invasion analysis of T24 cells treated with LEN-<i>NDRG2</i>. The invasion ability was estimated using Transwell coated with Matrigel. The invasiveness of LEN-<i>NDRG2</i> group cells was significantly lower than that in the other two groups. **p<0.001. (B) Migration analysis of T24 cells treated with LEN-<i>NDRG2</i>. The migration ability was estimated using Transwell uncoated with Matrigel. The migration ability of LEN-<i>NDRG2</i> group cells was significantly lower than that in the other two groups. **p<0.001. (C and D) Expression of MMP-2 and MMP-9 were measured after T24 cells were infected with LEN-<i>NDRG2</i>. LEN-<i>NDRG2</i> group reduced MMP-2 and MMP-9 expression compared to the other groups (** p<0.01).</p

The expression of <i>NDRG2</i> and c-Myc protein in bladder carcinoma tissues detected with immunohistochemistry staining

<p>(A) The expression levels of <i>NDRG2</i> protein decrease while the degree of malignancy of bladder carcinoma increases (x400)(1). Normal bladder tissue; (2) Bladder papilloma(T0-Ta); (3) High-level bladder cancer(T2-T4). (B) The expression levels of c-Myc protein increases while the degree of malignancy of bladder carcinoma increases (x400)(4). Normal bladder tissue; (5) Bladder papilloma(T0-Ta); (6) High-level bladder cancer(T2-T4). The sections of (B) (4–6) originated from the same paraffin blocks as (A) (1–3) respectively.</p

Differential expression of <i>NDRG2</i> among bladder cancer cell lines

<p>(A) Real-time PCR analysis of <i>NDRG2</i> mRNA expression. (B) Expression level of <i>NDRG2</i> protein as assayed by western blot. All the assays were repeated independently for at least three times. The results are shown as the mean ± SD (** p<0.001)..</p

The influences of <i>NDRG2</i> overexpression on T24 cells infected with lentivirus <i>in vivo</i>.

<p>(A) The effect of <i>NDRG2</i> overexpression on cell cycle, apoptosis and metastasis regulators as assayed by western blot. (B) Relative quantification of protein expression, normalized to GAPDH levels. Western blots were analyzed with Kodak Digital Science one-dimensional software. The figure shows the tendency of each group as indicated. All the assays were repeated independently for at least three times. The results are shown as the mean ± SD (* p<0.01).</p

pLEN-GFP-<i>NDRG2</i> infected T24 cells to increased <i>NDRG2</i> expression in bladder cancer cells

<p>(A) Detection of lentiviral infection efficiency, and phase contrast (left) or GFP (right) images were obtained four days after infection. (B) Real-time PCR analysis of <i>NDRG2</i> mRNA expression in T24 cells. (C) Western blot analysis of <i>NDRG2</i> protein expression. The results are shown as the mean ± SD (** p<0.01)..</p