Hyperthermia-induced NDRG2 upregulation inhibits the invasion of human hepatocellular carcinoma via suppressing ERK1/2 signaling pathway

Abstract

Hyperthermia (HT) has been proven to be able to alter the invasion capacity of cancer cells. However, the detailed mechanisms responsible for the anti-metastasis effects of HT have not been elucidated. N-myc downstream-regulated gene 2 (NDRG2), as a member of the NDRG family, has been suggested to be highly responsive to various stresses and is associated with tumor suppression. The present study aimed to investigate the biological role of NDRG2 in the invasion of human hepatocellular carcinoma (HCC) cells exposed to HT. We found that NDRG2 could be induced by HT at 45°C. In addition, NDRG2 overexpression inhibited the expression of matrix metallo proteinases-2 (MMP-2) and MMP-9 as well as the invasion of HCC cells, whereas knockingdown NDRG2 reversed the anti-invasion effect of HT in vivo. Further investigation revealed that the phosphorylation level of ERK1/2, but not that of JNK and p38MAPK, was reduced in NDRG2 overexpressing cells. Moreover, the knockdown of NDRG2 expression resulted in increased cell invasion, which was rescued by treating the HepG2 cells with the ERK1/2 inhibitor PD98059, but not with the p38MAPK inhibitor SB203580 or the JNK inhibitor SP600125. Finally, the synergistic cooperation of HT at 43°C and NDRG2 expression effectively reduced cytotoxicity and promoted the anti-invasion effect of HT at 45°C. Taken together, these data suggest that NDRG2 can be induced by HT and that it mediates the HT-caused inhibition of invasion in HCC cells by suppressing the ERK1/2 signaling pathway. The combined application of constitutive NDRG2 expression with HT may yield an optimized therapeutic benefit.

Figure 1. HT retards the invasion of HCC with up-regulation of NDRG2.

(A) HepG2 and Huh7 cells were exposed to different temperatures (37°C, 39°C, 41°C, 43°C and 45°C) for 30 min. Matrigel invasion assays were performed after 24 h of heat treatment in order to evaluate the effect of HT on invasiveness. Cell invasion is shown in phase contrast images and graphical representations. Invasion was assessed by counting cells in five microscopic fields per well at 400×magnification. The data are representative of triplicate experiments and calculated as the means ± SD. The invasion rate of cells at 37°C was defined as 100%, and *P<0.05 indicates the degree of statistical significance as compared to cells at 37°C. (B) Expression levels of NDRG2, MMP-2 and MMP-9 in heat-treated HepG2 and Huh7 cells were determined by Western blot after 24 h of heat treatment at different temperatures (37°C, 39°C, 41°C, 43°C and 45°C) for 30 min. Tubulin was used as the internal loading control.

Figure 2. NDRG2 overexpression enhances the anti-invasion effect of HT in HCC cells.

(A) Expression level of NDRG2 in Control, Cherry (transfected with overexpression control plasmid), and NDRG2 (transfected with NDRG2 overexpression plasmid) cells. (B) NDRG2 expression level in Cherry or NDRG2 cells was determined by Western blot after HT at 45°C for 30 min. (C) The anti-invasive effect of HT was studied in Cherry or NDRG2 cells after HT at 45°C for 30 min. Cell invasion is depicted in the phase contrast images and the graphical representation. Invasion was assessed by counting invasive cells in five microscopic fields per well at 400×magnification. The data are representative of triplicate experiments and were calculated as the means ± SD. The invasion rate of Cherry-37°C cells was defined as 100%. *P<0.05 or **P<0.01 indicatesthe degree of statistical significance compared to Cherry-37°C cells. (D) Expression levels of MMP-2 and MMP-9 in 45°C heat-treated Cherry or NDRG2 cells were assessed by Western blot analysis. Tubulin was used as the internal loading control.

Figure 3. Down-regulation of NDRG2 affects the invasiveness of heat-treated HCC cells in vivo.

(A) Expression level of NDRG2 in Scramble-HepG2 (transfected with silencing expression control plasmid) and shNDRG2-HepG2 (transfected with NDRG2 silencing plasmid) cells. HepG2 cells were transfected separately with the Scramble or shNDRG2 and then individually injected into the hind legs of the mice. After two weeks of implantation, mice in the heat-treated groups were subjected to a 45°C water bath for 30 min. Each experimental group contained 5 mice. Four weeks later, mice were sacrificed, and primary tumors were removed for histological examination. (B) Representative H&E staining of histological sections revealed histological destruction in each group. Arrows point to invasion areas. Stars mark the locations of muscle. (C) Immunohistochemistry showed expression levels of NDRG2, MMP-2 and MMP-9 in the tumor tissues of each group. Bar = 30 µm (magnification 400×).

Figure 4. NDRG2 inhibits HT-induced ERK1/2 activation in HCC cells.

Cherry (transfected with over expression control plasmid) and NDRG2 (transfected with NDRG2 expression plasmid) cells were subjected to a 45°C water bath for 30 min and then incubated at 37°C for the indicated periods of time. The cells were lysed and levels of ERK1/2, JNK, and p38MAPK were detected by Western blot. Levels of ERK1/2, p38MAPK and JNK phosphorylated proteins at the indicated time points after heat treatment were detected by Western blot and quantitated by measuring band intensities. The values of the fold activations were normalized to the total ERK1/2, p38MAPK or JNK values. The data are representative of three independent experiments and were calculated as the means ± SD. *P<0.05 indicates degrees of statistical significance as compared to Cherry cells. Tubulin served as the loading control.

Figure 5. NDRG2 mediates the anti-invasion effect of HT via the inhibition of ERK1/2 activation.

shNDRG2-HepG2 (transfected with NDRG2 silencing plasmid) cells were pre-incubated with 10 µmol/ml PD98059, 10 µmol/ml SB203580 or 50 µmol/ml SP600125 for 30 min before heat treatment at 45°C for 30 min. (A) Cell invasion is represented graphically. The data are representative of triplicate experiments and were calculated as the means±SD. *P<0.05 indicates the degree of statistical significance. (B)MMP-2 and MMP-9 expression levels were determined by Western blot. Tubulin served as the loading control.

Figure 6. NDRG2 enhances the efficacy of HT.

(A) The effect of HT on necrosis and apoptosis was studied in Cherry (transfected with over expression control plasmid) or NDRG2 (transfected with NDRG2 expression plasmid) cells after heat treatment at 43°C or 45°C for 30 min. Apoptosis was measured through FACS analysis of PI- or Annexin V-stained cells. Column data analysis of intact cells (Annexin V−, PI−), early apoptotic (Annexin V+, PI−) and late apoptotic/necrotic cells (Annexin V−/+, PI+) for each cell group. *P<0.05 indicates the degree of statistical significance compared to the Cherry-37°C group; ^# P<0.05 indicates the degree of statistical significance as compared to the Cherry-45°C group. (B)The anti-invasive effect of HT was studied in Cherry or NDRG2 cells after HT at 43°C or 45°C for 30 min. Cell invasion is depicted in the phase contrast images and the graphical representation. Invasion was assessed by counting cells in five microscopic fields per well at 400×magnification. The data are representative of triplicate experiments and were calculated as the means ± SD. The invasion rate of Cherry-37°C cells was defined as 100%. *P<0.05 indicates the degree of statistical significance as compared to Cherry-37°C cells. (C) Expression levels of MMP-2 and MMP-9 in 43°C or 45°C heat-treated Cherry or NDRG2 cells were assessed by Western blot. Tubulin was used as the internal loading control.