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. 2014 Sep 30;5(18):8478-91.
doi: 10.18632/onco_target.2328.

The iron chelator Dp44mT inhibits hepatocellular carcinoma metastasis via N-Myc downstream-regulated gene 2 (NDRG2)/gp130/STAT3 pathway

Jiabei Wang  1 , Dalong Yin  1 , Changming Xie  1 , Tongsen Zheng  1 , Yingjian Liang  2 , Xuehui Hong  2 , Zhaoyang Lu  2 , Xuan Song  2 , Ruipeng Song  2 , Haiyan Yang  2 , Boshi Sun  2 , Nishant Bhatta  2 , Xianzhi Meng  2 , Shangha Pan  2 , Hongchi Jiang  2 , Lianxin Liu  3
Affiliations

The iron chelator Dp44mT inhibits hepatocellular carcinoma metastasis via N-Myc downstream-regulated gene 2 (NDRG2)/gp130/STAT3 pathway

Jiabei Wang et al. Onco_target. .

Abstract

Here we showed that hepatocellular carcinoma (HCC) cell lines with high metastatic potential had low levels of NDRG2. The iron chelator Dp44mT up-regulated NDRG2, suppressed epithelial-mesenchymal transition (EMT) and inhibited tumor metastasis in HCC having high metastatic potential. Also Dp44mT attenuated the TGF-β1-induced EMT in HCC having low metastatic potential. In agreement, silencing endogenous NDRG2 with shNDRG2 in HCC cells attenuated the effect of Dp44mT. We showed that the NDRG2/gp130/STAT3 pathway can mediate Dp44mT effects. In agreement, we found that a combination of NDRG2 expression and p-STAT3 levels is a strong predictor of prognosis in HCC patients. We suggest that up-regulation of NDRG2 by Dp44mT is a promising therapeutic approach in HCC.

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Conflict of interest statement

Disclosure of potential conflicts of interest

No potential conflicts of interest were disclosed.

Figures

Figure 1
Figure 1. Levels of NDRG2 are decreased in HCC clinical samples, and decreased levels of NDRG2 indicate invasion/metastasis of HCC
(A) and (B) The protein and mRNA levels were evaluated in the indicated cell lines. The expression of NDRG2 was normalized against β-actin. (C) Cell invasion assay was performed in the indicated cells. The invading cells through the ECM were enumerated 48 hours later. The results represent means ± SD of experiments performed in triplicate. (D) Real-time PCR results of relative expression level of NDRG2 mRNA in 17 normal liver (Normal), 16 HCC without vascular invasion (Non-inv), and 15 HCC with vascular invasion (Inv) samples. mRNA levels of NDRG2 were normalized against β-actin. The results represent means ± SD of experiments performed in triplicate. (E) Protein levels of NDRG2 were determined in 15 Normal, 15 Non-inv, and 15 Inv samples. Box plot graph showed the statistical analysis of NDRG2 expression in all samples. The expression of NDRG2 was normalized against β-actin.
Figure 2
Figure 2. Dp44mT inhibits invasion and adhesion of cancer cells in vitro and inhibits metastasis in vivo through up-regulating NDRG2
(A) Dp44mT increased the expression of NDRG2 significantly in HCC-LM3 cells. Cell invasion experiment results showed Dp44mT (10 μM) or NDRG2 overexpression significantly inhibited the invasive capacity of HCC-LM3 cells, and NDRG2 knockdown attenuated the Dp44mT-induced inhibition of invasion in HCC-LM3 cells. The results represent means ± SD of experiments performed in triplicate. (B) 24 hours after Dp44mT (10 μM) treatment, adhesion to matrix proteins were performed in HCC-LM3 cells (which infected with NDRG2 or shNDRG2). Adhering cells were quantified by CytoSelectTM Cell Adhesion Assay Kit. The results represent means ± SD of experiments performed in triplicate. (C) 4 weeks after Dp44mT treatment, lung tissues were harvested. Representative lung tissue sections from each group were shown (hematoxylin and eosin stain; magnification, × 40). The number of lung metastatic foci in each group was calculated.
Figure 3
Figure 3. Dp44mT inhibits EMT in HCC cells
(A) 12 hours after Dp44mT (10 μM) treatment, real-time PCR was performed to assess mRNA levels of epithelial markers E-cadherin, cytokeratin-8, cytokeratin-17, cytokeratin-18, claudin-1, and claudin-8 and mesenchymal markers N-cadherin, vimentin, Jagged1, Jagged2, Goosecoid, and an EMT regulator TWIST1 in HCC-LM3 cells. Results were normalized against β-actin. The results represent means ± SD of experiments performed in triplicate. (B) 24 hours after treatment, immunoblotting showed Dp44mT (10 μM) or NDRG2 overexpression increased the expression of E-cadherin and decreased the expression of N-cadherin, vimentin and TWIST1 in HCC-LM3 and MHCC-97H cells. Knockdown of NDRG2 abrogated the effect of Dp44mT-induced reduction of EMT. β-actin was used as the internal control. All assays were done in triplicate. (C) Single and merged images were taken to show immunofluorescence staining of N-cadherin (green) and vimentin (red) accompanied by the cell nucleus (blue) stained by DAPI. (D) HCC-LM3 liver tumors from different groups were immunostained for indicated molecules. Pictures are representative of three independent experiments.
Figure 4
Figure 4. Dp44mT attenuates the TGF-β1-induced EMT in MHCC-97L and SMMC-7721 cells
(A) Cell invasion experiment results showed TGF-β1 (5 ng/ml) or NDRG2 knockdown significantly enhanced the invasive capacity of MHCC-97L cells, and Dp44mT (10 μM) and NDRG2 overexpression attenuated the TGF-β1/ NDRG2 knockdown induced activation of invasion in MHCC-97L and SMMC-7721 cells. The results represent means ± SD of experiments performed in triplicate. (B) Cells were pretreated in the presence or absence of TGF-β1 (5 ng/ml) for 48 hours and followed by co-incubation with Dp44mT (10 μM) for another 24 h. The expression of indicated proteins were detected by immunoblotting. (C) Iron (FeCl3) inhibits Dp44mT's ability to attenuate the TGF-β1-induced EMT. HCC cells were pre-treated in the presence or absence of TGF-β (5 ng/mL) for 48 h and followed by co-incubation with either: FeCl3 (20 μM), Dp44mT (10 μM) or Dp44mT (10 μM) + FeCl3 (20 μM) for another 24 h. (D) Immunoblotting showed that TGF-β1 or shNDRG2 treatment increases the expression of Snail, Slug and pSMAD3. Dp44mT or NDRG2 overexpression can attenuate the effect of TGF-β1. β-actin was used as the internal control. All assays were done in triplicate.
Figure 5
Figure 5. gp130/STAT3 pathway is involved in mediating Dp44mT action, and combination of NDRG2 expression and p-STAT3 signal is a powerful predictor of poor clinical outcome in HCCs
(A) 24 hours after indicated treatment (Dp44mT 10 μM), HCC cells were analyzed for the indicated protein by immunoblotting. β-actin was used as the internal control. (B) Cell invasion assay showed Dp44mT attenuated the IL-6 (20 ng/ml) induced invasive capacity in MHCC-97L cells. The results represent means ± SD of experiments performed in triplicate. (C) Dp44mT reduced IL-6-induced phosphorylation of STAT3 in MHCC-97L and SMMC-7721 cells. β-actin was used as the internal control. (D) Combination of NDRG2 and p-STAT3 enhanced correlation to clinical parameters and the significance for poor prognosis.
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References

    1. He J, Gu D, Wu X, Reynolds K, Duan X, Yao C, Wang J, Chen CS, Chen J, Wildman RP, Klag MJ, Whelton PK. Major causes of death among men and women in China. N Engl J Med. 2005;353(11):1124–1134. - PubMed
    1. El-Serag HB, Marrero JA, Rudolph L, Reddy KR. Diagnosis and treatment of hepatocellular carcinoma. Gastroenterology. 2008;134(6):1752–1763. - PubMed
    1. Portolani N, Coniglio A, Ghidoni S, Giovanelli M, Benetti A, Tiberio GA, Giulini SM. Early and late recurrence after liver resection for hepatocellular carcinoma: prognostic and therapeutic implications. Ann Surg. 2006;243(2):229–235. - PMC - PubMed
    1. Wu Q, Wang R, Yang Q, Hou X, Chen S, Hou Y, Chen C, Yang Y, Miele L, Sarkar FH, Chen Y, Wang Z. Chemoresistance to gemcitabine in hepatoma cells induces epithelial-mesenchymal transition and involves activation of PDGF-D pathway. Onco_target. 2013;4(11):1999–2009. - PMC - PubMed
    1. Kalinowski DS, Richardson DR. The evolution of iron chelators for the treatment of iron overload disease and cancer. Pharmacol Rev. 2005;57(4):547–583. - PubMed

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