The epithelial-mesenchymal transition (EMT) is a key step for cancer cell migration, invasion, and metastasis. Transforming growth factor-β (TGF-β) regulates the EMT and the metastasis suppressor gene, N-myc downstream-regulated gene-1 (NDRG1), could play a role in regulating the TGF-β pathway. NDRG1 expression is markedly increased after chelator-mediated iron depletion via hypoxia-inducible factor 1α-dependent and independent pathways (Le, N. T. and Richardson, D. R. (2004) Blood 104, 2967-2975). Moreover, novel iron chelators show marked and selective anti-tumor activity and are a potential new class of anti-metabolites. Considering this, the current study investigated the relationship between NDRG1 and the EMT to examine if iron chelators can inhibit the EMT via NDRG1 up-regulation. We demonstrated that TGF-β induces the EMT in HT29 and DU145 cells. Further, the chelators, desferrioxamine (DFO) and di-2-pyridylketone-4,4-dimethyl-3-thiosemicarbazone (Dp44mT), inhibited the TGF-β-induced EMT by maintaining E-cadherin and β-catenin, at the cell membrane. We then established stable clones with NDRG1 overexpression and knock-down in HT29 and DU145 cells. These data showed that NDRG1 overexpression maintained membrane E-cadherin and β-catenin and inhibited TGF-β-stimulated cell migration and invasion. Conversely, NDRG1 knock-down caused morphological changes from an epithelial- to fibroblastic-like phenotype and also increased migration and invasion, demonstrating NDRG1 knockdown induced the EMT and enhanced TGF-β effects. We also investigated the mechanisms involved and showed the TGF-β/SMAD and Wnt pathways were implicated in NDRG1 regulation of E-cadherin and β-catenin expression and translocation. This study demonstrates that chelators inhibit the TGF-β-induced EMT via a process consistent with NDRG1 up-regulation and elucidates the mechanism of their activity.

Iron (Fe) is essential for cellular metabolism e.g., DNA synthesis and its depletion causes G1/S arrest and apoptosis. Considering this, Fe chelators have been shown to be effective anti-proliferative agents. In order to understand the anti-tumor activity of Fe chelators, the mechanisms responsible for G1/S arrest and apoptosis after Fe-depletion have been investigated. These studies reveal a multitude of cell cycle control molecules are regulated by Fe. These include p53, p27Kip1, cyclin D1 and cyclin-dependent kinase 2 (cdk2). Additionally, Fe-depletion up-regulates the mRNA levels of the cdk inhibitor, p21CIP1/WAF1, but paradoxically down-regulates its protein expression. This effect could contribute to the apoptosis observed after Fe-depletion. Iron-depletion also leads to proteasomal degradation of p21CIP1/WAF1 and cyclin D1 via an ubiquitin-independent pathway. This is in contrast to the mechanism in Fe-replete cells, where it occurs by ubiquitin-dependent proteasomal degradation. Up-regulation of p...

Tumor metastasis is an important clinical problem, contributing to the majority of cancer-related deaths. The recent discovery of metastasis suppressor genes, such as N-myc downstream-regulated gene-1 (Ndrg-1), has introduced a novel approach to treating cancer and preventing metastasis. Ndrg-1 has been identified as a protein involved in the differentiation of epithelial cells. In addition, Ndrg-1 expression can be regulated by androgens and is involved in the pathology of the disease, Hereditary Motor and Sensory Neuropathy-Lom (HMSNL). However, one of the most well documented links between Ndrg-1 and pathophysiology is its association with inhibition of tumor metastasis. The expression of Ndrg-1 was found to be significantly downregulated in a variety of different neoplasms including breast, colon and prostate cancer. Furthermore, Ndrg-1 expression was shown to be negatively correlated with tumor metastasis. Studies in vitro and in vivo have demonstrated a significant reduction in the metastatic ability of cells overexpressing Ndrg-1. The ability of these cells to invade was also compromised. The Gleason grade of prostate and breast cancers was found to correlate with Ndrg-1 expression, with more advanced and poorly differentiated tumors having lower Ndrg-1 levels. Recently, Ndrg-1 expression was demonstrated to be regulated by cellular iron levels and induced by iron chelators. These latter compounds were recently identified as potential anticancer agents as they selectively prevent cancer cell proliferation and lead to apoptosis. The discovery that iron chelators also increase Ndrg-1 expression further augments their antitumor activity and provides a novel strategy for the treatment of cancer and its metastasis.

Pancreatic cancer is an aggressive neoplasm, with a mortality rate close to 100%. The most successful agent for pancreatic cancer treatment is gemcitabine, although the overall effect in terms of patient survival remains very poor. This study was initiated to evaluate a novel class of anticancer agents against pancreatic cancer. This group of compounds belongs to the dipyridyl thiosemicarbazone class that have been shown to have potent and selective activity against a range of different neoplasms in vitro and in vivo. We demonstrate for the first time in pancreatic cancer that these agents increase the expression of the growth and metastasis suppressor N-myc downstream-regulated gene 1 and its phosphorylation at Ser330 and Thr346 that is important for its activity against this tumor. In addition, these agents increased expression of the cyclin-dependent kinase inhibitor p21^CIP1/WAF1, whereas decreasing cyclin D1 in pancreatic cancer cells. Together, these molecular alterations account, in part, for the pronounced antitumor activity observed. Indeed, these agents had significantly higher antiproliferative activity in vitro than the established treatments for pancreatic cancer, namely gemcitabine and 5-fluorouracil. Studies in vivo demonstrated that a novel thiosemicarbazone, namely di-2-pyridylketone 4-cyclohexyl-4-methyl-3-thiosemicarbazone hydrochloride, completely inhibited the growth of pancreatic cancer xenografts with no evidence of marked alterations in normal tissue histology. Together, our studies have identified molecular effectors of a novel and potent antitumor agent that could be useful for pancreatic cancer treatment.