Transferrin iron uptake is stimulated by ascorbate via an intracellular reductive mechanism.

Although ascorbate has long been known to stimulate dietary iron (Fe) absorption and non-transferrin Fe uptake, the role of ascorbate in transferrin Fe uptake is unknown. Transferrin is a serum Fe transport protein supplying almost all cellular Fe under physiological conditions. We sought to examine ascorbate's role in this process, particularly as cultured cells are typically ascorbate-deficient. At typical plasma concentrations, ascorbate significantly increased (59)Fe uptake from transferrin by 1.5-2-fold in a range of cells. Moreover, ascorbate enhanced ferritin expression and increased (59)Fe accumulation in ferritin. The lack of effect of cycloheximide or the cytosolic aconitase inhibitor, oxalomalate, on ascorbate-mediated (59)Fe uptake from transferrin indicate increased ferritin synthesis or cytosolic aconitase activity was not responsible for ascorbate's activity. Experiments with membrane-permeant and -impermeant ascorbate-oxidizing reagents indicate that while extracellular ascorbate is required for stimulation of (59)Fe uptake from (59)Fe-citrate, only intracellular ascorbate is needed for transferrin (59)Fe uptake. Additionally, experiments with l-ascorbate analogs indicate ascorbate's reducing ene-diol moiety is necessary for its stimulatory activity. Importantly, neither N-acetylcysteine nor buthionine sulfoximine, which increase or decrease intracellular glutathione, respectively, affected transferrin-dependent (59)Fe uptake. Thus, ascorbate's stimulatory effect is not due to a general increase in cellular reducing capacity. Ascorbate also did not affect expression of transferrin receptor 1 or (125)I-transferrin cellular flux. However, transferrin receptors, endocytosis, vacuolar-type ATPase activity and endosomal acidification were required for ascorbate's stimulatory activity. Therefore, ascorbate is a novel modulator of the classical transferrin Fe uptake pathway, acting via an intracellular reductive mechanism.

Novel thiosemicarbazone iron chelators induce up-regulation and phosphorylation of the metastasis suppressor N-myc down-stream regulated gene 1: a new strategy for the treatment of pancreatic cancer.

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.

The metastasis suppressor, N-myc downstream regulated gene 1 (NDRG1), upregulates p21 via p53-independent mechanisms.

The metastasis suppressor, N-myc downstream regulated gene-1 (NDRG1), has been shown to markedly reduce metastasis of numerous tumors. The current study was focused on further elucidating the molecular mechanisms behind the antitumor function of NDRG1. We have identified for the first time that NDRG1 upregulates the potent cyclin-dependent kinase inhibitor, p21. This effect was observed in three different cancer cell types, including PC3MM and DU145 prostate cancer cells and H1299 lung carcinoma cells, and occurred independently of p53. In addition, reducing NDRG1 expression using short hairpin RNA in PC3MM and DU145 cells resulted in significantly reduced p21 protein levels. Hence, p21 is closely correlated with NDRG1 expression in these latter cell types. Examining the mechanisms behind the effect of NDRG1 on p21 expression, we found that NDRG1 upregulated p21 via transcriptional and posttranscriptional mechanisms in prostate cancer cells, although its effect on H1299 cells was posttranscriptional only. Further studies identified two additional NDRG1 protein targets. The dominant-negative p63 isoform, ΔNp63, which has been found to inhibit p21 transcription, was downregulated by NDRG1. On the other hand, a truncated 50 kDa MDM2 isoform (p50(MDM2)), which may protect p21 from proteasomal degradation, was upregulated by NDRG1. The downregulation of ΔNp63 and upregulation of p50(MDM2) are potential mechanisms by which NDRG1 increases p21 expression in these cells. Additional functional studies identified that NDRG1 inhibits cancer cell migration, suggesting that p21 is a molecular player in its antimetastatic activity.

The iron-regulated metastasis suppressor NDRG1 targets NEDD4L, PTEN, and SMAD4 and inhibits the PI3K and Ras signaling pathways.

AIMS: The metastasis suppressor gene, N-myc downstream regulated gene-1 (NDRG1), is negatively correlated with tumor progression in multiple neoplasms, including pancreatic cancer. Moreover, NDRG1 is an iron-regulated gene that is markedly upregulated by cellular iron-depletion using novel antitumor agents such as the chelator, di-2-pyridylketone 4,4-dimethyl-3-thiosemicarbazone (Dp44mT), in pancreatic cancer cells. However, the exact function(s) of NDRG1 remain to be established and are important to elucidate. RESULTS: In the current study, using gene-array analysis along with NDRG1 overexpression and silencing, we identified the molecular targets of NDRG1 in three pancreatic cancer cell lines. We demonstrate that NDRG1 upregulates neural precursor cell expressed developmentally downregulated 4-like (NEDD4L) and GLI-similar-3 (GLIS3). Further studies examining the downstream effects of NEDD4L led to the discovery that NDRG1 affects the transforming growth factor-ß (TGF-ß) pathway, leading to the upregulation of two key tumor suppressor proteins, namely phosphatase and tensin homolog deleted on chromosome 10 (PTEN) and mothers against decapentaplegic homolog-4 (SMAD4). Moreover, NDRG1 inhibited the phosphatidylinositol 3-kinase (PI3K) and Ras oncogenic pathways. INNOVATION: This study provides significant insights into the mechanisms underlying the antitumor activity of NDRG1. For the first time, a role for NDRG1 is established in regulating the key signaling pathways involved in oncogenesis (TGF-ß, PI3K, and Ras pathways). CONCLUSION: The identified target genes of NDRG1 and their effect on the TGF-ß signaling pathway reveal its molecular function in pancreatic cancer and a novel therapeutic avenue.