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Final Progress Reports: New York University School of Medicine: Metal-Induced Inflammatory Factors, Oxidative Stress and Suppression of their Effects

Superfund Research Program

Metal-Induced Inflammatory Factors, Oxidative Stress and Suppression of their Effects

Project Leader: Krystyna Frenkel
Grant Number: P42ES010344
Funding Period: 2000 - 2006

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Final Progress Reports

Year:   2005 

Studies continued on the effects of arsenite (As), a human carcinogen, on HOS cell transformation.  Caffeic acid phenethyl ester (CAPE), a naturally occurring immuno­modulating agent, was used to evaluate the role of inflammatory factors in the process of transformation and in As-transformed HOS (AsT-HOS) cells.  Co-incubation of HOS with As+CAPE prevented As-mediated decline in cytokine mRNAs and cell transformation.  CAPE (10 µM) had no effect on HOS cell growth, however, CAPE (1-10 µM) inhibited AsT-HOS cell growth, induced G2/M arrest, triggered apoptosis, and decreased cyclin B1 and cdc2 in comparison to HOS cells.  AsT-HOS cells were readily killed by CAPE following increases in p53 and p53-dependent genes.  Resveratrol and EGCG, anti­oxidants present in grape skin and green tea, respectively, were needed at 10- and 20-fold higher doses than CAPE (2.5 µM) to cause 50% growth arrest in AsT-HOS cells. 

Although arsenite itself does not participate in redox reactions, oxidative stress is important in As-induced cell transformation.  Gene arrays showed that As affected expression of antioxidant enzymes SOD and a quinone oxidoreductase (NQO) homologue.  Both mRNAs were 10-fold lower in AsT-HOS than in HOS.  A concomitant As+CAPE treatment inhibited As-mediated HOS transformation and, concurrently, prevented decline in SOD mRNA and enzyme levels in comparison to As-treated HOS.  p53 was decreased in AsT-HOS at mRNA and protein levels from those in HOS cells.  To establish whether As-induced reduction in NQO has an effect on p53, HOS cells were treated with dicoumarol (NQO inhibitor).  Dicoumarol caused a dose-dependent decrease in p53 protein, thus indicating that As-mediated suppression of antioxidant defenses, could affect function of p53 and, consequently, apoptosis and tumorigenesis. 

Presence of a low CAPE dose during As exposure prevented HOS cell transformation and minimized many of As-mediated genetic changes.  Interestingly, of the NF-κBs, only p105/p50 NF-κB1 subunits were decreased by As.  CAPE counteracted arsenite-mediated decreases in NF-κB p50 as well as cytokine mRNA down-regulation.  To prove the involvement of NF-κB1 in cell transformation and genetic changes, these genes were knocked-down in HOS cells using p50 siRNA.  The decreases were p50 siRNA dose-dependent and caused up-to 5-fold decline in p50 and even greater decrease in p105.  These cells were not analyzed yet for other changes.

Also investigated was whether altered iron (Fe) homeostasis could contribute to As-mediated cell transformation.  Ferritin was ~60-70 times lower in AsT-HOS and in 8-week As-treated HOS, while total Fe levels were higher in AsT-HOS cells in comparison to control HOS cells, as determined by atomic absorption (NIEHS shared facility).  CAPE counteracted As-induced ferritin decline during the 8-week exposure.  A lower ferritin in AsT-HOS cells was due to the decreased mRNA, which is likely because of As-mediated decline in NF-κB1 needed for ferritin gene expression.  The decline in TfR was due to a lowered Fe regulatory proteins binding activity.  These results suggest that altered Fe homeostasis could contribute to As-induced oxidative stress and, thus, may be involved in As-mediated cell transformation. 

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