Faculty Bibliography

ABSTRACT: BACKGROUND: Young women diagnosed with breast cancer are known to have a higher mortality rate from the disease than older patients. Specific risk factors leading to this poorer outcome have not been identified. In the present study, we hypothesized that iron deficiency, a common ailment in young women, contributes to the poor outcome by promoting the hypoxia inducible factor-1alpha (HIF-1alpha and vascular endothelial growth factor (VEGF) formation. This hypothesis was tested in an in vitro cell culture model system. RESULTS: Human breast cancer MDA-MB-231 cells were transfected with transferrin receptor-1 (TfR1) shRNA to constitutively impair iron uptake. Cellular iron status was determined by a set of iron proteins and angiogenesis was evaluated by levels of VEGF in cells as well as by a mouse xenograft model. Significant decreases in ferritin with concomitant increases in VEGF were observed in TfR1 knockdown MDA-MB-231 cells when compared to the parental cells. TfR1 shRNA transfectants also evoked a stronger angiogenic response after the cells were injected subcutaneously into nude mice. The molecular mechanism appears that cellular iron deficiency elevates VEGF formation by stabilizing HIF-1alpha. This mechanism is also true in human breast cancer MCF-7 and liver cancer HepG2 cells. CONCLUSIONS: Cellular iron deficiency increased HIF-1alpha, VEGF, and angiogenesis, suggesting that systemic iron deficiency might play an important part in the tumor angiogenesis and recurrence in this young age group of breast cancer patients

Inhibition of the TOR signalling pathway by genetic or pharmacological intervention extends lifespan in invertebrates, including yeast, nematodes and fruitflies; however, whether inhibition of mTOR signalling can extend lifespan in a mammalian species was unknown. Here we report that rapamycin, an inhibitor of the mTOR pathway, extends median and maximal lifespan of both male and female mice when fed beginning at 600 days of age. On the basis of age at 90% mortality, rapamycin led to an increase of 14% for females and 9% for males. The effect was seen at three independent test sites in genetically heterogeneous mice, chosen to avoid genotype-specific effects on disease susceptibility. Disease patterns of rapamycin-treated mice did not differ from those of control mice. In a separate study, rapamycin fed to mice beginning at 270 days of age also increased survival in both males and females, based on an interim analysis conducted near the median survival point. Rapamycin may extend lifespan by postponing death from cancer, by retarding mechanisms of ageing, or both. To our knowledge, these are the first results to demonstrate a role for mTOR signalling in the regulation of mammalian lifespan, as well as pharmacological extension of lifespan in both genders. These findings have implications for further development of interventions targeting mTOR for the treatment and prevention of age-related diseases

Estrogen and iron play critical roles in a female body development and were investigated in the present study in relation to in vitro cell proliferation. Prempro, a hormone replacement therapy drug, and 17beta-estradiol (E2) were shown to increase cell proliferations in estrogen receptor positive (ER+) cells independent of progesterone receptor (PR) status. For example, increased cell proliferation was observed in ER+/PR+ human breast cancer MCF-7, its matching non-cancerous human breast epithelial MCF-12A, and ER+/PR+ murine mammary cancer MXT+ cells, but not in ER-/PR- MDA-MB-231, its matching non-cancerous MCF-10A, and MXT- (ER-/PR+) cells. By mimicking post-menopausal conditions of high estrogen in local breast tissue and increased iron levels due to cessation of menstrual periods, E2 and iron were shown to exert synergistic effects on proliferation of MCF-7 cells and significantly increased Ki67 and proliferating cell nuclear antigen. Western blotting of E2-treated ER+ but not ER- cells showed that E2 also increased transferrin receptor (TfR). Further studies are needed to assess the mitogenic effects of iron and estrogen in normal post-menopausal breast

Quantitative cancer incidence data exist for various laboratory animal models, but little of this information is usable for estimating human risks, primarily because of uncertainties about possible mechanistic differences among species. Acceptance and utilization of animal data for human risk assessment will require a much better understanding of the comparative underlying mechanisms than now exists. A dual-lesion, radiation-track model in rat skin has proven to be consistent with tumor induction data with respect to acute radiation doses ranging from 0.5 up to 10 Gy and higher, and average LETs ranging from 0.34 to 150 keV microm(-1) according to the form neoplastic risk (D,L) = CLD + BD2. A recent result with the 56Fe ion beam showed dose-response consistency for malignant (carcinomas) and benign (fibromas) tumor induction with earlier results utilizing argon and neon ion beams. A discrepancy between the model and experiment was found indicating that proportionality of cancer yield with LET did not occur at 150 versus 125 keV microm(-1), i.e. tumor yield did not increase in spite of a 20% increase of LET, which suggests that a LET response maximum exists at or within this dose range. Concordance between the model and tumor induction data in rat skin implies that potential intervening complexities of carcinogenic progression fail to obscure the basic radiobiological assumptions underpinning the model. Gene expression microarray analysis shows that vitamin A inhibits the expression of about 80% of the inflammation-related genes induced by the radiation and prevents about 46% of the neoplasms associated with 56Fe ion radiation without appearing to interfere with the underlying dose and LET response patterns. Further validation is needed, but the model has the potential to provide quantitative estimates of cancer risk as a function of dose and LET for almost any type of radiation exposure and even for combinations of different radiations provided only three empirical parameters can be established for each type of radiation and organ system.

PURPOSE: The aim of the study is to determine the reliability during a 2-year period of several newly developed iron-related assays to assess their potential for use in prospective epidemiologic studies. METHODS: We assessed the temporal reliability of several iron-related assays by using three serum samples collected at yearly intervals from 50 postmenopausal participants in a large prospective study. RESULTS: We observed high reliability coefficients for ferritin (0.78; 95% confidence interval [CI], 0.67-0.86), soluble transferrin receptor (sTfR; 0.79; 95% CI, 0.69-0.87), sTfR/ferritin ratio (0.74; 95% CI, 0.62-0.83), and hepcidin (0.89; 95% CI, 0.84-0.94). In a subset of 30 women, lower reliability was observed for serum iron (0.50; 95% CI, 0.29-0.70), unsaturated iron-binding capacity (0.55; 95% CI, 0.34-0.73), total iron-binding capacity (0.60; 95% CI, 0.40-0.76), and serum transferrin saturation rate (0.44; 95% CI, 0.22-0.65). The reliability of anti-5-hydroxymethyl-2'-deoxyuridine autoantibody titers, a biomarker of oxidized DNA damage, one of the mechanisms by which iron is thought to impact disease risk, was very high (0.97, 95% CI, 0.5-0.99). CONCLUSIONS: Our results show that some newly developed iron-related assays could be useful tools to assess iron-disease associations in prospective cohorts that collect a single blood sample

Chronic exposure to low doses of arsenite causes transformation of human osteogenic sarcoma (HOS) cells. Although oxidative stress is considered important in arsenite-induced cell transformation, the molecular and cellular mechanisms by which arsenite transforms human cells are still unknown. In the present study, we investigated whether altered iron homeostasis, known to affect cellular oxidative stress, can contribute to the arsenite-mediated cell transformation. Using arsenite-induced HOS cell transformation as a model, it was found that total iron levels are significantly higher in transformed HOS cells in comparison to parental control HOS cells. Under normal iron metabolism conditions, iron homeostasis is tightly controlled by inverse regulation of ferritin and transferrin receptor (TfR) through iron regulatory proteins (IRP). Increased iron levels in arsenite transformed cells should theoretically lead to higher ferritin and lower TfR in these cells than in controls. However, the results showed that both ferritin and TfR are decreased, apparently through two different mechanisms. A lower ferritin level in cytoplasm was due to the decreased mRNA in the arsenite-transformed HOS cells, while the decline in TfR was due to a lowered IRP-binding activity. By challenging cells with iron, it was further established that arsenite-transformed HOS cells are less responsive to iron treatment than control HOS cells, which allows accumulation of iron in the transformed cells, as exemplified by significantly lower ferritin induction. On the other hand, caffeic acid phenethyl ester (CAPE), an antioxidant previously shown to suppress As-mediated cell transformation, prevents As-mediated ferritin depletion. In conclusion, our results suggest that altered iron homeostasis contributes to arsenite-induced oxidative stress and, thus, may be involved in arsenite-mediated cell transformation

Recent evidence suggests that inflammatory cytokines and growth factors contribute to arsenite (As)-induced human carcinogenesis. We investigated the expression of inflammatory cytokine mRNAs during the transformation process induced by chronic As exposure in non-tumorigenic human osteogenic sarcoma (N-HOS) cells using gene arrays, and results were confirmed by RT-PCR and protein arrays. Caffeic acid phenethyl ester (CAPE), a naturally occurring immunomodulating agent, was used to evaluate the role of inflammatory factors in the process of As-mediated N-HOS cell transformation and in As-transformed HOS (AsT-HOS) cells. We found that an 8-week continuous exposure of N-HOS to 0.3 microM arsenite resulted in HOS cell transformation. That exposure also caused substantial decreases in inflammatory cytokine mRNAs, such as interleukin (IL) IL-1alpha, IL-2, IL-8, IL-18, MCP-1, TGF-beta2, and TNF-alpha, while it increased c-jun mRNA in a time-dependent manner. Co-incubation of N-HOS with As and CAPE (0.5-2.5 microM) prevented As-mediated declines in cytokine mRNAs in the co-treated cells, as well as their transformation to anchorage independence, while it caused decreases in c-jun mRNA. CAPE (up to 10 microM) had no effect on growth of N-HOS cells. However, CAPE (1-10 microM) treatment of AsT-HOS cells inhibited cell growth, induced cell cycle G2/M arrest, and triggered apoptosis, accompanied by changes in cytokine gene expression, as well as decreases in cyclin B1 and cdc2 abundance. Resveratrol (RV) and (-)(.) epigallocatechin gallate (EGCG), preventive agents present in grapes and green tea, respectively, induced similar changes in AsT-HOS cell growth but required much higher doses than CAPE to cause 50% growth arrest (<2.5 microM CAPE versus 25 microM RV or 50 microM EGCG). Overall, our findings suggest that inflammatory cytokines play an important role in the suppressive effects of CAPE on As-induced cell transformation and in the selective cytotoxicity of CAPE to As-transformed HOS cells

Based on the first National Study of Coal Workers' Pneumoconiosis (CWP) and the U.S. Geological Survey database of coal quality, we show that the prevalence of CWP in seven coal mine regions correlates with levels of bioavailable iron (BAI) in the coals from that particular region (correlation coefficient r = 0.94, p < 0.0015). CWP prevalence is also correlated with contents of pyritic sulfur (r = 0.91, p < 0.0048) or total iron (r = 0.85, p < 0.016) but not with coal rank (r = 0.59, p < 0.16) or silica (r = 0.28, p < 0.54). BAI was calculated using our model, taking into account chemical interactions of pyrite, sulfuric acid, calcite, and total iron. That is, iron present in coals can become bioavailable by pyrite oxidation, which produces ferrous sulfate and sulfuric acid. Calcite is the major component in coals that neutralizes the available acid and inhibits iron's bioavailability. Therefore, levels of BAI in the coals are determined by the available amounts of acid after neutralization of calcite and the amount of total iron in the coals. Using the linear fit of CWP prevalence and the calculated BAI in the seven coal mine regions, we have derived and mapped the pneumoconiotic potencies of 7,000 coal samples. Our studies indicate that levels of BAI in the coals may be used to predict coal's toxicity, even before large-scale mining