Arsenic is well documented as a chemotherapeutic agent capable of inducing cell death; however, it is also considered as a human carcinogen. Although it has recently been shown that arsenite exposure can potentiate genotoxicity, little is known about its global effects exerted in cells at the proteome level. Immortalized human small airway epithelial cells exposed to arsenite were used to identify phosphoproteins of two major signaling cascades, such as the human phospho-receptor tyrosine kinase (Phospho-RTK) and the mitogen-activated protein kinases (MAPKs). These two arrays included several phosphoproteins, such as EGFR, ErbB2, ErbB4, InsulinR, Flt-3, extracellular signal-regulated kinases (ERK1/2), intracellular kinases such as AKT, GSK-3, c-Jun N-terminal kinases (JNK1-3) and different p38 isoforms (alpha/beta/delta/gamma). In arsenite-treated cells, phosphorylation of EGFR, InsulinR and Flt3R showed an increase when compared to their non-arsenite treated counterparts. Inhibitors of these proteins further confirmed the involvement of such proteins in the neoplasm transformation of arsenite-treated human small airway epithelial cells as seen in changes in plating efficiency, anchorage-independent growth and proliferation rate. It can be concluded that analysis of phosphoprotein by using phosphoproteomic profiling can be very useful to understand the mechanism of arsenite-induced carcinogenesis. less...

Based on epidemiological data, chronic exposure to high levels of inorganic arsenic in drinking water is carcinogenic to humans, inducing skin, urinary bladder and lung tumors. In vivo, inorganic arsenic is metabolized to organic methylated arsenicals including the highly toxic dimethylarsinous acid (DMA(III)) and monomethylarsonous acid (MMA(III)). Short-term treatment of rats with 100 microg/g trivalent arsenic (As(III)) as sodium arsenite in the diet or in drinking water induced cytotoxicity and necrosis of the urothelial superficial layer, with increased cell proliferation and hyperplasia. The objectives of this study were to determine if these arsenic-induced urothelial effects are dose responsive, the dose of arsenic at which urothelial effects are not detected, and the urinary concentrations of the arsenical metabolites. We treated female F344 rats for 5 weeks with sodium arsenite at dietary doses of 0, 1, 10, 25, 50, and 100 ppm. Cytotoxicity, cell proliferation and hyperplasia of urothelial superficial cells were increased in a dose-responsive manner, with maximum effects found at 50 ppm As(III). There were no effects at 1 ppm As(III). The main urinary arsenical in As(III)-treated rats was the organic arsenical dimethylarsinic acid (DMA(V)). The thio-metabolites dimethylmonothioarsinic acid (DMMTA(V)) and monomethylmonothioarsinic acid (MMMTA(V)) were also found in the urine of As(III)-treated rats. The LC(50) concentrations of DMMTA(V) for rat and human urothelial cells in vitro were similar to trivalent oxygen-containing arsenicals. These data suggest that dietary As(III)-induced urothelial cytotoxicity and proliferation are dose responsive, and the urothelial effects have a threshold corresponding to the urinary excretion of measurable reactive metabolites. less...

Arsenic is ubiquitously present in the environment; it is a known human carcinogen and paradoxically it is also a successful drug for the clinical remission of acute promyelocytic leukemia (APL). The cellular responses induced by arsenite treatment have been investigated for years; however, the precise mechanisms underlying its cytotoxicity and therapeutic activity remain unclear. Here we report the use of mass spectrometry together with stable isotope labeling by amino acids in cell culture (SILAC) for the comparative study of protein expression in HL-60 cells that were untreated or treated with a clinically relevant concentration of arsenite. Our results revealed that, among the 1067 proteins quantified in both forward and reverse SILAC measurements, 56 had significantly altered levels of expression induced by arsenite treatment. These included the up-regulation of core histones, neutrophil elastase, a-mannosidase as well as the down-regulation of fatty acid synthase and protein phosphatase 1a We further demonstrated that the arsenite-induced growth inhibition of HL-60 cells could be rescued by treatment with palmitate, the final product of fatty acid synthase, supporting that arsenite exerts its cytotoxic effect, in part, via suppressing the expression of fatty acid synthase and inhibiting the endogenous production of fatty acid. The results from the present study offered important new knowledge for gaining insights into the molecular mechanisms of action of arsenite. less...

Arsenicals have been used since ancient Greek and Roman civilizations and in the Far East as part of traditional Chinese medicine. In Western countries, they became a therapeutic mainstay for various ailments and malignancies in the 19th and early 20th centuries. Fowler's potassium bicarbonate-based solution of arsenic trioxide (As2O3)solution was the main treatment of chronic myeloid leukaemia until the 1930s. After a decline in the use of arsenic during the mid-20th century, arsenic trioxide was reintroduced as an anticancer agent after reports emerged from China of the success of an arsenic trioxide-containing herbal mixture for the treatment of acute promyelocytic leukaemia. Arsenic trioxide was first purified and used in controlled studies in China in the 1970s.Subsequently, randomised clinical trials performed in the United States led to FDA approval of arsenic trioxide in the treatment of patients with relapsed or refractory acute promyelocytic leukaemia. less...

Background: Inorganic arsenic is a ubiquitous environmental carcinogen affecting millions of people worldwide. Evolving theory predicts that normal stem cells (NSCs) are transformed into cancer stem cells (CSCs) that then drive oncogenesis. In humans, arsenic is carcinogenic in the urogenital system (UGS), including the bladder and potentially the prostate, whereas in mice arsenic induces multi-organ UGS cancers, indicating that UGS NSCs may represent targets for carcino-genic initiation. However, proof of emergence of CSCs induced by arsenic in a stem cell population is not available.Methods: We continuously exposed the human prostate epithelial stem/progenitor cell line WPE-stem to an environmentally relevant level of arsenic (5 microM) in vitro and determined the acquired cancer phenotype.Results: WPE-stem cells rapidly acquired a malignant CSC-like phenotype by 18 weeks of exposure, becoming highly invasive, losing contact inhibition, and hyper-secreting matrix metalloproteinase-9. When hetero-transplanted, these cells (designated As-CSC) formed highly pleomorphic, aggressive tumors with immature epithelial- and mesenchymal-like cells, suggesting a highly pluripotent cell of origin. Consistent with tumor-derived CSCs, As-CSCs formed abundant free-floating spheres enriched in CSC-like cells, as confirmed by molecular analysis and the fact that only these floating cells formed xeno-graft tumors. An early loss of NSC self-renewal gene expression (p63, ABCG2, BMI-1, SHH, OCT-4, NOTCH-1) during arsenite exposure was sub-sequently reversed as the tumor suppressor gene PTEN was progressively suppressed and the CSC-like phenotype acquired.Conclusions: Arsenite transforms prostate epithelial stem/progenitor cells into CSC-like cells, indicating that it can produce CSCs from a model NSC population. Editor's SummaryThe transformation of normal stem cells (NSCs) into cancer stem cells (CSCs) has been proposed as a primary initiating event in carcinogenesis, but direct evidence of such a mechanism has not been demonstrated. Tokar et al. (p. 108) studied effects of continuous exposure to environmentally relevant concentrations of arsenic on the human prostate epithelial stem/progenitor WPE-stem cell line. The authors report that the WPE-stem cells acquired a malignant CSC-like phenotype by 18 weeks of exposure and that the transformed cells produced aggressive highly pleomorphic tumors (consistent with a pluripotent cell of origin) when transplanted into male nude mice. In addition, expression of self-renewal genes (TP63, ABCG2, BMI1, SHH, OCT-4, and NOTCH-1) was inhibited during arsenite exposure but subsequently returned, coincident with progressive inhibition of the PTEN tumor suppressor gene. The authors conclude that findings are consistent with arsenic-mediated transformation of epithelial stem/progenitor cells into CSC-like cells. less...

Recently we showed that the polyunsaturated fatty acid (PUFA) docosahexaenoic acid (DHA) sensitizes arsenic trioxide (As2O3)-resistant tumor cells to a clinically achievable concentration (1 microM) of As2O3 via a reactive oxygen species (ROS)-dependent mechanism. The aim of the present study was to evaluate, whether this combined effect of As2O3 and DHA is also applicable to other PUFAs [i.e., eicospentaenoic acid (EPA), arachidonic acid (AA), and gamma-linolenic acid (GLA)]. Fourteen tumor cell lines were incubated with As2O3 (1 microM), PUFA (25-100 microM), or the combination thereof (+/- vitamin E). Cell viability (colorimetric), apoptosis (bivariate annexin V/propidium iodide staining, detection of hypodiploid DNA), and thiobarbituric acid reactive substances (TBARS) were evaluated. Twelve of 14 As2O3-resistant cell lines tested were resistant to PUFA monotherapy. However, combined treatment with As2O3 and either PUFA significantly reduced cell viability in a dose-dependent manner with AA being the most potent As2O3 enhancer. The combined cytotoxic effect of As2O3/AA treatment was due to induction of apoptosis, preceded by increased intracellular TBARS and was abolished by the antioxidant vitamin E. Importantly, the combined effect of As2O3 and AA was selectively toxic for malignant cells because no cytotoxic effect was observed in normal skin fibroblasts and human microvascular endothelial cells. In conclusion, our study shows that also other PUFAs than DHA-and in particular the omega-6-PUFA AA--can be used as effective modulators of tumor cell chemosensitivity to clinically achievable concentrations of As2O3. Enhanced lipid peroxidation most likely constitutes the key mechanism for the combined effect. less...

Lead, a ubiquitous heavy metal, is an important industrial and environmental pollutant that can target the vascular endothelium. To clarify the effects of lead on the unfolded protein response (UPR) and their significance in cytotoxicity, we examined the expression and function of endoplasmic reticulum (ER) chaperones glucose-regulated protein 78 (GRP78) and 94 (GRP94) in vascular endothelial cells. We used bovine aortic endothelial cells as an in vitro model of the vascular endothelium. Exposure of vascular endothelial cells to lead nitrate resulted in a marked induction of GRP78 and GRP94 mRNA levels. In response to lead, the expression of GRP78 and GRP94 proteins also significantly increased in a dose- and time-dependent manner. In addition, siRNA-mediated knockdown of GRP78 significantly enhanced lead-induced cytotoxicity. Compared with other metal(loid)s, including cadmium chloride, zinc sulfate, copper sulfate, and sodium arsenite, lead nitrate was found to be the most potent metal to induce these chaperones in endothelial cells. In the examined UPR pathways, lead increased the phosphorylation of IRE1 and JNK. Interestingly, the lead-induced upregulation of GRP78 and GRP94 was almost completely blocked by the JNK inhibitor SP600125 or AP-1 inhibitor curcumin. Taken together, these results suggest that lead induces ER stress, but the induction of GRP78 and GRP94 expression via the JNK-AP-1 pathway functions as a defense mechanism against lead-induced cytotoxicity in vascular endothelial cells. less...

A comprehensive method for simultaneously detecting species of arsenic and selenium including arsenite (As(III)), monomethylarsonic acid (MMA), dimethylarsinic acid (DMA), arsenate (As(V)), selenocystine (SeCys), selenomethionine (SeMet) and selenate (Se(IV)) was developed with high performance liquid chromatography-hydride generation-double channel atomic fluorescence spectrometry (HPLC-HG-AFS). An anion-exchange column (PRP-X100) with eluent of 10 mmol/L NH4H2PO4 containing 2.5% (v/v) methanol was employed to separate these species within 12 min. The detection limits of As(III), DMA, MMA, As(V), SeCys, SeMet and Se(IV) were 1, 3, 2, 3, 4, 18 and 3 microg/L (200 microL of injection), respectively. The relative standard deviations in five independent determinations varied from 1.9% to 6.1% for arsenic and selenium species at the concentration levels of 100 and 300 microg/L. The proposed method was applied to analyze the selenium yeast tablet and human urine samples. The recoveries from spiked selenium yeast tablet and urine samples ranged from 88% to 105% and from 83% to 108%, respectively. The results showed that this method can be used for determining arsenic and selenium species in urinary metabolites and drug samples in daily analysis conveniently. less...

Arsenic is well-known as a carcinogen predisposing humans to some severe diseases and also as an effective medicine for treating acute promyelocytic leukemia, syphilis, and psoriasis. Multiple active mechanisms, including cell cycle arrest and apoptosis, have been proposed in therapy; however, the opposing effects of arsenic remain controversial. Our previous study found that arsenic trioxide (ATO)-induced activation of p21(WAF1/CIP1) (p21) led to A431 cell death through the antagonistic effects of the signaling of ERK1/2 and JNK1. In the current study, the inhibitory effects of JNK1 on ATO-induced p21 expression were explored. Over-expression of JNK1 in A431cells could inhibit p21 expression, which was associated with HDAC1 and TGIF. Using the GST pull-down assay and fluorescence resonance energy transfer analysis, N-terminal domain (amino acids 1-108) of TGIF was critical to its binding with c-Jun, was found. Using reporter assays, requirement of the C-terminal domain (amino acids 138-272) of TGIF to suppress ATO-induced p21 expression was observed. Thus, the domains of TGIF that carried out its inhibitory effects on p21, were identified. Finally, treatment with JNK inhibitor SP600125 could enhance ATO-induced apoptosis of HaCaT keratinocytes by using flow cytometry. less...

When cultured human keratinocytes reach confluence, they undergo a program of changes replicating features of differentiation in vivo, including exit from the proliferative pool, increased cell size, and expression of specialized differentiation marker proteins. Previously, we showed that insulin is required for some of these steps and that arsenite, a human carcinogen in skin and other epithelia, opposes the differentiation process. In present work, we show that insulin signaling, probably through the IGF-I receptor, is required for the increase in cell size accompanying differentiation and that this is opposed by arsenite. We further examine the impact of insulin and arsenite on PKCdelta, a known key regulator of keratinocyte differentiation, and show that insulin increases the amount, tyrosine phosphorylation, and membrane localization of PKCdelta. All these effects are prevented by exposure of cells to arsenite or to inhibitors of downstream effectors of insulin (phosphotidylinositol 3-kinase and mammalian target of rapamycin) Retrovirally mediated expression of activated PKCdelta resulted in increased loss of proliferative potential after confluence and greatly increased formation of cross-linked envelopes, a marker of keratinocyte terminal differentiation. These effects were prevented by removal of insulin, but not by arsenite addition. We further demonstrate a role for src family kinases in regulation of PKCdelta. Finally, inhibiting epidermal growth factor receptor kinase activity diminished the ability of arsenite to prevent cell enlargement and to suppress insulin-dependent PKCdelta amount and tyrosine 311 phosphorylation. Thus suppression of PKCdelta signaling is a critical feature of arsenite action in preventing keratinocyte differentiation and maintaining proliferative capability. (c) 2010 Wiley-Liss, Inc. less...