Enhanced constitutive invasion activity in human nontumorigenic keratinocytes exposed to a low level of barium for a long time.

We have recently demonstrated that exposure to barium for a short time (≤4 days) and at a low level (5 µM = 687 µg/L) promotes invasion of human nontumorigenic HaCaT cells, which have characteristics similar to those of normal keratinocytes, suggesting that exposure to barium for a short time enhances malignant characteristics. Here we examined the effect of exposure to low level of barium for a long time, a condition mimicking the exposure to barium through well water, on malignant characteristics of HaCaT keratinocytes. Constitutive invasion activity, focal adhesion kinase (FAK) protein expression and activity, and matrix metalloproteinase 14 (MMP14) protein expression in primary cultured normal human epidermal keratinocytes, HaCaT keratinocytes, and HSC5 and A431 human squamous cell carcinoma cells were augmented following an increase in malignancy grade of the cells. Constitutive invasion activity, FAK phosphorylation, and MMP14 expression levels of HaCaT keratinocytes after treatment with 5 µM barium for 4 months were significantly higher than those of control untreated HaCaT keratinocytes. Taken together, our results suggest that exposure to a low level of barium for a long time enhances constitutive malignant characteristics of HaCaT keratinocytes via regulatory molecules (FAK and MMP14) for invasion.

Barium inhibits arsenic-mediated apoptotic cell death in human squamous cell carcinoma cells.

Our fieldwork showed more than 1 µM (145.1 µg/L) barium in about 3 µM (210.7 µg/L) arsenic-polluted drinking well water (n = 72) in cancer-prone areas in Bangladesh, while the mean concentrations of nine other elements in the water were less than 3 µg/L. The types of cancer include squamous cell carcinomas (SCC). We hypothesized that barium modulates arsenic-mediated biological effects, and we examined the effect of barium (1 µM) on arsenic (3 µM)-mediated apoptotic cell death of human HSC-5 and A431 SCC cells in vitro. Arsenic promoted SCC apoptosis with increased reactive oxygen species (ROS) production and JNK1/2 and caspase-3 activation (apoptotic pathway). In contrast, arsenic also inhibited SCC apoptosis with increased NF-κB activity and X-linked inhibitor of apoptosis protein (XIAP) expression level and decreased JNK activity (antiapoptotic pathway). These results suggest that arsenic bidirectionally promotes apoptotic and antiapoptotic pathways in SCC cells. Interestingly, barium in the presence of arsenic increased NF-κB activity and XIAP expression and decreased JNK activity without affecting ROS production, resulting in the inhibition of the arsenic-mediated apoptotic pathway. Since the anticancer effect of arsenic is mainly dependent on cancer apoptosis, barium-mediated inhibition of arsenic-induced apoptosis may promote progression of SCC in patients in Bangladesh who keep drinking barium and arsenic-polluted water after the development of cancer. Thus, we newly showed that barium in the presence of arsenic might inhibit arsenic-mediated cancer apoptosis with the modulation of the balance between arsenic-mediated promotive and suppressive apoptotic pathways.

A redox-linked novel pathway for arsenic-mediated RET tyrosine kinase activation.

We examined the biochemical effects of arsenic on the activities of RET proto-oncogene (c-RET protein tyrosine kinases) and RET oncogene (RET-MEN2A and RET-PTC1 protein tyrosine kinases) products. Arsenic activated c-RET kinase with promotion of disulfide bond-mediated dimerization of c-RET protein. Arsenic further activated RET-MEN2A kinase, which was already 3- to 10-fold augmented by genetic mutation compared with c-RET kinase activity, with promotion of disulfide bond-mediated dimerization of RET-MEN2A protein (superactivation). Arsenic also increased extracellular domain-deleted RET-PTC1 kinase activity with promotion of disulfide bond-mediated dimerization of RET-PTC1 protein. Arsenic increased RET-PTC1 kinase activity with cysteine 365 (C365) replaced by alanine with promotion of dimer formation but not with cysteine 376 (C376) replaced by alanine. Our results suggest that arsenic-mediated regulation of RET kinase activity is dependent on conformational change of RET protein through modulation of a special cysteine sited at the intracellular domain in RET protein (relevant cysteine of C376 in RET-PTC1 protein). Moreover, arsenic enhanced the activity of immunoprecipitated RET protein with increase in thiol-dependent dimer formation. As arsenic (14.2 microM) was detected in the cells cultured with arsenic (100 microM), direct association between arsenic and RET in the cells might modulate dimer formation. Thus, we demonstrated a novel redox-linked mechanism of activation of arsenic-mediated RET proto-oncogene and oncogene products.