NADPH Oxidase Activation Is Required in Reactive Oxygen Species Generation and Cell Transformation Induced by Hexavalent Chromium

Abstract

Hexavalent chromium [Cr(VI)] is a well-known human carcinogen associated with the incidence of lung cancer. Although overproduction of reactive oxygen species (ROS) has been suggested to play a major role in its carcinogenicity, the mechanisms of Cr(VI)-induced ROS production remain unclear. In this study, we investigated the role of NADPH oxidase (NOX), one of the major sources of cellular ROS, in Cr(VI)-induced oxidative stress and carcinogenesis. We found that short-term exposure to Cr(VI) (2μM) resulted in a rapid increase in ROS generation in Beas-2B cells, and concomitantly increased NOX activity and expression of NOX members (NOX1-3 and NOX5) and subunits (p22(phox), p47(phox), p40(phox), and p67(phox)). Cr(VI) also induced phosphorylation of p47(phox) and membrane translocation of p47(phox) and p67(phox), further confirming NOX activation. Knockdown of p47(phox) with a short hairpin RNA attenuated the ROS production induced by Cr(VI). Chronic exposure (up to 3 months) to low doses of Cr(VI) (0.125, 0.25, and 0.5μM) also promoted ROS generation and the expression of NOX subunits, such as p47(phox) and p67(phox), but inhibited the expression of main antioxidant enzymes, such as superoxidase dismutase (SOD) and glutathione peroxidase (GPx). Chronic Cr(VI) exposure resulted in transformation of Beas-2B cells, increasing cell proliferation, anchorage independent growth in soft agar, and forming aggressive tumors in nude mice. Stable knockdown of p47(phox) or overexpression of SOD1, SOD2, or catalase (CAT) eliminated Cr(VI)-induced malignant transformation. Our results suggest that NOX plays an important role in Cr(VI)-induced ROS generation and carcinogenesis.

Constitutive Activation of Epidermal Growth Factor Receptor Promotes Tumorigenesis of Cr(VI)-transformed Cells through Decreased Reactive Oxygen Species and Apoptosis Resistance Development

“…Phosphorylation of EGFR at tyrosine1068 was much higher at the exposure duration of 4 months and 6 months than that of 2 months and 3 months. At this stage of chronic Cr(VI) exposure (6 months), the cells were malignant transformed as examined by anchorage-independent cell growth (soft agar) assay (data not shown), similar to those observation in our previous study (17). Phosphorylation of EGFR at Tyr-1068 was highest in both two clones of transformed cells isolated from the cells exposed to Cr(VI) for 6 months compared with various durations of Cr(VI) exposure (Fig.…”

“…It has been reported that EGFR mRNA level was increased in BEAS-2B cells exposed to 1 M of Cr(VI) up to 8 culture passages (14). Our previous study has demonstrated that chronic exposure of BEAS-2B cells to Cr(VI) was able to induce malignant cell transformation (17). The present study has found that EGFR was constitutively activated in Cr(VI)-transformed BEAS-2B cells.…”

“…In leiomyoma smooth muscle cells, the inhibition of NOX impaired MAPK signaling activation and decreased the cell proliferation response to EGF (33). Our previous study has reported that Cr(VI) is able to activate NOX (17). It appears that that Cr(VI)-induced NOX activation may be one of the mechanisms for EGFR activation.…”

“…In contrast, expressions of anti-apoptotic proteins Bcl-2 and Bcl-xL were elevated, indicating that both apoptotic and anti-apoptotic proteins contribute to apoptosis resistance in Cr(VI)-transformed cells. It has been reported that short-term exposure to Cr(VI) generates reactive oxygen species (ROS), which is very important in Cr(VI)-induced carcinogenesis (17,(21)(22)(23). Although ROS generated by Cr(VI) are responsible for Cr(VI)-induced cell transformation (16), these species are also capable of causing apoptosis (24).…”

The role of p38 signaling and poly(ADP-ribosyl)ation-induced metabolic collapse in the osteogenic differentiation-coupled cell death pathway

“…Activity of NADPH oxidase was measured according to Wang et al [30]. The ratio of lucigenin oxidation was normalized to protein content (quantified with a Pierce BCA protein assay kit).…”