br Fig Promotion of Cr VI exposure on prostate cancer
Fig. 2. Promotion of Cr(VI) exposure on prostate cancer cell proliferation A. The cytotoxicity of Cr(III) and Cr(VI) exposure on PC3 U 46619 using Alamar Blue assay (n = 6). B. Eﬀects of Cr(VI) exposure on cell proliferation by MTT assay (n = 6). C. Colony-forming unit assays on cells exposed to Cr(VI). D. The tumor growth curves in mice that harbored tumor implants derived from PC3 cells with or without Cr(VI) exposure (n = 6). A representative image showing tumors from the 3 groups of mice (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article).
Fig. 3. Promotion of Cr(VI) exposure on prostate cancer cell migration.
A. Wound healing assay for PC3 cell migration upon Cr(VI) exposure (n = 3). B. Transwell migration assay for cell invasion upon Cr(VI) exposure (n = 6). C. Confocal imaging of F-actin stained with fluorescein isothiocyanate (FITC)-conjugated phalloidin in PC3 cells upon exposure to Cr(VI) for 24 h. The F-actin filaments are shown in green and the cell nuclei are shown in blue by counter staining with 4,6-diamidino-2-phenylindole (DAPI), ×400. D. The occurrence of tumor lung metastasis in mice with or without Cr(VI) exposure. A representative image of a lung with metastasis from a Cr(VI) exposed mouse. The lung was stained with Bouin's solution, red arrows show the metastatic tumor.
event responsible for triggering tumor metastasis. We found that mRNA expression of epithelial protein markers, such as E-cadherin (p = 0.0019), was markedly decreased under Cr(VI) exposure, while the expression of mesenchymal protein markers, such as N-cadherin (p < 0.001) and Snail (p < 0.001), were up-regulated (Fig. 5A). Western blot analysis gave similar results for protein levels (Fig. 5B). In sum, these data indicate that Cr(VI) can promote cell migration in prostate cancer via the EMT.
With the widespread industrial Cr usage, the genotoxic and carci-nogenic potential of Cr on humans and animals has become a global concern (Annangi et al., 2016; Chen and Costa, 2017). Cr mostly exists in two stable oxidation states, Cr(III) and Cr(VI) (Kirpnick-Sobol et al., 2006), but in nature, exists only in the Cr(III) form. It has been reported that Cr(III) accumulates in various tissues of the body after Cr supple-mentation (Collins et al., 2010; Stearns et al., 1995), and several studies suggest that Cr(III) could cause DNA damage by direct interaction with DNA (Kirpnick-Sobol et al., 2006). However, most genotoxicity tests demonstrate that Cr(III) exposure is not visibly genotoxic (De Flora et al., 1990), and no studies demonstrated carcinogenic potential upon Cr(III) ingestion both in vitro and in vivo (De Flora et al., 1990; Kirpnick-Sobol et al., 2006). In the current study, we found similar results in that Cr(III) exposure had no obvious carcinogenic eﬀects in prostate cancer.
In contrast, Cr(VI) and its compounds originate from industrial production, and have long been recognized as inhaled carcinogens of lung and liver cancer (Holmes et al., 2006; Park et al., 2004; Yang et al., 2013). Moreover, several epidemiologic studies presented that exposure to Cr(VI) in water could cause the development of oral cavity neoplasms
and small intestine neoplasms in rats and mice (Linos et al., 2011; Stout et al., 2009). In addition, Cr(VI) was identified as ‘likely to be a car-cinogen to humans’ following the 2005 USEPA Cancer Risk Assessment Guidelines and the cancer potency to humans was equal to 0.5 (mg/kg/ day)−1 (Stern, 2010). In 1965, Cr(VI) was classified as a human car-cinogen by the IARC (Goulart et al., 2005). However, our understanding of the potential role of Cr(VI) in prostate cancer is limited. An epide-miologic study of Spanish mainland towns showed that chronic ex-posure to Cr in topsoil was associated with the development of prostate cancer (Núñez et al., 2016). Additionally, high concentrations of Cr exposure induced cell apoptosis in nontumorigenic human prostate cells (El-Atta et al., 2014). In the current study, we demonstrated that Cr(VI) exposure promoted prostate cancer cell growth both in vivo and in vitro, confirming the pathogenic eﬀect of Cr(VI) as a carcinogenic risk factor.