From metal to malignancy: How ROS and inflammatory cytokines accelerate cadmium-induced prostate carcinogenesis

Cadmium (Cd) is a toxic metal ubiquitous in nature and an industrial pollutant. Exposure to elemental Cd or its salts lead to their high accumulation in tissues and clears very slowly, biological half-life; >30 years. Abnormally high levels of Cd are reported in human prostate tissues. The studies on rodents have shown that their exposure causes prostate cancer. The mechanism of carcinogenesis caused by Cd remains unclear. This study tested the hypothesis that continuous low-dose exposure to Cd induces chronic inflammation driven by high levels of Reactive Oxygen Species (ROS) which promote sustained up-regulation of proinflammatory cytokines and accelerating the transformation of healthy prostate epithelial cells to tumorigenic cells. We used two non-transformed prostate epithelial cell lines (RWPE-1 and NHPrE-1) and exposed to 10μM of Cadmium Chloride (CdCl 2 ) continuously up to one year. The CdCl 2 exposed cells and culture media were regularly analyzed for changes in oxidative stress, inflammatory cytokines, elevated carcinogenesis markers. Cd exposure significantly increased the cellular ROS levels beginning in the first week of exposure and remained elevated. Analysis of a repertoire of cytokines, pro-tumorigenic growth factors and several transcription factors using ELISA, qPCR, cytokine array and transcriptomic RNA-seq analysis showed a strong correlation with exposure time and an increase in the levels of key cytokines and transcription factors. We found IL-8 was the first cytokine to increase when exposed to CdCl 2 , followed by activation of NF-κB (p65-rel), VEGF-A, & B and other factors. Further, IL-1beta, IL-6, IFN-gamma, and TGF-beta and IL-8 receptors CXCR1, and CXCR-2 were also elevated with prolonged exposure to Cd. In addition, when compared to unexposed cells, we observed activation of Nuclear Factor-kB (NF-κB), and pro-tumorigenic growth factors involved in angiogenesis vascular endothelial growth factors VEGF-A and B); all at RNA and protein levels. The inflammatory cytokine array analysis also supported the occurrence of a “cytokine storm” in the CdCl 2 -exposed RWPE-1 and NHPrE-1 cells. Neutralizing oxidative stress with ROS scavenger [e.g. N-Acetyl cysteine (NAC)], significantly reduced the mRNA levels of cytokines and pro-inflammatory chemokines. RWPE-1 and NHPrE-1 cells exposed to CdCl 2 for a year were fully transformed into tumorigenic cells and formed subcutaneous tumors in less than 30 days in male NOD/SCID mice. In summary, IL-8 and NF-κB mediated signaling axis is critical for Cd-induced carcinogenesis and controlling this axis is a potential strategy to prevent metal-induced toxicity and carcinogenesis.