Promotional effects of aged ZnO nanoparticles on migration, invasion and metastasis of human breast cancer MCF-7 cells in vitro and in vivo.

BACKGROUND: The high success of ZnO nanoparticles (NPs), mainly associated with their unique physicochemical properties, biocompatibility, and cost-effective mass production, has led to an increasing application in our daily life, industry and biomedical fields. Although many studies have been carried out to detect potential toxicity of pristine ZnO NPs, most of them have been developed under acute and short-term exposure conditions. In terms of human risk, the evaluation of long-term exposures to sub-toxic doses of NPs, especially regarding environmental transformed (aged) ZnO NPs remains a challenge. Our previous findings have revealed that ZnO NPs underwent physicochemical transformations with aging and its genotoxicity/carcinogenicity change accordingly. METHODS: In the present work, we focused on to determine the cancer progression-related end-points under a scenario of four months exposure to non-cytotoxic aged ZnO NPs (1.5 μg/mL) in MCF-7 cells (a human breast cancer cell line with poor tumorigenicity in nude mice). We performed the in vitro assays regarding cancer cell progression such as plate cloning formation, soft agar colony formation, transwell migration and invasion. A mouse in situ xenograft model was constructed by intravenously injecting MCF-7 cells that had been chronically exposed to aged ZnO NPs for 4 months, to examine the effects of aged ZnO NPs on tumor metastasis in vivo. Hematoxylin-eosin (HE) staining and immunohistochemistry (IHC) were used to detect proliferation and the expression of Ki67 protein for evaluation of lung/liver metastases. RESULTS: The results showed that aged ZnO NPs significantly enhanced the migration and invasion ability of MCF-7 cells in vitro. Surprisingly, this cancer progression effect was also reflected by the fact that MCF-7 cells underwent specific hepatic metastasis rather than pulmonary metastasis in vivo. Mechanistically, this process was accompanied by up-regulation of Claudin-2 (CLDN2). The further in vitro experiments showed that CLDN2 knockdown significantly decreased the migration and invasion of MCF-7 cells. CONCLUSION: The study points out the potential cancer-promoting risks of exposure to ZnO NPs at low dose and under long-term exposure condition, and the importance of using real-life exposure scenarios to fully assess the safety of NPs.