Zinc oxide nanoparticles induce human multiple myeloma cell death via reactive oxygen species and Cyt-C/Apaf-1/Caspase-9/Caspase-3 signaling pathway in vitro

Abstract

Background: Human multiple myeloma (MM) is a malignant and incurable B cell tumor. Zinc oxide nanoparticles (ZnO NPs) have been widely used in biomedical fields including anti-bacterial and anti-tumor. However, the influence of ZnO NPs on MM cells is still unclear. The present study aimed to investigate the effect of ZnO NPs on MM cell (a human myeloma-derived RPMI8226 cell line) death in vitro and the underlying mechanism.

Methods: The morphology of ZnO NPs was characterized by transmission electron microscopy (TEM), and the inhibitory and apoptotic effect of ZnO NPs on human MM cells was monitored by a CCK-8 method and an Annexin V-FITC/PI assay. Meanwhile, the morphological change in the cells after exposure to ZnO NPs was observed by a light field microscope. Moreover, the effects of ZnO NPs on the ATP level, reactive oxygen species (ROS) generation, and apoptosis were separately explored by the DCFH-DA fluorescent probe, flow cytometry, and ATP bioluminescence assay. Moreover, the expression of cytochrome C (Cyt-C), Apaf-1, Caspase-9 and Caspase-3 at mRNA and protein levels was further determined by using quantitative PCR (Q-PCR) and western blotting. In the present study, the human peripheral blood mononuclear cells (PBMCs) were used as normal control samples for the relevant experiment.

Results: The results indicated that ZnO NPs could significantly inhibit human MM cell proliferation and cell death in a time- and dose-dependent manner in vitro, and this outcome can be confirmed by cell morphology and apoptosis assay. Meanwhile, the results also showed that ZnO NPs could effectively increase ROS production and decrease ATP levels in human MM cells. ZnO NPs could also significantly elevate the expression of Cyt-C, Apaf-1, Caspase-9 and Caspase-3 at mRNA and protein levels, leading to cell death. By contrast, ZnO NPs showed little cytotoxic influence on PBMCs.

Conclusion: ZnO NPs can significantly induce human MM cell death in a time- and dose-dependent manner in vitro, decrease the ATP production and enhance the ROS generation. ZnO NPs can also increase Cyt-C, Apaf-1, Caspase-9 and Caspase-3 expression at mRNA and protein levels in human MM cells, and initiate MM cell apoptosis, indicating that Cyt-C, Apaf-1, Caspase-9 and Caspase-3 play crucial roles in ZnO NPs-induced, mitochondria-mediated apoptosis in human MM cells. Overall, ZnO NPs may be a potential agent in treating human multiple myeloma in clinical practice.

Keywords: Apoptosis; Caspase signaling pathway; Multiple myeloma; Peripheral blood mononuclear cell; ZnO nanoparticle.