Cationic dendrimers possess attractive nano-sized architectures that make them suitable as targeted drug/gene delivery systems. However, very little is known about their mechanisms of cell death in cellular systems. In the current study, the apoptotic and necrotic effects of starburst polyamidoamine(PAMAM) and polypropylenimine (DAB) dendrimers in cultured RAW 264.7 murine macrophage-like cells were investigated. Cationic dendrimer treatment produced a typically dose-dependent cytotoxic effect on macrophage cells. RAW 264.7 cells exposed to cationic dendrimers exhibited morphological features of apoptosis. Apoptotic ladders were observed in DNA extracted from RAW 264.7 cells treated with cationic dendrimers. Analysis from flow cytometry demonstrated an increase in hypodiploid DNA population (sub-G1) and a simultaneous decrease in diploid DNA content, indicating that DNA cleavage occurred after exposure of the cells to cationic dendrimers. Also, cells treated with DAB dendrimer induced a higher percentage of sub-G1 population than those treated with PAMAM dendrimer at the same dose. In addition, it was shown that pre-treatment of RAW 264.7 cells with the general caspase inhibitor zVAD-fmk prevented some degree of apoptosis induced by cationic dendrimers, suggesting that apoptosis in macrophage cells involves a caspase dependent pathway. Macrophage cells were also found to be sensitive to induction of apoptosis by dendrimers, whereas NIH/3T3 cells (mouse fibroblast) and BNL CL.2 (mouse liver) cells did not undergo apoptosis. These results could be helpful for optimizing the biocompatibility of dendrimers used for targeted drug/gene delivery.

Because the role of the viral B2 protein in the pathogenesis of nervous necrosis virus infection remains unknown, the aim of the present study was to determine the effects of B2 protein on hydrogen peroxide (H2O2)-mediated cell death via mitochondrial targeting. Using a B2 deletion mutant, the B2 mitochondrial targeting signal sequence (41RTFVISAHAA50) correlated with mitochondrial free radical production and cell death in fish cells, embryonic zebrafish, and human cancer cells. After treatment of grouper fin cells (GF-1) overexpressing B2 protein with the anti-oxidant drug, N-acetylcysteine (NAC), and overexpression of the antioxidant enzymes, zfCu/Zn superoxide dismutase (SOD) and zfCatalase, decreased H2O2 production and cell death were observed. To investigate the correlation between B2 cytotoxicity and H2O2 production in vivo, B2 was injected into zebrafish embryos. Cell damage, as assessed by the acridine orange assay, gradually increased over 24 h post-fertilization, and was accompanied by marked increases in H2O2 production and embryonic death. Increased oxidative stress, as evidenced by the up-regulation of Mn SOD, catalase, and Nrf2, was also observed during this period. Finally, B2-induced dynamin-related protein 1 (Drp1)-mediated mitochondrial fragmentation and cell death could be reversed by NAC and inhibitors of Drp1 and Mdivi in GF-1 cells. Taken together, betanodavirus B2 induces H2O2 production via targeting the mitochondria, where it inhibits complex II function. H2O2 activates Drp1, resulting in its association with the mitochondria, mitochondrial fission and cell death in vitro and in vivo.

Our results indicate that tyloxapol induces apoptosis in RAW 264.7 and NIH/3T3 cells. These data provide a novel insight into the cytotoxic action of tyloxapol at the molecular level.

No abstract

The molecular functions of betanodavirus non-structural protein B and its role in host cell survival remain unclear. In the present study, we examined the roles of specific nuclear targeting domains in B1 localization as well as the effect of B1 nuclear localization on the cell cycle and host cell survival. The B1 protein of the Red spotted grouper nervous necrosis virus (RGNNV) was detected in GF-1 grouper cells as early as 24 hours post-infection (hpi). Using an EYFP-B1 fusion construct, we observed nuclear localization of the B1 protein (up to 99%) in GF-1 cells at 48 hpi. The nuclear localization of B1 was mediated by two arginine-rich nuclear targeting domains (B domain: 46RRSRR51; C domain: 63RDKRPRR70) and domain C was more important than domain B in this process. B1 nuclear localization correlated with upregulation of p53 and p21(wef1/cip1); downregulation of Cyclin D1, CDK4 and Mdm2; and G1/S cell cycle arrest in GF-1 cells. In conclusion, nuclear targeting of the RGNNV B1 protein via two targeting domains causes cell cycle arrest by up-regulating p53/p21 and down-regulating Mdm2, thereby regulating host cell survival.