Study On Modes Of Death In A549 Cells Induced By Silica Nanoparticles

In this study, human lung cancer cell line A549 was used as the cell model to detect modes of cell death after exposure to silica nanoparticles. Cell proliferation was detected by MTT assay. Morphological changes in A549 cells were observed under light microscopy after Haematoxylin and Eosin(HE) staining, cells apoptosis was detected by both AO/EB fluorescence staining and flow cytometry (FCM) with Annexin V-FITC/PI double staining; the expression of apoptosis related proteins were detected by immunocytochemistry. The autophagy was analyzed using fluorescent microscope and fluorospectrophotometer by monodansylcadaverin(MDC) staining. The changes of cell cycle were detected by FCM. The experimental results of the study were shown as followed:1.The cytotoxicity of A549 induced by silica nanoparticlesMTT assay was performed to detect the cytotoxicity after exposure to silica nanoparticles (12.5, 25, 50, 100, 200μg/mL respectively) for 24 h. The results showed that the survival rate was decreased with the increase of silica nanoparticles dosage. There was significant differences in silica nanoparticles exposure groups compared with counterpart in control group (P < 0.05). The results suggested that proliferation of A549 cells could be inhibited by silica nanoparticles in a dose-dependent manner.2.Morhpological changes in A549 cells induced by silica nanoparticleHE staining showed that after silica nanoparticles treatment with different concentrations, A549 cells exhibited morphological changes: cells in control group mostly were spindle shaped with uniform size. The nuclei were located in the center of the cell and nuclear/cytoplasm ratio was large. In the nanoparticle exposure groups, as exposure dose increased, cells with irregular shapes accounted increasingly, with larger nuclear/cytoplasm ratio, condensed cell size, increased cytoplasm and vacuolated nucleus, enhanced basophilic of chromatin as well as shrinkage, fragmentation or even collapse of nucleus, all of which were indicative of cell apoptosis.3.Apoptosis of A549 cells induced by silica nanoparticlesThe AO/EB fluorescence stain assay showed that after being treated with silica nanoparticles for 24h, A549 cells demonstrated decreased green fluorescence as exposure dose increased. The apoptotic cells were with rough membrane edges, and increased nucleus margination, fragmentation and collapse. Orange stain increased and even presented red necrotic cells.FCM with Annexin V-FITC/PI double staining was also performed to examine apoptosis. After exposure to silica nanoparticles (25, 50, 100 and 200μg/mL) for 24 h, apoptotic rate ascended with the increasing of silica nanoparticles except for 200μg/mL group. And differences were all significant compared with negative control groups (P < 0.05). Apoptotic rate reached the highest in 100μg/mL group. And apoptotic rate in 200μg/mL group was not significantly different compared with that in 100μg/mL group.The immunocytochemical method was performed to detected the expression of apoptosis related proteins after exposure to silica nanoparticles (25, 50, 100,and 200μg/mL) for 24 h. The results showed that the expression of bax and bcl-2 were enhanced with the increase of silica nanoparticles where bcl-2/bax ratio decreased gradually, and there were significant differences between exposure and negative control groups (P < 0.05). The expressions of cyt C, caspase-3 and caspase-9 increased as exposure dose increased in exposure groups and differences were significant compared with control group (P < 0.05).4.Autophagy changes of A549 cells induced by silica nanoparticlesResults of MDC staining showed that the autophagy in A549 cells was increased after exposure to silica nanoparticles (25, 50 and 100μg/mL) by 24 h whereas in 200μg/mL group, autophagosome declined, and fluorescence weakened.After the cells were dyed with MDC, they were detected and quantified for fluorescence intensity by fluorospectrophtometer. The results showed that fluorescence intensity in 25, 50 and 100μg/mL groups were enhanced and with significant difference compared with control group (P < 0.05). The fluorescence intensity was the highest in 50μg/mL group. No statistical significance was observed between 200μg/mL group and control group.5.Cell cycle changes of A549 induced by silica nanoparticlesA549 cells were stained with PI to analyze cell cycle by FCM. The results showed that cells treated with different doses of silica nanoparticles (25, 50, 100, 200μg/mL) were observed cell arrest in G2/M phase. And except for 200μg/mL group, cell percentage in G2/M phase increased as exposure dose increased and compared with control group, differences were significant (P < 0.05). As for 200μg/mL group, the percentage declined but was still higher than that in control group.In summary, silica nanoparticles could induce the cytotoxicity to A549 cells. It could change the cell morphology, induce cell apoptosis via mitchodrial pathway, as well as autophage level and cell cycle changes. As whether the changes were related with cell death of autophage or mitotic catastrophe, further investigations are required. This study was helpful for investigation of cellular toxicity of silica nanoparticles and related mechanism, and provide experimental basis for the mechanism of cell apoptosis along with as possible cell death pathways induced by silica nanoparticles.