Zinc oxide nanoparticles induce apoptosis and autophagy in human ovarian cancer cells

Abstract

Background: Zinc oxide nanoparticles (ZnO NPs) are frequently used in industrial products such as paint, surface coating, and cosmetics, and recently, they have been explored in biologic and biomedical applications. Therefore, this study was undertaken to investigate the effect of ZnO NPs on cytotoxicity, apoptosis, and autophagy in human ovarian cancer cells (SKOV3).

Methods: ZnO NPs with a crystalline size of 20 nm were characterized with various analytical techniques, including ultraviolet-visible spectroscopy, X-ray diffraction, transmission electron microscopy, Fourier transform infrared spectroscopy, and atomic force microscopy. The cytotoxicity, apoptosis, and autophagy were examined using a series of cellular assays.

Results: Exposure of cells to ZnO NPs resulted in a dose-dependent loss of cell viability, and the characteristic apoptotic features such as rounding and loss of adherence, enhanced reactive oxygen species generation, and loss of mitochondrial membrane potential were observed in the ZnO NP-treated cells. Furthermore, the cells treated with ZnO NPs showed significant double-strand DNA breaks, which are gained evidences from significant number of γ-H₂AX and Rad51 expressed cells. ZnO NP-treated cells showed upregulation of p53 and LC3, indicating that ZnO NPs are able to upregulate apoptosis and autophagy. Finally, the Western blot analysis revealed upregulation of Bax, caspase-9, Rad51, γ-H₂AX, p53, and LC3 and downregulation of Bcl-2.

Conclusion: The study findings demonstrated that the ZnO NPs are able to induce significant cytotoxicity, apoptosis, and autophagy in human ovarian cells through reactive oxygen species generation and oxidative stress. Therefore, this study suggests that ZnO NPs are suitable and inherent anticancer agents due to their several favorable characteristic features including favorable band gap, electrostatic charge, surface chemistry, and potentiation of redox cycling cascades.

Keywords: DNA fragmentation; apoptosis; autophagy; human ovarian cancer cells SKOV3; mitochondrial membrane potential; zinc oxide nanoparticles.

Figure 1

Characterization of ZnO NPs by UV-visible spectroscopy, XRD, TEM, FTIR, and AFM. Notes: (A) UV-visible spectroscopy, (B) XRD spectrum, (C) TEM, (D) FITR, and (E) AFM. At least three independent experiments were performed for each sample and reproducible results were obtained. The results of a representative experiment are presented. Scale bar 200 nm. Abbreviations: AFM, atomic force microscopy; FTIR, Fourier transform infrared; TEM, transmission electron microscopy; UV, ultraviolet; XRD, X-ray diffraction; ZnO NPs, zinc oxide nanoparticles.

Figure 2

Effects of ZnO NPs on cell viability and cell morphology. Notes: SKOV3 cells were incubated with different concentrations of ZnO NPs for 12 and 24 h, and then the viability of SKOV3 cells was determined using WST-8 assay. (A) The results are expressed as the mean ± standard deviation of three independent experiments. A significant difference was observed between control and treated cells. The viability of treated cells was compared to that of the untreated cells using Student’s t-test (^*P<0.05). (B) Phase contrast microscopy data showing the morphologic appearance of SKOV3 cells after treatment with ZnO NPs for 12 h. Scale bars =200 μm. Abbreviation: ZnO NPs, zinc oxide nanoparticles.

Figure 3

Evaluation of ROS level in SKOV3 cells after ZnO NP treatment. Notes: (A) Intracellular ROS levels were measured with fluorescence imaging using the DCFH-DA probe in cells cultured in the presence of ZnO NPs (0, 5, 10, 20, and 30 μg/mL) for 12 h. Scale bars =200 μm. (B) The average intensity of fluorescence in SKOV3 cells. The results are expressed as the mean ± standard deviation of three independent experiments. There was a significant difference in the ROS generation of treated cells compared to that of untreated cells, as assessed using the Student’s t-test (^*P<0.05; ^**P<0.01). (C) LDH leakage levels were measured in cells cultured in the presence of ZnO NPs (0, 5, 10, 20, and 30 μg/mL) for 12 h. Abbreviations: DCFH-DA, dichlorodihydrofluorescein diacetate; LDH, lactate dehydrogenase; ROS, reactive oxygen species; ZnO NPs, zinc oxide nanoparticles.

Figure 4

Effects of ZnO NPs on mitochondrial membrane permeability and apoptosis in SKOV3 cells. Notes: SKOV3 cells were treated with ZnO NPs (0, 5, 10, 20, and 30 μg/mL) for 12 h. (A) Mitochondrial membrane potential (∆ψm) was evaluated using JC-1 in treated cells. Red fluorescence indicates JC-1 aggregates within the mitochondria in healthy cells, whereas green fluorescence indicates JC-1 monomers in the cytoplasm and loss of ∆ψm. Scale bars =100 μm. (a) Ratio of JC-1 monomers to JC-1 aggregated. (B) Apoptosis was assessed in a TUNEL assay; the nuclei were counterstained with DAPI. Representative images show apoptotic (fragmented) DNA (red staining) and the corresponding cell nuclei (blue staining). (b) The average intensity of TUNEL fluorescence in SKOV3. Scale bars =100 μm. ^*P<0.05; ^**P<0.01. Abbreviations: DAPI, 4′,6-diamidino-2-phenylindole; TUNEL, terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling; ZnO NPs, zinc oxide nanoparticles.

Figure 5

Nuclear DNA damage in SKOV3 cells after ZnO NP treatment using immunocytofluorescense with γ-H2AX and Rad51 antibody. Notes: (A) Nuclear γ-H2AX foci in cells in vitro exposed to ZnO NPs (0, 5, 10, 20, and 30 μg/mL) for 12 h. The results are expressed as the mean ± standard deviation of three separate experiments. (a) The average intensity of γ-H2AX fluorescence in SKOV3. Scale bars =100 μm. (B) Nuclear Rad51 foci in cells in vitro exposed to ZnO NPs (0, 5, 10, 20, and 30 μg/mL) for 12 h. The results are expressed as the mean ± standard deviation of three separate experiments. (b) The average intensity of Rad51 fluorescence in SKOV3. Scale bars =100 μm. ^*P<0.05; ^**P<0.01. Abbreviation: ZnO-NPs, zinc oxide nanoparticles.

Figure 6

ZnO NP exposure increases apoptosis and autophagy in cultured SKOV3 cells in 12 h. Notes: The cells were treated with ZnO NPs (0, 5, 10, 20, and 30 μg/mL) for 12 h and then processed for immunofluorescence analysis. (A) p53-stained SKOV3: (a) the average intensity of p53 fluorescence in SKOV3. Scale bars =100 μm. (B) LC3-stained SKOV3: (b) the average intensity of LC3 fluorescence in SKOV3. Scale bars =100 μm. ^*P<0.05; ^**P<0.01. Abbreviation: ZnO NPs, zinc oxide nanoparticles.

Figure 7

Assessment by Western blotting. Notes: The SKOV3 cells were treated with ZnO NPs (30 μg/mL) for 12 h and the expression analysis of Bax, Bcl-2, caspase-9, Rad51, γ-H2AX, LC3, and p53 was performed by Western blot analysis. Data are presented from three independent experiments. Abbreviation: ZnO NPs, zinc oxide nanoparticles.