Zinc oxide nanoparticles selectively induce apoptosis in human cancer cells through reactive oxygen species

Abstract

Background: Zinc oxide nanoparticles (ZnO NPs) have received much attention for their implications in cancer therapy. It has been reported that ZnO NPs induce selective killing of cancer cells. However, the underlying molecular mechanisms behind the anticancer response of ZnO NPs remain unclear.

Methods and results: We investigated the cytotoxicity of ZnO NPs against three types of cancer cells (human hepatocellular carcinoma HepG2, human lung adenocarcinoma A549, and human bronchial epithelial BEAS-2B) and two primary rat cells (astrocytes and hepatocytes). Results showed that ZnO NPs exert distinct effects on mammalian cell viability via killing of all three types of cancer cells while posing no impact on normal rat astrocytes and hepatocytes. The toxicity mechanisms of ZnO NPs were further investigated using human liver cancer HepG2 cells. Both the mRNA and protein levels of tumor suppressor gene p53 and apoptotic gene bax were upregulated while the antiapoptotic gene bcl-2 was downregulated in ZnO NP-treated HepG2 cells. ZnO NPs were also found to induce activity of caspase-3 enzyme, DNA fragmentation, reactive oxygen species generation, and oxidative stress in HepG2 cells.

Conclusion: Overall, our data demonstrated that ZnO NPs selectively induce apoptosis in cancer cells, which is likely to be mediated by reactive oxygen species via p53 pathway, through which most of the anticancer drugs trigger apoptosis. This study provides preliminary guidance for the development of liver cancer therapy using ZnO NPs.

Keywords: ROS; ZnO nanoparticles; apoptosis; cancer therapy; p53.

Figure 1

UV-Visible spectrum of zinc oxide nanoparticles.

Figure 2

X-ray diffraction pattern of zinc oxide nanoparticles.

Figure 3

Electron microscopy characterization of zinc oxide nanoparticles. (A) Field emission scanning electron microscope image, (B) field emission transmission electron microscopy image (inset with higher magnification), (C) frequency of size distribution, and (D) energy-dispersive X-ray spectroscopy spectrum.

Figure 4

Effect of zinc oxide nanoparticles on the viability of three types of cancer cells (HepG2, A549, and BEAS-2B) and two types of normal cells (rat astrocytes and hepatocytes). Cells were treated with zinc oxide nanoparticles at the concentrations of 0 μg/mL, 5 μg/mL, 10 μg/mL, and 15 μg/mL for 24 hours. At the end of exposure, cell viability was determined using MTT assay, as described in Materials and methods. Data represented are mean ± standard deviation of three identical experiments made in triplicate. Note: *Statistically significant difference as compared with the controls (P < 0.05 for each).

Figure 5

Quantitative real-time polymerase chain reaction analysis of mRNA levels of apoptotic genes in human lung cancer (HepG2) cells treated with zinc oxide nanoparticles (ZnO NPs) at the concentration of 15 μg/mL for 24 hours. Quantitative real-time polymerase chain reaction was performed by QuantiTect SYBR Green PCR kit using an ABI PRISM 7900HT sequence detection system. The β-actin was used as the internal control to normalize the data. ZnO NP-induced alterations in mRNA levels are expressed in relative quantity compared with those for the respective unexposed control cells. (A) p53, (B) bax, (C) bcl-2, and (D) caspase-3. Data represented are mean ± standard deviation of three identical experiments made in triplicate. Note: *Statistically significant difference as compared with the controls (P < 0.05 for each).

Figure 6

Zinc oxide nanoparticles (ZnO NPs) induced apoptosis in human lung cancer (HepG2) cells. (A) Western blot analysis of p53, bax, bcl-2, and bax proteins in HepG2 cells treated with 15 μg/mL ZnO NPs for 24 hours. At the end of exposure, cells were lysed in RIPA buffer and cell extract subjected to Western blots antibodies, as described in Materials and methods. Immunoblot images are the representative of three identical experiments. The β-actin blot is a loading control. (B) Protein levels were also analyzed by desitometric analysis using AlphaEase TM FC StandAlone V.4.0.0 software. Results are expressed as a fold change over the control group. Bar diagrams are from mean ± standard deviation of protein levels from three blots. (C) ZnO NPs induced the activity of Caspase-3 enzyme in HepG2 cells. Caspase-3 data represented are mean ± standard deviation of three identical experiments made in triplicate. (D) ZnO NPs induced the apoptotic DNA fragmentation in HepG2 cells treated with 15 μg/mL ZnO NPs for 24 hours. Note: *Statistically significant difference as compared to the controls (P < 0.05 for each). (P < 0.05 for each). On the other hand, ZnO NPs induced GSH depletion (Figure 8A) along with the lower activity of antioxidant enzymes GPx, GR, SOD, and CAT in HepG2 cells (Figure 8B–E) (P < 0.05 for each).

Figure 7

Zinc oxide nanoparticles (ZnO NPs) induced oxidant generation in human lung cancer (HepG2) cells treated with 15 μg/mL ZnO NPs for 24 hours. At the end of treatment, reactive oxygen species (ROS) and malondialdehyde (MDA) levels were determined, as described in Materials and methods. (A) ROS and (B) MDA. Data represented are mean ± standard deviation of three identical experiments made in triplicate. Note: *Statistically significant difference as compared to the controls (P < 0.05 for each).

Figure 8

Zinc oxide nanoparticles (ZnO NPs) diminished the antioxidant defense system of human lung cancer (HepG2) cells treated with 15 μg/mL ZnO NPs for 24 hours. At the end of treatment, glutathione (GSH) level and antioxidant enzyme activity were determined, as described in Materials and methods. (A) GSH, (B) GSH peroxidise (GPx), (C) GSH reductase (GR), (D) superoxide dismutase (SOD), and (E) catalase (CAT). Data represented are mean ± standard deviation of three identical experiments made in triplicate. Note: *Statistically significant difference as compared to the controls (P < 0.05 for each).