Titanium dioxide nanoparticles induce mitochondria-associated apoptosis in HepG2 cells

Abstract

Widespread applications of nanosized materials over the past decade have prompted investigations of desirable properties and potential hazards to humans and the environment. Titanium dioxide (TiO₂) nanoparticles are one of the most widely used nanoparticles. To investigate the effect of biological functions induced by TiO₂ nanoparticles (10 nm: TiO₂ NPs) on human liver cell lines, normal liver cell line L02 and hepatoma cell line HepG2 were co-cultured with exogenous TiO₂ NPs. Cell growth and proliferation, cell cycle, and the apoptosis rate were analyzed. The effects of TiO₂ NPs on the expression levels of apoptosis-associated protein caspase-3 and the membrane channel protein αENaC and caspase-3/7 activity were determined. Moreover, the influence of TiO₂ NPs on the expression levels of the mitochondria-related proteins SIRT3, VDAC1, and ACSS1, the mitochondrial membrane potential and the ADP/ATP ratio were also examined. Our results revealed that TiO₂ NPs inhibited the growth and proliferation of HepG2 cells, suppressed the S phase of cell cycling, and induced apoptosis of HepG2 cells. Following an increase in concentration, the inhibitory effect induced by TiO₂ NPs on proliferation and cell cycle was more evident, and the apoptosis rate increased in a significant concentration-dependent manner, whereas there was no significant effect on the growth, proliferation, apoptosis, and cell cycle of L02 cells. In addition, the results of western blot showed that in HepG2 cells, TiO₂ NPs upregulated the expressions of the apoptosis-related protein caspase-3 and the membrane channel protein αENaC in a concentration-dependent manner. However, in L02 cells, there was no significant difference in the expression levels of caspase-3 or αENaC. Furthermore, TiO₂ NPs induced depolarization of the mitochondrial membrane, upregulated the expression levels of the mitochondria-related proteins SIRT3 and VDAC1, and downregulated the expression level of the key respiratory chain protein ACSS1 in HepG2 cells. However, in L02 cells, the expressions of SIRT3, VDAC1, and ACSS1 exhibited no clear change. The apoptosis of HepG2 cells induced by TiO₂ NPs may be achieved by regulating intracellular osmotic pressure; moreover, upregulating the expression of the channel protein αENaC or the mitochondrial porin VDAC1 and depolarizing the mitochondrial membrane of HepG2 cells resulted in the loss of Cyt-c and ATP and further activated caspase-3. To further confirm the above results, a nude mouse xenograft model was employed. After a certain period of treatment with TiO₂ NPs, the nude mice were sacrificed, tumors were removed, and the expression of related proteins was detected. Immunohistochemistry and western blot results showed that the expressions of the proteins VDAC1 and SIRT3 were clearly upregulated in tissues treated to TiO₂ NPs, whereas the expression of ACSS1 was downregulated. The results were consistent with the above in vitro results. All the above results confirmed that TiO₂ NPs can act as a safe antitumor agent.

Fig. 1. Elemental composition of TiO₂ NPs. The elemental composition of TiO₂ NPs used in this study was determined by Elementar Analysensysteme GmbH (elemental analyzer) and an S4 Explorer X-ray fluorescence spectrometer. The material contained 96.59% TiO₂ NPs.

Fig. 2. Characterization and analysis of TiO₂ NPs. (A) BJH pore size distribution and plot of the adsorption isotherm of TiO₂ NPs. The BET surface area was 79.299 m² g⁻¹. The total pore volume for pores with a diameter of less than 343.88 nm at P/P₀ = 0.9944002 was 0.5062 cm³ g⁻¹. (B) The average pore diameter of TiO₂ NPs was 25.54 nm.

Fig. 3. Particle size distribution of TiO₂ NPs on dishes. Dishes (cut to a size of 1 × 1 cm) coated with TiO₂ NPs were placed under a JEOL JSM-7401 (JEOL Ltd., Tokyo, Japan) scanning electron microscope (SEM) to analyze the morphology of NPs. The SEM was operated at 100 and 10 kV. Thermogravimetric analysis was carried out with a Q50TGA thermogravimetric analyzer (Thermal Analysis, Inc., New Castle, DE, USA) from room temperature to 1173 K at a rate of 10 K min⁻¹ under an air flow of 30 mL min⁻¹. Individual anatase-like spherical aggregates of TiO₂ nanoparticles with a diameter of 10–30 nm were observed.

Fig. 4. SEM measurements of TiO₂ NP-coated dishes. (A) SEM image of TiO₂ NPs with diameters of about 10 nm at a magnification of 30 000×. (B) SEM image of TiO₂ NPs with diameters of about 10 nm at a magnification of 2 000 000×. (C) Aggregates of TiO₂ NPs on the PS surface observed by SEM. (D) Elements present in TiO₂ NPs on the PS surface determined from the power spectrum.

Fig. 5. TiO₂ NPs inhibited the growth of HepG2 cells. L02 and HepG2 cells were added to 6-well plates and co-cultured with different concentrations of TiO₂ NPs for 48 h for cell counting. (A) After co-culturing with TiO₂ NPs for 48 h, the number of L02 cells underwent no clear change. (B) When HepG2 cells were co-cultured with TiO₂ NPs for 48 h, the number of cells decreased. All data are presented as mean ± standard error of the mean. *p < 0.05, **p < 0.01.

Fig. 6. TiO₂ NPs inhibited the proliferation of HepG2 cells. L02 and HepG2 cells were added to 96-well plates and co-cultured with different concentrations of TiO₂ NPs. A CCK-8 assay kit was used to determine the effect of TiO₂ NPs on the proliferation of L02 and HepG2 cells at different time points (12, 24 and 48 h). (A) When L02 cells were co-cultured with TiO₂ NPs, the OD values decreased slightly at each time point, but there was no statistically significant difference. (B) When HepG2 cells were co-cultured with TiO₂ NPs for 12 h and 24 h, the OD values decreased significantly, especially at 48 h. All data are presented as the mean ± standard error of the mean. *p < 0.05, **p < 0.01.

Fig. 7. TiO₂ NPs arrested the cell cycle of HepG2 cells at the S phase. L02 and HepG2 cells were added to 6-well plates and co-cultured with different concentrations of TiO₂ NPs for 48 h, and flow cytometry was used to determine the influence of TiO₂ NPs on the cell cycle in L02 and HepG2 cells. (A) The cell cycle in L02 cells underwent no obvious change. (B) The proportion of HepG2 cells in the S phase significantly decreased, whereas that in the G2 phase increased; both exhibited concentration dependence. All data are presented as mean ± standard error of the mean. *p < 0.05, **p < 0.01.

Fig. 8. TiO₂ NPs induced apoptosis in HepG2 cells. L02 and HepG2 cells were added to 6-well plates and co-cultured with different concentrations of TiO₂ NPs for 48 h. Flow cytometry was used to determine the influence of TiO₂ NPs on the apoptosis of L02 and HepG2 cells. (A and B) When L02 cells were co-cultured with TiO₂ NPs for 48 h, the apoptosis rate underwent no obvious change. (C and D) When HepG2 cells were co-cultured with TiO₂ NPs, the apoptosis rate increased significantly after 48 h. All data are presented as the mean ± standard error of the mean. *p < 0.05, **p < 0.01.

Fig. 9. TiO₂ NPs increased the activity of caspase-3/7. L02 and HepG2 cells were co-cultured with different concentrations of TiO₂ NPs in 96-well plates for 24 h and 48 h and then, a caspase-3/7 detection kit was used to analyze the activity of caspase-3/7 and further elucidate the influence of TiO₂ NPs on the apoptosis of HepG2 cells. (A) There was no significant change in the activity of caspase-3/7 in L02 cells. (B) TiO₂ NPs induced an increase in the activity of caspase-3/7 in HepG2 cells, which was more obvious after 48 h. All data are presented as the mean ± standard error of the mean. *p < 0.05, **p < 0.01.

Fig. 10. TiO₂ NPs depolarized the membrane of HepG2 cells. L02 and HepG2 cells were co-cultured with different concentrations of TiO₂ NPs in 6-well plates for 48 h and then, a JC-10 detection kit was used to analyze changes in the mitochondrial membrane of the cells. (A) In L02 cells, there was no significant decrease in the ratio of red to green fluorescence. (B) In HepG2 cells, the ratio of red to green fluorescence decreased in a concentration-dependent manner. All data are presented as the mean ± standard error of the mean. *p < 0.05, **p < 0.01.

Fig. 11. TiO₂ NPs increased the ADP/ATP ratio in HepG2 cells. L02 and HepG2 cells were added to 96-well plates and co-cultured with different concentrations of TiO₂ NPs for 24 and 48 h. Then, an ADP/ATP assay kit was used to determine the influence on the application of ADP/ATP induced by TiO₂ NPs. (A) After co-culture for 24 or 48 h, TiO₂ NPs had no significant effect on the ADP/ATP ratio in L02 cells. (B) In HepG2 cells, the ADP/ATP ratio increased significantly after co-culture for 24 h and became more obvious at 48 h. All data are presented as the mean ± standard error of the mean. *p < 0.05, **p < 0.01.

Fig. 12. TiO₂ NPs induced HepG2 cell apoptosis in vitro through a mitochondrial pathway regulated by αENaC. The expression levels of the apoptosis-related proteins caspase-3, the mitochondrial loading proteins SIRT3, VDAC1, and ACSS1 and the membrane channel protein αENaC in L02 and HepG2 cells were determined by western blotting. (A) In L02 cells, there was no obvious change in the levels of caspase-3, αENaC, SIRT3, VDAC1 and ACSS1. (B) In HepG2 cells, the levels of caspase-3, αENaC, SIRT3, and VDAC1 increased in a dose-dependent manner after co-culture with different concentrations of TiO₂ NPs, whereas the level of ACSS1 decreased in a dose-dependent manner.

Fig. 13. TiO₂ NPs suppressed tumor growth in xenografts of nude mice. When tumors in mice reached a size of 4–10 mm, the experiment was initiated, and this was regarded as the 1st day. TiO₂ was formulated at a concentration of 0.5 mg mL⁻¹ in PBS. The nude mice were randomly divided into 2 groups and were injected with either TiO₂ (50 μL) or vehicle once every two days 4 times. We measured the volumes of the tumors once every two days before the injections until the 21st day. The tumor volumes decreased after treatment with TiO₂ NPs in comparison with that of the vehicle group.

Fig. 14. TiO₂ NPs induced HepG2 cell apoptosis in vivo through a mitochondrial pathway. By constructing a model of a transplanted hepatoma, the biological effects induced by TiO₂ NPs on HepG2 cells in vivo were examined. (A) The results of immunohistochemistry showed that in comparison with the results of the control group, the protein expression of VDAC1 and SIRT3 in liver tissues treated with TiO₂ NPs was clearly upregulated, whereas the expression of ACSS1 was downregulated. (B) The results of western blot suggested that TiO₂ NPs significantly upregulated the protein expression of VDAC1 and SIRT3 and downregulated the expression of ACSS1.

Fig. 15. The pathway by which TiO₂ NPs induce mitochondria-associated apoptosis in HepG2 cells. The morphology of TiO₂ NPs determines how they enter cells. For example, the monomer may enter cells via free diffusion and αENaC channels, whereas the polymer enters cells via pinocytosis. After that, TiO₂ can accumulate in mitochondria, which depolarizes the mitochondrial membrane and opens VDAC1 channels. Moreover, TiO₂ NPs can reduce the expression level of ACSS1, block the tricarboxylic acid cycle and give rise to an unbalanced ADP/ATP ratio, which results in the extensive accumulation of pro-apoptotic substances (such as Cyt c) in the mitochondria. Afterwards, pro-apoptotic substances can enter the cytoplasm via mitochondrial VDAC1 channels, activate caspase-3/7, cause DNA degradation and promote apoptosis.