Zinc Oxide nanoparticles induce oxidative and proteotoxic stress in ovarian cancer cells and trigger apoptosis Independent of p53-mutation status

Abstract

Ovarian cancer continues to be the most lethal among gynecological malignancies and the major cause for cancer-associated mortality among women. Limitations of current ovarian cancer therapeutics is highlighted by the high frequency of drug-resistant recurrent tumors and the extremely poor 5-year survival rates. Zinc oxide nanoparticles (ZnO-NPs) have shown promise in various biomedical applications including utility as anti-cancer agents. Here, we describe the synthesis and characterization of physical properties of ZnO-NPs of increasing particle size (15 nm - 55 nm) and evaluate their benefits as an ovarian cancer therapeutic using established human ovarian cancer cell lines. Our results demonstrate that the ZnO-NPs induce acute oxidative and proteotoxic stress in ovarian cancer cells leading to their death via apoptosis. The cytotoxic effect of the ZnO-NPs was found to increase slightly with a decrease in nanoparticle size. While ZnO-NPs caused depletion of both wild-type and gain-of-function (GOF) mutant p53 protein in ovarian cancer cells, their ability to induce apoptosis was found to be independent of the p53-mutation status in these cells. Taken together, these results highlight the potential of ZnO-NPs to serve as an anti-cancer therapeutic agent for treating ovarian cancers independent of the p53 mutants of the cancer cells.

1. Introduction

With an estimated 22,440 new cases and 14,080 deaths in the United States alone in 2017, ovarian cancer continues to be the most lethal among all gynecologic malignancies and the fifth leading cause for cancer-associated deaths among women in the United States [1]. The absence of reliable early diagnostic markers and the lack of effective therapeutic options have contributed to the disappointing five-year survival rate for ovarian cancer patients (46%) [1–3]. Current treatment modality involves surgical cytoreduction followed by treatment with a combination of platinum and taxane based therapies [4]. Although effective in about 60–80% of patients during early stages, a large majority of patients exhibit tumor recurrence [4]. Compounding this situation further, almost all relapsed tumors exhibit resistance to the FDA-approved platinum-based chemotherapeutic drugs such as cisplatin and carboplatin and patients with resistant tumors have a median survival time of 6 months, with only 27% of patients living longer than 12 months. Thus, there is an urgent need to overcome the limitations in treatment options available for ovarian cancer, especially development of drug-resistance in recurring tumors.

Among factors that have been shown to contribute towards resistance to chemotherapy in ovarian cancer cells are presence of gain-of-function (GOF) mutations in the tumor protein 53 (TP53 or p53) gene. Mutations in the p53 gene are extremely frequent in high-grade serous ovarian carcinomas (HGSOC), the most malignant and common ovarian cancer subtype [5, 6]. While some of these mutations contribute to cancer progression as a result of loss of wild-type (WT) p53 activity, others result in the gain of an oncogenic function. These GOF oncogenic p53 mutant proteins have been shown to exhibit higher protein stability leading to their accumulation in cancer cells. These oncogenic proteins form stable protein aggregates, activate alternative gene expression programs, and contribute to carcinogenesis as well as development of drug resistance [6, 7]. While strategies capable of targeting the different p53 GOF mutant proteins have shown promise in selectively killing ovarian cancer cells, these strategies still need to be translated to a clinical setting and validated [8, 9]. Another approach to counter drug resistance observed in ovarian cancers is to develop therapeutics that induce cancer cell death independent of the p53 mutation status of tumor cells and thus can overcome the drug-resistance induced by the presence of specific p53 GOF mutants. Nanoparticles (particles in the size of 1–100 nm) are currently being used for many biomedical applications such as targeted drug delivery systems, bio-imaging, wound healing and bone regeneration [10–15]. The size of the nanomaterials has a direct influence on its properties and biological activity [16–18]. The reduction of materials to nanoscale often results in altered structural, morphological, physical (electric and magnetic), and chemical properties [16]. These alterations enable the nanoparticles to interact with different biomolecules in unique ways effecting specific responses [16]. Owing to their size and unique surface properties, nanoparticles offer several advantages that have helped propel them as an emerging class of novel anti-cancer therapeutics. Among them, metal oxide nanoparticles such as Zinc Oxide nanoparticle (ZnO-NP) have been shown to display several unique properties that are widely adapted as key components in various biomedical and cosmetic applications such as in sunscreens, foot care, and ointments [19–22]. Several studies have also shown ZnO-NPs exhibit antibacterial activities [23–26]. Nanoparticles like hydroxyapatite and titanium dioxide have found extensive use in drug targeting due to their biocompatibility [27, 28]. While the utility of metal oxide nanoparticles as a carrier in drug delivery systems has been widely explored, their potential to effect cancer cell death has emerged more recently.

Here, we describe the synthesis of ZnO-NPs of different sizes, their biophysical characterization and evaluation of their anti-cancer effects using human ovarian cancer cells carrying different p53 mutations. Our data show that ZnO-NP induce apoptosis in ovarian cancer cells in a p53-mutation independent manner, suggesting their applicability a potential anti-cancer agent in treating drug-resistant ovarian cancers.

2. Materials and Methods

2.1. Cell lines and cell culture:

SKOV3 and 3T3-L1 cells were purchased from ATCC. TYKNu cells were a kind gift from Dr. Hilary Kenny, Univ. of Chicago (Chicago, IL). ALST, OVCAR3, and OVCA420 cells were a kind gift from Dr. KK Wong (MD Anderson Cancer Center, Houston, TX). All ovarian cancer cells were cultured in RPMI-1640 media containing L-glutamine supplemented with FBS (10%), MEM vitamins (1%) and non-essential amino acids (1%). The p53 mutations in these cell lines were confirmed as described previously [29]. Standard cell culture techniques were followed, and all cells were verified to be mycoplasma free and maintained in a 37°C humidified atmosphere with 5% CO₂. All treatments mentioned in this study were also carried out at these conditions.

2.2. Reagents and Antibodies:

Details of antibodies used in this study and their dilutions used for Western blot analyses are as follows: p53 (Santa Cruz (FL-393); 1:1000), ß-actin (Sigma (A2228); 1:5000), Ubiquitin (Santacruz (P4D1, sc-8014); 1:1000), HSP105 (Santacruz (B-7, sc74550); 1:1000), HSP90α/β (Santa Cruz (F-8, sc-13119); 1:1000), HSP70 (Santa Cruz (W-27, sc-24); 1:1000), p-HSP27x (Santa Cruz (B-3, sc-166693); 1:1000), HSP10 (Santa Cruz (D-8, sc-376313); 1:1000), cleaved caspase-3 (D175) (Cell Signaling (D3E9, mAb#9795); 1:1000), BCL2 (Santa Cruz (sc-783); 1:1000).

2.3. Synthesis of Zinc Oxide nanoparticles:

Zinc Nitrate (0.5 M) is added drop-by-drop to a sodium hydroxide solution (1 M) and stirred continuously for 15 min. The white precipitate formed is centrifuged and washed with distilled H₂O repeatedly. The washed white powder (Zn(OH)₂) is dried in a hot air oven at 60°C. During drying Zn(OH)₂ is completely converted to ZnO. The dried ZnO powder is then annealed at different temperatures (300°C, 500°C, 700°C, and 900°C) for 3 hours yielding ZnO nanoparticles S1, S2, S3 and S4 respectively.

2.4. Cell viability analysis:

10⁴ cells were plated in each well of a 96-well plate and treated with different concentrations of ZnO-NPs as indicated in the figure legends. Cell viability was measured 24 hours post-treatment using WST1 reagent (Roche) as per manufacturer’s instructions. The values reported are mean ± SE of multiple experiments.

2.5. Western blot analysis:

Western blot analyses were performed as described elsewhere [30–32]. Briefly, cells were lysed in lysis buffer (150 mM NaCl, 50 mM Tris (pH 7.5), 0.5% NP-40, protease (Sigma) containing phosphatase inhibitors (Gene Depot)). Protein concentrations were determined using the Bio-Rad protein assay reagent. Equal amounts of total protein were loaded in each lane of either 12% or a 4–20% gradient SDS-PAGE gels. Proteins were resolved by electrophoresis at 150V for 60 minutes and transferred to polyvinylidene difluoride (PVDF) membranes. Western blot analyses were performed using the antibodies as indicated in the manuscript.

2.6. Measurement of intracellular GSH levels:

RT-AM (RealThiol AM ester) was a kind gift from Dr. Jin Wang (Baylor College of Medicine, Houston, TX). Intracellular GSH levels were measured as described previously [33]. In brief, ALST cells were grown as described earlier on glass bottom cell culture dishes compatible with confocal microscopy. Cells were treated with 5 μg/ml ZnO-NPs (S1, S2, S3 and S4) for 4 hours. Cells were treated with RT-AM (1 μM with 1% DMSO in RPMI-1640) for 10–15 min before imaging. Confocal images were acquired with 405 nm laser/418–495 nm filter and 488 nm laser/499–615 nm filter. All the microscope settings were kept consistent between each experiment.

2.7. Immunofluorescence study:

Ovarian cancer cells were cultured on coverslips. For detecting mitochondria, the cells were treated with mitotracker dye for 30 min. The cells were fixed with 4% paraformaldehyde and permeabilized with Triton X-100. Hoechst dye was used to stain the nuclei. Fluorescence images were captured using a GE Healthcare Delta Vision live high-resolution deconvolution microscope

2.8. Equipment used for physical characterization of the ZnO-NPs:

Powder X-ray Diffraction (XRD, Seifert, JSO-DE BYEFLEX 2002, Germany) was utilized to identify the crystalline phase composition. The morphology and crystal structure of the product were observed using JEOL 2000Fx-II Transmission Electron Microscopy (TEM) operated at 200kv High Resolution analtical TEM with a W-source and a point–point resolution of 2 A° The functional groups present in the hydroxyapatite were analyzed by FTIR (FTIR, Perkin Elmer Spectrum One). UV measurement is done with Perkin Elmer LAMBDA 950 UV-VIS-NIR Spectrophotometer.

3.0. Results and Discussion

3.1. Synthesis and characterization of ZnO-NPs by X-ray diffraction (XRD)

To generate ZnO nanoparticles, Zn(NO₃)₂ was added drop-wise to 1M NaOH solution and stirred continuously for 15 min (Fig. 1A). The white Zn(OH)₂ precipitate was washed repeatedly in distilled water and dried in a hot air oven at 60°C to yield ZnO powder (Fig. 1A). Since particle size has been shown to influence the properties of nanoparticles, the dried ZnO powder was annealed at different temperatures (300°C, 500°C, 700°C, and 900°C) to generate ZnO nanoparticles (ZnO-NPs) of different sizes (Fig. 1A). The purity of the synthesized ZnO nanoparticles was confirmed by UV spectroscopy. The UV spectrum showed a distinct peak at 360 nm, the characteristic absorption wavelength of ZnO-NPs (Fig. 1B). The absorption of photon energy by ZnO-NPs increases with increase in annealing temperature. A red shift is observed due to annealing because of the decrease in band gap with increase in particle size. X-ray diffraction (XRD) using these ZnO-NPs annealed at different temperatures revealed peaks at 2θ = 31.68° 34.3 ° 36.15° 47.4 ° 56.54° 6 .76° 66.30° 67.94°, 69.05°, and 72.50° representing the diffraction planes (1 0 0), (0 0 2), (1 0 1), (1 0 2), (1 1 0), (1 0 3), (2 0 0), (1 1 2), (2 0 1) and (004) respectively (Fig. 1C). The formation of ZnO-NPs in wurtzite structure was confirmed from XRD analysis (Fig. 1C). The nanoparticles were found to be polycrystalline in nature. When the nanoparticles are annealed at different temperatures there is an increase in the intensity and sharpening of XRD peaks. This confirms the increase in crystallinity of nanoparticles with annealing. The grain size also increased with the increase in annealing temperature. The merging of bonds at the grain boundaries leads to grain growth during annealing. The average grain size (d) of the ZnO-NPs was estimated b Scherrer’s formula, d = K λ / β½Cos θ; whereK= 0.9 is the shape factor λ is the X-ra wavelength of Cu Kα radiation (1.54 Å) θ is the Bragg diffraction angle and β is the full width at half maximum of the respective diffraction peak (Table 1). The decrease in FWHM leads to an increase in grain size associated with the increase in annealing temperature. Recrystallization is also enhanced by annealing process.

Figure 1.

X-ray diffraction (XRD) pattern of ZnO-NPs (S1–S4) annealed at various temperatures. (A) Cartoon illustrating the method used for preparation of ZnO-nanoparticles of different size. (B) UV spectra for the ZnO-NPs (S1–S4) showing the purity of the synthesized nanoparticle. (C) The XRD patterns demonstrate crystalline nature of ZnO. The peaks in order of increasing 2θ are assigned to the (100),(002),(101),(10),(110),(103),(200),(112), (201), and (004) reflection lines of hexagonal ZnO particles with wurtzite structure. No characteristic peaks of any impurities were detected.

Table 1:

Grain size of the ZnO-NPs annealed at different temperatures calculated using XRD

ZnO nanoparticles	Annealing Temperature (°C)	d(Å)	Grain size from XRD (nm)
S1	300	2.47731	15
S2	500	2.48676	28
S3	700	2.46411	39
S4	900	2.46325	55

3.2. Analysis of ZnO-NPs by Fourier-transform infrared spectroscopy (FITR)

Fourier-transform infrared (FTIR) spectroscopy analysis on the ZnO-NPs annealed at different temperatures (S1, S2, S3, and S4) revealed that annealing at higher temperatures induced a change in the structural and chemical nature of the nanoparticles (Fig. 2). FTIR shows the chemical bonds present in the nanoparticles. The presence of band at 444 – 555 cm⁻¹ confirms the wurtzite structure of ZnO-NPs. The vibrations due to the asymmetrical and symmetrical stretching of zinc carboxylate groups are observed at 1602 and 1386 cm⁻¹. The bending of the OH molecule, C-OH in plane bending and out plane bending is seen at 1643, 1386 and 1127 cm⁻¹ respectively. The band due to absorbed H₂O is at 3500 cm⁻¹. There is a decrease in the intensity of this peak due to annealing. There is also a change in distribution of peaks. A shift towards a higher wave number with an increase in annealing temperature is also observed. At 900°C the peak disappears. With the increase in annealing temperature the intensity of the peak at 1386 cm⁻¹ decreases. This is because at high annealing temperature the bonding between hydrogen and oxygen does not take place.

Figure 2.

FTIR image of ZnO-NPs annealed at different temperatures.

3.3. Transmission electron microscopy (TEM) analysis of ZnO-NPs annealed at different temperatures

Transmission electron microscopy (TEM) analyses on ZnO-NPs annealed at different temperatures revealed further that they are spherical in shape and mono dispersive in nature (Fig. 3A). Grain growth is observed with increase in the annealing temperature (Fig. 3). Annealing leads to increase in the crystallinity of the material and hence increases the number of crystallites. The mean size of the nanoparticles increases with the increase in annealing temperature (Fig. 3). The ZnO-NPs are very sensitive to the annealing process and aggregate during annealing due to the high surface energy. At higher annealing temperatures the nanoparticles were found to merge and form a neck between two particles. Based on TEM images, the average particle size of the ZnO nanoparticles annealed at 300°C, 500°C, 700°C, and 900°C were calculated as as 80 ± 10 nm, 160 ± 12 nm, 250 ± 15 nm and 400 ± 10 nm, respectively (Table 2).

Figure 3.

Transmission Electron Microscopy (TEM) of ZnO-NPs S1 (A), S2 (B), S3 (C), and S4 (D)

Table 2:

The size of the ZnO-NPs calculated using TEM.

ZnO nanoparticles	Annealing Temperature (°C)	Average particle size (nm)
S1	300	80 ± 10
S2	500	160 ± 12
S3	700	250 ± 15
S4	900	400 ± 10

3.4. ZnO-NPs causes cell death ovarian cancer cells expressing WT-p53

To determine the effect of ZnO-NPs of different sizes on human ovarian cancer cells, we treated ALST cells expressing WT-p53 with 5 μg/ml of S1, S2, S3, and S4, respectively, for 24 hours. Upon 24-hour treatment all the ZnO-NPs (S1–S4) induced dramatic cell death in ALST cells (Fig. 4A). To determine whether ZnO-NPs exhibited cytotoxicity in ALST cells at lower concentrations, we performed cell viability assays using increasing concentrations of S1, S2, S3, and S4 in 96-well plates. ALST cells were found to be extremely sensitive to all four ZnO-NPs even at lower concentrations (Fig. 4B). 70–80% cytotoxicity was observed using as low as 250 ng/mL ZnO-NPs for 24 hours. At lower concentrations (250 and 500 ng/mL), S4, the larger sized ZnO-NP, exhibited a slightly less cytotoxic effect compared to S1, S2 and S3, suggesting an inverse correlation between the nanoparticle size and its effectiveness to kill ovarian cancer cells (Fig. 4B). This difference in the cytotoxic effects between ZnO-NPs of different sizes was not observed at 1000 ng/ml (Fig. 4B). This may be because at higher concentrations the nanoparticles tend to aggregate resulting in an increase in their effective size. This change in effective particle size could potentially influence its properties including cytotoxicity. To further confirm the potential correlation between ZnO-NPs size and anti-cancer cytotoxicity, and to determine the how rapidly they induce cell death in the p53-WT expressing human ovarian cancer cells, we treated ALST cell with 5 μg/ml and 10 μg/ml S1, S2, S3, and S4, respectively, for a shorter duration (6 hours). Significant ALST cell death was observed by 6 hours of ZnO NP treatment (Fig. 4C). Consistent with our previous observation the cytotoxicity of the ZnONPs showed a slight decrease with increase in particle size. However, at higher nanoparticle concentration (10 μg/ml), the size-dependent cytotoxic effects of ZnO-NPs were less pronounced (Fig. 4C). In contrast to its effect on ALST cells, the ZnO-NPs exhibited much lower cytotoxic effects at the same doses on non-transformed 3T3-L1 fibroblast cells (Fig. 4D).

Figure 4.

Effect of ZnO-NPs on the viability of ALST (p53-WT) ovarian cancer cells. (A) ZnONPs (5 μg/ml, 24 hours) causes severe cell death in ALST cells. (B) Effect of low concentration of ZnO-NPs treatment (24 hours) on the viability of ALST cells. (C) Effect of short-term (6 hour) treatment of ZnO-NPs (5μg/ml and 10 μg/ml) on ALST cells. (D) Effect of ZnO-NPs treatment (24 hours) on the viability of 3T3-L1 fibroblast cells.

3.5. ZnO-NPs exert cytotoxic effects in ovarian cancer cells containing p53 GOF mutations

To determine the effects of ZnO-NPs on human ovarian cancer cells that harbor specific p53 GOF mutations we used established human ovarian cancer cells homozygous for the three most frequent p53 GOF mutations; R175H (TYK-Nu cells), R248Q (OVCAR3 cells), R273H (OVCA420 cells) (Fig. 5A). Similar to ALST cells (p53-WT), ZnO-NPs of different sizes (S1–S4) exhibited dramatic cytotoxic effects in TYK-Nu cells (p53-R175H) when treated for 24 hours (Fig. 5B). Around 70% reduction in cell viability was observed by 24-hours of ZnO-NP treatment (250 ng/ml) (Fig. 5B). Unlike in p53-WT expressing ALST cells, no statistically significant difference was observed in the cytotoxicity induced by ZnO-NPs of different sizes when treated for 24 hours at low concentrations of S1, S2, S3 and S4 (Fig. 5B). However, similar to ALST cells, short-term treatment (6 hours) using a higher concentration of ZnO-NPs (5 μg/ml) resulted in a nanoparticle-size dependent cytotoxicity (Fig. 5C). Compared to S1 and S2, larger sized ZnO nanoparticles (S3 and S4) exhibited significantly decreased cytotoxicity (Fig. 5C). Similar results were observed using OVCAR3 (p53-R248Q) cells (Fig. 5D and 5F). However, compared to ALST (p53-WT), TYK-Nu (p53-R175H), and OVCAR3 (p53-R248Q) cells, OVCA420 (p53-R273H) cells were more resistant to lower concentrations of ZnO-NPs, with only 30–50 percent cytotoxicity observed upon 24-hour treatment using 250 ng/ml ZnO-NPs (Fig. 5E). Increasing the concentration to 500 ng/ml, resulted in around 80% cytotoxicity in S1 and S2 treated OVCA420 cells while S3 and S4 were found to exert reduced cytotoxic effects on these cells (around 30–50 percent) (Fig. 5E). The cytotoxicity in S3-treated (24 hours) OVCA420 cells increased to 75% at 1000 ng/ml and that of the larger sized S4 nanoparticles increased to around 50% (Fig. 5E). Further, similar to ALST, TYK-Nu and OVCAR3 cells, short-term treatment (6 hours) using a higher concentration of ZnO-NPs (5 μg/ml) resulted in a nanoparticle-size dependent cytotoxicity in the OVCA420 cells (Fig. 5F). Taken together, these results suggest that the OVCA420 cells are slightly more resistant to ZnO-NPs compared to ALST, TYK-Nu and OVCAR3 cells and further show that smaller sized nanoparticles exert greater cytotoxic effects than larger nanoparticles in ovarian cancer cells.

Figure 5.

Effect of ZnO-NPs on ovarian cancer cells expressing distinct p53-GOF mutant proteins. (A) List of ovarian cancer cell lines used and the specific p53 mutations in these cells. (B) Effect of ZnO-NPs treatment (24 hours) on the viability of TYK-Nu cells (p53-R175H). (C) Effect of short-term (6 hour) treatment of ZnO-NPs (5μg/ml and 10 μg/ml) on TYK-Nu cells. (D) Effect of ZnO-NPs treatment (24 hours) on the viability of OVCAR3 cells (p53-R248Q). (E) Effect of ZnO-NPs treatment (24 hours) on the viability of OVCA420 cells (p53-R273H). (F) Effect of short-term (6 hour) treatment of ZnO-NPs (5μg/ml) on OVCAR3 and OVCA420 cells.

3.6. ZnO-NP mediated cytotoxicity in ovarian cancer cells is independent of the p53-status

While our data using human ovarian cancer cells expressing p53-WT and the different GOF mutants suggested that the cytotoxicity of ZnO-NPs on these cells is independent of the p53-mutation status, we wanted to establish conclusively whether p53-status had any role in mediating the cytotoxic effects of these ZnO-NPs. It had been previously reported that ZnO causes depletion of p53-R175H GOF mutant protein [34]. Consistent with this observation, treatment of TYK-Nu cells with ZnO-NPs (5 μg/ml; 4 hours) resulted in loss of p53-R175H protein (Fig. 6A). The effect on p53-R175H protein levels was evident within 2 hours of treatment with 5 μg/ml of these nanoparticles (Fig. 6B). Even at a much lower concentration (500 ng/ml) the ZnO-NPs were found to cause a decrease in p53-R175H levels (Fig. 6C). In contrast to its effect on p53-R175H mutant, no effect on p53-WT levels was observed upon treatment of ZnO-NPs at lower concentrations (500 ng/ml; 6 hours) or at higher concentrations for a short duration (5 μg/ml; 2 hours) (Fig. 6D and 6E). However, longer treatment (4 hours) with 5 μg/ml ZnO-NPs caused a dramatic decrease in p53-WT levels (Fig. 6F). ZnO-NPs (5 μg/ml, 4 hours) also caused a decrease in the level of p53-R248Q GOF mutant in OVCAR3 cells (Fig. 6G). By contrast, the levels of p53-R273H protein in OVCA420 cells did not change upon ZnO-NP treatment (Fig. 6G). Although depletion of p53 GOF mutants in cancer cells have been shown to induce cancer cell death, the effect of ZnO-NPs on p53-WT suggested that the major mechanism contributing to cytotoxicity in these cells is likely independent of p53.

Figure 6.

Effect of ZnO-NPs on ovarian cancer cells is independent of p53 mutation status. ZnO-NPs causes a decrease in p53-R175H levels in TYK-Nu cells when treated at (A) 5 μg/ml; 4 hours, (B) 5 μg/ml; 2 hours or (C) 500 ng/ml; 6 hours. ZnO-NPs does not cause a decrease in p53-WT levels in ALST cells treated at (D) 500 ng/ml; 6 hours or (E) 5 μg/ml; 2 hours. (F) 5 μg/ml ZnO-NP treatment for 4 hours causes a dramatic decrease in p53-WT levels in ALST cells. (G) 5 μg/ml ZnO-NP treatment for 4 hours causes a decrease in p53-R248Q levels in OVACR3 cells but does not affect the levels of p53-R273H in OVCA420 cells. (H) Effect of ZnO-NPs treatment (24 hours) on the viability of SKOV3 cells (p53-null). (I) Effect of short-term (6 hour) treatment of ZnO-NPs (5μg/ml) on SKOV3 cells (p53-null).

To further confirm this, we treated p53-null ovarian cancer cells SKOV3 with ZnO-NPs. Treatment of SKOV3 cells with ZnO-NPs of different grain size (S1–S4) at lower concentrations (250–1000 ng/ml) for 24 hours resulted in about 60% cytotoxicity (Fig. 6H). The nanoparticle size appeared to have no impact on the cytotoxic effects at these conditions (Fig. 6H). However, similar to previous results using ovarian cancer cells expressing either WT or GOF mutant p53, 5 μg/ml ZnO-NPs treatment for 6 hours resulted in severe decrease in cell viability in the p53-null SKOV3 cells (Fig. 6I). The nanoparticle S1 was found to have the most cytotoxic effect followed by S2, S3 and S4 respectively, again confirming our previous observation that there is an inverse correlation between ZnO-NP size and its cytotoxic effects on ovarian cancer cells (Fig. 6I). These results taken together reveal that while smaller ZnO-NPs have a higher anti-cancer cytotoxic potential, the effect is significant only in a specific concentration range and treatment windows which need to be determined empirically for the different cancer cells. Also, the different ovarian cancer cells exhibit different sensitivity towards ZnO-NPs, which is most likely mediated by a mechanism that is independent of the p53-mutation status of the cancer cells.

3.7. ZnO-NPs induces oxidative and proteotoxic stress in ovarian cancer cells

Since ZnO has been previously shown to cause oxidative stress in cells [17, 18], we determined the effect of the ZnO-NPs S1, S2, S3 and S4 on the oxidative environment of ovarian cancer cells. To determine the effect of ZnO-NPs on the cellular redox environment, we utilized the RealThiol (RT) reagent that enables precise real-time measurement of free glutathione levels in living cells [33]. Decreased glutathione level in cells indicates higher oxidative stress in cells. We observed that ZnO-NP treatment induced a dramatic decrease in free glutathione levels in the ALST cells, suggesting induction of oxidative stress upon ZnO-NP treatment (Fig. 7A). We also detected a steep increase in the accumulation of ubiquitinated proteins in ALST cells upon exposure to ZnO-NPs (5 μg/ml, 4 hours). Accumulation of ubiquitinated proteins was also observed upon shorter exposure to ZnO-NPs (2 hours) and upon treatment with lower concentration of ZnO-NPs (500 ng/ml, 6 hours) (Fig. 7C and 7D). Accumulation of ubiquitinated proteins was also observed in ovarian cancer cells expressing p53-R273H (OVCA420), p53-R175H (TYK-Nu) and p53-R248Q (OVCAR3) GOF mutants indicating that proteotoxic stress is a consistent response conserved across the different cell lines upon exposure to ZnO-NPs (Figs. 7E, 7F and 7G). To determine whether ZnO-NPs caused any changes to mitochondrial distribution in cells, we used a mitochondria-labeling dye mitotracker to probe the effect of ZnONPs on the mitochondria in ALST cells. ZnO-NP treatment caused a decrease in the number of mitochondria in these cells (Fig. 7H). The mitochondria were also found to exist as distinct speckles upon ZnO-NP treatment (Fig. 7H).

Figure 7.

ZnO-NPs induce severe oxidative and proteotoxic stress in ovarian cancer cells. (A) ZnO-NPs (5 μg/ml, 4 hours) cause a dramatic decrease in intracellular glutathione levels in ALST cells. ZnO-NPs cause increased accumulation of ubiquitinated proteins in ALST cells when treated at (B) 5 μg/ml; 4 hours, (C) 5 μg/ml; 2 hours or (D) 500 ng/ml; 6 hours. ZnO-NPs (5 μg/ml, 4 hours) cause increased accumulation of ubiquitinated proteins of OVCA420 (E), TYK-Nu (F), and OVCAR3 (G) cells. (H) Effect of ZnO-NPs (5 μg/ml, 4 hours) on mitochondria in ALST cells. DAPI (blue) and mitochondria (red).

3.8. ZnO-NPs causes changes in heat shock protein levels and induces apoptosis in ovarian cancer cells

Cellular stress is often accompanied with changes in the levels of heat shock proteins. Consistently, we observed that the ZnO-NPs caused changes in the levels of several key heat shock proteins such as HSP105, HSP90, p-HSP27 and HSP10 (Figs. 8A,8B, 8C, and 8D). The changes in levels of specific heat-shock proteins were found to be dependent on the concentration of ZnO-NPs, duration of treatment and the size of nanoparticles, suggesting that different heat shock proteins are induced in response to varying intensity of cellular stress (Figs. 8A, 8B, and 8C). Interestingly, no change was observed in the levels of HSP70 even upon 5 μg/ml ZnO-NP treatment for 4 hours (Fig. 8B).

Figure 8.

ZnO-NPs induce apoptosis in ovarian cancer cells. ZnO-NPs cause changes in the levels of heat-shock proteins in ALST cells treated at (A) 5 μg/ml; 4 hours, (B) 5 μg/ml; 2 hours or (C) 500 ng/ml; 6 hours. (D) ZnO-NPs (5 μg/ml, 4 hours) alter the levels of heat-shock proteins in OVCA420 cells. (E) ZnO-NP (S1) treatment (5 μg/ml, 6 hours) induces morphological changes in ALST cells suggesting apoptotic cell death. (F) ZnO-NP treatment (5 μg/ml, 4 hours) cause activation of caspase-3 as shown by accumulation of cleaved caspase-3 products in ALST cells. (G) ZnO-NPs cause a decrease in the levels of the anti-apoptotic protein BCL2 in ALST cells. (H) ZnO-NP treatment (5 μg/ml, 4 hours) causes activation of caspase-3 as shown by accumulation of cleaved caspase-3 products in TYK-Nu cells.

To identify the mechanism of cell death induced upon ZnO-NP treatment, we first looked for phenotypic features exhibited by ovarian cancer cells undergoing ZnO-NP induced cell death using microscopy. Microscopic examination revealed several morphological hallmarks of apoptosis such as shrinkage of the cells and fragmentation into membrane-bound apoptotic bodies (Fig. 8E). Internalized ZnO-NPs were also visible (Fig. 8E). Western blot analyses further confirmed apoptosis as the cell death mechanism induced by ZnONPs. Increased levels of pro-apoptotic cleaved caspase-3 levels and decreased levels of anti-apoptotic BCL2 was observed in ALST cells upon ZnO-NP treatment (Figs. 8F and 8G). Increased cleaved caspase-3 levels were also observed in p53-R175H expressing TYK-Nu cells confirming that the mechanism of cell death induced by ZnO-NPs is also conserved across ovarian cancer cells exhibiting different p53 mutation status (Fig. 8H).

4.0. Conclusions

Nanoparticles are increasingly being recognized for their potential utility in biological applications including use as anti-cancer therapeutics [35–39]. Although nanoparticles can be synthesized using many different types of materials, compatibility and toxicity are major factors that need to be considered while developing nanomaterials for biomedical applications. ZnO is considered to be a ‘GRAS’ (generally recognized as safe) substance by the Food and Drug Administration (FDA) [40]. Because of this, ZnO has become a highly attractive and preferred material in nanomedicine [40]. Recent studies have shown that ZnO-NPs exhibit selective cytotoxicity towards cancer cells [36,37,40]. However, the mechanism of cytotoxicity from ZnONPs is not completely understood [40]. In this report, we examined the therapeutic potential of ZnO-NPs as an anti-cancer agent for ovarian cancer. Our results show that ZnO-NPs trigger severe oxidative and proteotoxic stress in ovarian cancer cells resulting in apoptotic cell death. Interestingly, the in vitro cytotoxic effects of these ZnO-NPs on fibroblast cells were found to be much reduced when compared to cancer cells, suggesting the potential for these nanoparticles for in vivo translation.

Generation of reactive oxygen species (ROS) has been shown previously to be a feature of ZnO-NPs [36,37,40]. High and sustained ROS build-up can deplete the anti-oxidative capacity of cells and cause them to undergo cell death. Due to its higher metabolic demands and rapid rate of proliferation, cancer cells have higher cellular ROS levels compared to normal cells. This inherent elevated oxidative state makes cancer cells more sensitive to ZnO-NPs treatment. High ROS accumulation can cause oxidative damage to proteins resulting in their ubiquitination. Accumulation of ubiquitinated proteins in cells puts an increased load on the protein degradation machinery in cells leading to proteotoxic stress. We observe both a decrease in anti-oxidative capacity and an increase in proteotoxic stress in ovarian cancer cells upon ZnO-NP treatment. In response to metal-induced oxidative stress, cancer cells are known to assemble specialized proteasome isoforms that have increased proteolytic capacity [41]. Whether ZnO-NP treatment induces the assembly of these alternative proteasome isoforms in ovarian cancer cells is not known and could be a focus of research in future.

Since the size of nanoparticles dictate its properties and downstream functions, we synthesized nanoparticles of different sizes and evaluated their impact on the cytotoxicity towards ovarian cancer cells. The smaller nanoparticles have larger surface area and thereby have more molecules exposed to interact with surrounding biomolecules and trigger a biological response. Furthermore, cellular uptake could also be more efficient for smaller nanoparticles compared to ones that are larger in size. Due to these factors, the biological response of nanoparticles could significantly vary depending on their size. While smaller sized nanoparticles were found to be more cytotoxic than larger particles, the difference was found to be highly dependent on the concentration and duration of treatment. We hypothesize that this is likely due to the tendency of these nanoparticles to aggregate at higher concentrations when treated for longer time points. This issue can be overcome by coating the nanoparticles with distinct sized polymers [42]. Such a modification will also enable a more efficient uptake of nanoparticles by cells. However, the impact of such a modification on the inherent properties of the nanoparticle and its cytotoxicity towards ovarian cancer cells would need to be determined empirically.

GOF mutations in p53 have been shown to contribute towards the resistance of ovarian cancer cells to current therapeutics [43, 44]. Our results reveal that the cytotoxicity of ZnO-NPs towards ovarian cancer cells is independent of the p53 mutation status of these cells, demonstrating the promise of ZnO-NPs in treating drug-resistant ovarian cancers. Interestingly, ZnO-NPs induced depletion of p53-R175H, and p53-R248Q GOF mutants but not p53-R273H mutant protein. How these usually stable p53 GOF mutants are depleted in response to ZnO-NPs could potentially reveal new pathways and mechanisms through which these oncogenic proteins can be regulated in cancer cells and pave the way for new targeted approaches against these proteins. Taken together, the results so far convey great promise and enthusiasm for ZnO-NPs as an anti-cancer therapeutic agent. However, successful clinical translation of ZnO-NPs would require comprehensive understanding of the specific properties and characteristics of these nanoparticles, the mechanism behind their inherent toxicity against cancer cells, and a more rigorous toxicity analysis in in vivo models.

Highlights:

• ZnO nanoparticles (ZnO-NP) of different size was synthesized and its physical properties were characterized.

• ZnO-NP exhibit size-dependent cytotoxicity towards ovarian cancer cells.

• Cytotoxicity of ZnO-NP is independent of the p53 mutation status of the ovarian cancer cells.

• ZnO-NP induces oxidative and proteotoxic stress in ovarian cancer cells leading to cancer cell death via apoptosis.