Investigations on Effects of Titanium Dioxide (TiO2) Nanoparticle in Combination with UV Radiation on Breast and Skin Cancer Cells

doi:10.21203/rs.3.rs-2084950/v1

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Investigations on Effects of Titanium Dioxide (TiO2) Nanoparticle in Combination with UV Radiation on Breast and Skin Cancer Cells

https://doi.org/10.21203/rs.3.rs-2084950/v1

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In this study, we have investigated the chemotherapeutic potential of titanium dioxide (TiO₂) nanoparticles on skin and breast cancer cells. The cells have treated with a 75 µg/ml concentration of titanium dioxide and then exposed to UV-A radiation. The combined effects of titanium dioxide and UV-A radiation on cell viability, cell cycle, plasma membrane, mitochondrial membrane potentials and apoptotic activity of the cells were investigated. As a result of the research, an increase was observed in the viability of cells treated with 75 µg/ml titanium dioxide concentration, while a significant decrease in cell viability was observed for both cell types when UV-A radiation and TiO₂ were applied together. The results also showed that the percentage of apoptotic cells increased as a result of UV + TiO₂ exposure. Accordingly, it can be said that TiO₂ nanoparticles may research as potential chemotherapeutic agents for skin and breast cancers, especially in the presence of UV radiation.

Titanium dioxide nanoparticle

ultraviolet radiation

breast cancer

skin cancer

Titanium dioxide (TiO₂) is a type of photocatalyst that is widely used in scientific and commercial applications. It has high oxidizing activity and produces oxidizing radicals such as hydroxyl radicals, superoxide anion and singlet oxygen in aqueous medium as a result of UV photon absorption below 385 nm wavelength. It is known that these products, which oxidize proteins and lipids in the cell membrane and cellular components, trigger cell death due to apoptosis and necrosis. Due to its effects on cell death, TiO₂'s anticancer properties have been frequently investigated recently.

One of the most important products obtained by using TiO₂ is titanium dioxide nanoparticles (TiO₂-NPs). TiO₂-NPs are chemically inactive and water-insoluble solid semiconducting materials. They exhibit photocatalytic activity if there is light with the energy around or greater than the band gap energy. These properties proposal a wide range of efforts TiO₂-NPs are widely used in various usages such as biomedicine, pharmaceuticals and cosmetics. TiO₂-NPs have also important physical, chemical and biological properties such as chemical stability, good oxidation capacity, biocompatibility, low toxicity. On the other hand, due to the relatively low cost of the raw material and its processing, it has received widespread attention in recent years. Another important point about the use of TiO₂-NPs in scientific studies is that they are widely investigated in biomedical fields and cancer researches, including photodynamic therapy, drug targeting systems, cell imaging, bioengineering and biosensors (Yamaguchi et al., 2010; Chihara et al., 2007; Blake et al., 1999; Thevenot et al., 2008; et al., 2014). The high refractive index of TiO₂ can be expressed as its most important advantage. Thanks to this index, TiO₂ has very good hiding power and brightness.

Potential therapeutic effects of TiO₂–NPs and UV radiation in cancer treatment have been shown in different in-vitro studies in literature. TiO₂ colloidal suspension was found to be effective in killing HeLa cells (Fujishima et al., 2000). Cancer cell-specific photokilling was successfully illustrated using antibodyimmobilized TiO₂-NPs with 1 J/cm² UV irradiation (Matsui et al., 2010). Nasr et al., 2018 evaluated the effect of TiO₂ –NPs on apoptosis induction and invasion of gastric cancer cell line, MKN-45. TiO₂-NPs promote oxidative stress in cells and destroy them through the cell membrane and DNA damaging, finally result cell death. Kubata et al., (1994) showed that human T-24 bladder cancer cells were killed via to free radical production due to combined application of TiO₂–NPs and of UV radiation. Qingning et al., (2009) used one-dimensional titanium dioxide whiskers (TiO₂ Ws) and made a strategy to examine their drug delivery application and anti-tumor effect combined with daunorubicin (DNR) photocatalytic activity applied on human hepatocarcinoma cells (SMMC-7721 cells) under UV irradiation.

In this study, we aimed to investigate the chemotherapeutic potential of TiO₂–NPs on skin and breast cancer cells. The cells were treated with different concentrations of titanium dioxide and then exposed to UV-A radiation because it is known that TiO₂ exhibits intrinsically strong absorption of UV light [Yoon et all. 2018]. The study of UV-A in cell studies is also important because as sunlight passes through the atmosphere, almost all of the UV-C and UV-B radiation is absorbed by the ozone layer, water vapour, oxygen and carbon dioxide. However, since UV-A radiation is much less affected by the atmosphere and other conditions, almost all of the UV radiation that can reach the earth's surface consists of UV-A. The combined effects of TiO₂ and UV-A radiation on viability, cell cycle, mitochondrial membrane potential and apoptotic activity of cells were examined.

Figure 1

Supply of Titanium dioxide Nanoparticles

The TiO₂ nanoparticles used in the study were purchased from Aldrich and used without further purification or any additional chemical treatment.

Cell Culture

MCF-7 and SK-MEL-30 cells (HUKUK, Sap Institute Cell Culture Collection, Ankara, Turkey) were cultured in RPMI 1640 (Biological Industries, USA) supplemented with 10% fetal bovine serum (Capricorn Scientific, Germany), 1% penicillin-streptomycinantibiotic (Biological Industries, USA). The cultures were maintained at 37˚C in a 5% CO₂ incubator (New Brunswick Galaxy 170R). SK-MEL30 and MCF-7 cells, which were incubated with TiO₂ at 75 µg/ml concentration for 48 hours, were then exposed to UV-A radiation with a power density of 1.5 J/cm² with the UV exposure system.

In Vitro Cytotoxicity Assay

Cell-Quant MTT Cell Proliferation Assay Kit (Biosciences, USA) was used in the study. 75 µg/mL TiO₂ dose was applied to MCF-7 and SK-MEL-30 cells, approximately 5x10³-10x10³ per well. They were seeded into 96-well plates separately as UV and incubator groups. Cells were incubated for 48 hours at 37°C in incubator with 5% CO₂. Cell media were removed and incubated with 100 µL of cell medium and 10 µL of MTT Reagent (Component A) for 4 hours. Then, 100 µL of Detergent agent (Component B) was added and incubated again for 4 hours. The colour change in the wells was measured as absorbance with a microplate reader (Molecular Devices FilterMax F5) at a wavelength of 570 nm and the absorbance values of the groups were calculated as the percentage of cell proliferation.

Analysis Of Apoptosis By Flow Cytometry

The percentage of apoptotic cells was analyzed by Annexin V-FITC and propidium iodide (PI) staining using flow cytometer. Briefly; following to different concentrations of TiO₂ treatment and UV-A exposure, MCF-7 and SK-MEL-30 cells were washed with PBS by gentle shaking and pipetting up and down and then resuspended in binding buffer. 5 ml Annexin-V-FITC was added to 195 ml cell suspension. The cells were then vortexed and incubated for 10 min at room temperature. Cells were then washed again and resuspended in X ml binding buffer and X ml PI (X mg/ml) was added into solution. Samples were analyzed by a Flow Cytometer (marka) using the X software (X marka). Annexin–V stained cells were considered as apoptotic (Han et al., 2008; Sumit and Paul, 2005).

Jc-1 Assay

In MCF-7 groups; Mitochondrial Membrane Potential Assay Kit (with JC-1, Elabscience) was used in JC-1 study and we applied 2x10⁵ cells. Cell suspension was split into tubes. After discard the supernatant, PBS was added to wash the cells. 500 Μl JC-1 Staining Buffer added and incubate at 37°C, 5% CO₂ incubator for 15 ~ 20 min. Centrifuge at 300 g for 5 min was done to discard the supernatant and 500 µLpre-cold 1x JC-1 Assay Buffer was added to wash the cells twice. Add 500 µLpre-cold 1x JC-1 Assay Buffer to resuspend the cells and 100 µL was added to the wells of 96-well black plate. Red fluorescence (excitation 550 nm, emission 600 nm) and green fluorescence (excitation 485 nm, emission 535 nm) were measured using a fluorescence plate reader (Molecular Devices FilterMax F5). It was calculated the ratio of red fluorescence divided by green fluorescence.

JC-1 Mitochondrial Membrane Potential Detection Kit (Biosciences, USA) was used in SK-MEL-30 groups. TiO₂ doses (75 µg/mL and 0) were applied to approximately 5x10³-10x10³ cells in each well. They were inoculated into 96 plates separately as UV and incubator groups. The cell culture media is removed and replaced with 200 µL warm phosphate-buffered saline (PBS) after that 2 µL of JC-1 stock solution was added each well, and incubate the plate at 37°C, 5% CO₂ for 15 to 30 minutes. Wells were washed with 200 µL PBS twice. After 200 µL PBS was added each well. The cells were analysed immediately using a fluorescence plate reader with proper machine settings.

Statistical analysis

All experiments were performed under blind conditions. The data for each group was expressed as mean ± standard deviation (SD) of three independent experiments. Statistical analyses were carried out using One-way ANOVA with post-hoc Tukey HSD using SPSS. A difference at p < 0.05 was considered to be statistically significant.

It has been reported in previous studies that TiO2-NPs can have negative effects on cell viability, proliferation and cell cycle. However, such effects were not directly observed in our study. From in vitro cell analysis, cell viabilities were observed to be significantly increased in both MCF-7 and SK-MEL-30 cell lines in the presence of TiO₂ compared to the control group.

Figure 2

On the other hand, it is seen from Fig. 2 that exposed to UV-A radiation was lower than the cell viability of both control groups, which were not exposed to UV-A radiation. This is because UV light has lethal effects on living cells. The important result observed in the study is that exposure to UV-A + TiO₂ caused a decrease in cell viability of MCF-7 and SK MEL-30 cells. It was observed from experimental results that the decrease in cell viability of MCF-7 cells caused by UV-A + TiO₂ exposure was greater than the decrease in cell viability caused by UV-A exposure alone. In addition, there was no significant difference between cell viability of UV-A exposed SK-MEL-30 cells and cells viability of UV-A + TiO₂ exposed SK-MEL-30 cells.

It has known from the previous studies that TiO₂ strongly absorbs UV light [Hyewon et all. 2018]. In this case, it can be said that UV light activates TiO₂ nanoparticles. In addition, while the nanoparticles that are active under UV-A effect have serious effects on MCF-7 cells, they are ineffective on SK-MEL-30 cell line.

Figure 3

Figure 4

It is seen from Fig. 3 and Fig. 4 that UV-A radiation increased the apoptotic activities of MCF-7 cells. It was observed that the application of UV-A + TiO₂ caused a significant increase in the apoptotic cell numbers of MCF-7 cells. Similarly, it was observed that UV-A exposure caused apoptosis in SK-MEL-30 cells. Combined application of UV + TiO₂ caused a decrease in the number of apoptotic cells, but an increase in the number of necrotic cells.

Figure 5

Figure 5 shows the change in mitochondrial membrane potential of MCF-7 and SK MEL-30 cells which were determined by JC-1 fluorescent staining. An increase in the red/green fluorescence ratios (mitochondria hyperpolarization) was observed in the UV-A and UV-A + TiO₂ group. It was observed that UV-A exposure caused hyperpolarization in the mitochondrial membrane of SK MEL-30 cells.

Figure 6

In Fig. 6, it was observed that UV-A radiation and UV-A + TiO₂ applications had very little effects on the cell cycles of MCF-7 cells, and moderate effects on the cell cycles of SK-MEL-30 cells.

TiO₂ nanoparticles, which have a strong oxidizing effect under UV-A radiation, are excited at photon energy above 3.4 eV and may cause free radical formation such as hydroxy radical, superoxide anion and hydrogen peroxide (Yamaguchi et al., 2008; Olmedo et al., 2005; Dick et al., 2003; Long et al., 2006; Ogino et al., 2006). These radicals oxidize protein and lipid in the cell membrane and cellular components, causing cell death based on apoptosis and necrosis (Zhang et al., 2004). In this study we investigated the effects of UV-A radiation combined with TiO₂ on human skin melanoma (SK-MEL-30) and breast adenocarcinoma (MCF-7) cells.

SK-MEL-30 and MCF-7 cells incubated with TiO₂ at concentration of 75 µg/ml for 48 hours were then exposed to UV-A radiation at a power density of 1.5 J/cm². The results of our study showed that UV-A radiation exposure caused a decrease in cell viability of MCF-7 cells. We found a similar decrease in cell viability of the group to which we applied UV-A radiation and TiO₂ nanoparticles in combination. We observed that the inhibitory effect of the combined application of UV-A radiation and TiO₂ nanoparticles on cell proliferation of MCF-7 cells is greater than the inhibitory effect of the application of UV-A radiation alone.

In addition to the above results, we observed that the number of apoptotic cells in the UV-A radiation group was higher than the number of apoptotic cells in the control group. Similar to the cell viability results, we found that the number of apoptotic cells in the group treated with UV-A radiation and TiO₂ combined was much higher than the number of apoptotic cells in the control and UV-A radiation groups. These results show also that UV + TiO₂ causes a synergistic effect on cell viability and apoptotic activities of MCF-7 cells.

In SK-MEL-30 cells, it was seen that there was no significant difference between cell viability after UV-A radiation exposure and cell viability after UV-A + TiO₂ exposure. These results showed that the combined application of UV + TiO2 does not create a synergistic effect on SK-MEL-30 cells at the TiO₂ doses used in the study.

Change in mitochondrial membrane potential (ΔΨ_m) is one of the important indicators of mitochondria-dependent apoptosis. The change in ΔΨm of MCF-7 and SK-MEL-30 cells was detected by JC-1 fluorescent staining in our study. While we detected that UV-A radiation caused hyperpolarization of mitochondria in MCF-7 cells, we observed that UV-A + TiO₂ (75 µg/ml) application further increased this hyperpolarization. Similarly, we detected hyperpolarization in mitochondrial membranes of SK-MEL-30 cells due to UV-A radiation exposure. Some studies in the literature have reported a decrease in cell proliferation of cancer cells due to hyperpolarization of mitochondria (Kadenbach et al., 2004; Eşmekaya et al., 2017).

In this study, the chemotherapeutic effects of titanium dioxide (TiO2) nanoparticles on skin and breast cancer cells in the presence and absence of UV-A radiation were investigated. As a result of the experimental studies, it was determined that only TiO2 application increased the viability of both cell groups, while only UV-A radiation exposure decreased. However, drastic reductions in viability of both MCF-7 cells and SK-MEL cells were observed after exposure to both. This was interpreted as TiO₂-NP activated by UV-A and TiO₂-NP increased the efficacy of UV-A radiation on cell death. According to the results of this study, since it is seen that the combined application of UV-A + TiO2 has the potential to be used in the treatment of breast and skin cancer, it is anticipated that more and more advanced studies will be conducted on the anticancer effects of these two in the future.

Ethics Approval Not applicable.

Consent to Participate Yes.All authors agreed to participate in this research.

Consent for Publication Yes. All authors have approved the last version of the manuscript for its submission.

Author Contribution Conceptualization: Meriç Arda Eşmekaya, Mustafa Tuğfan Bilkan; methodology: Meriç Arda Eşmekaya, Mustafa Tuğfan Bilkan, Ayşe Gülnihal Canseven Kurşun; formal analysis and investigation: Meriç Arda Eşmekaya, Zehra Çiçek; writing–original draft preparation: Meriç Arda Eşmekaya, Mustafa Tuğfan Bilkan; writing–review and editing: Meriç Arda Eşmekaya, Mustafa Tuğfan Bilkan; funding acquisition: Meriç Arda Eşmekaya; resources: Meriç Arda Eşmekaya, Zehra Çiçek, Mehmet Özler; supervision: Meriç Arda Eşmekaya.

Funding This study was funded by the Gazi University BAP department (GrantNo: 01/2019-32).

Competing Interests The authors declare no competing interests.

Data Availability: The datasets generated and/or analyzed during the current study are not publicly available but are available from the corresponding author on reasonable request.

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You are reading this latest preprint version

Investigations on Effects of Titanium Dioxide (TiO2) Nanoparticle in Combination with UV Radiation on Breast and Skin Cancer Cells

Status:

Version 1

Abstract

Figures

Introduction

Material And Methods

Supply of Titanium dioxide Nanoparticles

Cell Culture

In Vitro Cytotoxicity Assay

Analysis Of Apoptosis By Flow Cytometry

Jc-1 Assay

Statistical analysis

Results And Discussions

Discussion

Conclusion

Declarations

References

Status:

Version 1