Original Article |
Corresponding author: Nasrin Ziamajidi ( n_ziamajidi@yahoo.com ) © 2022 Sajedeh Daei, Roghayeh Abbasalipourkabir, Korosh Khanaki, Fatemeh Bahreini , Nasrin Ziamajidi.
This is an open access article distributed under the terms of the Creative Commons Attribution License (CC BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Citation:
Daei S, Abbasalipourkabir R, Khanaki K, Bahreini F, Ziamajidi N (2022) Effects of gold nanoparticles on oxidative stress status in bladder cancer 5637 cells. Folia Medica 64(4): 641-648. https://doi.org/10.3897/folmed.64.e66784
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Introduction: Nanomedicine has recently been known as an emerging research area with promising applications in cancer diagnosis and treatment. Aside from this, gold nanoparticles (AuNPs), as one of the important components of nanomedicine, have attracted considerable attention due to their special physicochemical properties and lower toxicity than other nanoparticles. Despite the impressive advantages of AuNPs, it has not been yet determined whether oxidative stress contributes to the toxicity of AuNPs on bladder cancer.
Aim: The aim of this study was to address this issue by conducting experiments in order to investigate the effects of 20 nm AuNPs on human bladder cancer 5637 cells.
Materials and methods: The viability of 5637 cells was evaluated upon 24 hour exposure to different concentrations of AuNPs (0-50 µg/ml) by 3-(4, 5-dimethylthiazol, 2-yl)-2, 5-diphenyl tetrazolium bromide (MTT) assay. In order to evaluate oxidative stress status, total antioxidant capacity (TAC), total oxidant status (TOS), malondialdehyde (MDA) and also activities of antioxidant enzymes including glutathione peroxidase (GPx), catalase (CAT) and superoxide dismutase (SOD) were all determined by colorimetric assay kits.
Results: The results from our experiment showed that the cytotoxicity caused by AuNPs was dose-dependent and the IC50 value was found to be 43.14 µg/ml after 24-hour exposure. Furthermore, MDA and TOS levels were significantly increased in treated cells compared to untreated cells (p<0.05). In contrast, TAC level and the activities of SOD, GPx, CAT were significantly decreased in AuNPs-treated groups as compared with the untreated cells (p<0.05).
Conclusions: Overall, AuNPs decrease the cell viability and increase oxidative stress in bladder cancer 5637 cells.
bladder cancer 5637 cells, cell viability, oxidative stress, gold nanoparticles
Bladder cancer is one of the most commonly occurring human cancers linked with malignant tumors in the urinary tract. It affects both men and women. Despite the advances of surgery and chemotherapy, the specificity of these conventional therapies is decreased by dose-limiting toxicity. Furthermore, since using these therapy methods are limited due to their considerable side effects such as hemorrhagic cystitis, there are increasing attentions for alternative anticancer treatments. However, it has been a highly challenging task to find potential therapies for various types of cancer. Therefore, resorting to more effective yet less toxic therapies is inevitable for cancer treatment. It would probably seem that nanoparticles could revolutionize the treatment of cancer in the near future.[
The aim of this work was to address this issue by investigating the cytotoxicity and oxidative stress status in the presence of AuNPs.
Spherical gold nanoparticle (20 nm, citrate-stabilized) dispersed in deionized water (99.95% purity) was purchased from Iranian Nanomaterials Pioneers Company, NANOSANY (Mashhad, Iran). Transmission electron microscopy (TEM) and scanning electron microscopy (SEM) of AuNPs are shown in Figs
Human bladder cancer 5637 cell line was obtained from Pasteur Institute, (Tehran, Iran) and cultured in RPMI-1640 (KRPM500) including 10% FBS (KFBS100), 1% penicillin streptomycin (BI-1203) at 5% CO2 and 37°C. In order to subculture (passage) cells, 0.25% trypsin-EDTA solution (KRT100) was used. All experiments were done between passages 2 and 10. Different concentrations of AuNPs in serum-free RPMI-1640 were freshly prepared and were used to treat cells.
To evaluate AuNPs cytotoxic activity, the 3-(4, 5-dimethylthiazol, 2-yl)-2, 5-diphenyl tetrazolium bromide (MTT) (Sigma Aldrich, St. Louis, MO, USA) assay was employed.
Briefly, 5637 cells were grown in 96-well plates at a density of 2×104 cells per well. After 24 hours, the cells were treated with different concentrations of AuNPs (0, 1.5, 3.12, 6.25, 12.5, 25, 50 µg/ml). After 24 hours of incubation, MTT reagent (5 mg/ml in PBS) was added to each well and was then incubated for 4 hours. Next, the medium was depleted and 100 µl dimethyl sulfoxide (DMSO) (DNA biotech, Iran) was added to each well in order to dissolve the formazan crystals. Finally, the absorbance was evaluated at 570 nm using an ELISA plate reader (RT-2100C Microplate Reader, China). The experiments were run in triplicate. The 5637 cell viability was calculated using the following formula:
Lastly, IC50 was calculated from MTT results.
Cells were seeded in a 6-well plate at a density of 3×105 cells/well and treated with different concentrations of AuNPs (12.5, 25, and 50 µg/ml) determined by MTT assay. After 24 hours, the plate was observed under the inverted microscope (Nikon Eclipse TS 100) to evaluate morphological changes.
Cell lysates for evaluating TAC, TOS, MDA, GPx, catalase (CAT), SOD were prepared according to the Kiazist kit protocol (Hamedan, Iran). Overall, after 24 hours of treating with AuNPs (with concentrations of 12.5, 25, and 50 µg/ml), the 5637 cells were washed with cold PBS) pH 7.0) slowly and then dissociated with trypsin-EDTA, and collected by centrifugation at 1500 rpm for 5 min. Afterwards, the cells were resuspended in PBS or the buffer recommended by Kiazist company and lysed either by freeze-thaw or incubation at 4°C. Next, the cells were centrifuged at 12000×g for 15 min at 4°C. Finally, supernatants were separated and stored at −80°C. It is worth mentioning that protease inhibitor cocktail (PI; (KPIMM)) and butylated hydroxytoluene (BHT) solution were added in the lysis buffer for investigating antioxidant enzymes and MDA, respectively. Protein levels of each sample were estimated by the Bradford method[
The levels of TAC (Kiazist, KTAC-96), TOS (Kiazist, KTOS-96), MDA (Kiazist, KMDA-96), were determined via the aforementioned kits according to the manufacturer’s instructions. Briefly, TAC level was assessed based on CUPRAC assay in which the existing antioxidants make the cupric (Cu+2) reduce to cuprous (Cu+1) that produce color in the presence of chromogen. The TOS level was determined based on the oxidation of ferrous to ferric, which also produces color in presence of chromogen. In order to evaluate lipid peroxidation, MDA forms a complex with thiobarbituric acid (TBA), then absorbance was read at 532 nm. Oxidative stress index (OSI) was estimated according to the following formula:
The activity of antioxidant enzymes, [i.e., SOD (Kiazist, KSOD-96), GPx (Kiazist, KGPx-96), CAT (Kiazist, KCAT-96)], were determined using the aforementioned kits according to the manufacturer’s instructions.
All experiments were performed in triplicate. Statistical analyses were conducted using SPSS 26 software (SPSS Inc., Chicago, IL) and GraphPad Prism 8 software (San Diego, CA, USA). First, the normal distribution and homogeneity of data variances were evaluated by Shapiro-Wilk normality test. Then, one-way ANOVA followed by post-hoc Tukey test were employed for multiple comparisons. Results were reported as mean ± SD, and the obtained p values less than 0.05 were considered statistically significant.
As shown in Fig.
Inhibition of 5637 cell survival after 24-hour exposure to various concentrations of gold nanoparticles (AuNPs) (0, 1.56, 3.12, 6.25, 12.5, 25, and 50 µg/ml) according to the results of MTT assay. Asterisks indicate significant difference compared with the control cells (*p<0.05, **p<0.01, ***p<0.001). The results are shown as mean ± SD of three individual 96-well plates (n=3).
As shown in Fig.
Morphological alterations in 5637 cells treated with 12.5 (b), 25 (c) and 50 μg/ml (d) AuNPs in comparison with untreated cells (a). It is noteworthy that the cell density was decreased while the number of rounded and wrinkled cells was increased in AuNPs-treated cells in a dose dependent manner. Objective magnification is 10×.
Changes in MDA, TOS and TAC levels according to different concentrations of AuNPs in 5637 cells are shown in Fig.
As shown in Fig.
CAT activity was significantly decreased in 12.5, 25, and 50 µg/ml concentrations compared with the control cells (p<0.001) (Fig.
Effects of AuNPs on oxidative stress parameters in 5637 cells. a) total oxidant status (TOS), b) malondialdehyde (MDA), c) total antioxidant capacity (TAC), and d) oxidative stress index (OSI). Asterisks indicate significant differences relative to control (*p<0.05, **p<0.01, ***p<0.001). Number sign indicates significant differences between groups (#p<0.05, ##p<0.01, ###p<0.001).
Effects of AuNPs on antioxidant enzymes activity in bladder cancer 5637 cells. a) glutathione peroxidase (GPx), b) catalase (CAT), c) superoxide dismutase (SOD). Asterisks indicate significant difference compared with control (*p<0.05, **p<0.01, ***p<0.001). Number sign indicates significant differences between groups (#p<0.05, ##p<0.01, ###p<0.001).
It is well known that AuNPs originating from different sources such as biological[
According to the obtained results from oxidative stress parameters, the levels of TAC, TOS, and MDA were fully consistent with the ones obtained from MTT assay. In order to explain in more details, unsaturated fatty acids and lipids are oxidated in the presence of ROS which can cause lipid peroxidation (LPO). Furthermore, MDA, resulting from the breakdown of polyunsaturated fatty acids (PUFAs) in lipid membranes, is a strong indicator of LPO. [
Our results also showed that AuNPs can reduce antioxidant enzymes activity e.g. CAT, SOD, GPx. In consistency with our study, Costa et al.[
Our results emphasize that fact that the activity of SOD is decreased under treatment with AuNPs. Specifically, it is reported that SOD and GPx activities are usually decreased in cancers depending on excess production of ROS.[
Gold nanoparticles are one of the important metallic nanoparticles used in nanomedicine technology. According to our results, AuNPs can effectively reduce bladder cancer 5637 cells viability in a dose-dependent manner. We also found that AuNPs have the ability to reduce the antioxidant capacity of cancer cells and increase oxidative parameters. It seems that oxidative stress contributes to the toxicity of AuNPs. However, further investigations considering different signaling pathways involved in cancer development or progression are needed to prove it.
This study has been adapted from a MSc thesis at Hamadan University of Medical Sciences, Iran. The first author would like to thank Dr. Sajad Daei for editing this article.
Funding
This study was financially supported by Vice-chancellor for Research and Technology, Hamadan University of Medical Sciences.
Conflicts of Interest
There is no conflict of interest in this paper.