SPC212 human mesothelioma cells underwent apoptosis, oxidative stress, and morphological deformation following Astaxanthin treatment

Astaxanthin (ASX) is one of the keto‐carotenoids, which is biologically more active than other counterparts. Besides its variety of beneficial effects, it was reported to exert anticancer effects. Despite its utilization against different cancer types, the effect of ASX on mesothelioma has yet to be well‐studied. In this study, our goal is to ascertain how ASX will affect SPC212 human mesothelioma cells. First, the effective doses of ASX against SPC212 cells were investigated by the 3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyltetrazolium bromide (MTT) test. Thereafter, with flow cytometry analysis, Annexin‐V and caspase 3/7 assay were implemented for the evaluation of apoptotic cell death and an oxidative stress test was carried out to determine how the free radicals changed. Ultimately, the cells' morphology was examined under a light microscope. The effective doses of ASX were found as 50, 100, and 200 µM. In the Annexin V assay, the total apoptosis increased to around 12%, 30%, and 45% with increasing doses of ASX. In the caspase 3/7 assay, the total apoptosis was around 25% and 38% at 100 and 200 µM. In oxidative stress analysis, reactive oxygen species‐positive cells rose from 4.54 at the lowest dose to 86.95 at the highest dose. In morphological analysis, cellular shrinkage, decrease in cell density, swelling and vacuolations in some cells, membrane blebbing, and apoptotic bodies are observed in ASX‐treated cells. To conclude, the current study provided insights into the efficacy and effects of ASX against SPC212 mesothelioma cells regarding morphology, proliferation, and cell death for future studies.

Astaxanthin (ASX), a ketocarotenoid, was discovered by Kuhn and Sorensen who initially extracted it from lobster. ASX can be produced by some organisms including plants, bacteria, and microalgae. [4] ASX has various biological functions; immune response enhancing, protection against oxidative stress and ultraviolet, and providing the reproductive ability and stress tolerance. [5] Recently ASX is of great interest due to its possible versatile effects against cancer, diabetes, inflammation, and oxidative stress. [6] In the last 30 years of research, it was pointed out that oxidative stress causes chronic inflammation, which leads to chronic diseases such as cancer and skin damage. [7] ASX was shown to improve oxidative stress metabolism and prevent inflammation by downregulating and inhibiting inflammatory mediators. AST performed antimigration and anti-invasion activity in the treatment of colorectal cancer by increasing the miR-29a-3p and miR-200a levels and thus suppressing the MMP2 and ZEB1 protein levels. [8] In SCC131 and SCC4 oral cancer cells and cancer models, ASX was documented to inhibit PI3K/Akt and the associated nuclear factor-kappa B (NF-κB) and signal transducer and activator of transcription 3 signaling pathways; thereby, hampering the cancerrelated symptoms. [9] Utilization of ASX in experimental animals with sepsis and acute lung injury was suggested to hinder mitogenactivated protein kinase (MAPK)/NF-κB signaling pathway and inflammatory factors, and significantly enhance survival. [10] ASX is also documented to have some synergic effects when co-used with other proteins. For example, ASX was shown to increase its anticancer activity when used with human serum albumin (HSA), the most abundant blood protein in SKOV3 ovarian cancer cells. SKOV3 cells were given ASX + HSA to explore the effects of cell proliferation, cell cycle, and drug resistance. ASX + HSA treatment was shown to increase the anticancer effects of AST, reduce the G1 phase cell cycle and induce apoptosis in SKOV3 cells. [11] Another example of the synergic effect of ASX, Chen et al. evaluated ASX and erlotinib for Xeroderma pigmentosum C (XPC) expression-mediated cytotoxicity in non-small cell lung cancer (NSCLC) cells. Activation of p38 MAPK with ASX reduced XPC expression in lung adenocarcinoma cells. Inhibition of p38 MAPK activity decreases cytotoxicity and cell growth inhibition and destruction of XPC using siRNA increased the cytotoxic effects of ASX. [12] In another study, cotreatment of ASX and anticancer drug carbendazim (Carb) to MCF-7 breast cancer cells increased the antiproliferative effect of Carb treatment alone and reduced the G2/M phase cell cycle and decreased intracellular reactive oxygen species (ROS) levels. [13] SPC212 human mesothelioma cells are suitable cell lines being a model cell line for MPM, which are derived from malignant mesothelioma a 47-year-old female patient, exposed to asbestos and with a biphasic tumor of the pleural cavity. To the best of our knowledge, there are no studies investigating the effect of ASX on these particular cell lines and exploring its effective concentration. To fill this lacuna, we proposed carrying out research to unravel how different doses of ASX will affect SPC212 human mesothelioma cells.

| Cell culture
Human mesothelioma cells (SPC212) were grown in Dulbecco's modified Eagle medium (Gibco, 11966025) containing 1% penicillin-streptomycin, 10% fetal bovine serum at 5% CO 2, and 95% relative humidity at 37°C in culture medium. When the culture flask has a cell density of 70% (about 2-3 days) it is separated into subcultures. Before doing this, the old medium of SPC212 cells was removed by washing the adhered cells' surface 2 times with 1 mL of phosphate-buffered saline (Gibco, 10010023). Then the cells were dislodged by 1 min treatment with Trypsin-ethylenediaminetetraacetic acid (Gibco, R001100), and then appropriately subcultured.

| Cytotoxicity analysis
The effect of ASX on the cell viability of SPC212 cells was

| Annexin V test
Annexin V assay (Gibco, V13242) was accomplished in compliance with the manufacturer's instructions for the kit. The SPC212 cell line was cultivated in six-well plates and the cells were applied the different doses of ASX including 50, 100, and 200 µM.
After 24 h SPC212 cells were dislodged from plates with trypsin-EDTA and taken into 1.5 mL tubes. The SPC212 cells were gained in the bottom of the tubes by 5-min centrifugation at 300 × g.
Next, the pellet was resuspended by ready-to-use medium (100 μL) and blended with annexin V and left to a 20 min incubation at dark at 24°C. In the end, the results are evaluated by Muse™ Cell Analyser.

| Oxidative stress
Oxidative stress evaluation was carried out through a commercial oxidative stress kit (Muse ® Oxidative Stress Kit; Merck Millipore) and was gauged according to the manufacturer's instructions. The SPC212 cell line was cultivated in six-well plates and administered with 50, 100, and 200 µM of ASX. After 24 h, SPC212 cells were dislodged from plates with trypsin-EDTA and taken into 1.5 mL tubes. Once the SPC212 cells were gained in the bottom of the tubes by 5-min centrifugation at 300 × g, they were diluted with provided buffer in the kit at a cell density of 1 × 10 6 -1 × 10 7 /mL. Next, this mixture was blended with the working solution procured by the kit with a ratio of a ratio of 1:20 (10 µL + 190 µL, respectively).

| Hematoxylin-eosin staining
Hematoxylin-eosin stain is regularly exploited for investigating morphological alterations of cells regarding cytoplasmic and nuclear as well as extracellular matrix-related modifications. The morphological alterations of ASX-treated cells were observed by hematoxylin and eosin stain. Initially, SPC212 cells were grown in six-well plates at a density of 3 × 10 5 and treated with 50, 100, 200, and 300 μM of ASX for 24 h. After that, the cells were exposed to a 10 min-fixation with pure ice-cold methanol, followed by incubation in hematoxylin and eosin stains, respectively for 5 min each. Then the cells were dipped into 1% ammonia solution for 1 min. Finally, the cells were rinsed in PBS and examined under a light microscope.

| Statistical analysis
First, we checked the data whether it is normally distributed or not. Shapiro-Wilk test showed that all the data follows the normal distribution. Therefore, a one-way analysis of variance (ANOVA) was exploited to unravel the significant difference between the groups. Levene's test confirmed the equality of variance of the data; therefore, as a post hoc multiple comparison tests, we utilized Tukey's test. The p < 0.05 were deemed statistically significant. All statistical analyses were implemented employing the GraphPad Prism 7.0 statistical software. All statistical analyses were employed by one-way ANOVA followed by using p < 0.05 was considered significant.

| MTT assay results
The antiproliferative effect of ASX on SPC212 cells was ascertained through the MTT test, as displayed in Figure 1 and

| Annexin V assay results
According to the Annexin V findings (Figure 2 and

| Caspase 3/7 assay results
In an attempt to assess whether the inhibitory effects on cell proliferation were associated with effects on the caspase-mediated apoptosis pathway, caspase-3/7 activity was to measured by flow cytometry (Figure 3 and Table 3) As shown in Figure   Caspase 3/7 assay exhibited that SPC212 cells were noticed to be driven to apoptosis differently than control cells, which showed relatively higher viability and almost underwent no apoptosis.

| Oxidative stress results
As regards the oxidative stress results as shown in Figure 4 and

| DISCUSSION
Recently, the MPM prevalence is noted to reach a peak due to the difficulty in its early prognosis and unsatisfactory treatment modalities. [1] Although asbestos production and its commercial use are restricted or forbidden, its exploitation is still widespread. While males have a higher incidence of disease, females have a better prognosis. Hereditary background poses a risk for the predisposition to mesothelioma. [2] According to the results of our literature research; this is the first study to investigate the effect of ASX on human mesothelioma cells, SPC212.
ASX is a red-orange pigment found in marine organisms, especially salmonids, shrimps, and crayfish. [4] ASX, akin to other carotenoids including zeaxanthin, lycopene, and β-carotene, has many metabolisms and physiology-associated roles. In addition, ASX is a more bioactive compound than other carotenoids. Owing to the molecular arrangement, ASX has many characteristics that enable its utilization to improve human well-being. For example, it possesses a relatively stronger antioxidant capacity than its correspondences. [6]  levels. [14] Furthermore, ASX is also reported to significantly reduced in vitro proliferation and migration rates of breast cancer cells. [13] In the study of Kim et al. the MTT assay was performed for ASXtreated (50 and 100 µM) colon cancer cells to measure cell proliferation. [15] In another study, the cytotoxic activity of ASX on and H1703 cells to colonize. [16] Furthermore, ASX was shown to suppress the proliferation of LS-180 colon cancer cells and decreased the cell viability in a dose-dependent manner. [12] ASX exerted an inhibitory effect on NSCLC cell proliferation and viability. [14] ASX blocked the cell proliferation in the breast cancer cells investigated. [13] An appropriate concentration of ASX is effective for preventing the proliferative activity of Caco-2 cells. [17] The common point in ASX toxicity studies is that ASX causes dose and timedependent cytotoxic effects on cell lines. ASX decreased SPC212 cell viability dose-and time-dependently and the IC50 value for 24 h ASX treatment against SPC212 cells was detected as 241.7 µM.
Innate oxidative stress is considered to be the pathophysiology of cancers because of the potent factor of angiogenesis. ROS is generated by aerobic cellular metabolism and scavenged by antioxidant mechanisms. ROS production and antioxidant defense capability cause oxidative stress. Oxidative stress results in cell damage. [18] ASX has antioxidant, anti-inflammatory, and immunomodulatory properties. Antioxidants are the focus of research due to their anticancer properties. Previous studies of ASX reported reducing oxidative stress and preserving metabolism. [19] ASX has much more potent antioxidants than other carotenoids such as zeaxanthin, lutein, tunaxanthin, cantaxanthin and b-carotene, and α-tocopherol. ASX inhibited stress in the liver by reducing lipid peroxidation and NK cell activity. Also, ASX showed an inhibitory effect on hepatic metastases by its antioxidative properties. [20] ASX inhibited the cytotoxic effects of glutamate in mouse hippocampal HT22 cells. ASX reduced cell viability, glutamate-induced caspase activation, and ROS nuclear accumulation. [21] Oxidative stress is an important factor in diseases such as cancer and causes mitochondrial dysfunction. Wolf et al. analyzed basal oxidative stress, superoxide measurement, and mitochondrial membrane potential. They reported that ASX decreased oxidative stress and protected HeLa cells against oxidative stress. [22] ASX provided decreases in SOD2 level and SOD activity and reduced mitochondrial ROS in gastric epithelial AGS cells. [17] In rat glioma cells, ASX showed ROS and free radical scalvaging properties as a quencher of singlet reactive oxygen, nitrogen, and two-electron oxidants. [23] ASX is a potent antioxidant and has a terminal carbonyl group that is conjugated to a polyene.
It scavenges free radicals. So ASX protects cells from cancer. ASX may result variously from its antioxidant and anticancer properties interactions with altering gene expression and cellular signaling cascades. [24] In our oxidative stress analysis, consistent with the literature, ASX also decreased oxidative stress in a dose-dependent way in SPC212 cells.
Apoptosis is an organized programmed cell death that occurs in physiological and pathological conditions. Apoptosis can be triggered 3. [21] In vitro studies using cancer cell lines and in vivo tumor models have indicated the antiproliferative and proapoptotic effects of ASX. [25][26][27] ASX reduced oxidative stress and cell death in HT22 cells.
ASX decreased ROS and thereby aborted intrinsic apoptosis by upregulating antiapoptotic Bcl-2 expression and downregulating proapoptotic Bax expression. [21] Zhang et al. examined the effect of ASX and other carotenoids at different dose ranges on K562 cells.
They reported that carotenoids (b-carotene, ASX, capsanthin, and bixin) decreased the viability of K562 cells and induced cell apoptosis. [28] Kavitha et al. evaluated the effect of ASX in the hamster buccal pouch carcinogenesis model by caspase 3/9 test by enzymatic assay. ASX significantly increased the expression of caspase-9, -3, and poly (ADP-ribose) polymerase. [29] Meng et al.
reported ASX treatment is not a clear indicator of protective effects on apoptosis for RWPE-1 or PC-3 cells by Annexin V. [18] We analyzed ASX's apoptotic effect on SPC212 cells after the cytotoxic and antiproliferative effect analyses. We recognized this finding by Annexin V and caspase 3/7 analysis by flow cytometry.
Annexin V test and caspase 3/7 assays exhibited that SPC212 cells, to which ASX was applied, were noticed to be driven to apoptosis differently than control cells, which showed relatively higher viability and almost underwent no apoptosis.
In conclusion, we indicated that ASX induced growth inhibition, morphological deformation, oxidative stress, and apoptosis in human mesothelioma cells, and its IC50 value for SPC212 cells was found to be 241.7 µM for 24 h. We think that the present results will be an incentive for and provide new insights into future studies since there are no studies about ASX's effect on lung cancer cells. Varol Şahintürk contributed to supervision and project administration. All authors read and approved the manuscript. The authors declare that all data were generated in-house and that no paper mill was used.

CONFLICT OF INTEREST STATEMENT
The authors declare no conflict of interest.

DATA AVAILABILITY STATEMENT
All data analyzed during this study are included in this article. Author elects to not share data.

ETHICS STATEMENT
This is an in vitro study and no ethical approval is required.