Effect of evernic acid on human breast cancer MCF‐7 and MDA‐MB‐453 cell lines via thioredoxin reductase 1: A molecular approach

Thioredoxin reductase 1 (TrxR1) has emerged as an important target for anticancer drug development due to its overexpression in many human tumors including breast cancer. Due to the serious side effects of currently used commercial anticancer drugs, new natural compounds with very few side effects and high efficacy are of great importance in cancer treatment. Lichen secondary metabolites, known as natural compounds, have diverse biological properties, including antioxidant and anticancer activities. Herein, we aimed to determine the potential antiproliferative, antimigratory, and apoptotic effects of evernic acid, a lichen secondary metabolite, on breast cancer MCF‐7 and MDA‐MB‐453 cell lines and afterward to investigate whether its anticancer effect is exerted by TrxR1‐targeting. The cytotoxicity results indicated that evernic acid suppressed the proliferation of MCF‐7 and MDA‐MB‐453 cells in a dose‐dependent manner and the IC50 values were calculated as 33.79 and 121.40 μg/mL, respectively. Migration assay results revealed the notable antimigratory ability of evernic acid against both cell types. The expression of apoptotic markers Bcl2 associated X, apoptosis regulator, Bcl2 apoptosis regulator, and tumor protein p53 by quantitative real‐time polymerase chain reaction and western blot analysis showed that evernic acid did not induce apoptosis in both cell lines, consistent with flow cytometry results. Evernic acid showed its anticancer effect via inhibiting TrxR1 enzyme activity rather than mRNA and protein expression levels in both cell lines. In conclusion, these findings suggest that evernic acid has the potential to be evaluated as a therapeutic agent in breast cancer treatment.

Reactive oxygen species (ROS) trigger programmed cell death in normal cells, and an increase in ROS level leads to oxidative stress in cells (Perillo et al., 2020). Excessive production of ROS can damage essential cellular components and ultimately result in many biological disorders including neurodegenerative and cardiovascular diseases, aging, cell death, and cancer (Izzo et al., 2021;Martindale & Holbrook, 2002). In this case, the antioxidant system tries to cope with the neutralization of ROS against the damage caused by oxidative stress, which causes aforementioned disorders, especially cancer (Choi et al., 2002;DeBerardinis & Chandel, 2016;Lincoln et al., 2003;Sabharwal & Schumacker, 2014). Therefore, the protection of ROS homeostasis by natural or synthesized compounds has been proposed as an effective therapeutic strategy for cancer treatment (Kalın et al., 2022b;Zhang et al., 2019).
The thioredoxin system, which consists of thioredoxin reductase (TrxR), thioredoxin, and nicotinamide adenine dinucleotide phosphate (NADPH), is one of the enzymatic antioxidant systems. TrxR1 localized in the cytoplasm plays a critical role in cellular activities such as cell viability, proliferation, apoptosis, and regulation of cellular redox environment (Lillig & Holmgren, 2007;Lu et al., 2007;Tinkov et al., 2018).
Many studies have reported that TrxR1 is overexpressed in various types of tumor cells, so it is considered a diagnostic marker for anticancer therapy (Cheng et al., 2014;Lei et al., 2018;Shao et al., 2015).
In literature, evernic acid, one of lichen seconder metabolites, has moderate cytotoxic effect in various types of cancer cells, including breast cancer (Burlando et al., 2009;Kizil et al., 2014;Kosani c et al., 2013). Furthermore, our previous study revealed that evernic acid exerts an inhibitory effect on mitochondrial TrxR purified from rat lung (Ozgencli et al., 2019). However, the anticancer mechanism of evernic acid is not fully defined, and the primary cellular target and mode of action of evernic acid in breast cancer are still unclear. Herein, we investigated the cytotoxic, apoptotic, and migratory effects of evernic acid on breast cancer MCF-7 and MDA-MB-453 cell lines and also determined whether this effect occurs via the modulation of TrxR1 at the gene and protein expressions, as well as enzymatic activity levels in detail. and MDA-MB-453 cells in Eagle's Minimum Essential Medium (EMEM; Sigma-Aldrich). These media contained 10% (v/v) heatinactivated fetal bovine serum (FBS) (HyClone), 1% L-glutamine (Thermo Fisher Scientific), and 1% penicillin/streptomycin (Sigma-Aldrich). Both cell lines were cultured at 37 C in a 5% CO 2 atmosphere.

| Preparation of evernic acid
Evernic acid (C 17 H 16 O 7 ) was purchased from Cayman Chemical Company and was dissolved in dimethyl sulfoxide as a stock solution and stored at À20 C until use. Evernic acid was diluted with a medium at different concentrations (10,25,50,75,100, and 150 μg/mL).

| XTT assay
MCF-7 and MDA-MB-453 cells were seeded into 96-well plates at a density of 1 Â 10 4 cells per well in DMEM and EMEM, respectively, and allowed to attach overnight at 37 C and 5% CO 2 atmosphere.

| Wound healing assay
Cells were cultured in 6-well plates at 5 Â 10 5 cells/well to confluent monolayers. Straight wounds were made by using a sterile 10-μL pipette tips. After washing with Dulbecco's phosphate-buffered saline to remove cell debris, the cells were incubated in a complete growth medium with 10% FBS in the absence or presence of evernic acid. The wound surface was photographed via an inverted microscope at regular intervals (0, 12, and 24 h). The ratio of cell migration was calculated as the percentage of the remaining cell-free area compared with the area of the initial scratched area.
Results of assays were obtained from at least three independent experiments.

| Transwell migration assay
The

| Protein preparation and western blotting analysis
The cells were harvested and lysed in 400-μL radioimmunoprecipitation assay buffer (RIPA buffer) (9806, Cell Signaling Technology), including

| Statistical analysis
Data were obtained from three separate experiments and presented as the mean ± SD. The statistical comparison of the results was performed by unpaired t-test and two-way analysis of variance by using the GraphPad Prism Software version 5.0 for Windows (GraphPad Software). A symbol (*) indicates statistically significant changes. The symbol expressions are as follows: p > 0.05 (not significant, ns); *p < 0.05 (significant), **p < 0.01 (very significant), and ***p < 0.001 (extremely significant).

| RESULTS
3.1 | Antiproliferative effect of evernic acid in breast cancer cell lines To determine the cytotoxic ability of evernic acid in human breast cancer, the XTT assay was performed. The results showed that evernic acid exhibited a lower antiproliferative effect on MCF-7 cells up to 100 μg/mL at 24 h ( Figure 1A). However, treatment of MCF-7 cells with evernic acid for 52 h was resulted in a dose-dependent cytotoxicity, and the IC 50 value was calculated as 33.79 ± 4.25 μg/mL ( Figure 1C). On the other hand, the cytotoxic effect of evernic acid on MDA-MB-453 cells was seen to be slightly lower than that of MCF-7 cells ( Figure 1B), that is, it showed low-to-moderate effect at 48 h with an IC 50 value 121.40 ± 2.02 μg/mL in MDA-MB-453 cells ( Figure 1C). Also, the inverted microscope images of both cells by evernic acid were taken to observe the morphological changes ( Figure 1D). . All data represent the mean ± SD of three independent experiments. Scale bar, 100 μm. p > 0.05 (not significant, ns); *p < 0.05 (significant), **p < 0.01 (very significant), and ***p < 0.001 (extremely significant).

| Impact of evernic acid on apoptosis and necrosis in breast cancer cell lines
also BAX/BCL2 ratio in MCF-7 cells treated with IC 50 value of evernic acid for 52 h was found to be dramatically downregulated as 4.3-, 2.5-, and 2.4-fold, respectively, compared with the control group. However, the P53 gene expression level did not statistically change in evernic acid treated-MCF-7 cells ( Figure 2C). As for MDA-MB-453 cells, the expression of BAX, BCL2, and P53 genes and BAX/BCL2 ratio was observed to be downregulated as 2.1-, 1.2-, 1.9-, and 1.7-fold, respectively, compared with the control group at 48 h ( Figure 2D).    the control group and 20.8 ± 2.6% in evernic acid treated-MCF-7 cells for 12 h ( p > 0.05). When increasing the time to 24 h, wound closure was measured as 38.8 ± 3.2% in the control group, whereas this rate was 30.0 ± 5.1% after evernic acid treatment ( p < 0.001) ( Figure 3A).

| DISCUSSION
Breast cancer is one of the most challenging diseases with a high mortality rate among all cancer types (Becker, 2015). Different medicinal methods such as surgery, chemotherapy, radiotherapy, and hormone therapy have been used for a long time in the treatment of breast cancer. However, these methods especially chemotherapy are gradually losing their effectiveness due to severe side effects and multidrug resistance. Therefore, it is becoming important to figure out new natural compounds with little adverse effects and high efficiency (Mitra & Dash, 2018). Lichen secondary metabolites, known as natural products, have different biological properties including antioxidant and anticancer activities, based on the inhibition of cell proliferation and regulation of cancer-related pathways in breast cancer (Mayer et al., 2005;Molnár & Farkas, 2010;Solárová et al., 2020).
It is known that the proliferation of cancerous cells is generally mediated by several pathways and biomarkers are used for diagnostic purposes (Cheang et al., 2008;Yamane et al., 2007). TrxR1, one of the thioredoxin system enzymes, plays a critical role in cellular activities as well as the regulation of the redox environment and has been used as a marker due to its overexpression in different cancer cells including breast cancer (Jovanovi c et al., 2022;Lu et al., 2007;Ozgencli et al., 2019). Thus, inhibition of TrxR1 is widely thought to function as a mechanism of cell death in tumor cells (Kirkpatrick et al., 1998;Nguyen et al., 2006). In the literature, preliminary anticancer studies of evernic acid have been carried out in several different cancer types except for breast cancer (Burlando et al., 2009;Kizil et al., 2014).
Therefore, in the first part of this study, the cytotoxic, apoptotic, and , and the enzymatic activity of TrxR1 (E, F) in both cell lines. All data represent the mean ± SD of three independent experiments. p > 0.05 (not significant, ns); *p < 0.05 (significant), **p < 0.01 (very significant), and ***p < 0.001 (extremely significant). TrxR1, thioredoxin reductase 1.  (Burlando et al., 2009). In another study, evernic acid was reported to have a moderate cytotoxic effect on human cervical cancer (HeLa) (Kizil et al., 2014). In another study, the antiproliferative effects of evernic acid on human colon cancer (HCT-116), human embryonal kidney (HEK293T), HeLa, murine fibroblast (NIH3T3), and murine leukemic macrophage (RAW264.7) cell lines were investigated at different concentrations (0.3, 3, and 30 μg/mL) for 24 h, showing that evernic acid at 30 μg/mL was only able to prevent the growth of NIH3T3 cells (Roser et al., 2022). Also, our results indicated that MCF-7 cells were Apoptosis, controlled by Bcl-2 family proteins, including Bax (proapoptotic) and Bcl-2 (anti-apoptotic), as well as P53, is the process of programmed cell death (Elmore, 2007;Tzifi et al., 2012). Our results showed that evernic acid had no inducing effect on the apoptotic pathway in P53 levels and BAX/BCL2 ratio in both MCF-7 and MDA-MB-453 cells. Moreover, the flow cytometry results related to the apoptotic mechanism were observed to be strongly consistent with gene and protein expression results. A similar situation has been reported in the literature that evernic acid does not affect apoptosis in HCT-116 cells (Roser et al., 2022). In addition, Lee et al. (2021) reported Cell death occurs not only by apoptosis but also via nonapoptotic forms of cell death such as pyroptosis and necroptosis (Tsujimoto, 2012). Nonapoptotic forms of cell death can be triggered independently of apoptosis or can occur with the failure of apoptosis.
Moreover, it was emphasized that nonapoptotic cell death may be activated in the killing of apoptosis-resistant cancer cells, thus providing new opportunities to manipulate cell death therapeutically (Tait et al., 2014).
Metastasis, which is the spread of cancer cells to different tissues and organs, causes the formation of new tumors and leads to the death of most patients with cancer (Devita et al., 1975). Therefore, TrxR1 plays an important role in the redox regulation of the cells, but its overexpression promotes cell growth, progression, and metastasis in tumor cells (Bhatia et al., 2016;Powis & Montfort, 2001;Tonissen & Di Trapani, 2009). Cancer cells have higher endogenous oxidative stress and thus higher dependence on TrxR1 than normal cells. Therefore, targeting TrxR1 by chemotherapeutic agents with anticancer properties has been considered a plausible therapeutic approach (Arnér, 2017). Thus, inhibition of the TrxR1 by increased ROS production is aimed to cause more toxicity in cancer cells. In this line, we aimed to investigate whether the anticancer effect of evernic acid is related to the inhibition of TrxR1 in both cancer cell lines. Our results showed that evernic acid did not alter the protein expression of TrxR1 but significantly suppressed the enzyme activity in MCF-7 and MDA-MB-453 cells. In addition, evernic acid was found to increase gene expression despite suppressing TrxR1 enzyme activity in MCF-7 cells. In the literature, some studies have explained this situation through the result of negative feedback upregulation (Dong et al., 2016;Meiners et al., 2003). As a result, inhibition of TrxR1 activity by evernic acid in the current study may be due to an excessive increase in the amount of intracellular ROS, which could not be tolerated by cancer cells, and thus, evernic acid could be considered a novel TrxR1 enzyme inhibitor.

| CONCLUSIONS
The findings clearly demonstrated the cytotoxic and antimigratory effects of evernic acid in both MCF-7 and MDA-MB-453 cells.
Moreover, the results confirmed that the anticancer effect of evernic acid is mediated by the inhibition of TrxR1 activity in breast cancer ( Figure 5). Thus, it is claimed that evernic acid may act as a new candidate for TrxR1 inhibitors and also be considered a potent therapeutic agent for breast cancer treatment. Contributed reagents/materials/analysis tools: HB, AA, and ŞNK.

AUTHOR CONTRIBUTIONS
Wrote the paper: HB, AA, and ŞNK. The final version of the manuscript was read and approved by all authors.