Diffractaic acid exhibits thioredoxin reductase 1 inhibition in lung cancer A549 cells

Lung cancer is the leading cause of cancer‐related deaths all over the world. Therefore, it has gained importance in the development of new chemotherapeutic strategies to identify anticancer agents with low side effects, reliable, high anticancer potential, and specific to lung cancer cells. Thioredoxin reductase 1 (TrxR1) is an important therapeutic target for lung cancer treatment because of its overexpression in tumor cells. Here, we aimed to examine the anticancer effect of diffractaic acid, a lichen secondary metabolite, in A549 cells by comparing it with the commercial chemotherapeutic drug carboplatin and also to investigate whether the anticancer effect of diffractaic acid occurs via TrxR1‐targeting. The IC50 value of diffractaic acid on A549 cells was determined as 46.37 μg/mL at 48 h, and diffractaic acid had stronger cytotoxicity than carboplatin in A549 cells. qPCR results revealed that diffractaic acid promoted the intrinsic apoptotic pathway through the upregulation of the BAX/BCL2 ratio and P53 gene in A549 cells, which is consistent with the flow cytometry results. Furthermore, migration analysis results indicated that diffractaic acid impressively suppressed the migration of A549 cells. While the enzymatic activity of TrxR1 was inhibited by diffractaic acid in A549 cells, no changes were seen in the quantitative expression levels of gene and protein. These findings provide fundamental data on the anticancer effect of diffractaic acid on A549 cells targeting TrxR1 activity, suggesting that it could be considered a chemotherapeutic agent for lung cancer therapy.

exploration of new therapeutic agents with beneficial and less undesirable effects is indispensable for lung cancer treatment.
Lichens, which are organisms formed from a symbiotic association of mycobionts (fungi) and photobionts (green algae or cyanobacteria) are of great importance with their widespread use in traditional medicine in the treatment of various diseases such as respiratory diseases, arthritis, and cancer (Crawford, 2015;Molnár & Farkas, 2010).More than 1000 unique lichen secondary metabolites have been known for many biological functions such as anticancer, antioxidant, antifungal, antibiotic, and anti-inflammation (Kalın et al., 2022b;Solárová et al., 2020;Zambare & Christopher, 2012).Diffractaic acid, one of the secondary metabolites, is a depside derivative and has analgesic, antipyretic, antitumoral, antioxidant, and cytotoxic properties (Brisdelli et al., 2013;Odabasoglu et al., 2012).However, the anticancer effect and underlying mechanism of action of diffractaic acid on human lung cancer (A549) cells remain unclear.
Oxidative stress, an increased and common hallmark in many types of cancer, results from the overproduction of reactive oxygen species (ROS) by endogenous and exogenous factors.As cellular ROS at a certain level acts as a signal molecule in many vital physiological events, the enzymatic antioxidant system neutralizes excess ROS to maintain cellular redox balance (He et al., 2017).The thioredoxin (Trx) system is one of the main enzymatic antioxidant systems consisting of Trx, thioredoxin reductase (TrxR), and nicotinamide adenine dinucleotide phosphate (NADPH).TrxR, the homodimeric flavoprotein, catalyzes the reduction of oxidized Trx and has three isoforms in mammals: cytosolic (TrxR1), mitochondrial (TrxR2), and Trxglutathione reductase (TrxR3, also called TGR), which is mainly found in the testis (Lu & Holmgren, 2014).TrxR1 is overexpressed in many types of cancer and provides a contribution to the formation of lung cancer (Jovanovi c et al., 2020;Ozgencli, Kilic, et al., 2019).Hence, TrxR1 has been considered as a biomarker and target for anticancer agent development (Ye et al., 2019).The aim of this study was to examine the cytotoxic, apoptotic, and metastatic effects of diffractaic acid in A549 cells and also to investigate whether the anticancer effect of diffractaic acid occurs through TrxR1 targeting.

| Cytotoxicity assay
To perform the cell viability assay, A549 cells were seeded at a density of 7.5 Â 10 3 cells per well in 200 μL of the medium into 96-well plates and incubated overnight to attach before treatment.The cells were exposed to different concentrations of diffractaic acid and carboplatin in a time-dependent manner (24 and 48 h).Its cytotoxic effect was evaluated by 2,3-bis-(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide (XTT) assay (11465015001, Cell Proliferation Kit, Roche, Basel, Switzerland).After the medium was removed, 50 μL of XTT solution (5 mL XTT reagent:1 mL electron coupling reagent) and 100 μL medium were added to each well, and the plates were incubated at 37 C and 5% CO 2 .Absorbance was measured at 470 nm using an Epoch Microplate Reader (BioTek, Winooski, VT, USA).The results were represented with their IC 50 values (the concentration that reduces cell viability by 50%), and each value was presented with the standard deviation (±SD) of three independent experiments.

| Protein determination and western blotting analysis
A549 cells were seeded at a density of 1 Â 10 6 cells/well into a cell culture petri dish and incubated at 37 C. Afterward, the cells were treated with diffractaic acid at IC 50 value and further incubated for 48 h.The cells were then washed with cold DPBS and incubated on ice for 5 min by adding 400 μL of RIPA Buffer (9806S, Cell Signaling Technology, Danvers, MA, USA) containing 1 mM PMSF.The cells were scrapped, sonicated briefly, and centrifuged at 14,000 g for 10 min, and the resulting supernatant was stored at À80 C (Altay et al., 2022).Total protein amounts were determined spectrophotometrically following Bradford's method (Bradford, 1976) using bovine serum albumin (A9647, Sigma-Aldrich) as a standard.
For western blot analysis, 40 μg protein was separated by molecular weight on 12% SDS-PAGE gel and then transferred to polyvinylidene fluoride (PVDF) membranes.After blotting, the membranes were blocked with 5% skim milk powder in TBST buffer (Tris-buffered saline, 0.1% Tween 20) at room temperature for 1 h.

| Statistical analysis
The statistical comparison of the results was performed by unpaired ttest and two-way analysis of variance (ANOVA) by using the Graph-Pad Prism Software version 7.0 for Windows (GraphPad Software, La Jolla CA, USA).Data were acquired from three independent experiments and expressed as the mean ± SD of the mean.A symbol (*) displays statistically significant changes.

| Antiproliferative effect of diffractaic acid on human lung cancer cell line (A549)
The cytotoxic effects of diffractaic acid and carboplatin, frequently used as a chemotherapeutic drug, against A549 were determined by XTT assay.Accordingly, A549 cells were dose-dependently treated with diffractaic acid for 24 and 48 h and with carboplatin at 24 h.The results revealed that diffractaic acid and carboplatin decreased the viability of A549 cells dose-dependently, compared with the control group (Figure 1A,B).The IC 50 values of diffractaic acid for 24 and 48 h were calculated as 79.47 ± 1.35 μg/mL and 46.37 ± 1.92 μg/mL, respectively.Also, the IC 50 value of carboplatin was determined as 62.93 ± 1.96 μg/mL at 24 h (Figure 1C).Morphological changes of A549 cells treated with diffractaic acid at their IC 50 values were shown in Figure 1D.The obtained results indicate that the best IC 50 value for diffractaic acid was determined as 46.37 μg/mL at 48 h.

| Apoptotic effect of diffractaic acid on A549 cell line
To investigate the antiproliferative effect of diffractaic acid on A549 cells due to the induction of apoptosis, an Annexin V-FITC/PI staining assay was performed using flow cytometry analysis.According to the XTT assay results, A549 cells were treated with IC 50 value of diffractaic acid for 48 h and were compared with the untreated (control) group.
Flow cytometry analysis results revealed that diffractaic acid-treated cells reduced the percentage of viable cells by 17.44% when compared with the control group.Furthermore, this lichen acid increased the percentage of early apoptotic cells by 4.63% and late apoptotic cells by 16.14%.However, the rate of necrotic cells decreased by 3.32% (Figure 2A,B).These data, confirming the XTT assay results, show that diffractaic acid induces total apoptosis in A549 cells after 48 h of incubation with IC 50 value and especially proceeded through late apoptosis.

| Induction of quantitative expressions of apoptotic genes by diffractaic acid in the A549 cell line
To determine the impact of diffractaic acid on the molecular mecha-  observed.These results confirm the flow cytometric analysis showing that diffractaic acid directs A549 cells into apoptosis.

| The suppressive effect of diffractaic acid on the migration of the A549 cell line
To examine the anti-migratory ability of diffractaic acid against A549 cells, a wound healing assay was performed.Accordingly, A549 cells were treated with the IC 50 value of diffractaic acid; then, microscope images were taken at different time points (0, 6, 12, 24, and 48 h) (Figure 4A).The results showed that diffractaic acid significantly suppressed the migration of the cancer cells at 12, 24, and 48 h, that is, the migration of A549 cells was reduced by 4.90% at 12 h, 9.05% at 24 h, 20.18% at 48 h compared with the control group (Figure 4B).These findings confirm the anti-migrative power of diffractaic acid over A549 cells.

| Impact of diffractaic acid on the gene expression, protein expression, and enzymatic activity of TrxR1 in the A549 cell line
The effect of diffractaic acid on TrxR1 was investigated at gene, protein, and enzymatic activity levels as the inhibition of TrxR1 is considered as a promising target for cancer therapy.qPCR and western blot analysis results revealed that diffractaic acid could not significantly change the gene and protein expressions of TrxR1 compared with the control cells (Figure 5A,B).Whereas, the enzymatic activity of TrxR1 measured by the DTNB method indicated that diffractaic acid significantly inhibited TrxR1 activity in A549 cells (Figure 5C).

| DISCUSSION
Cancer is the second leading cause of death worldwide following cardiovascular diseases (Nagai & Kim, 2017).Lung cancer has one of the highest mortality rates among all cancer cases (Sung et al., 2021).
Although cancer cells have different genomic and proteomic features, standard procedures such as surgery, radiotherapy, and chemotherapy have been widely applied in the treatment of cancer patients.Chemotherapeutic treatments are known to be effective in some cancer types.However, many disadvantages occur during treatment, such as undesirable side effects and resistance of cancer cells to chemotherapeutic drugs (Holohan et al., 2013;Senapati et al., 2018).Therefore, it has gained importance in the development of new chemotherapeutic strategies to identify anticancer agents with low side effects and high anticancer potential and is reliable and specific to cancer cells (Hong et al., 2018).More than 60% of current anticancer drugs are derived from natural sources such as plants, bacteria, fungi, and marine organisms (Cragg & Pezzuto, 2016).These natural products have many advantages, such as being readily available and relatively safe and having a lower incidence of side effects.There are still many natural resources including lichens that have not been adequately researched (Dinçsoy & Cansaran Duman, 2017;Shrestha et al., 2015).Recent studies have proposed that lichen secondary metabolites could be used as new chemotherapeutic agents in cancer treatment (Nguyen et al., 2014;Ozgencli, Budak, et al., 2019;Solárová et al., 2020).Diffractaic acid, a lichen secondary metabolite, has been claimed to be an anticancer agent performed with different cancer cells.Some preliminary cytotoxicity studies with diffractaic acid have been carried out (Brisdelli et al., 2013;Kızıl & A gar, 2017;Truong et al., 2014).But, there is no detailed study that can reveal its anticancer mechanism of action in the literature.For this reason, we aimed to scrutinize the anticancer effect of diffractaic acid on A549 cells in detail and to determine the possible protein target in the anticancer effect.
Here, we first investigated the cytotoxic effect of diffractaic acid on A549 cells and compared it with the commercial chemotherapeutic drug carboplatin.As indicated in the Results section, our results showed that the A549 cell line was dose-and time-dependently sensitive against diffractaic acid and its effective IC 50 value was 46.37 μg/mL at 48 h.Although a biphasic curve was exhibited at the 24 h treatment period with diffractaic acid in A549 cells, a linear decrease was observed at the 48 h treatment period.In other words, diffractaic acid enhanced cell proliferation up to 25 μg/mL in 24 h and started to show an inhibition effect after this dose.A similar situation has also been reported in the literature (Kalın et al., 2022a;Kim et al., 2021;Martínez-Martínez et al., 2016).Several studies reported that diffractaic acid displays different cytotoxic effects depending on the cell lines.Truong et al. (2014) declared the potent cytotoxic effect of diffractaic acid on HeLa (human cervical carcinoma), NCI-H460 (human lung cancer), and MCF-7 (human breast cancer) cell lines at 100 μg/mL.In the literature, it has been reported that diffractaic acid has a significant antiproliferative effect on A549 (lung cancer) and AGS (gastric cancer) cell lines up to a concentration of 100 μg/mL (without calculating the IC 50 value), but has no toxic effect on healthy human umbilical cord vascular endothelial cells (HUVEC) up to 200 μg/mL for 48 h (Kızıl, 2016;Kızıl & A gar, 2017).
In another study, the IC 50 value of diffractaic acid on glioblastoma cells was reported as 35.67 μg/mL (Emsen et al., 2018).Kalın et al. (2022a) reported that diffractaic acid significantly inhibited cell viability in human breast cancer MCF-7 and MDA-MB-453 cells, with IC 50 values of 51.32 μg/mL and 87.03 μg/mL, respectively.The cytotoxic effect of diffractaic acid on A549 cells obtained from this study is closely consistent with the literature.Also, the IC 50 value of diffractaic acid against A549 cells was determined for the first time.Compared with carboplatin, the antiproliferative effect of diffractaic acid on A549 cells had an IC 50 value close to carboplatin after 24 h incubation, while diffractaic acid had a more effective IC 50 value after 48 h incubation.
This result reveals the cytotoxic strength of diffractaic acid.
Apoptosis is a physiological programmed cell death required to eliminate damaged cells and triggered by both intrinsic (mitochondrial) and extrinsic (death receptor) molecular signaling pathways.The mitochondrial pathway is regulated by BCL2 family members including proapoptotic (such as BAX) and anti-apoptotic (such as BCL2) factors.The BAX/BCL2 ratio is associated with the susceptibility of cells to apoptosis, and an increase in this level induces apoptosis.Moreover, tumor suppressors including P53 play a critical role in cancer prevention by triggering apoptosis (Khodapasand et al., 2015;Pistritto et al., 2016).Necrosis is an unregulated and accidental cell death morphologically defined by an increase in cell volume, swelling of organelles, rupture of the plasma membrane, and loss of intracellular contents (Kroemer et al., 2009).In the literature, controlled death by induction of apoptosis rather than necrosis is a desired outcome in chemotherapy (Jung et al., 2022;Nabavinia et al., 2022;Valcourt et al., 2019).Although the research on the apoptotic effects of lichen secondary metabolites has been declared in the literature, the study investigating the effect of diffractaic acid is limited.In this study, we examined the apoptotic properties of diffractaic acid in A549 cells by flow cytometric analysis, as well as by measuring the expression of the apoptotic pathway genes BAX, BCL2, and P53.In  (Guan, 2015).To date, there is no study indicating the effect of diffractaic acid on the migration of lung cancer cells.Thus, in this stage, we investigated the anti-migrative effect of diffractaic acid on A549 cells and our results revealed the strong anti-migrative activity of diffractaic acid on the A549 cells.These findings suggest the idea of examining the metastatic pathway for the anti-migratory activity of diffractaic acid in further studies.
In normal cell metabolism, the presence of ROS is essential for various processes such as cell proliferation, differentiation, and signal transduction (Reczek & Chandel, 2015).However, its overproduction leads to the dysfunction of vital cellular processes and carcinogenesis with cell proliferation, invasiveness, and metastatic potential (Sharifi-Rad et al., 2020).To maintain the redox balance, the cells have enzymatic antioxidant systems including the Trx (TrxR, Txn, and NADPH).
Although TrxR1 has diverse functions in healthy organisms, including cell death, proliferation, and protection of cells against oxidative damage, overexpression of TrxR1 in cancer cells causes tumor progressions via cell proliferation, resistance to apoptosis, and metastasis.Thus, TrxR1 has been used as a prognostic marker in various cancer treatments (Chen et al., 2016;Rajavel et al., 2019;Ye et al., 2019).In our previous study, we indicated the inhibitory effect of diffractaic acid on mitochondrial TrxR purified from rat lung and found it to be much stronger than the commercial anticancer drugs doxorubicin and cisplatin (Ozgencli, Budak, et al., 2019).Next, we investigated for the first time the effect of diffractaic acid on gene expression, enzyme activity, and protein expression of TrxR1 on A549 cells.Our results revealed that diffractaic acid significantly inhibited the enzymatic activity of TrxR1 in A549 cells, but a non-significant change was observed in gene and protein expressions.The overall results suggest that diffractaic acid exerts its anticancer effect on A549 cells, especially via inhibition of TrxR1.

| CONCLUSIONS
Our current study revealed the cytotoxic, apoptotic, and antimigrative activities of diffractaic acid on A549 cells.The inhibition of TrxR1 enzyme activity by diffractaic acid could be considered as an inhibitor that binds to the active site of the enzyme or inhibits the enzyme with a covalent or non-covalent modification.This situation needs to be detailed in future studies.As a result, it can be said that diffractaic acid may be a new inhibitor for TrxR1.These findings also suggest that diffractaic acid may be a candidate chemotherapeutic agent as a TrxR1 inhibitor for the treatment of lung cancer.
control and diffractaic-treated A549 cells were harvested and lysed in 400 μL of RIPA buffer containing 1 mM PMSF.Two hundred microliters of K-phosphate buffer containing 100 mM potassium phosphate (Sigma-Aldrich, pH 7), 10mM ethylene diamine tetra acetic acid (EDTA, Sigma-Aldrich, pH 7), 100 μL NADPH (0.2 mM, Sigma-Aldrich), 100 μL BSA (0.2 mg/mL, Sigma-Aldrich), 100 μL DTNB (5 mM, Sigma-Aldrich), and 20 μL of supernatant were added to the test tubes, and the final volume was completed to 1000 μL with dH 2 O.The changes in TrxR1 enzyme activity were monitored spectrophotometrically at a wavelength of 412 nm every 3 min.TrxR1 activity is based on the detection of TNB (2-nitro-5-thiobenzoic acid) resulting from the reduction of DTNB per min.The molar extinction coefficient for TNB was 14.15 M À1 cm À1 .The production of 1 μmol of TNB per min under these conditions was used as an enzyme unit (EU) of TrxR1 activity.The specific activity of TrxR1 was measured as EU/mg of protein and expressed as a percentage of the control(Hukkamlı et al., 2023;Kalın et al., 2023;Sönmez Aydın et al., 2021).
nism of the apoptosis pathway, quantitative mRNA levels of BAX, BCL2, and P53 were examined by qPCR.Following the 48 h treatment of A549 cells with the effective IC 50 value of diffractaic acid, all of the gene expression levels were significantly elevated compared with the control group (Figure3A-C).As the increase in BAX/BCL2 ratio shows the apoptotic tendency of living cells, this ratio was also calculated.As shown in Figure3D, the elevated BAX/BCL2 ratio wasF I G U R E1 Effects of diffractaic acid and carboplatin on the viability of A549 cells.(A-B) Percentages of cell viability of human lung cancer cell line (A549) cells were determined by 2,3-bis-(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide (XTT) assay after treatment with different concentrations of diffractaic acid and chemotherapeutic drug carboplatin at 24 and 48 h.The control group was compared with the test groups and the cell viability of the treatment groups was normalized to the control groups.(C) The IC 50 values belong to diffractaic acid and carboplatin.(D) Inverted light microscope images (10Â objective) of A549 cells taken at 24 and 48 h after treatment with IC 50 concentrations of diffractaic acid.Data show the mean ± SD of three independent experiments.Scale bar, 100 μm.Statistical significance degrees (two-way analysis of variance [ANOVA]): p > 0.05 = (ns = not significant); *p < 0.05 significant; **p < 0.01 very significant; ***p < 0.0005; ****p < 0.0001 highly significant.