The expression profiles of apoptosis-related genes induced usnic acid in SK-BR-3 breast cancer cell

Abstract

This study aims to determine whether usnic acid (UA) could induce the expression of apoptosis-related genes in apoptosis pathway. The current study has enabled us to better understand the target of UA in the treatment of breast cancer. Cell viability was determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. Based on the previous study and the results of this study, UA had the most antiproliferative effect on SK-BR-3 breast cancer cell line. We examined differential expression of 88 apoptosis-related genes by quantitative real-time polymerase chain reaction using the apoptosis primary library panel in SK-BR-3 breast cancer cell. We observed a difference in the significant differential expression of 74 apoptosis-related genes in breast cancer after SK-BR-3 cells applied to UA (7.21 µM) for 48 h. The expression level of 56 of these 74 differentiated apoptosis-related genes increased (p < 0.05), but the expression level of the other 18 related genes decreased (p < 0.05). In order to evaluate the mechanism of apoptosis of UA, Western blot analysis was performed with Bcl-2, Bax, Caspase-3, and Caspase-9 antibodies. According to the Western blot analysis, we obtained similar results with gene-expression data. These results suggest that UA showed a cytotoxic effect in SK-BR-3 cells through activation of the mitochondrial apoptotic pathway. The obtained results from gene expression revealed that the effect of UA on apoptosis pathway is critical for clinical research.

Keywords

Usnic acid apoptosis qRT-PCR breast cancer

Introduction

Among noncommunicable diseases, cancer can be considered as unique and the most malignant disease that displays exponentially increasing incidence in recent years.¹ With the data from Globocan 2018, breast cancer is the most commonly diagnosed cancer for woman.¹ Breast cancer can be classified as Luminal A, Luminal B with HER-2 negative, Luminal B with HER-2 positive, HER-2 enriched, and basal-like (triple negative) according to the molecular structure.² Therapeutic approach should be designed regarding molecular structures of breast cancer subtypes.³ Most of the developed and investigated therapeutic approach should not be considered as just one solution for breast cancer treatment. Therapeutic approaches are classified into radiation therapy, surgery, and systematic therapies, which consist of immunotherapy, chemotherapy, hormonal and target therapy.³ Chemotherapy has been considered as the main solution of cancer treatment, but damage to healthy cells during treatment can cause many physical and psychological side effects on patients. For preventing drug resistance and increasing the side effect of the treatment, the cocktail of the chemotherapeutics has been used.⁴ However, researchers have been investigating different methods and alternative drug molecules to reduce the side effects of chemotherapeutics.

Many alternative pharmaceuticals candidate molecules have also developed for cancer treatment in recent years. Hu et al. determined apoptosis and inhibitory effect of flaxseed (Linum usitatissimum) on MCF-7 breast cancer cells.⁵ In their study, they presented decrease in cell viability after flaxseed extract application with dose-dependent manner.⁵ Wang et al. revealed the increased sensitivity of breast cancer to TNF-related apoptosis inducing ligand (TRAIL), which is a TNF superfamily member with a significant role in induction of tumor-selective cell death. They resulted in enhanced TRAIL induced apoptosis for gambogic acid in a dose-dependent manner.⁶ In recent years, lichens and their secondary metabolites have attracted attention as novel candidate molecules for cancer treatment.^7,8 Usnic acid (UA) is the most abundant lichen secondary metabolite, which plays significant roles in different biological activities, mainly antibacterial, antifungal, antiviral, cytotoxic, and antioxidant activity in addition to antiherbivore and insecticidal activity.^7,9 Its chemical property can be described as 2,6-diacetyl-7,9-dihydroxy-8,9b-dimethyldibenzofuran-1,3(2H,9bH)-dione.¹⁰ The ability to easily isolate high purity UA from different types of lichen species makes UA an important drug candidate molecule. Especially in recent studies, UA shows antiproliferative effects on different cancer cell lines.^10
–12 It has been reported that UA shows antiproliferative activity against MCF-7 and MDA-MB-231 breast cancer cells in recent studies.^13,14 Eskiler et al. investigated the effects of UA and tamoxifen or enzalutamide on MCF-7 breast cancer cells and LNCaP prostate cancer cells.¹⁵ The examined cancer cells have been treated with UA combined with tamoxifen or enzalutamide or tamoxifen and enzalutamide. According to study result, they determined a combination of drugs with UA can be considered as potential therapeutic treatment.¹⁵ Qi et al. showed that (+)-UA takes part in inducing ROS production by reducing Nrf2 stability in a way of stimulating PI3K/Akt pathway.¹⁶

The success of chemotherapy application for patients with late-stage metastatic breast cancer is dependent on cells being able to maintain their ability to undergo apoptosis.¹⁷ However, tumor cells inherit the ability to avoid apoptosis, and this is an underlying molecular reason contributing to disease progression and chemotherapy resistance.¹⁷ Development of therapeutic options that focus on apoptosis mechanisms is thus essential and crucial for cancer treatment. Since synthetic drugs have side effects on breast cancer treatment, natural compounds and apoptosis status have attracted much attention in recent years. Recent research studies were based on inhibiting the carcinogenesis through apoptosis of novel candidate molecules.¹⁸

There are many studies that determine the apoptotic effect of UA.^{10,11,15,16,19

–22} In these studies, the apoptotic effect of UA was generally determined by flow cytometry and Western blot methods, and in the literature there are no studies in which the apoptotic effect is defined on mRNA level of UA on breast cancer cells. We showed more reliable results by analyzing data based on gene expression modification after applied to UA on breast cancer by quantitative real-time polymerase chain reaction (qRT-PCR).

In this study, it was aimed to demonstrate the inhibitory proliferative activity of the UA on SK-BR-3 breast cancer and noncancerous breast epithelial cells (MCF-12A). Moreover, we also determined the gene expression level of 88 apoptosis-related genes on breast cancer cell line. This is the first article that fully screened the apoptosis pathway to determine the effect of UA on breast cancer cells. The results obtained from apoptosis primary library array are verified by Western blot analysis. By depending on the results, UA can be considered as a potential apoptosis-inducing agent and might be classified as a favorable drug candidate for breast cancer treatment.

Materials and methods

In previous study, the antiproliferative effect of UA was determined in BT-474, MCF-7, and MDA-MB-431 cells.¹² In this study, we evaluated the antiproliferative effect of UA on SK-BR-3 cancer cell and MCF-12A noncancerous breast epithelial cell. In order to develop a more detailed and effective treatment method based on UA on breast cancer, we determined the effect of UA on four different types of breast cancer subtypes.

Usnic acid

UA was obtained from Sigma (St. Louis, USA) and UA stock concentration (100 µM) was prepared in 0.1% dimethyl sulfoxide (DMSO) (v/v). Appropriate dilutions of stock solution (50, 25, 15, 12.5, 6.25, 3.125, and 1.562 μM) were made with DMSO.

Cell culture

Human SK-BR-3 breast cancer and MCF-12A noncancerous breast epithelial cell lines have been obtained from American Type Culture Collection (Manassas, Virginia, USA). The breast cancer cells were maintained in Dulbecco’s modified Eagle’s Medium (DMEM) (Sigma) supplemented with 10% fetal bovine serum (FBS) (Biological Industries, Israel) and 1% antibiotics (100 µg/mL penicillin and 100 µg/mL streptomycin) (Biowest, Nuaillé, France). MCF-12A noncancerous breast epithelial cells were cultured in DMEM Ham’s F12 (Sigma) with % 1 penicillin/streptomycin, 10 μg/ml insulin, 0.5 mg/ml hydrocortisone (PubChem, Washington, USA), 10% FBS, and 20 ng/ml epidermal growth factor (EGF, Goquick, Massachusetts, USA). Both cells were incubated at 37°C in a humidified atmosphere of 5% CO₂.

Cell proliferation assay

The viability of cells was measured by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) method (Sigma). Cells (1 × 10⁵) were seeded in 96-well plates and incubated for 24 h. Then different concentrations of UA (100, 50, 35, 25, 15, 12.5, 6.25, 3.125, and 1.562 µL) were added to each well for incubation at 37°C in 5% CO₂ for 48 h. Nontreated cells were used as experimental controls. After 48 h, MTT solution was added to the plate (0.5 mg/mL final concentration), examined cells were incubated for 4 h. At the end of 4 h, 100 µL of DMSO has been added to each well. Absorbance level was measured as 570 nm by using microplate reader (PerkinElmer 1420 Multilabel Counter, Massachusetts, USA). The experiments were analyzed according to the mean ± standard deviation of the cell growth inhibition percentage and GraphPad Prism 7.01 program.

Total RNA extraction and cDNA synthesis

Cells (5 × 10⁵ cells/well) were cultured in six-well plates and were treated with IC₅₀ concentrations of UA for 48 h. SK-BR-3 breast cancer and MCF-12A noncancerous breast epithelial cells were harvested for total RNA extraction by using Genezol Reagent (Geneaid, Taiwan) following manufacturer’s protocol. RNA samples were measured using Nanodrop ND-1000 Spectrophotometer (Thermo Fisher Scientific, Illinois, USA) and RNA integrity was evaluated by 1% agarose gel electrophoresis. Reverse transcription process was performed by using NG dART Kit (EURx, Poland) following manufacturer’s protocol. The conditions for this step were determined as 10 min at 25°C initially, 30 min at 48°C for amplification step, and 5 min at 95°C for the final step.

qRT-PCR assay

cDNA samples have been used for further analysis of 88 human apoptosis library primers and were determined by RT-PCR system (Roche, OR, USA). PCR cycling conditions are determined as follows: 95°C for 10 min, for 40 cycles amplification 10 s at 95°C, 30 s at 60°C, and 8 s at 72°C. The expression levels of these 88 apoptosis-related genes were standardized to GAPDH housekeeping genes. ΔΔCt method was used for quantification.²³ All reactions were performed in triplicate. The data analyzed with analysis of variance test and value of p < 0.05 was considered as statistically significant.

Western blot analysis

SK-BR-3 breast cancer cells were treated with IC₅₀ concentrations of UA for 48 h. Cells were harvested and washed with cold phosphate-buffered saline. Proteins from cell pellets were isolated by applying lysis buffer, which contains 7 M urea, 2 M thiourea, 4% chaps, 1% DDT, 2% ampholyte, and supplemented with protease inhibitor. The percentages of the proteins were quantified by using BCA^TM Protein Assay Kit (Merck, Germany). By using 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis, equal amounts of protein obtained from each sample have been separated. Following the separation process, isolated proteins were transferred to nitrocellulose membrane (Thermo Fisher Scientific, Illinois, USA) and then blocked for 1 h using 5% nonfat milk. As control, membrane was incubated at 4°C including diluted anti-Bcl-2, Bax, Caspase-3 and Caspase-9 primary antibodies (Santa Cruz Biotechnology, Texas, USA) and GAPDH primary antibodies (Cell Signaling, Massachusetts, USA). Then, by using Tris-Buffered Saline Tween, membranes were washed for three times. After that step, membranes were incubated again at room temperature for 1.5 h with the application of horseradish peroxide conjugated secondary antibodies (Abcam, Cambridge, MA, USA). In the end of Western blot step, for visualization of the immunoreactive bands, chemiluminescence kit (Thermo Fisher Scientific, Illinois, USA) was used. The immunoreactive bands have been visualized and evaluated with ImageJ program version 1.41.

Results

Cell viability analysis

In order to determine the effect of UA, MTT assays were performed to four different breast cancer cell lines. We revealed the proliferative effect of UA on three breast cancer cells (BT-474, MCF-7, and MDA-MB-431) in our previous study.¹² Additionally, in this study, we included the SK-BR-3 breast cancer cell with different receptor status and noncancerous breast epithelial cell (MCF-12A). Thus, we determined the effect of UA with a detailed approach, using four breast cancer cells with different subtypes. In the result of the MTT assay, IC₅₀ values have been determined as 13.11, 12.65, and 12.84 for MCF-7, BT-474, and MDA-MB-431, respectively, in the previous studies.¹² In order to determine and prove the effect of UA on SK-BR-3 breast cancer cell and MCF-12A noncancerous breast epithelial cells, MTT assay used for time- and concentration-dependent manner. It should be noted that the 6.25 and 12.5 μM concentrations of UA treatments after 48 h showed significant antiproliferative effects on the SK-BR-3 breast cancer cell line (p < 0.05) (Figure 1). Using the GraphPad Prism 7.01 program, IC₅₀ values were calculated as 7.21 μM and 3.4 μM for UA on the SK-BR-3 and MCF-12A noncancerous breast epithelial cell lines, respectively. There was no significant decrease in cell viability of the MCF-12A cell lines at the increasing concentrations/times of UA application, as shown in Figure 1. We determined that UA showed 67.4% cell death rate on SK-BR-3 breast cancer cell lines (Figure 1). It was a promising result for UA to have a high antiproliferative effect on SK-BR-3 ER(−) and HER2(+) breast cancer subtype. UA breast cancer is very effective on the breast cancer cell and its low destructive effect on the noncancerous cell is an indication that UA can be a promising candidate molecule in the treatment of breast cancer.

Figure 1.

Effect of UA on the viability of the SK-BR-3 cancer cell line and MCF-12A noncancerous breast epithelial cell. Cells were treated with different concentrations of UA derivatives for 48 h. Each value is the mean (±SE) of three experiments performed in duplicate. Statistical significance was determined with ANOVA and is marked with *. After 48 h, three concentrations of UA (6.25, 12.5, and 25 μM) showed significant effect on SK-BR-3 breast cancer cell (*p < 0.05). While UA showed 67.4% cell death on SK-BR-3 breast cancer cell, there was no significant reduction in cell viability of MCF-12A noncancerous breast epithelial cell. ANOVA: analysis of variance; UA: usnic acid.

Gene expression analysis

In the present study, we analyzed the expression of 88 genes (apoptosis primary library array) following treatment of SK-BR-3 breast cancer cell line with UA (Table A1 in the Online Supplemental Material). The seven genes in the apoptosis array primary library, BAG3, TRAF6, TNFSF7, TNFSF1, TNFSF15, ATM, and TP73L, showed no expression changes after application of UA.

As a result of this study, apoptosis-related genes were divided into six groups depending on the gene families. In Caspase family, seven upregulated (CASP1, CASP2, CASP3 (p < 0.05), CASP4 (p < 0.05), CASP5, CASP10 (p < 0.05), and CASP8AP2 (p < 0.05)) and four downregulated (CASP6, CASP7 (p < 0.05), CASP8 (p < 0.05), and CAPS9 (p < 0.05)) genes were identified in SK-BR breast cancer cell upon UA treatment (Figure 2(a)). CASP3 gene has highest fold change level (15.21) (p < 0.05) (Figure 2(a)). On the other hand, among downregulated genes, CASP9 is the most downregulated gene with the fold change level of −11.49 (p < 0.05) (Figure 2(a)). Among Kinase family (Figure 2(b)), no downregulation has been observed in SK-BR cell in the presence of UA. In addition, among those upregulated genes (CHEK1, CHECK2, DAPK2, and RIPK2), RIPK2 showed higher fold change rate in comparison to other apoptosis-related genes (p < 0.05) (Figure 2(b)).

Figure 2.

UA induces apoptosis in SK-BR-3 breast cancer cells at mRNA level. The ratio of fold change has been determined by qRT-PCR assay. Relative fold change of (a) Caspase family consists 11 genes, 7 genes were upregulated and 4 genes were downregulated, (b) Kinase family, all of the 5 genes were upregulated, (c) Bcl-2 family, 7 genes were upregulated and 6 genes were downregulated, (d) TRAF family in which all of the genes were upregulated, (e) TNF family,18 genes were upregulated and 3 genes were downregulated, (f) different genes family, 15 genes were upregulated and 5 genes were downregulated. Results are statistically significant when *p < 0.05 in comparison to control group.

BCL2 family consists of 13 genes, which are divided into two subgroups as 7 upregulated (BCL2L2, BCL10, BAK1, BAX, BOK, MCL1, and HRK) and 6 downregulated (BCL2, BCL2A1, BCL2L11, BAG1, BAG3, and BAG4) genes (Figure 2(c)). MCL1 mRNA level was significantly higher (fold change 13.91) in the SK-BR cell (p < 0.05). UA did not significantly change the expression level of the BCL2LA1 gene, but this gene was found to show a −5.37 fold change compared to other downregulated genes in the BCL2 family (p < 0.05) (Figure 2(c)). In the Kinase family, TRAF genes only consist of upregulated genes after application of UA. Of these four upregulated genes (TRAF3, TRAF5, TRAF6, and TANK), TRAF6 showed the highest upregulation with 17.42 fold change levels (p < 0.05) (Figure 2(d)).

In the apoptosis array library, 21 genes belonging to the TNF family were determined after the expression of UA (TNF, TNFRSF11B, TNFRSF1A, TNFRSF21B, TNFSF6, TNFSF7, TNFRSF8, TNFSF8, TNFRSF9, TNFSF10, TNFRSF10A, TNFSF8, TNFRSF9, TNFSF10, TNFRSF10A, TNFSF10, TNFFFF TNFSF15, TNFSF4, CD40, FASLG, LTBR). In these genes, only the expression level of TNF, TNFSF7, and TNFRSF10D has been downregulated (Figure 2(e)). Among these genes, TNFSF8 gene showed significant upregulation with the fold change level of 21.44 (p < 0.05) (Figure 2(e)). Among all of these 73 apoptosis-related genes, TNFSF8 gene has the highest upregulation regarding fold change rate (p < 0.05) (Figure 2(e)). In addition, TNFSF6 can be considered as the most downregulated gene among all of those genes (p < 0.05) (Figure 2(e)).

For those which cannot be classified into a family, we grouped them as different genes. In different genes group, there are 20 genes, which consist of 15 upregulated (APAF1, ATM, BFAR, CARD4, BIK, CRADD, DFFA, DFFB, TP53, FADD, TP73L, GADD45A, RPA3, CIDEA, and CIDEB) and 5 downregulated genes (BRE, BIRC1, BIRC2, BIRC3, and BIRC5) (Figure 2(f)). After UA application on SK-BR-3 cells, GADD45A showed significantly highest up regulation (p < 0.05) (Figure 2(f)) among upregulated genes, while, BIRC2 was presented as most down regulated gene (p < 0.05) (Figure 2(f)) among all genes.

Western blot analysis

To determine the mechanisms underlying the apoptosis induced UA, we evaluated the expression of Bcl-2, Bax, Caspase-3, and Caspase-9 protein markers in SK-BR-3 (Figure 3(a)). As shown in Figure 3(b), treatment of UA on SK-BR-3 cell led to decreased Bcl-2 level. Bax was upregulated in SK-BR-3 cell treatment with UA (Figure 3(b)). Activation of apoptosis signaling by UA Caspase-3 and Caspase-9 protein levels was increased (Figure 3(b)).

Figure 3.

Apoptosis-related proteins were analyzed by Western blot. (a) The expression levels of apoptosis-related proteins after application of UA on SK-BR-3 cells. (b) Relative protein change of Bcl-2, Bax, Caspase-3, and Caspase-9 in comparison to GADPH (control). *p < 0.05 in comparison to control group.

Discussion

About 50% of natural products were approved by FDA and these natural products have been used in the drug market for human health.²⁴ However, candidate drug molecules could be limiting for treatment since they cannot be easily produced in their pharmacologically active form in higher quantities.^25,26 Researchers are increasingly searching for new effective candidate drug molecules from biological organisms instead of synthetic molecules. Lichens, which are the most efficient biological organisms, are the symbiotic organisms with mycobiant, algae or cyanobacterium.²⁷ In recent years, researchers have shown that lichen secondary metabolites have biological activity and their metabolites have advantages and become an important reason for preference for treatment against cancer disease.²⁴ One of the advantages, lichen secondary metabolite stems from never-ending potential biological source of chemical content with a variety of pharmacological activities of drug industries. Lichen secondary metabolites could play a significant role especially in cancer treatment because they are usually less toxic than traditional chemotherapy agents such as tamoxifen and paclitaxel, effective, inexpensive, and easily available.²⁷ Many studies indicated that UA has anticancer properties on different cancer cells. In this study, we evaluated antiproliferative effect of UA on SK-BR-3 breast cancer cell and noncancerous breast epithelial cell (MCF-12A). The results showed that UA exhibited moderate to high levels of anticancer activity on mainly SK-BR-3 breast cancer cell, without damaging MCF-12A noncancerous breast epithelial cell.

Carcinogenesis is related to an inequality between cell apoptosis and proliferation stage. In this study, we showed that UA induced apoptosis in breast cancer cell as confirmed by qRT-PCR and Western blot assay. Many studies have been reported to play an important role in apoptosis applied to UA. Previous studies have determined that UA inhibits high proliferation and induces apoptosis in various cancer cells, such as lung cancer cell (A549)²²; human melanoma (HTB-140) and human prostate cancer (DU-145, PC-3) cell lines²⁸; LUSC cells¹⁶; human gastric cancer AGS and SNU-1 cells¹⁹; breast (MCF-7), cervical (HeLa), and prostate cancer (PC-3) cell lines¹⁰; K562/ADR cells²⁹; HEPG2 and SNU-449 hepatocarcinoma cells³⁰; HTB-140 (Hs 294T)-human melanoma derived from metastasis to lymph node; DU145 (HTB-81)-human carcinoma derived from brain metastasis and PC-3 (CRL-1435)-human adenocarcinoma derived from bone metastasis and two normal types of cells: human skin fibroblasts and normal human prostate epithelium (PNT2)²⁸; MCF-7³¹; CaCo2, HepG2, Hep2C, Rd, Wehi¹¹; HCT116 and LS174³²; T47D and MCF-7³³; MCF-7, MDA-MB-231, BT-474¹². This is the first study in which the anti proliferative effect of UA on SK-BR-3 breast cancer cell lines determined with the contribution of our previously studied results. Furthermore, previously mentioned studies explain the induced effect of UA on apoptosis pathway with flow cytometry analysis on cell culture-based and protein results by performing Western blot analysis. Despite increasing research on the apoptotic effect of UA to the different cancer cells, there is not available explored on full apoptotic pathway bu using qRT-PCR on breast cancer. Thus, we evaluated transcriptional modulations induced of breast cancer cell to UA treatments. We have also found crucial therapeutic strategies by depending on the effect of UA on apoptosis pathway.

It has been proved that the damaging effect of UA on breast cancer cell lines is more considerable rather than human noncancerous breast epithelial cell. Studzińska-Sroka et al. examined the effect of physodic acid on MCF-10A cell and Ebrahim et al. determined the effect of norstictic acid on MCF-10A.^34,35 These studies suggested that there is no significant cytotoxic effect on noncancerous MCF-10A cells when physodic acid and norstictic acid applied. In this study, the effect of UA on MCF-12A noncancerous breast epithelial cell was obtained for the first time in the literature and it was determined that UA did not harm noncancerous breast epithelial cell while showing antiproliferative effect on breast cancer.

Ebrahim et al. demonstrated that norstictic acid, which is ethanol extracted of Usnea strigosa, has anticancer effect on MDA-MB-468, BT-474, MCF-7, MDA-MB-231, T-47D, and SK-BR-3 cells.³⁵ The highest antiproliferative effect of norstictic acid among all of these six breast cancer cell lines can be demonstrated in MDA-MB-231 and MDA-MB-468 cells. The invasion of MDA-MB-231 cells through basement of membrane and the migration of MDA-MB-468 cells are inhibited by norstictic acid. In addition, norstictic acid has no crucial cytotoxic effect on human nontumorigenic MCF-10A cells. Furthermore, c-Met, STAT3, paxillin/Rac-1, and FAK phosphorylation have also been suppressed by norstictic acid in MDA-MB-231 cells.³⁵ In contrast to Ebrahim et al.³⁵, we have studied similar breast cell lines but different lichen secondary metabolite. We have found the antiproliferative effect of UA on SK-BR-3 cells and there was no crucial cytotoxic effect on MCF-12A noncancerous breast epithelial cells. In addition, the major difference between our study and Ebrahim et al. can be considered as the effect of the UA specified study on SK-BR-3.³⁵

Through the regulation of gene expression, which are related to apoptosis such as Bcl-2 family products anti/pro-apoptotic proteins, p38, and p53,^8,11 lichens play crucial role as apoptosis activators in various cancer cell lines by depending on autonomous cell death.^8,36 Dinçsoy and Cansaran-Duman studied the antiproliferative effect of UA on nonmalignant cell lines (Vero, L929) and cancer cell lines (CaCo2, RD, Hep2C, HepG2, and Wehi).¹¹ After application of UA on Hep2C cell line, they observed threefold decrease in Bax gene while Bcl-2 antiapoptotic gene and p53 tumor suppressor gene decrease fivefold. In addition, in RD cell line, there was 8-fold and 2.5-fold decrease in Bcl-2 and p53 genes, while Bax gene increased in 2-fold. In Wehi cell line, it was 2.5-fold increase in Bax gene while they observed 8.5-fold and 9.5-fold decrease in p53 and Bcl-2 genes, respectively. Kılıç et al. examined the effect of vulpinic acid on breast cancer cells (MDA-MB-231, MCF-7, SK-BR-3, and BT-474) and noncancerous cell lines (MCF-12A).¹² After vulpinic acid treatment on SK-BR-3 cell line, they have resulted in 14-fold increase in p53 and Bax gene expression and additional 3-fold increase in BIRC-5 gene. For Caspases, a threefold increase in Casp-8, fourfold increase in Casp-7, and fivefold changes in Casp-3 have been observed. Casp-9 gene showed least change among them with twofold increase. In comparison to Kılıç et al.¹², in this study, UA lichen secondary metabolite has been examined on the same breast cancer and noncancerous cell lines. Du et al. have investigated the effects of berberine and evodiamine on MCF-7 cells by performing MTT assay.³⁷ They have resulted in increased expression in p53 and Bax and reduced expression on Bcl-2 when applied combined treatment. Kumar et al. discussed the anticancer effect of the UA and molecular alterations of human gastric adenocarcinoma AGS and gastric carcinoma SNU-1 cells.¹⁹ By applying UA, they have resulted in significant increase in mitochondrial membrane depolarization and apoptotic cells. In addition, apoptosis induction has been linked with the increased ratio of Bax:Bcl2 expression and PARP cleavage.¹⁹ Kumar et al. clarified the significant role of UA in DNA damage with increased results of DNA damage response proteins including DNA-PKcs, pATM (Ser1981), Chk-2 and p53 quantities.¹⁹ The results we obtained from this study were similar to the literature.

In the study by Geng et al., the expression levels of apoptosis-related proteins (activated caspase-3 and PARP, Bax, and Bcl2) and autophagy-associated proteins (LC3-II and p62) were verified through Western blot analysis.³⁸ Western blot analysis revealed that the protein expression levels of Bax, activated caspase-3, and PARP were increased, but the level of Bcl-2 in GC cells was decreased, which led to cell apoptosis.³⁸ Similarly with Geng et al. results, in our study we have observed increase in Bax and activated Caspases while reduction in Bcl-2 by Western blot analysis.

The results of our study suggest that UA could be a promising molecule for the treatment of breast cancer, because UA could significantly prevent growth by inducing apoptosis in breast cancer cells. The side effect and insufficient of drugs used in breast cancer treatment, UA as a natural agent has a strong possibility to be developed into an effective therapeutics for breast cancer. Further studies are still needed to determine the potential clinical application and clarify their beneficial effects in breast cancer.

Supplemental material

Supplemental_material_Table_1_25.03.2020 - The expression profiles of apoptosis-related genes induced usnic acid in SK-BR-3 breast cancer cell

Supplemental_material_Table_1_25.03.2020 for The expression profiles of apoptosis-related genes induced usnic acid in SK-BR-3 breast cancer cell by RŞ Özben and D Cansaran-Duman in Human & Experimental Toxicology

Footnotes

Acknowledgment

The authors thank Ankara University, Management of Scientific Research Projects, for the financial support.

Declaration of conflicting interests

The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Funding

The author(s) disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: This work was financially supported by Ankara University, Management of Scientific Research Projects (Project No. 16H0415002).

ORCID iD

D Cansaran-Duman

Supplemental material

Supplemental material for this article is available online.

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