• Synergistic effects of ursolic acid and selenium nanoparticles on the inhibition of metastasis-associated gene expression in MDA-MB-231 breast cancer cells
  • Farnaz Azimi,1 Marzieh Sanaei,2 Ahmad Reza Shahverdi,3 Mohammad Hossein Yazdi,4,*
    1. Department of Pharmaceutical Biotechnology and Biotechnology Research Center, Faculty of Pharmacy, Tehran University of Medical Sciences, Tehran, Iran.
    2. Department of Pharmaceutical Biotechnology and Biotechnology Research Center, Faculty of Pharmacy, Tehran University of Medical Sciences, Tehran, Iran.
    3. Department of Pharmaceutical Biotechnology and Biotechnology Research Center, Faculty of Pharmacy, Tehran University of Medical Sciences, Tehran, Iran.
    4. Department of Pharmaceutical Biotechnology and Biotechnology Research Center, Faculty of Pharmacy, Tehran University of Medical Sciences, Tehran, Iran.


  • Introduction: Breast cancer, accounting for 11.7% of all cancer cases in 2020 according to the World Health Organization, remains one of the leading causes of cancer-related mortality among women (Dai et al. 2017) . Breast cancer is further classified into subtypes based on receptor expression, including progesterone receptor (PR), human epidermal growth factor 2 receptor (HER2), and estrogen receptor (ER). Among these, triple-negative breast cancer (TNBC) is particularly aggressive due to the lack of these receptors, affecting 15–20% of patients. TNBC tumors typically exhibit a high histologic grade and an increased rate of early relapses, often diagnosed at an advanced stage (Singh and Yadav 2021) . In contrast to receptor-positive tumors, TNBC tumors are considered more aggressive and incapable of treatment. Patients with TNBC who developed distant metastases had a low overall survival rate of around eighteen months (Vagia, Mahalingam, and Cristofanilli 2020) . Breast tumor mortality is often caused by its ability to spread to other parts of the body, known as metastasis. This process consists of various stages including (i) tumor cells’ transformation by degradation of extracellular matrix (ECM), (ii) intravasation that is denoted by tumor cells’ migration to vessels, (iii) extravasation, and (iv) proliferation in the target organ (Van Zijl, Krupitza, and Mikulits 2011) , during which cancerous cells spread from the primary tumor site to other parts of the body via the lymphatic system, bloodstream, or direct extension. The poor prognosis associated with TNBC stems from its aggressive nature and the limited success of conventional treatments, such as chemotherapy, which often result in significant toxicity and minimal improvement in survival rates. Moreover, it harbors noticeable toxicity. Therefore, discovering a new TNBC treatment strategy is critical to increasing life expectancy (Chien et al. 2018; Wang et al. 2019) . Natural products, derived from plants, animals, and microorganisms, have shown significant potential in cancer prevention and treatment (Yao et al. 2020) . They interfere with cancer initiation, development, and progression through mechanisms such as cellular proliferation, differentiation, apoptosis, angiogenesis, and metastasis (Muhammad et al. 2022) . These compounds offer a multi-targeted approach, modulating various signaling pathways involved in cancer, such as NF-κB (Nuclear Factor Kappa B), PI3K/Akt (Phosphatidylinositol 3- Kinase/Protein Kinase B), MAPK (Mitogen-Activated Protein Kinase), and JAK/STAT (Janus Kinase/Signal Transducer and Activator of Transcription) pathways, and can induce apoptosis, inhibit angiogenesis, and prevent metastasis. Combining natural compounds with conventional therapies can enhance efficacy, reduce toxicity and overcome resistance compared to monotherapy (Naeem et al. 2022; Deng et al. 2020) . Ursolic acid, a pentacyclic triterpenoid, is a versatile compound found in various medicinal plants such as Prunella vulgaris, Nepeta cataria and Lavandula stoechas, fruits and vegetables with a wide range of pharmacological activities, including anti-cancer, anti- inflammatory, antioxidant, and neuroprotective effects (Ryu et al. 2000) . It demonstrates significant potential in inhibiting metastasis across various cancer types, including colorectal cancer, non-small cell lung cancer, and breast cancer through various mechanisms, including induction of apoptosis, cell cycle arrest, and inhibition of key signaling pathways (Yin et al. 2018; Zou et al. 2019) . According to a study in 2010, UA has shown anti-invasive effects on highly metastatic breast MDA-MB-231 cells by inhibiting the phosphorylation of Jun N- terminal kinase, Akt, and the mammalian target of rapamycin. In addition, it appears to reduce nuclear NF-κB protein levels, followed by downregulation of MMP-2 and u-PA expression (Yeh, Wu, and Yen 2010) . Furthermore, it has been shown that UA inhibits the proliferation of colorectal cancer cells by targeting multiple cells signaling pathways, including NF-κB, STAT3, and β-catenin, and downregulating proteins associated with cell survival, and metastasis (Prasad et al. 2012) . Thus, the use of UA could enhance such therapeutic strategies by amplifying targeted effects on cancer cells while reducing side effects, positioning it as a valuable natural component in cancer treatment (Shanmugam et al. 2013; Wang et al. 2012) . Cell adhesion molecules (CAMs) are considered crucial to preserving tissue structure and function. CAMs participate as a binding agent in which they mediate cell-to-cell or cell-to- extracellular matrix (ECM) binding (Ren, Roberts, and Shi 2011) . Among them, intercellular adhesion molecule-1 (ICAM-1), a transmembrane glycoprotein of the immunoglobulin (Ig)- like superfamily, demonstrates overexpression in highly metastatic cancers (Schröder et al. 2011) such as TNBC, around 8 to 25-fold higher (Guo et al. 2014) . It has been claimed that ICAM-1 lead to the interaction between leukocytes and endothelium, which facilitates tumor cells’ extravasation (Figenschau et al. 2018; Reina and Espel 2017) . Matrix metalloproteinases (MMPs), other biomarkers of invasive cancers, are from the zinc- dependent endopeptidases involved in tissue remodeling, and cancer development. MMPs elevate the cancer invasion rate by reducing cell adhesion through the degradation of the ECM which acts as a physical barrier to tumor metastasis (Tang, McMullen, and Brindley 2019) . As reported in several studies, MMP-2 and MMP-9 are significantly increased in various metastatic cancers, leading to the eradication of basement membrane (BM) and ECM and, in turn, cancer metastasis (Vizoso et al. 2007; Katunina et al. 2011; Parsons et al. 1998) . Selenium (Se) is a trace element that plays a crucial role in living organisms. Among different forms of Se, red elemental selenium (Se 0 ), known as selenium nanoparticle (SeNP) has greatly attracted attention due to its therapeutic advantages, including anti-cancer, antioxidant, anti-inflammatory and antimicrobial effects. Moreover, nanosized Se is more bioavailable, affordable, and less toxic than other selenium-containing compounds (Forootanfar et al. 2014) . SeNPs can be synthesized through different methods, including physical, chemical, and biological. Chemical synthesis is mostly a common and rapid method. In contrast, microbes and plants are used for the biological synthesis of SeNPs (Skalickova et al. 2017) . Reports suggested that oral administrations of SeNP in affected mice by metastatic breast tumors enhanced their immune response and reduced metastasis to the liver (Faghfuri et al. 2015; Yazdi et al. 2012) . In this study, we aimed to examine the potential synergistic effects of Ursolic acid in combination with SeNPs on the reduction of invasion rate of MDA-MB-231 cells as a highly invasive breast cancer cell line and a good model of TNBC (ER − , PR − , HER2). To evaluate this hypothesis, the expression levels of MMP2, MMP9, and ICAM-1 were investigated by quantitative real-time PCR.
  • Methods: Cell culture condition Human breast cancer cell line MDA-MB-231 (IBRC C11325) was obtained from the Iranian Biological Resource Center (IBRC), (Tehran, Iran). The cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM) (Gibco, Germany) supplemented with 10% fetal bovine serum (FBS) (Gibco, Germany), 100 Units of penicillin/ml (Gibco, Germany), and 100 μg of streptomycin/ml (Gibco, Germany), then incubated at 37°C with 5% CO 2 . Selenium nanoparticles’ preparation According to previous study, biological SeNPs were produced using Lactobacillus plantarum (Zare et al. 2023) . Briefly, 1 mL of a 254 mM stock solution of SeO2 along with 1 mL of L. plantarum culture with OD 600 = 1 was added to 100 mL of fresh MRS broth and incubated at 37°C for 72h. To release the intracellular red elemental selenium, L. plantarum cell pellets were broken with liquid nitrogen and ultrasound treatment. SeNPs were purified using an n- Octyl alcohol/water extraction system (Shakibaie et al. 2010) . The SeNPs solution was stored at 4° C until use. Ursolic acid preparation 10 mg of ursolic acid was solved in 500 µL DMSO to prepare a stock solution. In the next steps, high glucose DMEM culture medium without FBS was used to dilute the stock solution and prepare the desired concentrations. MDA-MB-231 cell viability assay Cell viability was evaluated by MTT assay (Sigma, United Kingdom). To this end, 2×10 4 MDA-MB-231 cells/well were seeded in 96-well cell culture plates and incubated at 37° C for 24 h. Then, the medium was changed with the fresh one containing different concentrations of SeNPs (50,100,150,200,250 µg/mL) and ursolic acid (5,10,15,20,25 µg/mL). The treated cells were incubated separately for 24, 48, and 72 h to evaluate the time dependency of the treatment. 20µL MTT reagent (5 mg/mL) was added to each well after the mentioned times. Then the plates were further incubated for 2 h in darkness. Finally, the complete cell culture medium was replaced with 200 µL of dimethyl sulfoxide (DMSO) (Sigma, United Kingdom). After 15 minutes, the absorbance was measured at 570 nm to quantify the amount of formazan crystals. The cell viability percentage of treated cells was calculated using the following formula: %Cell viability ×100 RNA extraction and cDNA synthesis Approximately 2.5 × 10 5  MDA-MB-231 cells/well were seeded in three separate six-well plates and incubated for 4h. Each plate was treated with one of three protocols including 1) SeNPs group (150 µg/mL SeNPs), 2) ursolic acid group (20 µg/mL ursolic acid), and 3) combined group (150 µg/mL SeNPs + 20 µg/mL ursolic acid) and incubated for 24, 48, and 72 h. Total RNA was extracted using TRIzol reagent (Sinaclon, Tehran, Iran) according to the method previously described (Sanaei et al. 2021) . Isolated RNA was evaluated using Nanodrop with a spectrophotometer. The complementary DNA (cDNA) was synthesized from 3 μL of total RNA by incubation in a thermocycler for 1 h at 43°C with M-MLV reverse transcriptase enzyme (Fermentas, Lithuania) according to the manufacturer's instruction. Then, to visualize the results of the PCR product, amplificated cDNA with gene-specific primers (Table 1) were run through 1.5% agarose gel electrophoresis with ethidium bromide staining (Meryer, Shanghai). Real-time polymerase chain reaction Quantitative real-time PCR was performed with 25 μL of the PCR reaction mix, according to the manufacturer’s instructions. Briefly, each PCR reaction contains 12 μL of SYBR Green master mix, 2 μL of primers (Table 1), 3 μL of cDNA template, and 8 μL doubled distilled water. The PCR procedure consisted of 95°C for 10 min followed by 40 cycles of 95°C for 15 seconds, 60°C for 1 minute, and 72°C for 5 min. All the tests were run in triplicates on an ABI 7500 Fast Real-time PCR system. Data were analyzed by a comparative threshold (C t ), using the 2 −ΔΔCT method (Livak and Schmittgen 2001) and the mean fold inductions of samples were compared with the untreated samples. Scratch assay Cells were plated into a 6-well cell culture plate and incubated up to the approximate confluence of 90%. To minimize cell proliferation, 5 percent of FBS was used in culture media. Then, cells were carefully scratched using a yellow pipette tip, and cellular debris was washed away with PBS (Di et al. 2016) . Subsequently, cells were treated with 150 μg/mL SeNPs, 20 μg/mL ursolic acid, or their combination of them. Each cell-containing well was monitored during the following time (0, 24, 48, and 72 h) by taking the photos under an inverted microscope (Olympus Corp.), and the closure of the wound in each treated cell was compared with the control to observe the potential of cell migration. Statistical analysis Statistical analysis was performed using GraphPad Prism software version 8.4.3 (GraphPad Software, San Diego, CA, USA). Data were reported as group means ± SEM and were analyzed via two-way ANOVA followed by Tukey’s test. Differences were considered significant when the p-value was < 0.05.
  • Results: Cell viability by MTT assay To evaluate the cytotoxic effect of different doses of SeNPs (50-250 µg/mL), ursolic acid (5- 25 µg/mL), and the combination of them (SeNPs 50-200 μg/mL+ UA 20 μg/mL) on MDA- MB-231 cells, MTT assay was performed. A significant decrease in MDA-MB-231 cell viability was observed in cells treated with 150 μg/mL of SeNPs (Fig.1a), 20 μg/mL of UA (Fig.1b), and the combination dose of 150 μg/mL SeNPs + 20 μg/mL UA (Fig.1c). The percentage reduction in cell viability for SeNPs, UA, and combined group treatments was about 90%, 70%, and 80%, respectively compared to the control group (Fig 1). Therefore, the most effective doses of SeNPs and UA on cell viability were obtained from MTT analysis and are considered for performing the following experiments on expression and metastasis development. Effect of SeNPs and Ursolic acid on MMP-2, MMP-9, and ICAM-1 expression levels Our study aimed to investigate the effect of SeNPs and UA on tumor metastasis and invasion. In this regard, the expression levels of genes involved in tumor invasion, including MMP-2, MMP-9, and ICAM-1 were measured by real-time PCR. MDA-MB-231 cells were incubated in three separate cell culture plates with selective concentrations of 150 μg/mL SeNPs, 20 μg/mL ursolic acid, and a combination of 150 μg/mL SeNPs + 20 μg/mL UA for 24, 48, and 72 h (Fig 2). As shown in Fig. 2a, MMP-9 expression level was significantly decreased in all treated cells compared to the control group (**** p < 0.0001). Likewise, MMP-2 expression level indicates significant reduction after treatment with 20 μg/mL UA and the combination of 150 μg/mL SeNPs plus 20 μg/mL UA (**** p < 0.0001) and 150 μg/mL SeNPs (** p < 0.001, * p < 0.05) (Fig. 2b). Additionally, the results of ICAM-1 expression showed a significant reduction after incubation with the combination of 150 μg/mL SeNPs plus 20 μg/mL UA (**** p < 0.0001) and 20 μg/mL UA alone (** p < 0.001). However, 150 μg/mL SeNPs did not significantly affect ICAM-1 expression levels (Fig. 2c). Of note, the most reduction of MMP-9, MMP-2, and ICAM-1 gene expression belonged to the combination-treated compared with 150 μg/mL SeNPs or 20 μg/mL UA alone (Fig 2). Moreover, the inhibition of MMP-2, MMP-9, and ICAM-1 gene expression using UA, SeNPs, and their combination showed a time-dependent manner, in which treated cells indicated significant results after 72 h of incubation with all treatments (Fig.2). The relative expression data were normalized using β-actin as a housekeeping gene. Scratch assay To investigate the effect of SeNPs and ursolic acid on cell migration, the scratch assay was performed. As shown in Fig. 3, the motility of treated cells with 150 μg/mL SeNPs, 20 μg/mL UA, and the combination of them were reduced, according to the density of cells that filled the gap, compared to the control group. In addition, the combination of 150μg SeNPs plus 20μg UA demonstrates the lowest invasion rate rather than other treated cells with single groups. It was also obtained that all treated groups exhibited the highest inhibition of cell motility after 72 h of incubation.
  • Conclusion: Although advancements in cancer therapies have improved outcomes for some subtypes, metastasis in TNBC remains a critical challenge, necessitating innovative approaches, as explored in this study. Cancer metastasis requires a cascade of events such as cell adhesion, extracellular matrix (ECM) degradation, and invasion. Our findings confirm that MMP-9 and MMP-2, well-established drivers of cancer invasion through ECM degradation, were significantly downregulated by the combination of SeNPs and UA, highlighting their potential as therapeutic targets. In the tumor microenvironment of the highly metastatic carcinomas, MMP-9 and MMP-2 are up-regulated, which leads to Epithelial-mesenchymal transition (EMT) events and metastasis (Creighton, Chang, and Rosen 2010; Li et al. 2017) . Furthermore, tumor cells must reduce cell adhesion to promote migration. ICAM-1, a glycoprotein that belongs to the immunoglobulin superfamily and is involved in a variety of immune and inflammatory responses, lymphoid trafficking, and several malignancies, including TNBC, has the potential to be positively correlated with tumor metastasis and invasion through modulating cell-cell adhesion. Also, ICAM-1 harbors the potential to be detected as a molecular target for treating patients suffering from TNBC (Aysola et al. 2013) . Hence, downregulation of MMP2, MMP-9, and ICAM-1 could practically suppress the metastatic potential of malignant carcinomas. The present study investigated the effect of SeNPs, UA, and their combination on the proliferation and viability of MDA-MB-231 cells; and gene expression levels of MMP-9, MMP-2, and ICAM-1. Nanoparticle-sized selenium showed the most significant anti- proliferative effect on MDA-MB-231 cells at the concentration of 150 μg/mL rather than other treatment and control groups (Fig. 1a). UA inhibited cell proliferation significantly at the dose of 20 μg/mL in comparison to other doses of UA and the control group (Fig. 1b). Interestingly, the combination of 150 μg/mL SeNPs and 20 μg/mL UA indicated a significant effect on reducing cell viability compared with other mixed doses and control group (Fig. 1c). Results of gene expression level assay by Real-time PCR (Fig. 2) showed a time-dependent reduction in the expression level of MMP-9 and MMP-2 after treatment with 150 μg/mL SeNPs, 20 μg/mL UA, and their combination at mentioned doses. The fold induction of MMP-2 was decreased by SeNPs, UA, and the combination group by about 0.4, 0.5, and 0.6 compared with the control group, and the reduction of MMP-9 was about 0.5, 0.6, and 0.7 respectively. The outcomes indicated that the combination treatment with SeNPs and UA resulted in the utmost decrease in MMP-9 and MMP-2 gene expression levels in contrast to applying each of them separately. Thus, based on the obtained data, it can be concluded that SeNPs and UA possess the potential synergistic effect on the reduction of cell viability and motility. This synergy allows for lower effective doses of both agents, minimizing side effects while enhancing the efficacy. Selenium nanoparticles, due to their nanoscale size, large surface area, and superior cellular membrane penetration, significantly improve bioavailability and targeted drug delivery. This allows effective therapeutic action at reduced dosages, potentially minimizing toxicity (Bisht, Phalswal, and Khanna 2022) . Furthermore, ICAM-1 expression was significantly reduced with UA by about 0.3-fold and 0.5-fold with a combination of SeNPs and UA, while administration of 150 μg/mL SeNPs alone indicated the lowest effect on ICAM-1 expression with less than 0.2-fold reduction, which was not statistically significant. This observation highlights the fact that UA can target multiple molecular pathways involved in cancer progression including proinflammatory transcription factors, cell cycle proteins, growth factors, kinases, cytokines, chemokines, adhesion molecules, and inflammatory enzymes. This broad targeting can inhibit cancer initiation, promotion, and metastasis. The scratch assay was carried out to clarify the potential relationship between cell migration and metastasis with the downregulation of MMPs and ICAM-1 on treated cells. As shown in Figure 3, all treated cells demonstrated lower mobility than the control group, which exhibits that migration in drug-exposed cells has occurred at a lower rate. As concluded in this study, although applying 150 μg/mL SeNPs and 20 μg/mL UA separately revealed more cell inhibition rate, a combination group of SeNPs and UA not only decreases the viability of cancer cells in comparison to the control group, but also harbors synergistic actions in reducing the migration and metastasis of MDA-MB-231 cells by their most effective downregulation of MMP-9, MMP-2, and ICAM-1 expression. Also, previous studies proved that the high expression of these markers is positively associated with aggressive tumor phenotypes and a lower prognosis of breast cancer (Guo et al. 2014) .These promising findings position the combination of SeNPs and UA as a novel therapeutic strategy for TNBC. However, further validation in preclinical models is essential to unravel the precise mechanisms and evaluate clinical applicability.
  • Keywords: Ursolic acid, Biological selenium nanoparticles, Triple-negative breast cancer, Metastasis