# Effect of gaillardin on the metastatic capacity of breast cancer cells and its underlying mechanism

Sadegh Rajabi, Akram Shahhosseini, Mahboubeh Irani, Marc Maresca, Maryam Hamzeloo-Moghadam

► To cite this version:

Sadegh Rajabi, Akram Shahhosseini, Mahboubeh Irani, Marc Maresca, Maryam Hamzeloo-Moghadam. Effect of gaillardin on the metastatic capacity of breast cancer cells and its underlying mechanism. Advances in Cancer Biology - Metastasis, 2025, 15, pp.100164. 10.1016/j.adcanc.2025.100164. hal-05491887

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# Effect of gaillardin on the metastatic capacity of breast cancer cells and its underlying mechanism

Sadegh Rajabi<sup>a</sup>, Akram Shahhosseini<sup>a</sup>, Mahboubeh Irani<sup>a</sup>, Marc Maresca<sup>b, *, ID</sup>, Maryam Hamzeloo-Moghadam<sup>c, **</sup>

## ARTICLE INFO

### Keywords:

- Breast cancer

- Gaillardin

- Metastasis

- Epithelial-mesenchymal transition

- Angiogenesis

## ABSTRACT

The metastasis process plays an important role in the outcome of all cancers, including breast cancer, a leading cause of cancer mortality in women. This study assessed the effects of gaillardin on the metastatic activity of two different breast cancer cell lines. The MTT assay was used to obtain the IC₅₀ concentrations. Migration or metastatic capability of MCF7 and MDA-MB231 cell lines was assayed using the wound scratch assay. The real-time PCR was utilized to quantify the gene expression of epithelial-mesenchymal transition (EMT) markers CDH1, CDH2, VIM, and FN1, along with angiogenesis-related markers VEGFA and THBS1. Western blotting was conducted to estimate the protein expression of E-cadherin, N-cadherin, vimentin, fibronectin 1, VEGFA, and thrombospondin 1. Treatment of the MCF7 cell line with different concentrations of gaillardin revealed no significant effect on the metastatic capacity of these cancer cells compared with the controls. However, the migratory activity and aggressiveness of MDA-MB231 cells were significantly hindered compared to the control cells. The results of gene expression data revealed the upregulating effect of gaillardin on the expression of CDH1 and THBS1 genes. Conversely, this phytochemical significantly downregulated CDH2, VIM, FN1, and VEGFA transcripts. Western blotting results showed a similar effect of gaillardin on the expression levels of the above-mentioned markers. The present data highlight the anti-metastatic activity of gaillardin in breast cancer in a receptor-independent manner. These results also indicate gaillardin as a potential anti-metastatic natural compound against triple-negative breast cancer cells, via two mechanisms that act by suppressing EMT and angiogenesis.

## 1. Introduction

Breast cancer is a leading cause of cancer mortality in women [1]. Epidemiological data from the past two decades indicate a significant increase in breast cancer mortality rates globally [2]. Breast cancer can be classified into three distinct subtypes: estrogen receptor-positive/progesterone receptor-positive, HER2-positive, and triple-negative [3]. These subtypes exhibit distinct risk profiles, and therapeutic strategies are tailored to each patient based on tumor subtype, disease stage, and patient preferences [4]. Several classical therapeutic methods are available for curing patients with breast cancer, including surgery, radiotherapy, hormone therapy, and chemotherapy

[5]. Triple-negative breast cancer (TNBC) is the most aggressive form of this cancer and has a poor prognosis [6].

Given the emergence of therapeutic resistance and adverse events associated with conventional breast cancer treatments, disease recurrence and relapse remain significant clinical challenges. Consequently, the identification of efficacious and better-tolerated therapeutic strategies with reduced toxicity profiles is imperative [7]. Recent research indicates that the development of targeted molecular therapies, which selectively engage specific molecules within cancer cells, represents a promising and well-tolerated strategy for cancer management. For instance, the application of plant-derived compounds offers an efficacious therapeutic modality for certain cancers, often associated with a

<footnote><sup>a</sup> Traditional Medicine and Materia Medica Research Center, Shahid Beheshti University of Medical Sciences, Tehran, 1434875451, Iran</footnote>

<footnote><sup>b</sup> Aix Marseille Univ, CNRS, Centrale Marseille, iSm2, 13013, Marseille, France</footnote>

<footnote><sup>c</sup> Traditional Medicine and Materia Medica Research Center and Department of Traditional Pharmacy, School of Traditional Medicine, Shahid Beheshti University of Medical Sciences, Tehran, 1516745811, Iran</footnote>

<footnote>* Corresponding author.</footnote>

<footnote>** Corresponding author.</footnote>

<footnote>E-mail addresses: m.maresca@univ-amu.fr (M. Maresca), mhmoghadam@sbmu.ac.ir (M. Hamzeloo-Moghadam).</footnote>
reduced incidence of adverse effects [8].

Gaillardin, a sesquiterpene lactone, has been isolated from the aerial parts of *Inula oculus-christi* [9]. Our previous investigations have demonstrated that this compound exhibits anti-neoplastic activity and possesses the capacity to significantly induce apoptosis in a range of cancer cell lines [10–13]. Although sesquiterpene lactones have been shown as promising anti-metastatic and anti-angiogenic natural compounds against various cancer types [14], only one publication describes the effect of gaillardin on metastasis and angiogenesis in the context of ovarian cancer cells [15].

Metastasis refers to the process by which cancer cells disseminate from the primary tumor site to establish secondary tumors in distant organs [16]. Metastasis is the primary cause of mortality in cancer patients, rendering treatment significantly challenging [17]. Effective therapeutic interventions aimed at preventing metastasis should target the principal signaling pathways implicated in the various stages of the metastatic process [18]. Epithelial-mesenchymal transition (EMT), a process by which cancer cells acquire invasive properties through phenotypic conversion, is a critical pathway involved in the initiation of cancer cell metastasis [19]. Angiogenesis is a key process that is significantly upregulated during tumor metastasis, facilitating cancer cell migration and dissemination [20]. Considering the crucial roles of EMT and angiogenesis in the initiation and progression of metastasis, and given the absence of prior research evaluating the effects of gaillardin on metastasis and angiogenesis in breast cancer cells, this study was designed to investigate the impact of this compound on migration, EMT markers, and angiogenesis-related factors in two metabolically distinct breast cancer cell lines, the estrogen receptor (ER) and progesterone receptor (PR) receptor positive cell line MCF-7 and the triple-negative cell line MDA-MB231.

## 2. Materials and methods

### 2.1. Plant material and isolation of gaillardin

*Inula oculus-christi* L. (Asteraceae) specimens were collected in July 2023 from Mazandaran Province, Iran. The taxonomic identity of the plant material was confirmed by voucher specimen (TMRC-4630) deposited at the Herbarium of the Traditional Medicine and Materia Medica Research Center (TMRC), Shahid Beheshti University of Medical Sciences. Gaillardin was subsequently isolated from the aerial parts of the collected plant material, utilizing the extraction method previously described by Hamzeloo-Moghadam et al. [21].

### 2.2. Cell culture and preparation of phytochemical solution

MDA-MB231 and MCF-7 human breast cancer cells, exhibiting distinct metabolic profiles, were obtained from the Pasteur Institute (Tehran, Iran). Cells were maintained in Dulbecco's Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum (FBS; Gibco, Germany) and penicillin/streptomycin (100 U/ml; Sigma, Germany). Cells were cultured under controlled conditions of 98% humidity and 5% CO2 at 37 °C. Gaillardin, isolated and purified, was prepared as a stock solution in dimethyl sulfoxide (DMSO).

### 2.3. Cell viability assay

The proliferation of MDA-MB231 and MCF-7 cells was assessed using MTT (3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide) assay. Briefly, cells were seeded in 96-well microplates at a density of approximately 8000 cells per well. Subsequently, cells were treated with gaillardin at concentrations ranging from 0 to 50 µM for 24 and 48 h. Control wells received standard DMEM, while vehicle control wells were treated with DMSO (maximal final volume 1%). Following treatment, cells were incubated with MTT solution (0.5 mg/mL final concentration in the wells) for 4 h). At the end of the incubation, wells were emptied

and cells were lysed with 100 µL of DMSO per well. The absorbance of the wells was finally measured at 570 nm to determine cell proliferation percentages.

### 2.4. Scratch assay

The impact of gaillardin on the invasive and migratory capabilities of MDA-MB231 and MCF-7 cells was evaluated using the scratch assay, as previously described [22]. In brief, 500,000 cells per well were seeded in 6-well cell culture plates and incubated until they reached 60–70% confluency. Subsequently, three horizontal scratches were introduced across each well using 200 µl sterile pipette tips. A single vertical reference line was created in each well to ensure consistent photographic documentation of the scratches. Following the removal of cellular debris via washing with phosphate-buffered saline (PBS) buffer solution (pH ~ 7.4), wells were filled with complete DMEM medium. Control wells were then treated with DMSO, while test wells received gaillardin. Images of the scratches were then captured at 0, 9, 24, and 48 h, and the wound areas were quantified using ImageJ software.

### 2.5. Gene expression analysis

The effect of gaillardin on EMT and angiogenesis marker mRNA expression in MDA-MB231 cells was assessed using quantitative reverse transcriptase polymerase chain reaction (qRT-PCR). Briefly, MDA-MB231 cells were treated with gaillardin for 48 h, after which total RNA was extracted using the RNeasy mini kit (Qiagen, Korea). RNA absorbance was measured using a Nanodrop 2000c spectrophotometer, and cDNA was synthesized using a cDNA Synthesis Kit (BioFact, Korea). qRT-PCR was performed using an ABI PRISM7900HT (Applied Biosystems, USA) with SYBR GREEN PCR master mix (Ampliqon, Denmark) to quantify the expression of EMT markers including Cadherin1 (CDH1), N-cadherin or Cadherin2 (CDH2), Vimentin (VIM), Fibronectin1 (FN1), the pro-angiogenic gene Vascular Endothelial Growth Factor A (VEGFA), and the anti-angiogenic gene Thrombospondin1 (THBS1). Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) expression was used as an internal control for data normalization. Primer sequences for all gene transcripts are presented in Table 1.

### 2.6. Protein expression analysis

Western blot analysis was performed to evaluate the impact of gaillardin on the protein expression of EMT- and angiogenesis-related markers in the MDA-MB231 cells. Cells were treated with gaillardin for 48 h, then trypsinized and lysed using radioimmunoprecipitation assay (RIPA) buffer. Protein concentration was determined via the Bradford method [23]. Subsequently, 40 µg of total protein was separated by SDS-PAGE, followed by transfer to a PVDF membrane (Roche, Germany). Membranes were blocked with 5% non-fat milk for 1 h at room temperature and then incubated with primary antibodies (Santa Cruz Co., USA) against E-cadherin, vimentin, N-cadherin, fibronectin 1, vascular endothelial growth factor A (VEGFA), and thrombospondin 1 for 24 h at 4 °C. Beta-actin was used as a housekeeping protein to normalize protein expression. Following three washes with TBST buffer, membranes were incubated with horseradish peroxidase (HRP)-conjugated secondary antibody (Abcam, USA) for 1 h at room temperature. Protein bands were visualized using enhanced chemiluminescence solution (ECL, Amersham Pharmacia, Freiburg, Germany) and exposure to radiography film.

### 2.7. Statistical analysis

Statistical analysis of all data was performed using one-way ANOVA, with Duncan's multiple range test employed for post hoc comparisons. GraphPad Prism software was utilized for these analyses. Significance was defined as P < 0.05. All experiments were conducted in triplicate,
<table><caption>**Table 1**

Primer sequences used in the present study.</caption><thead><tr><td>Gene name</td><td>Forward primer</td><td>Reverse primer</td></tr></thead><tbody><tr><td>CDH1</td><td>5'-GGGGTCTGTCATGGAAGGTG-3'</td><td>5'-CGACGTTAGCCTCGTTCTCA-3'</td></tr><tr><td>CDH2</td><td>5'-GCGTCTGTAGAGGCTTCTGG-3'</td><td>5'-GCCACTTGCCACTTTTCCTG-3'</td></tr><tr><td>FN1</td><td>5'-ACAAGCATGTCTCTCTGCCAA-3'</td><td>5'-TCAGGAAACTCCCAGGGTGA-3'</td></tr><tr><td>VIM</td><td>5'-TCCGCACATTCGAGCAAAGA-3'</td><td>5'-ATTCAAGTCTCAGCGGGCTC-3'</td></tr><tr><td>VEGFA</td><td>5'-GAGCAAGACAAGAAAATCCC-3'</td><td>5'-CCTCGGCTTGTCACATCTG-3'</td></tr><tr><td>THBS1</td><td>5'-CCCTTGTGCTCAGAGTGGAT-3'</td><td>5'-GCCAGTAGAGAACAAATAAGCATGG-3'</td></tr><tr><td>β-actin</td><td>5'- CACCATTGGCAATGAGCGGTTTC-3'</td><td>5'- AGGTCTTTGCGGATGTCCACGT-3'</td></tr></tbody></table>

and data are presented as mean ± standard deviation (SD).

# 3. Results

## 3.1. Effect of gaillardin on the viability of MCF-7 and MDA-MB231 cells

Gaillardin's half-maximal inhibitory concentration (IC₅₀) values were determined in MDA-MB231 and MCF-7 cells using the MTT assay. After 24 h, gaillardin (0–50 µM) did not significantly reduce the viability of MCF-7 cells below 50 µM (Fig. 1A) or MDA-MB231 cells below 40 µM (Fig. 1C). However, a 48-h treatment significantly decreased MCF-7 cell viability at concentrations above 10 µM, yielding an IC₅₀ of 15.32 µM (Fig. 1). Similarly, 48-h treatment of MDA-MB231 cells significantly reduced viability, with an IC₅₀ of 28.21 µM (Fig. 1D). Consequently, subsequent experiments treated MDA-MB231 cells with 28 µM (IC₅₀) and 14 µM (IC₂₅) gaillardin for 48 h, and MCF-7 cells with 15 µM (IC₅₀) and 7 µM (IC₂₅) gaillardin for 48 h for the scratch assay.

## 3.2. Effect of gaillardin on the migration capacity of MCF-7 cells

To investigate the influence of gaillardin on the healing potential of MCF-7 cells, a scratch assay was conducted. As depicted in Fig. 2A, control MCF-7 cells, cultured in standard MDEM medium, exhibited

migration towards the scratch area beginning 9 h post-treatment. The scratched area was completely repopulated by these cells within 48 h. Likewise, treatment of MCF-7 cells with gaillardin at concentrations of 7 and 14 µM (corresponding to IC₂₅ and IC₅₀ on cell proliferation of MCF-7 cells) did not impede cell invasion into the central region of the scratch. Quantitative analysis of the scratched area, as illustrated in Fig. 2B, revealed that gaillardin treatment at either concentration failed to inhibit wound closure in MCF-7 cells.

## 3.3. Effect of gaillardin on the migration capacity of MDA-MB231 cells

The effect of gaillardin on MDA-MB231 cell migration was assessed via the wound scratch assay. Control cells treated with DMEM exhibited migration into the scratch area, resulting in wound closure at 48 h post-treatment, as shown in Fig. 3A. In contrast, treatment of MDA-MB231 cells with 14 µM and 28 µM of gaillardin (corresponding to IC₂₅ and IC₅₀ on cell proliferation of MDA-MB231 cells) inhibited cell migration. Quantitative analysis of scratch area percentages indicated that a 28 µM concentration of gaillardin significantly impeded wound closure in MDA-MB231 cells (Fig. 3B).

<figure><img src="image_4.png" /><figcaption>**Fig. 1.** The effects of gaillardin on the viability of MCF-7 (A and B) and MDA-MB231 (C and D) cells. Cells were treated with increasing concentrations of gaillardin (0, 2.5, 5.10, 20, 30, 40, and 50 µM) for 24 and 48 h. The proliferation of treated and control cells was assayed by the MTT method. The data are expressed as mean ± SD of three independent experiments. Significant differences were found between the treatment and control groups at *P*<0.05 and **P*<0.01.</figcaption></figure>
<figure><img src="image_5.png" /><figcaption>**Fig. 2.** (A) The effect of gaillardin treatment on the invasion and migration of the MCF-7 cells. The cultured cells were scratched with a sterile pipette tip after gaillardin treatment at doses of 7 and 15 µM. Photographs of the wound closure process were captured at 0, 9, 24, and 48 h after gaillardin treatment. (B) Data are expressed as the percentage of empty areas compared with the control (mean ± SD). \*P<0.05 and \*\*P<0.01 vs. 0h.</figcaption></figure>

### 3.4. Expression of EMT- and angiogenesis-related genes following gaillardin treatment of MDA-MB231 cells

The effect of gaillardin treatment on the expression of EMT and angiogenic gene markers in MDA-MB231 cells was measured using qRT-PCR (Fig. 4). Treatment with 14 and 28 µM gaillardin for 48 h resulted in a significant reduction in the expression of CDH2, VIM, FN1, and VEGFA genes in comparison to control cells. Furthermore, a 14 µM dose of gaillardin significantly upregulated CDH1 expression, while THBS1 levels were increased at both 14 and 28 µM doses compared to the control group.

### 3.5. Expression of EMT- and angiogenesis-related proteins following gaillardin treatment of MDA-MB231 cells

Western blot analysis was performed on MDA-MB231 cells to confirm the effects of gaillardin on EMT and angiogenic gene markers at the protein level. The results, presented in Fig. 5, demonstrate that treatment with gaillardin at 14 and 28 µM upregulated the protein expression of E-cadherin and thrombospondin 1 compared to control cells. Conversely, the expression of N-cadherin, vimentin, fibronectin 1, and VEGFA proteins was downregulated in gaillardin-treated cells relative to the control group.
<figure><img src="image_6.png" /><figcaption>Fig. 3. (A) The effect of gaillardin treatment on the invasion and migration of the MDA-MB231 cells. The cultured cells were scratched with a sterile pipette tip after gaillardin treatment at doses of 14 and 28 µM. Photographs of the wound closure process were captured at 0, 9, 24, and 48 h after gaillardin treatment. (B) Data are expressed as the percentage of empty areas compared with the control (mean ± SD). \*P<0.05 and \*\*P<0.01 vs. 0h.</figcaption></figure>

4. Discussion

The present study initially employed an MTT assay on two breast cancer cell lines (the receptor-positive cells MCF-7 and the triple-negative cells MDA-MB231) to determine the antiproliferative activity of gaillardin. Following the determination of IC₅₀ and IC₂₅ values, subsequent experiments were conducted utilizing these concentrations to evaluate the effect of gaillardin on the metastatic capacity of MCF-7 and MDA-MB231 cells. Results indicated that MCF-7 human breast cancer cells were more sensitive to the sesquiterpene lactone gaillardin, with an IC₅₀ on cell proliferation of 15 µM. However, migration assay showed that gaillardin did not significantly impact the metastatic capacity of MCF-7 cells, thus precluding further experimentation with this cell line. MTT assay results for the MDA-MB-231 cells demonstrated a lower sensitivity compared to MCF-7 cells, with an IC₅₀ value on cell

division of 28 µM. But unlike MCF-7 cells, gaillardin treatment significantly inhibited the migration capacity of MDA-MB231 cells at its IC₂₅ and IC₅₀ values, suggesting an inhibition of its metastatic potential. The potential anti-metastatic effect of gaillardin on MDA-MB231 cells was further investigated through RT-PCR and Western blot analysis. The expression levels of genes involved in two critical processes related to cancer cell metastasis, known as EMT and angiogenesis, were assessed. Analysis of EMT markers at the mRNA level revealed a significant upregulation of CDH1 gene expression and a marked downregulation in the expression of CDH2, VIM, and FN1 genes in gaillardin-treated MDA-MB231 cells. Gaillardin also diminished the expression of the pro-angiogenic gene marker VEGFA and augmented the anti-angiogenic gene THBS1 in the MDA-MB231 cell line. Western blot analysis corroborated gene expression data, demonstrating increased E-cadherin expression and reduced N-cadherin, vimentin, and fibronectin 1
<figure><img src="image_7.png" /><figcaption>Fig. 4. The effect of gaillardin treatment at the doses of 14 and 28 µM for 48 h on the mRNA levels of epithelial-mesenchymal transition (CDH1, CDH2, VIM, and FN1) and angiogenesis (VEGFA and THBS1) markers in the MDA-MB231 cells. The data are represented as means ± SD for the three independent experiments (*P<0.05 and **P<0.01, compared with the control).</figcaption></figure>

expression in gaillardin-administered MDA-MB231 cells compared to untreated cells.

Given that gaillardin is a novel sesquiterpene lactone, a very limited number of studies in the existing literature have investigated its anti-cancer effects. Gaillardin was found toxic to AGS and MKN45 human gastric cancer cell lines, the mechanism of action involving ROS-dependent suppression of the NF-κB pathway, resulting in the down-regulation of COX-2, MMP-9, TWIST-1, and BCL-2 genes [9]. An investigation into gaillardin's effects, both alone and with vincristine, on acute lymphoblastic leukemia (ALL) cell lines (NALM-6 and MOLT-4) revealed that gaillardin inhibited proliferation, induced G0/G1 cell cycle arrest, and promoted apoptosis in both cell lines. Moreover, gaillardin enhanced the sensitivity of these cell lines to vincristine [12]. Another in-vitro study investigated the effects of gaillardin on the HL-60 human myeloid leukemia cell line and demonstrated an antiproliferative activity linked to the induction of G1 cell cycle arrest and apoptosis, mediated by the production of reactive oxygen species (ROS) [24]. Similarly, a study conducted by Karami et al., it was shown that gaillardin is capable of initiating apoptosis and suppressing the proliferation in the Nalm-6 acute lymphoblastic leukemia cell line. These findings highlight the potential of gaillardin as an agent that can both trigger cell death and limit the rapid growth of leukemic cells [25]. Additionally, previous study has demonstrated that gaillardin inhibits the proliferation and causes apoptosis of the ovarian cells SKOV3 through an upregulation of CASP3, CASP8, CASP9, and BAX, while downregulating BCL-2 [26]. Gaillardin also reduced phosphorylated JAK2 and STAT3

proteins, suggesting its potential anti-ovarian cancer activity by a mechanism that suppresses JAK/STAT signaling [26].

Previous study has shown an anti-proliferative action of gaillardin on breast cancer cells occurring through a mechanism involving apoptotic cell death [21]. The induction of apoptosis by gaillardin in the human breast cancer cell line MCF-7 is related to its ability to inhibit the JAK/STAT signaling pathway, alteration of mitochondria, and suppression of autophagy [13].

To our knowledge, although previous study has demonstrated the anti-metastatic effect of gaillardin in the context of ovarian cancer cells [15], this study extends this finding to breast cancer cells. The results of the present study indicate that gaillardin does not exert any anti-metastatic activity against MCF-7 cells, a human breast cancer cell line with estrogen, progesterone, and glucocorticoid receptors. However, this phytochemical significantly hindered the metastatic capacity of the MDA-MB-231 cell line, a triple-negative breast cancer cell type. This suggests that the anti-metastatic activity of gaillardin in breast cancer may be receptor-independent.

As mentioned earlier, gaillardin treatment of the MDA-MB-231 cells inhibited the expression of EMT and angiogenesis-associated markers. Cancer cells undergo EMT to acquire mesenchymal characteristics to dissociate from the primary site to invade the nearby tissue and migrate to another organ [27,28]. These cancer cells lose cell-cell adhesion capacity and cell polarity, remodel their cytoskeleton, and change cell-matrix adhesion to obtain mesenchymal characteristics [29]. According to the literature, E-cadherin, N-cadherin, vimentin, and fibronectin 1 are well-known markers of the EMT process [30]. It is now clearly evident that invading cells downregulate E-cadherin while upregulate N-cadherin, vimentin, and fibronectin 1 to promote the EMT process, by which the cancer cell can detach from the primary tissue and migrate to the secondary site [31–33]. Hence, the impact of gaillardin on the MDA-MB231 cell line that led to the enhancement of E-cadherin and suppression of N-cadherin, vimentin, and fibronectin 1 in the present research indicated blockage of EMT and inhibition of invasion and migration.

Another critical phenomenon by which the invading cancer cells traverse the primary organ to disseminate to secondary organ(s) [34]. Angiogenesis is a biological phenomenon in which new blood vessels are formed by a complicated process that is controlled by various pro- and anti-angiogenic markers [35]. One of the key positive regulators of angiogenesis is the VEGF family, which is secreted by cancer cells to activate neovascularization by binding their receptors, VEGFRs [36]. VEGFA is the most important activator of angiogenesis and a critical target for anticancer drugs [37]. Thrombospondin-1, as a well-recognized anti-angiogenic factor, is involved in the stimulation of

<figure><img src="image_8.png" /><figcaption>Fig. 5. (A) The effect of gaillardin at the concentrations of 14 and 28 µM for 48 h on the protein expression of E-cadherin, N-cadherin, vimentin, fibronectin 1, VEGFA, and thrombospondin 1 in the MDA-MB231 cells. (B) Semiquantification of each protein band. *P<0.05, **P<0.01, and ***P<0.001.</figcaption></figure>
apoptotic cell death in endothelial cells, inhibition of proliferation and migration of endothelial cells, and modulation of the bioavailability and function of various VEGF family proteins [38,39]. As indicated above, gaillardin downregulated VEGFA and upregulated thrombospondin 1 in MDA-MB231 cells. This may propose a potential anti-angiogenic capacity for this phytochemical compound.

## 5. Conclusion

Our data indicated that gaillardin has no remarkable effect on the migratory and metastatic properties of MCF-7 cells, as a receptor-positive breast cancer cell line. However, this phytochemical significantly repressed the migration and metastasis of the triple-negative breast cancer cells MDA-MB-231. This may suggest a receptor-independent manner for gaillardin in prohibiting metastasis in these cells. Our further experiments unraveled that gaillardin has hindered MDA-MB231 cell migratory capacity by involving two critical cascades that help cancer cells promote dissemination to the distant organs. These cascades are EMT and angiogenesis that were inhibited in MDA-MB231 cells after treatment with gaillardin. Taken together, these data may provide evidence to suggest gaillardin as a potential drug with anti-metastatic and anti-angiogenic activity in triple-negative breast cancer cells.

## 6. Limitations of the study

Our study focused primarily on demonstrating the anti-metastatic efficacy of gaillardin in-vitro. While this study provides strong evidence for the anti-metastatic potential of gaillardin, it is important to acknowledge its limitations. The present study conducted in-vitro demonstrates the potential of gaillardin as an anti-metastatic and angiogenic compound in breast cancers. The absence of in-vivo data represents a limitation, impacting the translational relevance of our findings. Future studies should thus prioritize validation using in vivo models or advanced 3D models, such as 3D spheroid invasion assays, culture models, or in-vivo CAM assays, to strengthen the conclusions and support translational claims.

### CRediT authorship contribution statement

**Sadegh Rajabi:** Writing – review & editing, Writing – original draft, Supervision, Project administration, Methodology, Investigation, Funding acquisition, Formal analysis, Data curation, Conceptualization.
**Akram Shahhosseini:** Writing – review & editing, Writing – original draft, Methodology, Investigation, Formal analysis. **Mahboubeh Irani:** Writing – review & editing, Writing – original draft, Methodology, Investigation, Formal analysis. **Maryam Hamzeloo-Moghadam:** Writing – review & editing, Writing – original draft, Supervision, Project administration, Methodology, Conceptualization.

### Funding

This work was supported by Shahid Beheshti University of Medical Sciences, Tehran, Iran, under grant number 03-43009184.

### Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

### Acknowledgments

The authors would like to thank the Vice Chancellor for Research, Shahid Beheshti University of Medical Sciences, Tehran, Iran.

### Data availability

Data will be made available on request.

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