PPE preparation

Pomegranate skins were isolated, dried, ground into powder, and extracted with methanol. The extracts were concentrated under vacuum at 40°C and stored at -20°C.

Cell culture

The human metastatic breast cancer cell line, MDA-MB-231, was obtained from the National Cell Bank of Iran (Pasteur Institute of Iran, Tehran, Iran). Cells were cultured at 37 °C in a 5% CO2 humidified atmosphere in Dulbecco’s Modified Eagle Medium (DMEM) containing 10% fetal calf serum (FBS) and 1% penicillin-streptomycin at 10,000 units/ml.

Apoptosis assay

The cells were seeded in 6-well microplates and allowed to proliferate until the confluency reached 70–80%. Cells were incubated with 12.5, 25, 50, 100, 250, 500, and 1000 µg/ml of PPE for 24h, 48h, and 72h. Then the media were removed and cells were suspended in 500 µL of binding buffer. The cells were then incubated with annexin V-FITC and propidium iodide (PI) in the dark for 15 min at room temperature. Early and late apoptotic and necrotic cell populations were determined by constructing a dot-plot with the aid of fluorescence-activated cell sorting.

Migration assay

The transwell migration assay was performed as described before (11). Briefly, MDA-MB-231 cells were plated on the upper side of a polycarbonate membrane of a transwell chamber in medium without serum. The cells were washed twice with PBS, incubated with 12.5, 25, 50, 100, 250, 500, and 1000 µg/ml of PPE for 24 h, fixed and then stained with 4′,6-diamidino-2-phenylindole (DAPI). Four random fields of cells migrating through the membrane were counted on an Olympus IX71 fluorescence microscope.

RNA extraction and real-time PCR

Total RNA was isolated from the PPE-treated MDA-MB-231 cells using TriPure Isolation Reagent (Roche, Germany) following the manufacturer’s instructions. To remove genomic DNA contamination, the extracted RNAs were treated with DNase I. To synthesize cDNA, 1 µg of RNA was mixed with 1 µl of oligo (dT) (0.5 µg/µl) and incubated at 65°C for 5 min. After cooling on ice, each sample was mixed with 4 µl of 5X buffer, 2 µl of 10 mM dNTPs, 0.5 µl of RiboLock, and 1 µl of M-MLV-RTase, and incubated at 42 °C for 60 min, followed by 70°C for 10 min. The synthesized cDNAs were diluted in distilled water (1:3) and 2 µl of the diluted cDNA samples were mixed with real-time PCR reaction mixture containing 10 µl of 2X SYBR Green PCR Master Mix (Parstous, Iran) and 1 µl of specific primer pairs in final 20 µl volumes. Samples were incubated at 95 °C for 10 min, then amplified by 40 cycles of 95 °C for 15 sec, 60 °C for 30 sec, and 72 °C for 30 sec (12). Primers used in this study are shown in Table 1. The housekeeping gene, glucuronidase beta (GusB), was used to normalize target gene expression. The comparative expression level of each target gene was calculated by 2^-ΔΔCT. Melting curves were used to determine non-specific amplification.

Statistical analysis

The data were analyzed and visualized using GraphPad Prism version 6.0. Differences between the groups were assessed by one-way ANOVA followed by Dunnett’s test. Data are presented as means ± standard deviations (SDs).

Results

The effects of PPE on apoptosis of MDA-MB-231 cells

To investigate whether PPE affects TNBC cell apoptosis, cells were dual-labeled with Annexin V-FITC and PI. After PPE treatment for 24, 48, and 72 h, the percentages of the early and late apoptotic and necrotic cells were increased (Fig. 1).

Fig. 1

Flow cytometry with Annexin V and propidium iodide staining. Cells were treated with PPE at the indicated doses for 24, 48, and 72 h and apoptosis was assessed by flow cytometry. The four quadrants represent living cells (Annexin V-PI-), early apoptotic (Annexin V+PI-), late apoptosis (Annexin+PI+) or necrotic (Annexin V-PI+) stages. Values shown are percentages of each quadrant.

Cells treated with 500 or 1000 µg/ml of PPE for 24, 48, and 72 h were significantly more apoptotic than controls (data not shown).

The effects of PPE on migration motility in MDA-MB-231 cells

Cell migration was evaluated using a two-chamber transwell system. Cells were cultured with the various doses of PPE for 24 h and cells that had traversed the membrane to the bottom side were fixed and stained. The results showed that PPE inhibited cell migration in a dose-dependent manner (Fig. 2).

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Fig. 2

PPE inhibits MDA-MB-231 cell migration. (A) Cells on the transwell filter that migrated to the lower surface of the filter were fixed and then stained with DAPI and photographed on a light microscope. The bars represent 200 µm. (B) The results are an average of the number of migrated cells on the underside of the filter. Significance was set at *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

PPE altered metastasis-associated gene expression

Exposure of MDA-MB-231 cells to PPE for 48 and 72 h increased ICAM-1 and decreased MMP9, fibronectin, and VEGF mRNA expression in a dose-dependent manner. The 72-hour result is shown in Figure 3 (Fig. 3). PPE had no effect onVCAM-1 expression (data not shown).

Fig. 3

PPE modulates gene expression in MDA-MB-231 cells. Total RNA from PPE-treated and untreated MDA-MB-231 cells was extracted after 72 h of incubation, cDNA was synthesized, and real-time PCR was performed. ICAM-1 was upregulated while MMP9, fibronectin, and VEGF were downregulated by PPE in a dose-dependent manner. The data represent the means ± SDs (*P < 0.05, **P < 0.01).

Discussion

This study identified genes that might serve as biomarkers involved in the antimetastatic property of PPE on TNBC cells. We showed that PPE inhibits TNBC cell migration and stimulates the expression of genes involved in cell adhesion while reducing the expression of genes involved in cell invasion and migration.

Recently, it has been found that expression changes of several cell adhesion molecules alter the metastatic potential of cells. Among these is ICAM-1, a transmembrane glycoprotein in the immunoglobulin superfamily. In a clinicopathologic analysis of colorectal cancer, a correlation was found between ICAM-1 expression and patients’ prognoses. Patients with ICAM-1-negative tumors had significantly poorer prognoses than those with ICAM-1-positive tumors (13). Injection of colon cancer cells into ICAM-1-deficient mice promoted tumor progression and metastasis, whereas no change in tumor cell proliferation was reported (14). These findings indicate that ICAM-1 may have an important role in initiation and progression of metastasis. Wang et al. showed that treatment of prostate cancer cells with pomegranate juice upregulated genes involved in cell adhesion such as ICAM-1 (15). In our study, the finding that PPE

upregulates ICAM-1 suggests that the anti-metastatic effect of PPE may be partially due to increased ICAM-1 expression.

Vascular cell adhesion molecule 1, expressed on endothelial cells under inflammatory conditions, mediates leukocyte rolling, adhesion, and transmigration from the inner surface of blood vessels into tissue (16). Moreover, some cancer cell types express VLA-4, a ligand for VCAM-1. Interaction of VLA-4-positive tumor cells in the circulation with VCAM-1 on the activated endothelium enhances their transmigration into the subendothelium to make a new tumor site (17). In addition to endothelial expression of VCAM-1 and its role in metastasis, some cancer cell types also express VCAM-1. These cancer cells, by attracting VLA-4-positive monocytes and macrophages, are protected from immune surveillance (18). Additionally, binding of macrophages to VCAM-1 enhances survival signals in breast cancer cells that invade the lungs (19). Significant reductions in serum (soluble) VCAM-1 was reported after pomegranate juice intake (20). We found no decrease in VCAM-1 expression in TNBC cells. Further investigation should be performed to determine the mechanism by which pomegranate alters VCAM-1 expression.

The extracellular matrix (ECM) is a protein-rich environment that serves many functions including tissue support, regulation of cell-cell adhesion and communication, and support for cell chemotaxis and migration (21). A large body of evidence demonstrates that ECM proteins are aberrantly expressed in human cancers and actively contribute to tumor progression. In particular, the matrix metalloproteinases (MMPs), as major ECM components, have received increasing interest due to their participation in multiple stages of tumor progression. MMPs degrade and remodel the ECM, allowing tumor cell migration (22, 23). MMP9, an MMP family member, was introduced as a poor prognosis signature for breast cancer patients (24). MMP9 knockdown and gene silencing experiments showed reduced invasiveness and tumor growth in TNBC cells (24). Our data, in which PPE decreased MMP9 expression, suggests PPE as a drug that targets MMP9.

Fibronectin, another structural ECM component, is over-expressed in several cancers and has been shown to promote tumor growth and invasion (25). Overexpression of SOX2, through upregulation of fibronectin, increased cell migration and invasion in ovarian cancer cells (26). Wei et al. showed that nicotine increased colon cancer cells migration through upregulation of fibronectin. It was also shown that COX-2 signaling was involved in this induction (27). Knowles et al. found that fibronectin and integrin αvβ3 induced lung metastasis through upregulation of the transcription factor Slug, which mediates epithelial-mesenchymal transition (28). Balanis et al.

showed that adhesion of MDA-MB-231 breast cancer cells to fibronectin promotes cancer progression through a fibronectin-dependent STAT3 signaling pathway (29). Here, decreased expression of fibronectin may indicate its effect on metastasis inhibition after PPE exposure in TNBC cells.

Targeting VEGF signaling is an attractive approach to prevent tumor metastasis because cancer cells require angiogenesis to metastasize. Angiogenesis provides nutrients to tumor cells and provides them the opportunity to enter the circulation to form distant metastases. Tumor angiogenesis is elicited and regulated by several factors including VEGF-A, which is secreted by tumor cells and plays an important role in angiogenesis (30). Evidence from preclinical and clinical studies indicates that VEGF is predominantly associated with the formation of metastatic foci and poor prognosis (39, 31).VEGF binding to its receptor induces receptor dimerization and autophosphorylation, leading to activation of some signaling pathways including MAPK and ERK, which are involved in cell proliferation, survival, and migration (31). Decreased VEGF expression after treatment with PPE treatment suggests it may contribute to PPE’s antimetastatic effects.

The outlook for pomegranate as an antimetastatic drug can be improved by unraveling the mechanisms that mediate its antimigratory properties and identifying its novel targets. To our knowledge, this is the first report on the transcriptional changes of some key genes involved in metastasis after exposure of TNBC cells to PPE, and these results may at least partly explain the underlying mechanisms of this effect.

Acknowledgements

References