Cytotoxicity of Pomegranate Polyphenolics in Breast Cancer Cells in Vitro and Vivo - Potential Role of miRNA-27a and miRNA-155 in Cell Survival and Inflammation

Botanical extracts

Stiebs Pomegranate Products (Madera, CA) provided the pomegranate juice concentrate (Pg) utilized in this study from the 2009 California crop (Sample # 0622-33808). Pg was stored at 4°C upon receipt and isolated within 48 h. Polyphenols from Pg were diluted with water to facilitate loading and partitioning from a Sep Pack Vac 20 g C18 cartridge (Waters Corp. Milford, MA) previously conditioned with 100% methanol containing 0.01% HCl. Compounds were eluted with 100% methanol and solvent removed by rotary evaporation (Büchi Labortechnik AG, Flawil, Switzerland) at 40°C. Residual water was removed on a speedvac system (Savant, Thermo Scientific Inc, Pittsburgh, PA). The dried extract stored at −80°C prior to weighing and dissolution in dimethyl sulfoxide (DMSO) for cell culture and analytical procedures.

HPLC-PDA Analysis

The pomegranate polyphenolics were analyzed in negative ESI-MS and quantified against a standard of punicalagins A and B and cyanidin-3-gluoside. Separations were made on a SunFireTM C₁₈ column (Supelco Inc. Bellefonte, PA) (250 × 4.8 mm, 5 µm) at room temperature. A mobile phase of water/acetic acid was run (98:2) in Phase A and acetonitrile/water/acetic acid was run (68:30:2) in Phase B. A gradient program at 0.4 mL/min initially ran Phase B at 0%, from 0 to 5% Phase B in 1 min, from 5 to 30% Phase B in 15 min, from 30 to 65% Phase B in 40 min, and 65 to 95% Phase B in 50 min before returning to initial conditions. Detection was at 280 and 520 nm for ellagitannins and anthocyanins, respectively. Compounds were tentatively identified based on mass spectrometric analysis. This was performed on a Thermo Finnigan LCQ Deca XP Max ion trap mass spectrometer with an electrospray ionization probe in negative ion mode under the following conditions: sheath gas (N₂), 60 units/min; auxiliary gas (N₂), 5 units/min; spray voltage, 3.3 kV; capillary temperature, 250°C; capillary voltage, 1.5 V; tube lens offset, 0 V. Standard compounds for the identification and quantitative analysis of ellagitannins and anthocyanins were obtained from Acros Organics (Morris Plains, NJ.) and ChromaDex (Irvine, CA), respectively.

Reagents

Antibodies against cleaved caspase-3, poly-(ADP-ribose)-polymerase (PARP), NF-κB (p65), phosphorylated NF-κB (p65) as well as SHIP-1 were purchased from Cell Signaling Technology (Beverly, MA). All other antibodies and SHIP-1 small interfering RNA (SiRNA) were purchased from Santa Cruz Biotechnology Inc. (Santa Cruz, CA). Reporter lysis buffer and luciferase reagent for luciferase studies were obtained from Promega (Madison, WI). Invitrogen (Grand Island, NY) supplied LipofectAMINE 2000 reagent. Western lighting chemiluminescence reagent was purchased from Perkin-Elmer Life Sciences (Waltham, MA). Primers for Sp1, VEGF, VEGFR-1, survivin, ZBTB10, SHIP-1 were purchased from Integrated DNA Technologies (San Diego, CA). Primers for Sp3 and Sp4 were obtained from Qiagen; antagomers of miR-27a (inhibitor) and miR155 (inhibitor), as well as scrambled miRNA were from Dharmacon, Inc. (Lafayette, CO). mirVana TM extraction kit, reverse transcription (RT) and real-time PCR amplification kits were purchased from Applied Biosciences (Foster City, CA). Sp1, Sp3 and NF-κB promoter constructs were kindly provided by Dr. Yanan Tian (Texas A&M University).

Cell culture

Human mammary carcinoma cell lines BT474 and MDA-MB-231 as well as non-cancer breast fibroblasts MCF-10F and MCF-12F, were obtained from ATCC and maintained according to the suppliers guidelines (American Type Tissue Collection (ATCC, Manassas, VA).

Cell proliferation

The cell proliferation was assessed with an electronic cell counter at 48 h (Z1™ Series, Beckman Coulter, Inc, Fullerton, CA), as previously described, [31] and are presented as net growth.

Real-Time PCR analysis of mRNA and miRNA

BT474 and MDA-MB-231 cells were seeded (3 × 10⁵ cells onto a 6-well plate) and incubated for 24h to allow cell attachment. Cells were treated with Pg extract and after 24 h mRNA was extracted for gene expression analysis as previously described [31]. RNA-quality and quantity was assessed using the NanoDrop® ND-1000 Spectrophotometer (NanoDrop Technologies, Wilmington, DE). Reverse transcription (Invitrogen Corp., Grand Island, NY) and qRT-PCR were carried out as previously described [31] on an ABI Prism 7900 Sequence Detection System (Applied Biosystems Inc, Foster City, CA). Primers for TBP, ZBTB10, Sp1, Survivin, VEGF and VEGFR-1 have been previously described [31]. The primer-sequences for SHIP-1were F:5’-GTG-GAG-AGA-TCT-GGC-CTC-AG-3’ and R:5’-GGG-AGC-AAC-AGC-AAA-GAC-TC-3’, for VEGF, the sequence were F:5’-AAG GAG GAG GGC AGA ATC AT-3’, and R:5’-ATC TGC ATG GTG ATG TTG GA-3’. Integrated DNA Technologies, Inc. (San Diego, CA). Primers were designed using Primer Express software (Applied Biosystems, Foster City, Ca), homology-searched by an NCBI BLAST and examined by dissociation curve analysis. Primers for Sp3 and Sp4 were purchased from Qiagen. microRNA was extracted, reverse transcribed, and qRT-PCR reaction was performed as previously performed [30, 31].

Transfection with antagomers of miR-155 and miR-27a (inhibitors), miR-27a mimic, or small interfering RNA (siRNA) against SHIP-1 gene

Cells seeded (1×105) into 12-well plates were incubated for 24h to allow cell attachment. Transfections with 10nM siRNA against SHIP-1 gene Santa Cruz Biotechnology Inc. (Santa Cruz, CA) and antagomers (inhibitors) of 20 nM, 40 nM and 80 nM miR-27a and miR-155, and also mimic of 20 nM miR-27a (Dharmacon, Lafay ette, CO) were performed as previously described [6, 31]

Reporter gene transfection and luciferase assays

Cells were transfected with constructs as previously described [6, 9, 31].

Western blotting

Cells (4 × 105) were seeded in 6-well plates and incubated 24 h to allow cell attachment. They were also treated with Pg extract (0–10µg/mL) for 24 h. Cells were harvested and prepared for Western Blotting as previously performed [30, 31]. Proteins were visualized with a Kodak Molecular Imaging System (Carestream Health, Rochester, NY).

Xenograft study

Female athymic BALB/c nude mice (age 3–4 weeks) from Harlan laboratory (Houston, TX) were implanted with BT474 cells (2×10⁶ cells) in matrigel (BD Bioscience San Jose, CA) s.c into the flank [31]. After 10 days mice were treated with either 100 µl vehicle (1% DMSO in corn oil) by oral gavage, or 0.8mg gallic acid equivalent (GAE)/kg/day of Pg extract/d for 35 days after approval by the Institutional Animal Care and Use Committee at Texas A&M University (College Station, TX). Final body and tumor weights were determined. Tissues were flash-frozen in liquid nitrogen and stored at −80°C for mRNA and protein analysis. 4-AM-thick paraffin-embedded tumor sections were cut and stained with Hematoxylin and Eosin (H&E) for bright field microscopy.

Statistical analysis

Quantitative data represent mean values with the respective standard deviation (SD) or standard error of the mean (SE) corresponding to 3 or more replicates. Data were analyzed by one-way analysis of variance (ANOVA) using SAS version 9.3 (SAS institute Inc., Cary, NC). Tukey’s Post Hoc multiple comparisons were used (p<0.05) to establish significant statistical difference.

Polyphenolic Composition of Pomegranate Extract Determined by HPLC-MS

The polyphenolic profile of pomegranate varities was previously reported as being rich in ellagitannins and anthocyanins [19, 20, 25, 45]. The primary ellagitannin in pomegranate juice is punicalagin [45], that can be converted into free ellagic acid upon hydrolysis. In this study, a polyphenolic extract was prepared from a pomegranate juice extract (Pg) that was kindly provided by Stiebs LLC (Kirkland, WA). The chromatographic profiles (Fig. 1a and 1b) show the ellagitannins punicalagin A (peak 1), punicalagin B (peak 2), anthocyanins delphinidin 3-glucoside (peak 3) and cyanidin-3-glucoside (peak 4). Low concentrations of ellagic acid glucoside (peak 5) and free ellagic acid (peak 6) were also detected.

Anti-proliferative and Pro-apoptotic Activities of Pomegranate Extract

The cytotoxic activities of Pg were investigated in human BT474 and MDA-MB-231 breast cancer cell-lines and non-cancer breast fibroblasts MCF-10F and MCF-12F (Fig. 2a). In both cancer cell lines, there was a concentration-dependent decrease in cell viability after treatment with Pg (2.5–25 µg/ml) after 48 h. In contrast, no significant cytotoxic effects were observed in non-transformed cells treated under the same conditions (Fig. 2a). Similar results were observed after 72 h (data not shown). Pg-induced cytotoxicity was accompanied by activation of capase-3, a primary apoptosis-executing enzyme, and the cleaved product of the substrate Poly (ADP-ribose)-polymerase1 (PARP) [46]. Full lengths PARP protein was not decreased by Pg whereas the highest concentration of Pg decreased full length Caspase-3 (Fig. 2b).

Modulation of Sp transcription factors and Sp-regulated genes and disruption of miR-27a:ZBTB10

Treatment of BT474 breast cancer cells with Pg (2.5–10 µg/ml) decreased expression of Sp1, Sp3, and Sp4 mRNA (Fig. 3a) and protein (Fig. 3b). Pg also decreased luciferase-activity in BT474 cells transfected with a plasmid construct containing the GC-rich regions from the Sp1 and Sp3 promoters [31] (Fig. 3c). Additionally, Pg induced expression of the Sp-repressor ZBTB10 (Fig. 3d) and decreased VEGF, VEGFR-1 and survivin m RNA and protein (Fig. 3e) and (Fig. 3f). [31].

The basal expression of miR27a was higher in BT474 and MDA-MB-231 breast cancer cells compared to the non-cancer breast fibroblasts (Fig. 3g). Pg (2.5–10 µg/ml) significantly decreased miR-27a expression only in the cancer cell lines in a concentration-dependent manner, in the non-cancer fibroblasts minimal effects on miR-27a were observed (Fig. 3h). When BT474 cells were transfected with the antagomir (Ant.) of miR-27a, miR-27a was decreased and ZBTB10 mRNA was induced and this was comparable to the effects of Pg extract (Fig. 3d and 3h) Both the miR-27a antagomir and Pg extract alone and in combination decrease miR-27a (Fig. 3i) and induce ZBTB10 (Fig. 3j). Transfected of BT474 cells with miR27a mimic increased miR-27a level and this was partially reversed by the treatment with Pg, indicating that Pg directly or indirectly targets miR27a (Fig. 3k). We also observed the Pg extract decreased Sp1, NF-κB (p65) and VEGF protein (Fig. 4a) and mRNA (Fig. 4b) expression in MDA-MB-231 cells and this was accompanied by induction of ZBTB10 mRNA (Fig. 4c) and this is similar to the effects of Pg extract in BT474 cells.

Pg-induced Modulation of SHIP-1, PI3K and AKT and Involvement of SHIP-1 and miR-155 in the Modulation of AKT and NF-κB

Pg also induced the upregulation of SHIP-1 mRNA and protein in BT474 (Fig. 5a, 5b) and this was accompanied by a decrease in pPI3K and pAKT while but not AKT (total protein) (Fig. 5b). Pg extract also caused similar effects on SHIP-1, pAKT, pPI3K and AKT in MDA-MB-231 breast cancer cells (Fig. 5c). To better understand the effects of Pg on SHIP-1, PI3K and AKT, BT474 cells were transfected with the siRNA of SHIP-1. SiRNA SHIP-1 increased pAKT protein levels. In contrast, Pg decreased pAKT protein expression (Fig. 5b) and these effects were partially reversed by siRNA SHIP-1 (Fig. 5d). MiR-155 is overexpressed in several breast cancer cell lines [47–50]. It was previously reported that polyphenolic extracts decrease miR-155 [40, 41, 51]. Basal expression of miRNA-155 was elevated in BT474 and MDA-MB-231 cells compared to the non-cancer cell lines (Fig. 5e). Pg (2.5–10 µg/ml) significantly decreased miR-155 (Fig. 5f) in the cancer cell lines (compared MCF-10F and MCF-12F) and this correlates with the upregulation of SHIP-1 mRNA levels in BT474 and MDA-MB-231 cells (Fig. 5a and 5c). Transfection of BT474 cells with miR-155 antagomir of treatment with 10 µg/ml Pg alone or in combination decreased miR-155 expression (Fig. 5g) and increased SHIP-1 mRNA level (Fig. 5h). Further confirming that Pg extract disrupts miR155: SHIP-1 interaction in BT474 cells.

Effects of Pg on NF-κB

Pg significantly inhibited the constitutive expression and phosphorylation of NF-κB p65 in BT474 cells (Fig. 6a) and in MDA-MB-231 cells was also decreased by Pg (Fig. 4a) Furthermore, Pg extract decreased luciferase activity in BT474 cells transfected with pNF-κB-Luc (Fig. 6b) and similar results were observed in cell co transfected with miR-155 (Fig. 6c) or miR-27a (Fig 6d) antagomirs.. These results suggest that both microRNAs are involved in Pg-induced repression of NF-κB.

Xenograft Study in Athymic Female Nude Mice

The clinical relevance of the cytotoxic activities of Pg observed in vitro was investigated in an orthotopic model of breast cancer in athymic female nude mice with BT474 cells as xenografts. Treatment with Pg (0.8mg GAE/kg/day/) significantly decreased tumor volume and weight (Fig. 7a and 7b). Histopathologic evaluations of tumors indicated that tumors of Pg-treated animals exhibited with increased apoptotic lesions compared to the control group (Fig. 7c). Moreover, Pg decreased the expression of Sp1, mRNA and Sp1, Sp3, and Sp4 protein level (Fig. 7d and 7e) and significantly decreased miR-27a (Fig. 7f) whereas ZBTB10 mRNA was upregulated (Fig. 7d). In addition, Sp-regulated genes including VEGF, survivin and NFkB p65 were also decreased at the protein and mRNA level in tumors from mice treated with Pg compared to the control tumors (Fig. 7d and Fig. 7e).In addition SHIP-1 mRNA (Fig. 7d) and protein (Fig. 7e) levels were induced in tumors. This was accompanied by a reduction of SHIP-1-regulated proteins pAKT and pPI3K in mice exposed to Pg (Fig. 7e) and miR-155 (Fig. 7f) was also decreased.