Piperine inhibits the growth and motility of triple-negative breast cancer cells

ARTICLE IN PRESS Cancer Letters ■■ (2014) ■■–■■

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Cancer Letters j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / c a n l e t

Original Articles

Piperine inhibits the growth and motility of triple-negative breast cancer cells Anna L. Greenshields a,1, Carolyn D. Doucette a,1, Kimberly M. Sutton a, Laurence Madera b, Henry Annan c, Paul B. Yaffe d, Allison F. Knickle b, Zhongmin Dong c, David W. Hoskin a,b,c,* a

Department of Pathology, Faculty of Medicine, Dalhousie University, Halifax, Nova Scotia, Canada B3H 4R2 Department of Microbiology and Immunology, Faculty of Medicine, Dalhousie University, Halifax, Nova Scotia, Canada B3H 4R2 c Department of Biology, Saint Mary’s University, Halifax, Nova Scotia, Canada B3H 3C3 d Department of Surgery, Faculty of Medicine, Dalhousie University, Halifax, Nova Scotia, Canada B3H 4R2 b

A R T I C L E

I N F O

Article history: Received 1 August 2014 Received in revised form 6 November 2014 Accepted 7 November 2014 Keywords: Apoptosis Breast cancer Metastasis Piperine Xenograft

A B S T R A C T

Piperine, an alkaloid from black pepper, is reported to have anticancer activities. In this study, we investigated the effect of piperine on the growth and motility of triple-negative breast cancer (TNBC) cells. Piperine inhibited the in vitro growth of TNBC cells, as well as hormone-dependent breast cancer cells, without affecting normal mammary epithelial cell growth. Exposure to piperine decreased the percentage of TNBC cells in the G2 phase of the cell cycle. In addition, G1- and G2-associated protein expression was decreased and p21Waf1/Cip1 expression was increased in piperine-treated TNBC cells. Piperine also inhibited survival-promoting Akt activation in TNBC cells and caused caspase-dependent apoptosis via the mitochondrial pathway. Interestingly, combined treatment with piperine and γ radiation was more cytotoxic for TNBC cells than γ radiation alone. The in vitro migration of piperine-treated TNBC cells was impaired and expression of matrix metalloproteinase-2 and -9 mRNA was decreased, suggesting an antimetastatic effect by piperine. Finally, intratumoral administration of piperine inhibited the growth of TNBC xenografts in immune-deficient mice. Taken together, these findings suggest that piperine may be useful in the treatment of TNBC. © 2014 Elsevier Ireland Ltd. All rights reserved.

Introduction Mounting evidence that ingestion of certain culinary herbs and spices is associated with reduced cancer risk has generated tremendous interest in defining and exploiting the relatively nontoxic bioactive components of these natural products for their possible use in cancer prevention and/or treatment [1]. Black pepper, often referred to as “the king of spice”, is an important component of the Ayurvedic herbal formulation known as ‘Trikatu’, which is used in traditional Indian medicine for the treatment of a host of diseases that include gastrointestinal and respiratory ailments [2]. The medicinal effects of black pepper are at least in part attributable to the actions of piperine (1-piperoyl piperidine) [3], an amide alkaloid

Abbreviations: Ab, antibody; CDK, cyclin-dependent kinase; DiOC 6 , 3,30dihexyloxacarbocyanine iodide; GSH, reduced glutathione; HER2, human epidermal growth factor receptor 2; HRP, horseradish peroxidase; IAP, inhibitor of apoptosis protein; mAb, monoclonal antibody; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5diphenyltetrazolium bromide; PI, propidium iodide; ROS, reactive oxygen species; TNBC, triple-negative breast cancer. * Corresponding author. Tel.: +1 902 494 6509; fax: +1 902 494 5125. E-mail address: [email protected] (D.W. Hoskin). 1 Equal contributors.

found in the fruits of black pepper (Piper nigrum Linn.) and long pepper (Piper longum Linn.) plants [4]. A number of physiologic effects have been reported for piperine, including pain reduction [5], blood pressure lowering and associated vasomodulation [6], and enhanced digestion due to stimulation of gastric acid secretion and increased activity of intestinal and pancreatic lipases [7–9]. Piperine also increases the bioavailability of nutrients and drugs by increasing their absorption [10], as well as suppressing the action of the drug transporter P-glycoprotein [11] and decreasing drug metabolism by inhibition of cytochrome P450/CYP3A4 [12,13]. In addition, piperine is neuroprotective [14]. Importantly, piperine prevents tumor development [15], likely in part because of its antiinflammatory actions [16] and ability to interfere with angiogenesis [17]. Moreover, recent studies show that piperine is cytotoxic for mouse and human cancer cells, including 4T1 mouse mammary carcinoma cells [18] and PC3 human prostate cancer cells [19], and significantly increases the survival of mice injected with highly metastatic B16F10 mouse melanoma cells [20]. Taken together, these findings suggest that piperine may have potential as a therapeutic agent for the prevention and/or treatment of human cancers. Breast cancer is the most commonly diagnosed cancer in North American and European women, many of whom will succumb to metastatic disease in spite of receiving aggressive treatment [21].

http://dx.doi.org/10.1016/j.canlet.2014.11.017 0304-3835/© 2014 Elsevier Ireland Ltd. All rights reserved.

Please cite this article in press as: Anna L. Greenshields, et al., Piperine inhibits the growth and motility of triple-negative breast cancer cells, Cancer Letters (2014), doi: 10.1016/ j.canlet.2014.11.017

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Current approaches to the treatment of breast cancer are determined on the basis of whether the tumor cells are estrogen receptorpositive, overexpress human epidermal growth factor receptor 2 (HER2/ErbB2), or are triple-negative, i.e., fail to express estrogen receptor, progesterone receptor and HER2 [22]. However, a recent molecular analysis of tumors and germ-line DNA from 825 breast cancer patients has revealed the existence of four main breast cancer subtypes (Luminal A, Luminal B, Basal-like and HER2-enriched) that are heterogeneous with respect to the three major therapeutic breast cancer groups [23]. Advanced breast cancer that cannot be cured by surgery alone is typically treated with adjunct chemotherapy [24]. Unfortunately, chemotherapeutic drugs can cause secondary malignancies [25] and often have severe side effects due to their inability to discriminate between rapidly proliferating cancer cells and healthy dividing cells [26]. Moreover, breast cancers frequently become resistant to chemotherapy due to the emergence of variants that express multidrug resistance proteins such as P-glycoprotein [27]. This is a particular challenge for the treatment of recurrent triplenegative breast cancer (TNBC), which is hormone-independent and therefore refractory to hormonal-based therapy with tamoxifen or aromatase inhibitors, and fails to respond to HER2-targeted treatment [28]. The utilization of dietary phytochemicals with anticancer properties is currently being considered as a possible new approach to breast cancer treatment since these natural products are predicted to have fewer adverse side effects and to be effective against all breast cancer cell subtypes. With this in mind, we investigated the effect of the dietary phytochemical piperine on the growth of TNBC cell lines (MDA-MB-231 and MDA-MB-468) and estrogen receptor-expressing breast cancer cell lines (MCF-7 and T-47D) [29]. In addition, we determined the mechanism by which piperine inhibits TNBC cell growth and motility in vitro, as well as the in vivo effectiveness of piperine in a mouse model of TNBC. Materials and methods Animals Female 6–8 week old NOD-SCID mice were purchased from Charles River Canada (Lasalle, QC, Canada). Mice were housed in the Carleton Animal Care Facility at Dalhousie University and fed a standard diet of sterilized rodent chow and water ad libitum. The protocol (13–093) for in vivo studies was approved by the Dalhousie University Committee on Laboratory Animals and was in accordance with Canadian Council of Animal Care guidelines. Antibodies Anti-phospho-Akt (serine 473) rabbit antibody (Ab), anti-Akt rabbit Ab, antip21Waf1/Cip1 mouse monoclonal antibody (mAb), anti-cyclin D3 mouse mAb, and anticyclin-dependent kinase (CDK) 4 mouse mAb were purchased from Cell Signaling Technology Inc. (Beverly, MA). Anti-matrix metalloproteinase (MMP)-9 rabbit Ab, anti-E2F-1 mouse mAb, anti-CDK1 rabbit Ab, anti-cyclin B mouse mAb, and antiCdc25C mouse mAb were from Millipore Corporation (Temecula, CA). Anti-MMP-2 rabbit Ab, anti-actin goat Ab, horseradish peroxidase (HRP)-conjugated bovine antigoat IgG Ab, HRP-conjugated goat anti-mouse IgG Ab, and HRP-conjugated donkey anti-rabbit IgG Ab were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Anti-Smac/DIABLO rabbit mAb was purchased from Epitomics (Burlingame, CA). Anticytochrome c mouse mAb was purchased from BD Biosciences (Mississauga, ON). Reagents Dulbecco’s Modified Eagle’s Medium (DMEM), DMEM/F12, reduced glutathione (GSH), 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), aprotinin, bovine serum albumin, dimethyl sulfoxide (DMSO), leupeptin, Nonidet P-40, pepstatin A, phenylarsine oxide, phenylmethylsulfonyl fluoride, digitonin, sucrose, phosphate buffered saline (PBS), sodium deoxycholate, sodium fluoride, Triton X-100, mitomycin C, capsazepine, and piperine (purity ≥ 97%) were all purchased from SigmaAldrich Canada (Oakville, ON). Oregon Green-488 dye, 3,30-dihexyloxacarbocyanine iodide (DiOC6), fetal bovine serum, L-glutamine, penicillin/streptomycin solution, 4-(2hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) buffer solution, 0.25% trypsinEDTA, TrypLE Express, B27 serum-free supplement, and propidium iodide (PI) were purchased from Invitrogen Canada Inc. (Burlington, ON). DNase-free RNase A was

purchased from Qiagen Inc. (Mississauga, ON). Sodium orthovanadate, zVAD-fmk, and calcium chloride were purchased from EMD Chemicals, Inc. (Gibbstown, NJ). Ethylene diamine tetraacetic acid was purchased from EM Industries Inc. (Hawthorne, NY). Dithiothreitol, ethylene glycol tetraacetic acid, paraformaldehyde, sodium chloride, sodium dodecyl sulfate, Tris base, and Tween-20 detergent were purchased from Bio-Shop Canada Inc. (Burlington, ON). Basic fibroblast growth factor and epidermal growth factor were from PeproTech (Dollard des Ormeaux, QC). Cell culture MDA-MB-231 breast cancer cells were a kind gift from Dr. S. Drover (Memorial University of Newfoundland, St. John’s, NL, Canada). MDA-MB-468, T-47D, and MCF-7 breast cancer cells were generously provided by Drs. P. Lee, J. Blay, and K. Goralski, respectively (Dalhousie University, Halifax, NS, Canada). All breast cancer cell lines were authenticated by short tandem repeat profiling performed by ATCC (Manassas, VA). Human mammary epithelial cells were purchased from Lonza Inc. (Walkersville, MD) and maintained in MEGM medium (Lonza) at 37 °C in a 5% CO2 humidified atmosphere. Breast cancer cell lines were maintained in DMEM with 10% heatinactivated (56 °C for 30 min) fetal bovine serum, 5 mM HEPES buffer (pH 7.4), 2 mM L-glutamine, 100 U/ml penicillin, and 100 μg/ml streptomycin, henceforth referred to as complete DMEM (cDMEM). Cells were propagated in tissue culture flasks and maintained at 37 °C in a 10% CO2 humidified atmosphere. MTT assay Mitochondrial succinate dehydrogenase activity was used to assess cell growth using a modified MTT assay. Briefly, cells were seeded into quadruplicate wells (5 × 103 cells/well) of a 96-well flat-bottom tissue culture plate and allowed to adhere overnight. Cells were then treated with piperine or its vehicle (DMSO) for the desired time. In some experiments, cells were then treated with piperine or its vehicle for 1 h prior to exposure to 2–6 Gy of γ radiation delivered with a 137Cs source GammaCell GC3000 irradiator (MDS Nordion, Ottawa, ON). For the last 2 h of culture, MTT was added at a final concentration of 0.5 μg/ml, then plates were centrifuged at 1400 g for 5 min, the supernatant was discarded and formazan crystals were solubilized in 0.1 ml DMSO. The optical density of each well was determined at 490 nm using an ELX800 UV Universal Microplate Reader (BIO-TEK Instruments Inc., Winooski, VT). Spheroid culture and acid phosphatase assay MCF-7 spheroids were cultured in mammosphere medium (F12 medium supplemented with 20 ng/ml basic fibroblast growth factor, 20 ng/ml epidermal growth factor, 100 U/ml penicillin, 100 μg/ml streptomycin and B27 serum-free supplement) at 37 °C in a 10% CO2 humidified atmosphere. MCF-7 cells were seeded at 3 × 104 cells/well into ultra-low attachment 6-well Costar plates and spheroids were allowed to develop for 7 d prior to use. Spheroids were fed every 72 h. Following spheroid development, spheroids were treated with piperine or vehicle (DMSO) for 72 h. Following incubation, spheroids were washed in PBS and then resuspended in a 1:1 ratio with PBS and phosphatase solution (0.1 M sodium acetate at pH 5.5, 0.1% Triton X-100, 4 mg/ml phosphatase substrate) and incubated for 90 min at 37 °C in the dark. Following the incubation, each tube received 50 μl 1 N NaOH/ml and was centrifuged at 1000 g for 5 min. The supernatant was transferred to a 96-well plate and the absorbance was determined at 405 nm. Flow cytometric measurement of cell proliferation MDA-MB-468 cells were treated with 1.25 μM Oregon Green-488 dye in serumfree DMEM and incubated for 10 min on a rocker at room temperature. After removing excess dye by the addition of heat inactivated-fetal bovine serum, cells were pelleted and resuspended in cDMEM and incubated for 30 min at 37 °C. Cells were then washed, resuspended in cDMEM, and seeded at 5 × 104 cells/well into 6-well flatbottom tissue culture plates and allowed to adhere overnight. The following day, some cultures were harvested with TrypLE, fixed in 1% paraformaldehyde and stored at 4 °C for use as baseline controls. The remaining cultures were treated with the desired concentration of piperine or its vehicle (DMSO). After 72 h incubation, cells were harvested and analyzed by flow cytometry using a FACSCalibur flow cytometer (BD Biosciences). The number of cell divisions (n) was calculated using the mean channel fluorescence (MCF) of the sample (MCFsample) and the MCF of the baseline control (MCFbaseline) as follows: MCFbaseline = (2n)(MCFsample). Cell cycle analysis MDA-MB-468 cells were serum-starved overnight and seeded at 5 × 104 cells/ well in 6-well flat-bottom tissue culture plates and allowed to adhere overnight prior to treatment with piperine or its vehicle (DMSO) for 72 h. Cells were then harvested, washed, and resuspended in PBS, followed by the slow addition of ice cold 70% ethanol. Cells were stored for at least 24 h at −20 °C, then washed with PBS, resuspended in PI solution (0.1% v/v Triton X-100 in PBS containing 0.2 mg/ml DNasefree RNase A and 0.02 mg/ml PI) and incubated for 30 min at room temperature before

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analysis with a FACSCalibur flow cytometer. The DNA content of individual viable cells was quantified using ModFit LT 3.0 (Verity Software House, Topsham, ME). Apoptosis assay Flow cytometric analysis of Annexin-V-FLUOS/PI-stained cells was used to determine whether cell death occurred by apoptosis or necrosis. MDA-MB-468 cells were seeded at 5 × 104 cells/well into 6-well flat-bottom plates and allowed to adhere overnight prior to the addition of piperine or its vehicle (DMSO). After 72 h culture, nonadherent cells in the supernatant were collected and combined with adherent cells that were harvested using TrypLE Express. Cells were washed and then labeled with Annexin-V-FLUOS (Roche Diagnostics, Laval, QC) diluted as per the manufacturer’s instructions and PI (1 μg/ml) in detection buffer (10 mM HEPES, 140 mM NaCl, and 5 mM CaCl2) for 15 min at room temperature, followed by analysis using a FACSCalibur flow cytometer. Determination of mitochondrial membrane integrity MDA-MB-468 cells were seeded at 5 × 104 cells/well into 6-well flat-bottom plates and allowed to adhere overnight prior to the addition of piperine or its vehicle (DMSO). After 72 h culture cells were harvested and incubated for 15 min at 37 °C with 40 nM DiOC6 in cDMEM, then analyzed using a FACSCalibur flow cytometer. Western blot analysis Approximately 7 × 105 MDA-MB-468 cells were seeded into 75 mm2 tissue culture flasks and allowed to adhere overnight, then treated with piperine or its vehicle (DMSO). After 48 h culture, total protein was obtained by lysing cells on ice for 30 min in RIPA lysis buffer (1% Nonidet P-40, 0.5% sodium deoxycholate, 0.1% sodium dodecyl sulfate, 20 mM Tris, 150 mM NaCl, 1 mM ethylene diamine tetraacetic acid, 1 mM ethylene glycol tetraacetic acid at pH 7.5 with 5 μg/ml leupeptin, 5 μg/ml pepstatin A, 10 μg/ ml aprotinin, 100 μM sodium orthovanadate, 1 mM dithiothreitol, 10 mM sodium fluoride, 10 μM phenylarsine oxide, and 1 mM phenylmethylsulfonyl fluoride). For isolation of cytoplasmic proteins, cells were lysed on ice for 15 min with digitonin lysis buffer (75 mM NaCl, 1 mM NaH2PO4, 8 mM Na2HPO4, 250 mM sucrose and 190 μg/ ml digitonin with 5 μg/ml leupeptin, 5 μg/ml pepstatin, 10 μg/ml aprotinin, 100 μM sodium orthovanadate, 1 mM dithiothreitol, 10 mM sodium fluoride and 10 μM phenylarsine oxide). Following incubation, samples were centrifuged at 1000 g at 4 °C for 5 min to remove intact cells. Cell lysates were cleared by centrifugation at 14,000 g for 10 min at 4 °C and the supernatant collected. The protein concentration in cell lysates was determined using a protein assay kit (Bio-Rad, Hercules, CA). Equal amounts of denatured protein were resolved on 15, 12, or 7.5% sodium dodecyl sulfate–polyacrylamide gels (depending on desired protein size) for 1 h at 200 V. Protein was then transferred onto a nitrocellulose membrane using the iBlot transfer system (Invitrogen). Membranes were blocked for 1 h in Tris buffered saline (200 mM Tris, 1.5 M NaCl pH 7.6) containing 0.05% Tween-20 (TTBS) with 5% w/v skim milk powder. Blots were then washed in TTBS and incubated overnight at 4 °C with the desired primary Ab in either 5% w/v skim milk powder in TTBS or 5% w/v bovine serum albumin in TTBS, depending on the manufacturer’s recommendation. Blots were washed in TTBS and incubated with the appropriate secondary Ab in 5% w/v skim milk powder in TTBS. After additional washes, protein bands on the membrane were detected using ECL-plus western blotting detection reagent (GE Healthcare, Baie d’Urfe, QC) and visualized by X-ray film exposure. To confirm even protein loading, blots were reprobed with goat anti-actin Ab in 5% w/v skim milk powder and TTBS for 1 h, washed, and incubated for 1 h with secondary Ab in 5% w/v skim milk powder in TTBS. Protein bands were visualized as described above. Wound-healing assay Sterile culture-inserts (Ibidi, Munich, Germany) were placed in 6-well plates and MDA-MB-231 cells were seeded in each culture-insert well at a concentration of 1 × 104 cells in 0.1 ml cDMEM. Plates were incubated overnight at 37 °C under 10% humidified CO2. The next day, cells were treated with 10 μg/ml mitomycin C in cDMEM for 2 h, after which mitomycin C was removed and replaced with cDMEM. Cells were then cultured for 12 h. Culture-inserts were lifted from the wells using sterile forceps to create a uniform cell-free strip. Cultures were washed with cDMEM, then cultured in the absence or presence of 50 μM piperine for 17 or 33 h. Images were captured using a Nikon® Digital Sight camera head connected to a Nikon® Eclipse t5100 microscope (Nikon Canada, Mississauga, ON). Transwell cell migration assay MDA-MB-231 cells were cultured for 24 h in serum-free DMEM, then washed twice with PBS and resuspended in migration medium (DMEM plus 0.1% bovine serum albumin), prior to treatment with 50 μM of piperine or vehicle (DMSO). Treated cells were added to the upper wells (5 × 104 per well) of a 48-well microchemotaxis chamber (Neuro Probe, Gaithersburg, MD). Lower wells contained migration medium, as a baseline control, or DMEM plus 10% fetal bovine serum as a chemoattractant. The upper and lower wells were separated by a polycarbonate membrane with 8-μm-

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diameter pores (Neuro Probe). After 24 h of incubation, non-migrated cells were mechanically removed using a rubber blade scraper. Cells that adhered to the membrane were stained with the Diff-Quik Staining Kit (VWR, Radnor, PA). Migration for each treatment was measured by averaging the number of migrated cells per high powered field (200× magnification) of 5 random fields, with each treatment condition done in quadruplicate. Quantitative real-time PCR MDA-MB-231 cells were seeded at 5 × 104 cells/well into 6-well flat-bottom plates and allowed to adhere overnight prior to the addition of piperine (25 or 50 μM) or its vehicle (DMSO). After 48 h of culture, RNA was extracted from cells using a Qiagen RNeasy® minikit (Quiagen Inc., Toronto, ON) according to the manufacturer’s instructions. Purity of the extracted RNA was confirmed by spectrophotometric analysis. First-strand cDNA synthesis was performed using 667 ng RNA in 10 μl RNase-free water plus 1 μl of 50 μM oligo-dT and 1 μl of 100 mM dNTP (both from Invitrogen). The mixtures were heated to 65 °C in a Bio-Rad T100 thermal cycler and then chilled immediately on ice for 5–10 min. Four microliters of 5X First-Strand Buffer and 2 μl of 0.1 M dithiothreitol were added to each mixture, which was then incubated for 2 min at 42 °C, after which 1 μl of SuperScript® reversetranscriptase was also added (all from Invitrogen). Reaction mixtures were incubated at 42 °C for 50 min and at 70 °C for 15 min. Reaction products were stored at −20 °C. PCR reaction mixtures contained 5 μl of Qiagen Quantifast SYBR® Green, 1 μl of 1 μM sample cDNA, 2 μl RNase-free water and 1 μl of forward and reverse primers. Quantitative real-time PCR reactions were performed in triplicate using a Corbett Research Rotor Gene 6000 series analyzer (Corbett Robotics Inc., San Francisco, CA). The cycling conditions were: 5 min initial heat activation at 95 °C, 10 sec denaturation at 95 °C and 30 sec extension at 60 °C. Forty cycles were used and a melt curve analysis was performed. Forward and reverse primers with the following sequences for use in quantitative real-time PCR were synthesized by Integrated DNA Technologies, Inc. (Coralville, IA); hMMP2 – 5′-TGG CAA GTA CGG CTT CTG TC-3′ (forward), 5′-TTC TTG TCG CGG TCG TAG TC-3′ (reverse); hMMP9 – 5′-GAA GAG AAA TTC CAT GGA GCC AGG-3′ (forward), 5′-AGA AAT AAA AGA ACC CAA ATT CTT CAA AAA CA-3′ (reverse); hGAPDH – 5′-GAG TCA ACG GAT TTG GTC GT-3′ (forward), 5′-TTG ATT TTG GAG GGA TCT CG-3′ (reverse). Data were analyzed using the Rotor-Gene 6000 series software program. Tumor xenografts MDA-MB-468 breast cancer cells (2 × 106) mixed 1:1 with phenol red-free highconcentration Matrigel (BD Biosciences) were injected into the upper left mammary fat pad of female NOD/SCID mice. Once tumors reached a volume of at least 100 mm3 mice were randomized into control and treatment groups. Mice then received 15 μl of 0.7% DMSO in PBS (vehicle) or 0.2 mg/kg piperine in 0.7% DMSO in PBS via intratumoral injection every second day for a total of 3 injections. Tumor volume was calculated using the equation (L × P2)/2, where L and P denote the longest diameter and the diameter perpendicular to the longest diameter, respectively. On day 23, mice were euthanized by cervical dislocation, and tumors were excised and weighed. Statistical analysis Statistical analysis was performed by one-way analysis of variance (ANOVA) with the Bonferroni multiple comparison post-test, using InStat software version 3.00 (GraphPad Software Inc., San Diego, CA, USA). Differences were considered statistically significant when at p < 0.05.

Results Selective inhibition of breast cancer cell growth by piperine Four different human breast carcinoma cell lines that were HER2negative and differed with respect to estrogen receptor expression, as well as p53 status (see Table 1), were cultured for 72 h in the absence (vehicle only) or presence of piperine (50, 100 or 150 μM).

Table 1 Breast cancer cell line characteristics. Cell line

Estrogen receptor

HER2

p53 status

MCF-7 T-47D MDA-MB-231 MDA-MB-468

Positive Positive Negative Negative

Negative Negative Negative Negative

Wild-type Mutant Mutant Mutant

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150 μM piperine for 72 h (Appendix: Supplementary data 1), indicating that prolonged exposure to piperine resulted in an increased inhibitory effect on breast cancer cell growth. Importantly, primary cultures of normal human mammary epithelial cells were relatively resistant to piperine since 72 h treatment with the highest concentrations of piperine caused only a modest reduction in cell number (Fig. 1B). Piperine also inhibited the growth of MCF-7 mammospheres, as determined by an acid phosphatase assay (Fig. 1C); however, the effect was less marked than that observed in conventional cultures of MCF-7 cells (Fig. 1A). Piperine-treated mammospheres were smaller and less compact than control mammospheres, and were surrounded by a large number of floating cells with morphology characteristic of dead or dying cells. Since piperine mediates some of its physiologic effects via the TRPV1 receptor [30], we determined the effect of the TRPV1 receptor antagonist capsazepine on piperine-mediated inhibition of MDAMB-468 cell growth; however, TRPV1 receptor blockade failed to diminish the inhibitory effect of piperine (not shown). Piperinemediated inhibition of breast cancer cell growth therefore did not involve TRPV1 receptor signaling. Piperine suppresses TNBC cell proliferation To determine whether piperine inhibited breast cancer cell growth by interfering with cell proliferation, MDA-MB-468 cells were stained with Oregon Green 448 fluorescent dye prior to culture in the absence or presence of 100 or 150 μM piperine for 72 h. Flow cytometric analysis of the cells showed a dose-dependent increase in the fluorescence of piperine-treated MDA-MB-468 cells in comparison to the vehicle control (Fig. 2A), indicating that breast cancer cells underwent significantly fewer rounds of division in the presence of piperine. Cell cycle analysis of piperine-treated MDA-MB-468 cells showed a statistically significant decrease in the percentage of cells in the G2 phase of the cell cycle (Fig. 2B). In addition, western blot analysis showed reduced expression of G1-associated (cyclin D3, CDK4, E2F-1) and G2-associated (cyclin B, CDK1, Cdc25C) proteins, as well as increased expression of p21Waf1/Cip1 following treatment with piperine (Fig. 2C). Piperine-treated TNBC cells undergo apoptosis

Fig. 1. Piperine selectively inhibits breast cancer cell growth. (A) MDA-MB-231, MCF7, T-47D, MDA-MB-468 breast cancer cells or (B) human mammary epithelial cells were cultured in the presence of medium, vehicle (DMSO) or the indicated concentrations of piperine. After (A) 72 h or (B) 24, 48 and 72 h, cell growth was then assessed by MTT assay and the results were normalized with respect to cells cultured in medium alone. (C) MCF-7 breast cancer cells were seeded into ultra-low attachment 6-well plates and mammospheres were allowed to develop over 7 d, after which mammospheres were cultured in the absence or presence of 150 μM piperine for 72 h. Mammospheres were then photographed and viable cell number was determined using a modified phosphatase assay. (A–C) Data shown are the mean ± standard error of the mean (SEM) of at least 3 independent experiments; *p < 0.05, #p < 0.01 compared to the vehicle control by ANOVA with Bonferroni multiple comparison post-test.

Mitochondrial succinate dehydrogenase-mediated reduction of MTT was then used to determine the effect of piperine on breast cancer cell metabolic activity, which is a reflection of cell number. A dosedependent reduction in the number of TNBC cells (MDA-MB-231, MDA-MB-468) and estrogen receptor-expressing breast cancer cells (MCF-7, T-47D) was observed following piperine treatment; however, MD-MB-231 cells were least sensitive to growth inhibition by piperine, showing a statistically significant reduction in cell number only following exposure to 150 μM piperine (Fig. 1A). In addition, MDA-MB-468 cells that were treated with 75 μM piperine for 144 h showed similar growth inhibition to cells that were treated with

The contribution of cell death to piperine-mediated inhibition of breast cancer cell growth was determined by culturing MDAMB-468 cells in the absence or presence of piperine (50, 100 or 150 μM) for 72 h, and then staining the cells with Annexin-VFLUOS and PI. Flow cytometric analysis of piperine-treated TNBC cell cultures showed a dose-dependent increase in the percentage of Annexin-V-positive/PI-negative cells (lower right quadrant), indicative of early apoptosis, and Annexin-V-positive/PI-positive cells (upper right quadrant), indicative of late apoptosis/necrosis (Fig. 3A). Phase-contrast photomicrographs of piperine-treated MDA-MB468 cells showed a reduction in cell number and increase in rounded cells with morphology characteristic of apoptosis (Fig. 3B). We also examined the effect of piperine on the phosphatidylinositol-3kinase/Akt (protein kinase B) signaling pathway, which is implicated in breast cancer cell survival [31]. MDA-MB-468 cells that were cultured for 48 h in the presence of 150 μM piperine showed a reduction in phospho-Akt relative to control cells (Fig. 3C), indicating a reduction in pro-survival signaling in piperine-treated breast cancer cell cultures. Mitochondrial transmembrane potential is lost by piperine-treated TNBC cells Since mitochondria participate in apoptosis via the release of mitochondrial proteins into the cytoplasm [32], we used a membrane

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Fig. 2. Antiproliferative effect of piperine on TNBC cells. (A) MDA-MB-468 breast cancer cells were stained with Oregon Green 488 dye and cultured in the presence of medium, vehicle (DMSO) or the indicated concentrations of piperine for 72 h. Oregon Green-488 fluorescence was quantified by flow cytometry as a measure of cell proliferation and representative data are shown. The number of cell divisions was calculated and data shown are the mean of 3 independent experiments ± SEM. (B) MDA-MB-468 cells were cultured for 72 h in the absence or presence of 100 or 150 μM piperine, then stained with PI and analyzed by flow cytometry to determine cell cycle distribution. Data represent the mean ± SEM of at least 3 independent experiments. (A, B) Statistical significance in comparison to the vehicle control was determined by ANOVA and the Bonferroni multiple comparison post-test; *p < 0.05. (C) MDA-MB-468 cells were cultured in the presence of medium (M), the DMSO vehicle (V), or piperine (100 or 150 μM) for 48 h. Cells were then collected and lysed to obtain total cell protein. Equal amounts of protein were electrophoresed and transferred to a nitrocellulose membrane, which was then probed with anti-cyclin D3, anti-CDK4, anti-E2F-1, anti-cyclin B, anti-CDK1, anti-Cdc25C, or anti-p21Waf1/Cip1 Abs. The membrane was then washed and probed for actin to confirm equal protein loading. Results from a representative experiment are shown (n = 3).

potential-sensitive dye, DiOC6, to monitor changes in the mitochondrial transmembrane potential following piperine treatment of TNBC cells. As shown in Fig. 4A, MDA-MB-468 cells that were cultured in the presence of 100 or 150 μM piperine for 72 h experienced a significant reduction in mitochondrial membrane integrity. Consistent with compromised mitochondrial membrane integrity, western blot analysis of the cytosolic fraction from control and piperinetreated MDA-MB-468 cells showed that exposure to piperine caused the release of cytochrome c and Smac/DIABLO from the mitochondria of breast cancer cells (Fig. 4B). Caspases but not ROS participate in TNBC cell killing by piperine We next asked whether ROS and/or caspases participated in the piperine-induced apoptotic death of breast cancer cells.

Addition of the antioxidant GSH at 10 mM to MDA-MB-468 cell cultures prior to treatment with piperine for 72 h did not decrease the percentage of cells in early apoptosis or late apoptosis/necrosis, as determined by flow cytometric analysis of Annexin-V-FLUOS/PI-stained cells (Fig. 5A). These data indicate that piperine’s mode of action was ROS-independent. In contrast, addition of the pan-caspase inhibitor zVAD-fmk at 50 μM to MDA-MB-468 cell cultures prior to treatment with piperine for 72 h resulted in a substantial reduction in the percentage of cells undergoing early apoptosis, as determined by flow cytometric analysis of Annexin-V-FLUOS/PI-stained cells (Fig. 5B). Interestingly, zVAD-fmk did not decrease the percentage of piperine-treated cells undergoing late apoptosis/necrosis, suggesting a caspase-independent component of piperine-induced cytotoxicity.

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Fig. 3. Piperine induces apoptosis in TNBC cells. (A) MDA-MB-468 breast cancer cells were cultured in the absence or presence of the indicated concentrations of piperine for 72 h. Cells were then labeled with Annexin-V-FLUOS and PI, and analyzed by flow cytometry. Data shown are from a representative experiment (n = 3). (B) MDA-MB468 cells were cultured in the absence or presence of 150 μM piperine for 72 h, and then photographed under phase contrast at 200× magnification. (C) MDA-MB-468 cells were cultured in the presence of medium (M), the DMSO vehicle (V), or 150 μM piperine for 48 h. Cells were then collected and lysed to obtain total cell protein. Equal amounts of protein were electrophoresed and transferred to a nitrocellulose membrane, which was then probed with anti-phospho-Akt antibody. The membrane was probed for total Akt, then probed for actin to confirm equal protein loading. Results from a representative experiment are shown (n = 3).

Enhanced killing of TNBC cells by piperine in combination with ionizing radiation To determine whether piperine could potentiate the cytotoxic effect of ionizing radiation on TNBC cells, MDA-MB-468 cells were pretreated for 1 h with the DMSO vehicle or 25 μM piperine prior to exposing the cells to γ radiation. An MTT assay was used to measure changes in the relative cell number after 72 h of culture. As shown in Fig. 6, prior exposure to low-dose piperine enhanced the anti-proliferative effect of 4 and 6 Gy γ radiation on TNBC cells.

Piperine interferes with the metastatic potential of TNBC cells To investigate the anti-metastatic effects of piperine on TNBC cells, we performed wound-healing and cell migration assays with MDAMB-231 cells. The relative resistance of MDA-MB-231 cells to the growth inhibitory action of piperine allowed us to exclude this possible confounding effect from our experiments. As shown in Fig. 7A, wound healing was dramatically impaired in the presence of piperine at a concentration (50 μM) that was not cytotoxic for MDAMB-231 cells (Fig. 1A). In addition, the same concentration of piperine significantly inhibited chemoattractant-induced migration of MDAMB-231 cells in a cell migration assay (Fig. 7B). We next determined the effect of piperine on the expression of MMP-2 and MMP-9, which have been implicated in breast cancer metastasis because of their important role in mammary carcinoma cell motility and invasion [33]. As shown in Fig. 7C, MMP-2 and MMP-9 mRNA levels were significantly reduced in MDA-MB-231 cells treated with 50 μM piperine. MMP-2 protein was also decreased in piperine-treated MDA-MB231 cells relative to control cells (Fig. 7D); however, it was not possible to evaluate the effect of piperine on MMP-9 protein expression since MMP-9 synthesis by untreated MDA-MB-231 cells was below the level of detection for western blot analysis.

Piperine suppresses TNBC xenograft growth in mice Finally, we tested the effect of piperine on the growth of MDAMB-468 breast cancer cells implanted into the mammary fat pad of immune-deficient NOD/SCID mice. Intratumoral injections of piperine (0.2 mg/kg) into fully developed tumors (approximately 100 mm3 in volume) every other day for a total of three injections resulted in a statistically significant (p < 0.01) reduction in tumor size by day 23 compared to vehicle-treated tumors (Fig. 8A). The average weight of piperine-treated tumors was 54% that of vehicletreated tumors (Fig. 8B). In addition, there was no evidence of toxicity due to piperine treatment. The average weight of control and piperine-treated mice was comparable throughout the experiment and necropsies did not show any gross anatomical differences between the two groups.

Discussion Piperine from the fruits of black and long pepper plants shows promise as a natural source anticancer agent because of its reported ability to inhibit carcinogenesis, angiogenesis, tumor growth, and metastasis [15,17–20]; however, little is known about the effect that piperine has on breast cancer cells, particularly TNBC cells that lack functional p53. In this study, we show that piperine inhibited the in vitro growth of p53-deficient MDA-MB-231 and MDA-MB468 TNBC cells, as well as p53-deficient and estrogen receptorpositive T-47D breast cancer cells and estrogen receptor-positive MCF-7 cells that express wild-type p53 [34]. A recent study shows that piperine also suppresses proliferation and causes cytotoxicity in cultures of HER2-overexpressing SKBR3 and BT-474 breast cancer cells [35]. Collectively, these findings indicate that all three therapeutic groupings of breast cancer are potentially responsive to piperine treatment. In our hands, T-47D and MDA-MB-468 cells were more sensitive to piperine than MCF-7 cells, indicating that

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Fig. 5. Piperine-induced apoptosis is ROS-independent and caspase-dependent. (A) MDA-MB-468 breast cancer cells were pretreated for with 10 mM glutathione (GSH) for 30 min, then cultured in the absence or presence of 150 μM piperine for 72 h. (B) MDA-MB-468 breast cancer cells were pretreated for 1 h with 50 μM zVADfmk, then cultured in the absence or presence of 150 μM piperine for 72 h. (A, B) The percentage of cells in early apoptosis versus necrosis/late apoptosis was determined by flow cytometric analysis of PI- and Annexin V-FLUOS-stained cells. Data are shown as % cell death ± SEM; * p < 0.05 compared to total cell death in the presence of the vehicle (DMSO) alone, # p < 0.01 compared to the total cell death caused by piperine in the absence of zVAD-fmk, as determined by a one-way ANOVA with Bonferroni multiple comparison post-test. Fig. 4. Piperine causes mitochondrial membrane destabilization and release of cytochrome c and Smac/DIABLO. (A) MDA-MB-468 breast cancer cells were cultured in the absence (filled peak) or presence (open peak) of the indicated concentrations of piperine for 72 h. Cells were then harvested, labeled with 40 nM DiOC6 and analyzed by flow cytometry to assess mitochondrial membrane integrity. Data from a representative experiment are shown. Percent mitochondrial membrane integrity loss was calculated and data shown are the average of 3 experiments ± SEM;* p < 0.001 compared to the vehicle control, as determined by a one-way ANOVA with Bonferroni multiple comparison post-test. (B) MDA-MB-468 cells were cultured in the presence of medium (M), the DMSO vehicle (V), or 150 μM piperine for 48 h. Cells were then lysed and the cytoplasmic protein fraction was isolated and equal amounts of protein were electrophoresed and transferred to a nitrocellulose membrane, which was then probed with antibodies against cytochrome c or Smac/ DIABLO. The membrane was then probed for actin to confirm equal protein loading. Results from a representative experiment are shown (n = 3).

piperine-mediated growth inhibition did not require functional p53, even though cell cycle arrest and apoptosis induction in response to genotoxic stress is typically p53-dependent [36]. The relative resistance of MDA-MB-231 cells to growth inhibition by piperine was therefore most likely due to some factor other than a lack of functional p53. The fact that piperine was able to inhibit the growth of breast cancer cell lines that lack functional p53 is clinically important since p53 is often mutated and not functional in breast tumors [23]. Furthermore, piperine suppressed the growth of MCF-7 cells

in mammospheres, albeit to a lesser degree than its effect on monolayer cultures of MCF-7 cells. Mammosphere formation by normal human breast epithelial cells is also impaired in the presence of piperine [37]. Interestingly, MCF-7 cells that form mammospheres express typical cancer stem cell markers [38], while the 3-dimensional structure of multicellular tumor spheroids is more representative of the tumor microenvironment [39]. It is therefore reasonable to speculate that piperine may be able to interfere with the proliferation of cancer stem cells within breast tumors. Importantly, the growth of normal human mammary epithelial cells was only minimally affected by the highest concentrations of piperine used in this study, indicating a selective effect on the growth of neoplastic cells. This is in line with our earlier finding that piperine is not toxic to normal human endothelial cells [17]. Nevertheless, further study is needed to determine whether it is safe to administer high-dose piperine to breast cancer patients since acute and subacute toxicity has been reported in rodents following systemic administration of large quantities of piperine [40]. Inhibition of MDA-MB-468 breast cancer cell growth in the presence of piperine was in part due to decreased cell proliferation. In addition, cell cycle analysis of piperine-treated MDA-MB-468 cells

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Fig. 6. Combined treatment with low-dose piperine and ionizing radiation increases TNBC cell death. MDA-MB-468 breast cancer cells were treated with the vehicle (DMSO) or 25 μM piperine for 1 h prior to exposure to the indicated doses of γ radiation (2–6 Gy). Cells were then cultured for 72 h and relative changes in cell number were determined by MTT assay. Data shown are an average of 3 independent experiments ± SEM; * p < 0.01 compared to the vehicle control, # p < 0.01 compared to non-irradiated, piperine-treated cells, as determined by one-way ANOVA with a Bonferroni multiple comparison post-test.

showed a reduction in the percentage of cells in the G2 phase of the cell cycle. Although there was a consistent trend toward accumulation of piperine-treated MDA-MB-468 cells in the G1 and S phases of the cell cycle, this effect was not statistically significant; nevertheless, the additive effect of G1 and S phase accumulation likely accounts for the observed decrease in G2 phase accumulation. Expression of proteins associated with the G1 (cyclin D3, CDK4, E2F-1) and G2 (cyclin B, CDK1, Cdc25C) phases of the cell cycle decreased following exposure of MDA-MB-458 cells to piperine; however, the greatest effect appeared to be on G1-associated proteins. Binding of D-type cyclins to CDK4 and CDK6 is required for G1 phase activity [41], whereas E2F-1 is a transcription factor involved in G1/S transition [42]. CDK1, in complex with A- or B-type cyclins, is important for G2/M transition [41]. Cdc25C is a phosphatase that is active at the onset of mitosis but is also involved in G1/S and G2/M transitions [43]. Furthermore, increased expression of p21Waf1/Cip1 by piperine-treated MDA-MB-468 cells was consistent with inhibition of cell cycle progression since p21Waf1/ Cip1 is a CDK inhibitor [41] and also suppresses DNA synthesis by binding to and inhibiting proliferating cell nuclear antigen [44]. Taken together, our findings suggest that piperine had a broad inhibitory effect on both G1/S and G2/M transition, which may account for the failure of piperine-treated TNBC cells to show significant accumulation at a particular phase of the cell cycle. In contrast, G1 phase cell cycle arrest has been reported in a panel of piperine-treated human prostate cancer cell lines [45], whereas 4T1 mouse mammary carcinoma cells show G2/M arrest and reduced expression of cyclin B1 but not cyclin D1 following exposure to piperine [18]. The cell cycle regulators that are targeted by piperine therefore appear to vary depending on the cancer cell type. MDA-MB-468 breast cancer cells that were treated with piperine also died by apoptosis, as shown by the dose-dependent increase in the percentage of cells that were stained with Annexin V-FLUOS but not PI (early apoptosis) and with Annexin V-FLUOS and PI (late apoptosis/necrosis). In addition, changes in MDA-MB-468 cell morphology following exposure to piperine were consistent with the induction of apoptosis. Interestingly, constitutive phosphorylation of Akt on Ser473 was diminished when MDA-MB-468 cells were cultured in the presence of a cytotoxic concentration of piperine. Survival-promoting Akt signaling is also inhibited in SKBR3 breast cancer cells [35], indicating that this effect of piperine is not restricted to a single breast cancer cell type. This is of considerable interest because of the observation that the phosphatidylinositol-

Fig. 7. Piperine inhibits TNBC cell migration and expression of MMP-2 and MMP-9 mRNA. (A) MDA-MB-231 breast cancer cells were seeded into culture-inserts and treated with 10 μg/ml mitomycin C. Then culture-inserts were removed and cultures were treated with medium alone, the vehicle (DMSO) or 50 μM piperine for the indicated times. Images shown are representative of 3 independent experiments. (B) MDA-MB-231 cells were treated with 50 μM of piperine or vehicle (DMSO), and then added to the upper wells of a 48-well microchemotaxis chamber. Lower wells contained migration medium, as a baseline control, or DMEM plus 10% fetal bovine serum, as a chemoattractant. After 24 h, non-migrated cells were removed and membrane-adherent cells were stained and counted. Migration for each treatment was measured by averaging the number of migrated cells per high powered field (200× magnification) of 5 random fields, with each treatment condition done in quadruplicate. (C) MMP-2 and MMP-9 mRNA expression in MDA-MB-231 cells that were cultured for 48 h in the absence or presence of the indicated concentrations of piperine was determined by quantitative real-time PCR. Data shown are an average of 3 independent experiments ± SEM; * p < 0.05 compared to the vehicle control, as determined by one-way ANOVA with a Bonferroni multiple comparison post-test. (D) MDA-MB-231 cells were cultured in the presence of medium (M), the DMSO vehicle (V), or piperine (100 or 150 μM) for 72 h. Cells were then collected and lysed to obtain total cell protein. Equal amounts of protein were electrophoresed and transferred to a nitrocellulose membrane, which was then probed with anti-MMP-2 Ab. The membrane was then washed and probed for actin to confirm equal protein loading. Results from a representative experiment are shown (n = 3).

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Fig. 8. Piperine inhibits the growth of TNBC xenografts. MDA-MB-468 breast cancer cells in Matrigel were implanted into the mammary fat pad of female NOD/SCID mice. Upon reaching a size of 100 mm3 tumors were injected every second day for a total of 3 injections (indicated by arrows) with vehicle (0.7% DMSO in PBS) or 0.2 mg/kg piperine. (A) Tumor volume was measured over the course of the experiment. Data shown are the mean tumor volume and SEM (vehicle, n = 6; piperine, n = 5); * p < 0.01 compared to the vehicle-treated mice, as determined by Student’s t-test. (B) Mice were euthanized on day 23 following initiation of treatment, tumors were excised and weighed. Data shown are the individual and the mean weight of tumors in vehicle- or piperine-treated mice.

3-kinase/Akt signaling pathway is frequently dysregulated in breast cancer cells, leading to their enhanced survival and proliferation [31]. Indeed, the phosphatidylinositol-3-kinase/Akt signaling pathway is currently seen to be an important therapeutic target for breast cancer treatment [46]. Our current findings and those of others [35] suggest that piperine may have clinical utility as a phosphatidylinositol-3kinase/Akt inhibitor. Piperine treatment of MDA-MB-468 breast cancer cells resulted in diminished mitochondrial membrane integrity and the subsequent release of cytochrome c and Smac/DIABLO from mitochondria into the cytosol. This observation implicates the intrinsic pathway of apoptosis as the mechanism of piperine-induced cytotoxicity since cytosolic cytochrome c is essential for apoptosome formation, which results in the activation of initiator caspase-9 [32]. In addition, cytosolic Smac/DIABLO promotes apoptosis by binding to inhibitor of apoptosis proteins (IAPs), thereby preventing their suppression of caspase-activation [47]. Consistent with the involvement of caspases in piperine-induced apoptosis, we found that the percentage of early apoptotic MDA-MB-468 cells following piperine treatment was significantly reduced in the presence of the pancaspase inhibitor zVAD-fmk. In support of our findings, caspase activation by piperine has been reported in other cancer cell lines [18,19,35]. Our data show that oxidative stress was not a factor in the killing of breast cancer cells by piperine. Although we did not

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measure ROS production in piperine-treated MDA-MB-468 cells, the inability of the antioxidant GSH to reduce the percentage of early apoptotic or late apoptotic/necrotic MDA-MB-468 cells following piperine treatment excluded ROS as a mediator of cytotoxicity. This was in contrast to the ROS-dependent cytotoxic effect of piperine on rectal cancer cells [48], indicating that the mechanism of action is cell typespecific. Furthermore, the increased killing of MDA-MB-468 cells that we observed after combination treatment with γ radiation and piperine may be due to the ROS-independent cytotoxic effect of piperine complementing ROS-dependent killing of cancer cells by ionizing radiation [49]. Degradation of extracellular matrix components by tumor cellelaborated proteolytic enzymes and tumor cell locomotion are important steps in cancer invasion and metastasis [50], which remains a major cause of breast cancer mortality [51]. A noncytotoxic concentration of piperine suppressed metastatic activities of MDAMB-231 breast cancer cells, even though these cells were relatively resistant to the growth inhibitory effect of piperine. In this regard, piperine inhibited MDA-MB-231 cell motility in both woundhealing and cell migration assays. This finding is in line with reports that piperine reduces the in vitro migration of mouse mammary cancer cells [18] and other human cancer cell lines [19,35], indicating that this effect of piperine is not cell type-specific. A molecular mechanism that might account for diminished breast cancer cell motility in the presence of piperine is suggested by the finding that migration-related MMP-2 and MMP-9 mRNA expression by MDAMB-231 cells was inhibited in the presence of piperine. MMP-2 protein levels were also reduced in piperine-treated MDA-MB231 cells. MMP-9 protein expression was below the limit of detection for western blot analysis; however, this does not exclude the possibility of an inhibitory effect of piperine on MMP-9 protein levels under conditions that increase MMP-9 synthesis. MMP-2 and MMP-9 are among the MMPs that are overexpressed in breast tumor tissue [52] and have been implicated in breast cancer metastasis because of their important role in mammary carcinoma cell motility and invasion [33]. Although piperine-mediated inhibition of MMP-9 expression and/or activity has previously been reported in other mouse and human cancer cell lines [18,35,53], to our knowledge this is the first report that piperine also suppressed MMP-2 expression. Reduced expression of MMP-2 and MMP-9 mRNA by piperine-treated MDA-MB-231 cells may be due to the inhibitory effect of piperine on Akt activation since phosphatidylinositol-3kinase/Akt signaling is important for MMP-2 and MMP-9 activity in other human tumor cell types [54,55]. Taken together, these findings suggest that piperine-mediated inhibition of cancer cell motility and expression of MMPs may account for the ability of piperine to interfere with lung metastasis in mouse models of metastatic melanoma and mammary cancer [18,20]. The growth inhibitory effect of piperine on MDA-MB-468 breast cancer cells was confirmed in our in vivo studies, which showed that three intratumoral injections of piperine were sufficient to cause a marked reduction in the growth of MDA-MB-468 tumors that were already established in the mammary fat pad of immune-deficient mice. Interestingly, we achieved a statistically significant effect using a much lower dose of piperine (0.2 mg/kg) than that used by Lai and colleagues (2.5 and 5 mg/kg) to suppress the in vivo growth of 4T1 mouse mammary cancer cells, even though the route of administration and number of treatments were the same [18]. The reason for this discrepancy is not clear since the IC50 values of piperine for in vitro growth inhibition of the two breast cancer cell lines after 72 h treatment are comparable (90 μM piperine for MDA-MB468 cells, as shown in Fig. 1A, versus 79 μM piperine for 4T1 cells [18]). Intratumoral administration of phytochemicals is a useful approach to demonstrate in vivo activity without the confounding influence of the poor bioavailability of many of these agents, which

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Fig. 9. Scheme for piperine-induced inhibition of TNBC cell growth and motility. Treatment of TNBC cells with piperine inhibits cell cycle progression and induces the mitochondrial pathway of caspase-dependent apoptosis, involving the release of mitochondrial SMAC/Diablo (suppresses the function of IAPs) and cytochrome c (promotes apoptosome formation) into the cytoplasm. Piperine-mediated inhibition of MMP-2 and MMP-9 gene expression may contribute to decreased TNBC migration, which is important for metastasis.

can restrict their usefulness for oral consumption [56]. Given that average daily consumption of piperine in the form of black pepper varies between 16.5 and 29.7 mg [57], it is not feasible to reach bioactive levels of piperine by diet alone. Nevertheless, it is reasonable to expect that health benefits can be obtained by ingesting piperine supplements since approximately 96% of administered piperine is absorbed following oral intake by rats [58]. Although plasma concentrations of piperine on the order of 150 μM, which was the dose used in most of our cell culture experiments, may be difficult to attain by oral administration, our data show that longer exposure to concentrations of piperine as low as 25 μM also significantly inhibited the growth of breast cancer cells. It should be possible to achieve such biologically active plasma concentrations of piperine by oral consumption since pharmacokinetic studies in rats show that oral gavage of piperine at 54 mg/kg results in peak plasma concentrations of approximately 20 μM [59]. Moreover, administration of 10 mg/kg piperine by oral gavage is sufficient to inhibit the growth of LNCaP human prostate cancer cells in nude mice, even though maximal inhibition of LNCaP cell growth occurs following 72 h exposure to 100–200 μM piperine [19], which is similar to the concentrations of piperine used in most of our in vitro studies. Nanotechnology-based drug delivery systems show considerable promise as a means of enhancing the oral delivery of phytochemicals for use in the prevention and treatment of cancer [56]. For example, encapsulation of phytochemicals in nanoemulsions allows for a substantial 3-fold increase in oral bioavailability [60]. It is therefore possible that relatively high plasma concentrations of piperine might be achieved by oral supplementation using a nanotechnology-based delivery system. Although we cannot specify the exact amount of piperine that would need to be ingested in order to maintain an effective concentration in the blood, it seems likely that breast cancer patients could benefit from the sustained consumption of piperine in a form that would maximize its bioavailability; however, further study is required before

undertaking clinical trials because of the potential for toxic side effects [40]. In conclusion, as shown in Fig. 9, our findings suggest that piperine selectively inhibited the growth of breast cancer cell lines derived from patients with difficult-to-treat TNBC, as well as those with estrogen receptor-positive breast cancer, via an antiproliferative effect and induction of caspase-dependent apoptosis, without the need for functional p53. In addition, piperine interfered with breast cancer cell activities (migration and MMP expression) that are associated with metastasis. A reduction in constitutive Akt activation following piperine treatment of TNBC cells may represent the molecular basis for these diverse effects. Although early stage TNBC typically responds to initial chemotherapy, advanced or recurrent disease tends to resist current chemotherapeutic regimens [28]. Our in vitro and in vivo findings suggest that further investigation of piperine is warranted as a potential drug candidate for the treatment of p53-deficient breast cancers, particularly those with a triplenegative phenotype.

Acknowledgements This work was supported by a grant (R12F13) to D. Hoskin from the Canadian Breast Cancer Foundation-Atlantic Region. C. Doucette, A. Greenshields, and K. Sutton were recipients of Natural Sciences and Engineering Research Council Postgraduate Studentships. C. Doucette was also supported by a Nova Scotia Health Research Foundation Studentship. A. Greenshields, K. Sutton, L. Madera, and A. Knickle were recipients of Trainee Awards from the Cancer Research Training Program. L. Madera was also supported by a Canadian Breast Cancer Foundation-Atlantic Region Postdoctoral Fellowship. P. Yaffe was the recipient of a Resident Research Award from the Department of Surgery, a Killam Scholarship, and a Nova Scotia Health Research Foundation Studentship. H. Annan was

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