Metastasis and disease relapse are the major causes of morbidity and mortality among patients with triple-negative breast cancer (TNBC). Novel therapeutics that interfere with the process of metastasis, including epithelial-to-mesenchymal transition (EMT), are thus urgently required. Piperlongumine (PL) is a component of the fruits of the long pepper plant (
Breast cancer is classified into 5 distinct molecular subgroups: Normal-like, basal-like, luminal A, luminal B and human epidermal growth factor receptor (HER) 2-enriched (
Despite advances in treatment strategies and early detection, metastases remain as the primary cause of cancer-related mortality (
Certain bioactive dietary phytochemicals have attracted interest over the past few decades due to their potential for use in cancer prevention and treatment (
The present study compared the effects of free PL and PL-loaded mPEG-PLGA NPs (PL-NPs) on the
Dimethyl sulfoxide (DMSO), 3-(4,5-demethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), phenylmethylsulfonyl fluoride, Triton X-100, sodium deoxycholate, aprotinin, leupeptin, sodium fluoride, pepstatin A, dithiothreitol, polyamine oxidase, dichloromethane and gelatin were purchased from Sigma-Aldrich; Merck KGaA. L-glutamine, 10,000 units/ml penicillin/10,000 µg/ml streptomycin solution, 1M N-2 hydroxyethylpiperazine-N-2-ethane sulfonic acid (HEPES) buffer solution, fetal bovine serum (FBS), and Dulbecco's modified Eagle medium (DMEM) were from Invitrogen; Thermo Fisher Scientific, Inc. mPEG-PLGA (5-10 kDa) was from Akina Inc. Acrylamide/bis-acrylamide (29: 1, 30% solution), ammonium persulfate, sodium dodecyl sulfate, Tris-base, Tween-20, tetramethylethelyenediamide, ethylenediaminetetraacetic acid (EDTA) and ethylene glycol-bis(β-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA) were purchased from BioShop Canada Inc. Bio-Rad protein assay dye reagent concentrate and SsoFast EvaGreen™ Supermix® were from Bio-Rad Laboratories, Inc. PL, fibronectin and sodium orthovanadate were from EMD Millipore (Etobicoke, ON). ARP 100 was from Santa Cruz Biotechnology Inc..
Horseradish peroxidase (HRP)-conjugated rabbit anti-human β-actin monoclonal antibodies (Abs; cat. no. 12620), and rabbit monoclonal Abs against human β-catenin (cat. no. 8480), Slug (cat. no. 9585), ZEB1 (cat. no. 3396), pan-cadherin (cat. no. 4073), Smad3 (cat. no. 9520), NDRG1 (cat. no. 9485), and DNMT-1 (cat. no. 5032), were purchased from Cell Signaling Technology, Inc. Donkey anti-rabbit HRP-conjugated Abs (cat. no. sc-2313) were from Santa Cruz Biotechnology Inc. All Abs were diluted in 5% w/v fat-free milk or 5% w/v BSA, in Tween-TBS [20 mM Tris-HCl (pH 7.6), 200 mM NaCl, 0.05% Tween-20].
MDA-MB-231 human breast adenocarcinoma cells were kindly provided by Dr S. Drover (Memorial University of Newfoundland, St. John's, NL, Canada). MDA-MB-468 human breast adenocarcinoma cells were a generous gift from Dr P. Lee (Dalhousie University, Halifax, NS, Canada). BT-549 human breast ductal carcinoma cells were kindly provided by Dr P. Marcato (Dalhousie University, Halifax, NS, Canada). All breast cancer cell lines were free of mycoplasma contamination and were authenticated by the American Type Culture Collection (ATCC) using the short tandem repeat method. Breast cancer cells were cultured in DMEM that was supplemented with 10% heat-inactivated FBS, 100 U/ml penicillin, 100 µg/ml streptomycin, 2 mM of L-glutamine, and 5 mM of HEPES; henceforth, known as complete DMEM. Cells were maintained at 37˚C in a humidified 10% CO2 incubator.
NPs were prepared from mPEG-PLGA polymers using the thin-film hydration method, as previously described (
A JEM 1230 transmission electron microscope (JEOL Ltd.) and AMT Image Capture Engine (version 7.0; AMT Imaging) was used to image and measure 90 random particles, yielding an average NP size of 52.8±1.2 nm. Prior to use, the PL-NPs were filter-sterilized using a 0.20 µm syringe filter, which also removed polymer aggregates and any remaining PL crystals. The encapsulation efficiency was 20%, as determined by spectrophotometric analysis and a standard curve based on the absorbance of PL at 346 nm.
MDA-MB-231, MDA-MB-468 and BT549 cells were seeded into quadruplicate wells of a 96-well flat bottom cell culture plate at a concentration of 5x103 cells/well and incubated overnight to allow cell attachment. Cells were then cultured for 48 h in the presence of medium alone, vehicle (DMSO) alone, 2.5-10 µM free PL (dissolved in DMSO) or PL-NPs, or empty NPs. MTT solution was added to each well to a final concentration of 0.5 µg/ml. Cell-free supernatant was removed and formazan crystals were solubilized in DMSO. The absorbance was measured at 570 nm using an Expert 96 microplate reader (Biochrom ASYS) and the percentage metabolic activity was determined.
MDA-MB-231 cell monolayers were cultured for 36 h in the presence of the vehicle (DMSO) alone, 2.5 µM of free PL or PL-NPs, or empty NPs. The cells were then serum-starved for 12 h, harvested and resuspended at 1x106 cells/ml in 1 ml of appropriate treatment made in serum-free DMEM. A 50 µl aliquot of the cell suspension was loaded into the upper chamber of a Transwell migration apparatus. The cells migrated through an 8 µm porous membrane that was uncoated for migration assays or coated with fibronectin (0.05% w/v) or gelatin (0.01% w/v) for invasion assays. Growth medium containing 10% FBS was used as a chemoattractant. Migrated cells were stained for 45 sec at room temperature with a Diff-Quik™ staining kit (Siemens Inc.), photographed using a Nikon Eclipse TS 100 phase contrast microscope and Infinity 1 camera (Nikon Canada Inc.), and quantified using ImageJ software (version 1.51; National Institutes of Health).
MDA-MB-231 cell monolayers were cultured for 48 h in the presence of the vehicle (DMSO) alone, 2.5 or 5 µM of free PL or PL-NPs, or empty NPs. Cells were collected and resuspended in 50 µl of cold lysis buffer [0.1% v/v NP-40, 0.25% w/v sodium deoxycholate, 50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 5 mM EDTA, 5 mM EGTA pH 7.5] with a mixture of 1 mM phenylmethylsulfonyl fluoride, 5 µg/ml leupeptin, 5 µg/ml pepstatin, 10 µg/ml apotinin, 100 µM sodium orthovanadate, 1 mM dithiothreitol, 10 mM sodium fluoride and 10 µM polyamine oxidase. Following 15 min of incubation at 4˚C, debris was removed by centrifugation at 14,000 x g for 10 min at 4˚C. The supernatant containing total cellular proteins was collected and quantified by Bradford protein assay. Protein samples (30 µg) were loaded into wells of a 10% sodium dodecyl sulfate-polyacrylamide gel and proteins were separated by electrophoresis. Proteins were transferred to a nitrocellulose membrane and blocked with 5% fat-free milk for 1 h at room temperature to avoid non-specific binding. Blots were incubated overnight at 4˚C with primary Abs (anti-Slug, anti-ZEB1, and anti-pan-cadherin, 1:500; anti-β-catenin, anti-Smad3, anti-NDRG1, anti-DMNT1, anti-β-actin, 1:1,000). The membranes were thoroughly washed for 30 min with Tweet-TBS, and then incubated for 2 h at room temperature with the HRP-conjugated secondary Ab (1:1,000), followed by washing with Tween-TBS. Equal protein loading was confirmed by probing for β-actin expression. Protein bands were visualized with chemiluminescent HRP substrate Luminata™ (Merck KGaA) and Amersham high performance chemiluminescence film (GE Healthcare). Image Lab software (version 5.2; Bio-Rad) was used for densitometric analysis.
MDA-MB-231 cell monolayers were cultured for 48 h in the presence of the vehicle (DMSO) alone, the indicated concentrations of free PL or PL-NPs, or empty NPs. Total RNA was isolated from cells using the RNeasy mini kit (Qiagen Inc.) according to the manufacturer's instructions. RNA quantity and purity were determined by spectrophotometric analysis. Approximately 500 ng of RNA were reverse transcribed to cDNA using the iScript™ cDNA synthesis kit (Bio-Rad Laboratories, Inc.), according to the manufacturer's instructions. Appropriately diluted cDNA samples were combined with primer mix (10 µM forward and reverse primers), nuclease-free water, and SsoFast EvaGreen™ Supermix® (Bio-Rad Laboratories, Inc.) or SYBR®-Green PCR master mix (Qiagen Inc.) at a 1: 1: 3: 5 ratio, respectively. Samples were transferred to a Multiplate™ 96-well unskirted polypropylene PCR plate in triplicates and placed in the CFX Connect™ RT-PCR detection system (B Bio-Rad Laboratories, Inc.). The reaction steps were as follows: 10 min of activation at 95˚C, 40 cycles of 10 sec of denaturation at 95˚C, and 20 sec at the primer-specific annealing temperature. β-actin was also amplified at the same time and used as a reference gene. Data obtained from the RT-qPCR reaction were analyzed using CFX Manager software (version 3.1, Bio-Rad Laboratories, Inc.). The sequences of the primers were as follows: MMP2 (63˚C) forward, 5'-TGGCAAGTACGGCTTCTGTC-3' and reverse, 5'-TTCTTGTCGCGGTCGTAGTC-3'; E-cadherin (64˚C) forward, 5'-CAGCCACAGACGCGGACGAT-3' and reverse, 5'-CTCTCGGTCCAGCCCAGTGGT-3'; and β-actin forward, 5'-AAGATCAAGATCATTGCTCCTC-3' and reverse, 5'-CAACTAAGTCATAGTCCGCC-3'.
Statistical analysis was performed using one-way analysis of variance (ANOVA) with the Tukey-Kramer or Bonferroni multiple comparisons post hoc test, where appropriate, using GraphPad Prism analysis software (version 5.0, GraphPad Software Inc.). Differences were considered statistically significant at P<0.05.
The cytotoxicity of free PL and PL-NPs was compared using 3 different TNBC cell lines (
Transwell assays revealed that 2.5 µM of free PL or an equivalent concentration of PL-NPs significantly reduced the chemoattractant-induced migration of MDA-MB-231 TNBC cells through an uncoated membrane (
Western blot analysis revealed that culture in the presence of 2.5 µM free PL or an equivalent concentration of PL-NPs significantly reduced the MDA-MB-231 TNBC cell expression of the EMT-promoting transcription factor, Slug (
The effects of free PL and PL-NPs on the expression of TGFβ/Smad signaling pathway-associated Smad-3 and anti-metastatic NDRG1 in MDA-MB-231 cells were then determined. MDA-MB-231 cells that were cultured in the presence of 5 µM free PL or an equivalent concentration of PL-NPs exhibited decreased Smad3 expression (
Subsequently, the expression of DNMT1 was examined following treatment of the MDA-MB-231 TNBC cells with free PL or PL-NPs to determine whether there may be an effect on the hypermethylation of TNBC cell DNA. As shown in
PL has been shown to exert potent cytotoxic effects on TNBC cells (
Tumor cell migration and invasion through the basement membrane and ECM are essential components of tumor metastasis (
The present study also demonstrated that free PL and PL-NPs interfered with MDA-MB-231 TNBC cell expression of Slug and ZEB1, which are EMT-promoting transcription factors (
In the present study, the MDA-MB-231 TNBC cells treated with free PL or PL-NPs exhibited a reduced expression of Smad3, which is a key component of the TGFβ/Smad signaling pathway involved in the initiation of EMT (
EMT and the metastasis of TNBC cells is associated with the DNMT1-mediated hypermethylation of DNA (
In conclusion, the present study demonstrates, for the first time, to the best of our knowledge, that NPs formed from biocompatible mPEG-PGLA can deliver PL to cultures of TNBC cells without any loss of efficacy in comparison to free PL. In this regard, the growth of TNBC cells in monolayer cultures was inhibited by PL-NPs to the same extent as free PL. In addition, TNBC migration/invasion and the expression of EMT-promoting proteins was markedly decreased in the presence of PL-NPs. By contrast, TNBC cell expression of the tumor suppressor, NDRG1, and E-cadherin, which is associated with a less invasive epithelial phenotype, was upregulated by PL-NP treatment. Moreover, PL-NPs have the potential to prevent the hypermethylation of DNA via the PL-mediated inhibition of DNMT1 expression. Further analysis of the effects of PL-NPs on the epigenome is important, considering the interest in compounds that block the epigenetic modification of DNA as chemotherapeutic agents (
The authors would like to thank Ms. Mary Ann Trevors (Faculty of Medicine Electron Microscopy Core Facility, Dalhousie University) for providing assistance with NP characterization.
The present study was funded by a grant (no. 314347) to DWH from the Canadian Cancer Society. JGR was funded by a Nova Scotia Graduate Scholarship and a Cancer Research Training program award.
All data generated or analyzed during this study are included in this published article or are available from the corresponding author on reasonable request.
JGR performed all the assays. JGR and WF performed data analysis. JGR and WF drafted the manuscript. DWH designed the research. DWH and WF revised the manuscript. All authors read and approved the final manuscript.
Not applicable.
Not applicable.
The authors declare that they have no competing interests.
TNBC cell growth inhibition by PL and PL-NPs. (A) MDA-MB-231, (B) MDA-MB-468, and (C) BT-549 TNBC cells were seeded into 96-well flat bottom culture plates and cultured for 48 h in the presence of the vehicle (DMSO), empty NPs, or the indicated concentrations of free PL or PL-NPs. Cytotoxicity/growth inhibition was evaluated by the MTT assay. Data represent mean values ± standard error of the mean (SEM) of 3 independent trials. Statistical significance was determined using one-way ANOVA with the Tukey multiple comparisons post hoc test; *P<0.01, **P<0.001; ns, not significant; TNBC, triple-negative breast cancer; PL, piperlongumine; NPs, nanoparticles.
PL and PL-NPs inhibit the migration and invasiveness of MDA-MB-231 TNBC cells and their expression of MMP2. MDA-MB-231 cells were cultured in the presence of vehicle (DMSO), empty NPs, or 2.5 µM free PL or PL-NPs in serum-supplemented complete DMEM for 36 h followed by washing and culture for 12 h in serum-free complete DMEM. Cells were loaded into the upper chamber of a Transwell migration apparatus. Cells that moved through (A) uncoated 8 µm porous membranes used to assess migration, or (B) fibronectin-coated, and (C) gelatin-coated 8 µm porous membranes used to assess invasiveness, were stained and membranes were photographed at x20 magnification. Data shown are the mean number of migrating cells ± SEM of 3 (uncoated), 5 (fibronectin-coated) and 4 (gelatin-coated) independent experiments. (D) MDA-MB-231 cells were cultured for 48 h in the presence of vehicle (DMSO), empty NPs, or 2.5 µM free PL or PL-NPs. Total RNA was isolated from cells and mRNA was converted to cDNA. MMP2 mRNA expression was determined by RT-qPCR. β-actin was used as the reference gene. Data shown are mean MMP2 mRNA expression ± SEM of 3 independent experiments. (A-D) Statistical significance was determined by one-way ANOVA with the Bonferroni multiple comparisons post-test; *P<0.05, **P<0.01, ***P<0.001; ns, not significant. TNBC, triple-negative breast cancer; PL, piperlongumine; NPs, nanoparticles.
PL and PL-NPs suppress MDA-MB-231 TNBC cell expression of mesenchymal markers but upregulate E-cadherin expression. MDA-MB-231 cells were cultured for 48 h in the presence of the vehicle (DMSO), empty NPs, or the indicated concentrations of free PL or PL-NPs. (A and B) Total protein was isolated from lysed cells and subjected to western blot analysis. Equal protein loading was confirmed by probing for β-actin. Data shown are mean (A) Slug and β-catenin, and (B) N-cadherin expression ± SEM of 3 independent experiments. Representative blots are shown. (C) Total RNA was isolated from the cells and mRNA was converted to cDNA. E-cadherin mRNA expression was determined by RT-qPCR; β-actin was used as the reference gene. Data shown are mean E-cadherin mRNA expression ± SEM of 3 independent experiments. (A-C) Statistical significance was determined by one-way ANOVA with the Bonferroni multiple comparisons post-test; *P<0.05, **P<0.01, ***P<0.001; ns, not significant. TNBC, triple-negative breast cancer; PL, piperlongumine; NPs, nanoparticles.
PL and PL-NPs downregulate Smad-3 expression and upregulate NDRG1 expression by MDA-MB-231 TNBC cells. MDA-MB-231 cells were cultured for 48 h in the presence of the vehicle (DMSO), empty NPs, or 5 µM free PL or PL-NPs. Total protein was isolated from lysed cells and subjected to western blot analysis. Equal protein loading was confirmed by probing for β-actin. Data shown are mean (A) Smad-3 and (B) NDRG1 expression ± SEM of 3 independent experiments. Representative blots are shown. Statistical significance was determined by one-way ANOVA with the Bonferroni multiple comparisons post-test; *P<0.05, **P<0.01, ***P<0.001; ns, not significant. TNBC, triple-negative breast cancer; PL, piperlongumine; NPs, nanoparticles.
PL and PL-NPs inhibit DNMT-1 expression by MDA-MB-231 TNBC cells. MDA-MB-231 cells were cultured for 48 h in the presence of the vehicle (DMSO), empty NPs, or 5 µM free PL or PL-NPs. Total protein was isolated from lysed cells and subjected to western blot analysis. Equal protein loading was confirmed by probing for β-actin. Data shown are mean DNMT1 expression ± SEM of 3 independent experiments. A representative blot is shown. Relative protein expression level were compared using one-way ANOVA with the Bonferroni multiple comparisons post-test; *P<0.01; ns, not significant. TNBC, triple-negative breast cancer; PL, piperlongumine; NPs, nanoparticles.