Tumor Biol. DOI 10.1007/s13277-014-1665-y
RESEARCH ARTICLE
Human Sprouty1 suppresses growth, migration, and invasion in human breast cancer cells Ahmed H. Mekkawy & Mohammad H. Pourgholami & David L. Morris
Received: 22 August 2013 / Accepted: 17 January 2014 # International Society of Oncology and BioMarkers (ISOBM) 2014
Abstract Breast cancer is the most common cancer and the leading cause of cancer death in women worldwide. Expression of human Sprouty1 (hSpry1) gene is downregulated in most breast cancer patients, implicating it as an important tumor suppressor gene. So, we hypothesized that overexpression of hSpry1 gene may suppress breast cancer cell growth, migration, and invasion. Here, we demonstrate that in breast cancer cell lines, MDA-MB-231 and T47D, transfectioninduced overexpression of hSpry1 reduced cell population, proliferation, and colony formation in vitro without affecting cell apoptosis. Adhesion molecules act as both positive and negative modulators of cellular migration and invasion. Here, we found that overexpression of hSpry1 enhances the initial establishment events in breast cancer cell adhesion to type IV collagen and vitronectin. Moreover, the overexpression of hSpry1 in the highly invasive MDA-MB-231 breast cancer cells causes a significant reduction in cellular migration and invasion through Matrigel membranes. In addition, we showed that hSpry1 overexpression prevents VEGF secretion. VEGF is essential for primary tumor growth, migration, and invasion. Thus, our study provides a novel mechanism of tumor suppression activity of hSpry1.
Keywords Breast cancer . Sprouty . Proliferation . Adhesion . Migration . Invasion . VEGF
A. H. Mekkawy : M. H. Pourgholami : D. L. Morris Cancer Research Laboratories, University of New South Wales Department of Surgery, Sydney NSW 2217, Australia D. L. Morris (*) Department of Surgery, St George Hospital, University of New South Wales, Sydney NSW 2217, Australia e-mail: [email protected]
Introduction Sprouty (Spry) proteins were first identified as inhibitors of the receptor tyrosine kinase (RTK)-mediated Ras signaling in Drosophila [1]. The mammalian Spry family contains four homology proteins (Spry 1–4) with a conserved cysteine-rich region termed Spry domain [2]. Several studies on the Spry protein family have indicated its role as an inhibitor of the RTK in the Ras/mitogen-activated (MAP) kinase pathway involved in signaling of the fibroblast growth factor receptor (FGFR) [1] and the epidermal growth factor receptor (EGFR) [3]. However, other reports showed that mammalian Spry proteins can amplify EGF-mediated ERK/MAP kinase signaling [4, 5]. In addition, it has been shown that expression of human Sprouty1 (hSpry1) inhibits cellular proliferation and differentiation in noncancerous NIH3T3 cell line and human umbilical vein endothelial cells (HUVECs) [6, 7]. Since its role in the central of Ras/MAP kinase pathway, SPRY can act as putative tumor suppressor gene, and that loss of expression or function may allow the cell to be hypersensitive to growth signals [8]. Further, SPRY1 gene has been implicated with tumorigenesis of different types of cancers. hSpry1 protein was downregulated in 40 % of human prostate cancers when compared to normal prostate [9]. However, the expression levels of Spry1 were higher in hepatocellular carcinoma tissues than those in nontumor tissues [10, 11]. We have shown previously that hSpry1 interacts with the urokinase-type plasminogen activator receptor (uPAR), a receptor implicated in tumor growth, migration, and invasion [12] and that hSpry1 negatively regulates uPAR-stimulated cellular migration and invasion [13]. In 79 % of human breast cancers, the expression of hSpry1 has been found to be downregulated as compared to normal breast tissues [8]. Such observations suggest that hSpry1 may function as tumor suppressors in human breast cancer.
Tumor Biol.
Breast cancer is the most frequently diagnosed cancer and the leading cause of cancer death in females worldwide [14]. In this study, we hypothesized that hSpry1 could contribute to suppression of growth, migration, and invasion in human breast cancer cells. Confirming such hypothesis will improve our understanding of breast cancer onset and progression. This may lead to development of new molecular markers for predicting the onset of breast cancer as well as the identification of new anticancer therapies. In the current study, we overexpressed hSpry1 in MDA-MB-231 and T47D breast cancer cell lines to investigate the potential role of hSpry1 in suppression of human breast cancer. We found that hSpry1 overexpression suppresses breast cancer by modulating proliferation, adhesion, migration, and invasion of breast cancer cells. In addition, we revealed a novel mechanism by which hSpry1 exerts its inhibitory role in which hSpry1 overexpression prevents VEGF secretion.
Materials and methods
on the gel was verified by stripping and re-probing the blots with a mouse anti-GAPDH (Sigma-Aldrich).
Immunocytochemistry Cells transfected with either pCDNA3.1/hSpry1 or pCDNA3.1 empty plasmid were seeded into sterilized glass coverslips. Cells were washed with phosphate-buffered saline (PBS), fixed, and permeabilized with ice-cold methanol for 10 min at −20 °C. Cells were then washed, blocked with 1 % bovine sreum albumin (BSA), and incubated with anti-hSpry1 (Abnova), anti-caspase-3, anti-cytochrome-c (Cell Signaling Technology), or anti-VEGF (Santa Cruz Biotechnology) primary antibodies in 1 % BSA, followed by Alexa Fluor 488®conjugated secondary antibodies (Invitrogen) in 1 % BSA. Nuclei were counterstained with propidium iodide (PI, Sigma). Cells were then washed, mounted, and visualized using confocal laser scanning microscopy (Olympus IX71 Laser Scanning Microscope) and × 60 oil immersion lens.
Cell lines and culture
Cell viability assay
The human breast cancer cell lines MDA-MB-231 and T47D used in this study were obtained from the American Type Culture Collection (ATCC). Cells were maintained in Dulbecco’s modified Eagle medium supplemented with 10 % fetal calf serum (FCS) and 1 % antibiotics.
For viability experiments, cells were seeded into six-well plates for 24 h. At the end of the treatment period, cells were washed with PBS, trypsinized, and counted using trypan blue and hemocytometer. All experiments were set up in triplicate, and each experiment was performed three times.
Plasmid transfection The recombinant pCDNA3.1/hSpry1 was kindly provided by Dr. Bernard Kwabi-Addo, Department of Pathology, Baylor College of Medicine, USA. All plasmid DNAs were prepared using an EndoFree Plasmid Kit (Qiagen) following the manufacturer’s protocol. All transfections were performed using Nucleofector™ (Lonza) according to the manufacturer’s instructions. For generation of stable expression clones, transfected cells were seeded into six-well plates and transfected with pCDNA3.1/hSpry1 or control vector. Cells were maintained in a growth medium supplemented with 400–450 μg/ml Geneticin® (Gibco) for 14 days. More than 200 drug-resistant colonies were pooled to generate each individual polyclonal stable cell line. Expression of hSpry1 was confirmed using western blot and immunocytochemistry. Western blot analysis Western blot analysis was performed as previously described [15]. Briefly, cell lysates were probed with mouse anti-hSpry1 monoclonal antibody (Abnova, Taiwan) or mouse anti-VEGF (Santa Cruz Biotechnology). Comparable loading of proteins
Cell proliferation assay To evaluate the effect of hSpry1 overexpression on cell growth, cells transfected with pCDNA3.1 or pCDNA3.1/ hSpry1 were examined using MTT [3-(4,5-dimethylthiazol2-yl)-2,5-diphenyltetrazolium bromide] proliferation assay as previously described [16]. Briefly, cells were cultured (30,000/well, n=7) into 96-well plates. After incubation at 37 °C in 5 % CO2 for 20 h, 10 μl of MTT mixture was added to each well and incubated for 4 h at 37 °C in 5 % CO2, followed by addition of 100 μl of DMSO solution. Absorbance was measured at 570 nm using a 96-well plate reader.
Colony assay For colony formation assay, 500 cells were transferred into each well of six-well plates. Media were changed twice weekly for 3 weeks. Following this, plates were gently washed with PBS and cells were fixed with 100 % ethanol and stained with a 0.5 % solution of filtered crystal violet. Colonies consisting of more than 50 cells were counted under an inverted microscope.
Tumor Biol.
Internucleosomal DNA fragmentation Genomic DNA was isolated from cells using apoptotic a DNA ladder kit (Millipore), subjected to electrophoresis through a 1.5 % agarose gel in Tris-borate EDTA buffer, and stained with SYBR® Safe DNA Gel Stain (Invitrogen). In situ detection of fragmented DNA In situ labeling of DNA fragments was conducted using the terminal deoxyribonucleotidyl transferase (TdT)-mediated dUTP nick end labeling (TUNEL) assay (DeadEnd™ Fluorometric TUNEL system, Promega, Madison, WI, USA) in accordance with the instructions of the manufacturer. TUNEL labeling was observed by detecting the green fluorescence of apoptotic cells using confocal laser scanning microscopy (Olympus IX71 Laser Scanning Microscope). One thousand nuclei in each slide were analyzed for the number of TUNELpositive nuclei. Centrifugal force adhesion assay Cell adhesion to the extracellular matrix (ECM) is an essential process during cancer migration and invasion. To explore the consequence of hSpry1 overexpression on establishment events of adhesion, centrifugal force assay was used as previously described [17] with some modifications. Briefly, to measure the receptor-ligand affinity in the initial cell adhesion events, 96-well microplates were coated by addition of 100 μl of type IV collagen (10 μg/cm2), vitronectin (2 μg/ml), or heat-denatured BSA (10 μg/ml) in quadrate and then incubated overnight at 4 °C. Then, plates were placed on ice. Cells (105 cells/well) were added to the wells, and then, all wells were topped up completely with a tissue culture medium. Plates were covered with a parafilm and centrifuged for 4 min at 40×g/4 °C. Following that, additional plates were separately incubated for 4 min at 37 °C in order to follow the progress of adhesive strengthening events due to cytoskeletal engagement. Then, all plates were inverted and centrifuged for 4 min at 40×g/4 °C. Cells were gently washed with PBS, fixed with 2 % formaldehyde/PBS, stained with Giemsa, and counted in five different fields under the light microscope. Analysis of cell migration Cell migration was measured by using a wound healing assay as previously described [16]. Briefly, cells were seeded into six-well plates and incubated for 24 h at 37 °C. Cells transfected with either pCDNA3.1/hSpry1 or pCDNA3.1 empty plasmid were incubated to create a subconfluent monolayer. A wound was created manually by scrapping the cell monolayer with a yellow pipette tip. After washing, media were replaced with fresh one, cells were incubated in the
incubator, and images were taken at 0 and 16 h at four different focal areas in each well. These images were analyzed quantitatively by measuring the distance of cell migration in the wounded region. In addition, to assess the effect of hSpry1 in regulating cell migration, we used a 24-well Transwell® system with polycarbonate membranes of 8.0-μm pore size (#3422, Corning). Transfected cells (2×105/well) were seeded onto the upperside chambers in 0.2 ml of serum-free DMEM medium, and 0.6 ml of the same medium containing 1 % FCS was added to the lower chamber. At the end of the incubation period (18 h), cells remaining in the upper chamber were scraped. The number of cells that penetrate through the membrane to the lower surface is the migration number. These cells were fixed, Giemsa-stained, mounted on coverslips, and counted in five different fields under the light microscope.
Invasion assay Cell invasion assay is similar to that of cell migration assay; however, it requires cells first to enzymatically penetrate a barrier of an ECM or basement membrane extract and then to migrate through it. Here, invasion of breast cancer cells was assessed using 24-well plates with filters of 8.0-μm pore size and coated with Matrigel™ (#354480, BD Biosciences). Transfected cells (2×105/well) were seeded onto the upperside chambers in 0.25 ml of serum-free DMEM medium, and 0.75 ml of the same medium containing 10 % FCS was added to the lower chamber. At the end of the incubation period (18 h), cells remaining in the upper chamber were scraped. Cells that invaded and penetrated through the Matrigel-coated membrane to the lower surface were fixed, Giemsa-stained, mounted, and counted in five different fields under the light microscope.
Enzyme-linked immunosorbent assay Cells were seeded in six-well plates in 10 % FBS media for 24 h. VEGF produced in the culture medium was quantified using specific an enzyme-linked immunosorbent assay (ELISA) kit according to the manufacturer’s instructions (BioLegend Inc., San Diego, CA, USA). The attached cells were counted to normalize VEGF concentrations against number of cells.
Statistical analysis Data is expressed as percent of control (mean ± SEM). In addition, Student’s unpaired t test was used to determine probability values and significance differences.
Tumor Biol.
Results Expression and localization of hSpry1 in breast cancer cells permanently transfected with pCDNA3.1 or pCDNA3.1/hSpry1 To investigate the potential role of hSpry1 in breast cancer, we first confirmed the expression and localization of the protein in the transfected MDA-MB-231 and T47D breast cancer cell lines using both western blot and immunocytochemical analysis. Expression of hSpry1 in cells transfected with pCDNA3.1/hSpry1 was increased in comparison to cells permanently transfected with the control plasmid pCDNA3.1 using western blotting (Fig. 1a, b). In addition, immunocytochemical analysis showed that hSpry1 staining was prominent in the cytoplasm of the breast cancer cells permanently transfected with pCDNA3.1 (Fig. 1c, d). However, expression of hSpry1 was not detected in cells permanently transfected with pCDNA3.1 empty plasmid.
Fig. 1 Overexpression of hSpry1 in MDA-MB-231 and T47D cells stably transfected with pCDNA3.1/hSpry1. Lysates of MDA-MB-231 (a) and T47D (b) cells stably transfected with pCDNA3.1 or pCDNA3.1/hSpry1 were prepared for western blotting and then probed with an antibody specific for hSpry1. Equal protein loading was determined by stripping and reprobing the blots with an antibody against
hSpry1 suppress breast cancer growth without affecting cell apoptosis Figure 2 outlines the first set of experiments to test the hypothesis that hSpry1 would suppress breast cancer growth. The count of viable MDA-MB-231 and T47D cells overexpressing hSpry1 was significantly lower compared to control (p
RESEARCH ARTICLE
Human Sprouty1 suppresses growth, migration, and invasion in human breast cancer cells Ahmed H. Mekkawy & Mohammad H. Pourgholami & David L. Morris
Received: 22 August 2013 / Accepted: 17 January 2014 # International Society of Oncology and BioMarkers (ISOBM) 2014
Abstract Breast cancer is the most common cancer and the leading cause of cancer death in women worldwide. Expression of human Sprouty1 (hSpry1) gene is downregulated in most breast cancer patients, implicating it as an important tumor suppressor gene. So, we hypothesized that overexpression of hSpry1 gene may suppress breast cancer cell growth, migration, and invasion. Here, we demonstrate that in breast cancer cell lines, MDA-MB-231 and T47D, transfectioninduced overexpression of hSpry1 reduced cell population, proliferation, and colony formation in vitro without affecting cell apoptosis. Adhesion molecules act as both positive and negative modulators of cellular migration and invasion. Here, we found that overexpression of hSpry1 enhances the initial establishment events in breast cancer cell adhesion to type IV collagen and vitronectin. Moreover, the overexpression of hSpry1 in the highly invasive MDA-MB-231 breast cancer cells causes a significant reduction in cellular migration and invasion through Matrigel membranes. In addition, we showed that hSpry1 overexpression prevents VEGF secretion. VEGF is essential for primary tumor growth, migration, and invasion. Thus, our study provides a novel mechanism of tumor suppression activity of hSpry1.
Keywords Breast cancer . Sprouty . Proliferation . Adhesion . Migration . Invasion . VEGF
A. H. Mekkawy : M. H. Pourgholami : D. L. Morris Cancer Research Laboratories, University of New South Wales Department of Surgery, Sydney NSW 2217, Australia D. L. Morris (*) Department of Surgery, St George Hospital, University of New South Wales, Sydney NSW 2217, Australia e-mail: [email protected]
Introduction Sprouty (Spry) proteins were first identified as inhibitors of the receptor tyrosine kinase (RTK)-mediated Ras signaling in Drosophila [1]. The mammalian Spry family contains four homology proteins (Spry 1–4) with a conserved cysteine-rich region termed Spry domain [2]. Several studies on the Spry protein family have indicated its role as an inhibitor of the RTK in the Ras/mitogen-activated (MAP) kinase pathway involved in signaling of the fibroblast growth factor receptor (FGFR) [1] and the epidermal growth factor receptor (EGFR) [3]. However, other reports showed that mammalian Spry proteins can amplify EGF-mediated ERK/MAP kinase signaling [4, 5]. In addition, it has been shown that expression of human Sprouty1 (hSpry1) inhibits cellular proliferation and differentiation in noncancerous NIH3T3 cell line and human umbilical vein endothelial cells (HUVECs) [6, 7]. Since its role in the central of Ras/MAP kinase pathway, SPRY can act as putative tumor suppressor gene, and that loss of expression or function may allow the cell to be hypersensitive to growth signals [8]. Further, SPRY1 gene has been implicated with tumorigenesis of different types of cancers. hSpry1 protein was downregulated in 40 % of human prostate cancers when compared to normal prostate [9]. However, the expression levels of Spry1 were higher in hepatocellular carcinoma tissues than those in nontumor tissues [10, 11]. We have shown previously that hSpry1 interacts with the urokinase-type plasminogen activator receptor (uPAR), a receptor implicated in tumor growth, migration, and invasion [12] and that hSpry1 negatively regulates uPAR-stimulated cellular migration and invasion [13]. In 79 % of human breast cancers, the expression of hSpry1 has been found to be downregulated as compared to normal breast tissues [8]. Such observations suggest that hSpry1 may function as tumor suppressors in human breast cancer.
Tumor Biol.
Breast cancer is the most frequently diagnosed cancer and the leading cause of cancer death in females worldwide [14]. In this study, we hypothesized that hSpry1 could contribute to suppression of growth, migration, and invasion in human breast cancer cells. Confirming such hypothesis will improve our understanding of breast cancer onset and progression. This may lead to development of new molecular markers for predicting the onset of breast cancer as well as the identification of new anticancer therapies. In the current study, we overexpressed hSpry1 in MDA-MB-231 and T47D breast cancer cell lines to investigate the potential role of hSpry1 in suppression of human breast cancer. We found that hSpry1 overexpression suppresses breast cancer by modulating proliferation, adhesion, migration, and invasion of breast cancer cells. In addition, we revealed a novel mechanism by which hSpry1 exerts its inhibitory role in which hSpry1 overexpression prevents VEGF secretion.
Materials and methods
on the gel was verified by stripping and re-probing the blots with a mouse anti-GAPDH (Sigma-Aldrich).
Immunocytochemistry Cells transfected with either pCDNA3.1/hSpry1 or pCDNA3.1 empty plasmid were seeded into sterilized glass coverslips. Cells were washed with phosphate-buffered saline (PBS), fixed, and permeabilized with ice-cold methanol for 10 min at −20 °C. Cells were then washed, blocked with 1 % bovine sreum albumin (BSA), and incubated with anti-hSpry1 (Abnova), anti-caspase-3, anti-cytochrome-c (Cell Signaling Technology), or anti-VEGF (Santa Cruz Biotechnology) primary antibodies in 1 % BSA, followed by Alexa Fluor 488®conjugated secondary antibodies (Invitrogen) in 1 % BSA. Nuclei were counterstained with propidium iodide (PI, Sigma). Cells were then washed, mounted, and visualized using confocal laser scanning microscopy (Olympus IX71 Laser Scanning Microscope) and × 60 oil immersion lens.
Cell lines and culture
Cell viability assay
The human breast cancer cell lines MDA-MB-231 and T47D used in this study were obtained from the American Type Culture Collection (ATCC). Cells were maintained in Dulbecco’s modified Eagle medium supplemented with 10 % fetal calf serum (FCS) and 1 % antibiotics.
For viability experiments, cells were seeded into six-well plates for 24 h. At the end of the treatment period, cells were washed with PBS, trypsinized, and counted using trypan blue and hemocytometer. All experiments were set up in triplicate, and each experiment was performed three times.
Plasmid transfection The recombinant pCDNA3.1/hSpry1 was kindly provided by Dr. Bernard Kwabi-Addo, Department of Pathology, Baylor College of Medicine, USA. All plasmid DNAs were prepared using an EndoFree Plasmid Kit (Qiagen) following the manufacturer’s protocol. All transfections were performed using Nucleofector™ (Lonza) according to the manufacturer’s instructions. For generation of stable expression clones, transfected cells were seeded into six-well plates and transfected with pCDNA3.1/hSpry1 or control vector. Cells were maintained in a growth medium supplemented with 400–450 μg/ml Geneticin® (Gibco) for 14 days. More than 200 drug-resistant colonies were pooled to generate each individual polyclonal stable cell line. Expression of hSpry1 was confirmed using western blot and immunocytochemistry. Western blot analysis Western blot analysis was performed as previously described [15]. Briefly, cell lysates were probed with mouse anti-hSpry1 monoclonal antibody (Abnova, Taiwan) or mouse anti-VEGF (Santa Cruz Biotechnology). Comparable loading of proteins
Cell proliferation assay To evaluate the effect of hSpry1 overexpression on cell growth, cells transfected with pCDNA3.1 or pCDNA3.1/ hSpry1 were examined using MTT [3-(4,5-dimethylthiazol2-yl)-2,5-diphenyltetrazolium bromide] proliferation assay as previously described [16]. Briefly, cells were cultured (30,000/well, n=7) into 96-well plates. After incubation at 37 °C in 5 % CO2 for 20 h, 10 μl of MTT mixture was added to each well and incubated for 4 h at 37 °C in 5 % CO2, followed by addition of 100 μl of DMSO solution. Absorbance was measured at 570 nm using a 96-well plate reader.
Colony assay For colony formation assay, 500 cells were transferred into each well of six-well plates. Media were changed twice weekly for 3 weeks. Following this, plates were gently washed with PBS and cells were fixed with 100 % ethanol and stained with a 0.5 % solution of filtered crystal violet. Colonies consisting of more than 50 cells were counted under an inverted microscope.
Tumor Biol.
Internucleosomal DNA fragmentation Genomic DNA was isolated from cells using apoptotic a DNA ladder kit (Millipore), subjected to electrophoresis through a 1.5 % agarose gel in Tris-borate EDTA buffer, and stained with SYBR® Safe DNA Gel Stain (Invitrogen). In situ detection of fragmented DNA In situ labeling of DNA fragments was conducted using the terminal deoxyribonucleotidyl transferase (TdT)-mediated dUTP nick end labeling (TUNEL) assay (DeadEnd™ Fluorometric TUNEL system, Promega, Madison, WI, USA) in accordance with the instructions of the manufacturer. TUNEL labeling was observed by detecting the green fluorescence of apoptotic cells using confocal laser scanning microscopy (Olympus IX71 Laser Scanning Microscope). One thousand nuclei in each slide were analyzed for the number of TUNELpositive nuclei. Centrifugal force adhesion assay Cell adhesion to the extracellular matrix (ECM) is an essential process during cancer migration and invasion. To explore the consequence of hSpry1 overexpression on establishment events of adhesion, centrifugal force assay was used as previously described [17] with some modifications. Briefly, to measure the receptor-ligand affinity in the initial cell adhesion events, 96-well microplates were coated by addition of 100 μl of type IV collagen (10 μg/cm2), vitronectin (2 μg/ml), or heat-denatured BSA (10 μg/ml) in quadrate and then incubated overnight at 4 °C. Then, plates were placed on ice. Cells (105 cells/well) were added to the wells, and then, all wells were topped up completely with a tissue culture medium. Plates were covered with a parafilm and centrifuged for 4 min at 40×g/4 °C. Following that, additional plates were separately incubated for 4 min at 37 °C in order to follow the progress of adhesive strengthening events due to cytoskeletal engagement. Then, all plates were inverted and centrifuged for 4 min at 40×g/4 °C. Cells were gently washed with PBS, fixed with 2 % formaldehyde/PBS, stained with Giemsa, and counted in five different fields under the light microscope. Analysis of cell migration Cell migration was measured by using a wound healing assay as previously described [16]. Briefly, cells were seeded into six-well plates and incubated for 24 h at 37 °C. Cells transfected with either pCDNA3.1/hSpry1 or pCDNA3.1 empty plasmid were incubated to create a subconfluent monolayer. A wound was created manually by scrapping the cell monolayer with a yellow pipette tip. After washing, media were replaced with fresh one, cells were incubated in the
incubator, and images were taken at 0 and 16 h at four different focal areas in each well. These images were analyzed quantitatively by measuring the distance of cell migration in the wounded region. In addition, to assess the effect of hSpry1 in regulating cell migration, we used a 24-well Transwell® system with polycarbonate membranes of 8.0-μm pore size (#3422, Corning). Transfected cells (2×105/well) were seeded onto the upperside chambers in 0.2 ml of serum-free DMEM medium, and 0.6 ml of the same medium containing 1 % FCS was added to the lower chamber. At the end of the incubation period (18 h), cells remaining in the upper chamber were scraped. The number of cells that penetrate through the membrane to the lower surface is the migration number. These cells were fixed, Giemsa-stained, mounted on coverslips, and counted in five different fields under the light microscope.
Invasion assay Cell invasion assay is similar to that of cell migration assay; however, it requires cells first to enzymatically penetrate a barrier of an ECM or basement membrane extract and then to migrate through it. Here, invasion of breast cancer cells was assessed using 24-well plates with filters of 8.0-μm pore size and coated with Matrigel™ (#354480, BD Biosciences). Transfected cells (2×105/well) were seeded onto the upperside chambers in 0.25 ml of serum-free DMEM medium, and 0.75 ml of the same medium containing 10 % FCS was added to the lower chamber. At the end of the incubation period (18 h), cells remaining in the upper chamber were scraped. Cells that invaded and penetrated through the Matrigel-coated membrane to the lower surface were fixed, Giemsa-stained, mounted, and counted in five different fields under the light microscope.
Enzyme-linked immunosorbent assay Cells were seeded in six-well plates in 10 % FBS media for 24 h. VEGF produced in the culture medium was quantified using specific an enzyme-linked immunosorbent assay (ELISA) kit according to the manufacturer’s instructions (BioLegend Inc., San Diego, CA, USA). The attached cells were counted to normalize VEGF concentrations against number of cells.
Statistical analysis Data is expressed as percent of control (mean ± SEM). In addition, Student’s unpaired t test was used to determine probability values and significance differences.
Tumor Biol.
Results Expression and localization of hSpry1 in breast cancer cells permanently transfected with pCDNA3.1 or pCDNA3.1/hSpry1 To investigate the potential role of hSpry1 in breast cancer, we first confirmed the expression and localization of the protein in the transfected MDA-MB-231 and T47D breast cancer cell lines using both western blot and immunocytochemical analysis. Expression of hSpry1 in cells transfected with pCDNA3.1/hSpry1 was increased in comparison to cells permanently transfected with the control plasmid pCDNA3.1 using western blotting (Fig. 1a, b). In addition, immunocytochemical analysis showed that hSpry1 staining was prominent in the cytoplasm of the breast cancer cells permanently transfected with pCDNA3.1 (Fig. 1c, d). However, expression of hSpry1 was not detected in cells permanently transfected with pCDNA3.1 empty plasmid.
Fig. 1 Overexpression of hSpry1 in MDA-MB-231 and T47D cells stably transfected with pCDNA3.1/hSpry1. Lysates of MDA-MB-231 (a) and T47D (b) cells stably transfected with pCDNA3.1 or pCDNA3.1/hSpry1 were prepared for western blotting and then probed with an antibody specific for hSpry1. Equal protein loading was determined by stripping and reprobing the blots with an antibody against
hSpry1 suppress breast cancer growth without affecting cell apoptosis Figure 2 outlines the first set of experiments to test the hypothesis that hSpry1 would suppress breast cancer growth. The count of viable MDA-MB-231 and T47D cells overexpressing hSpry1 was significantly lower compared to control (p
