Boyden Chamber Migration Assay Research Articles

Clinical Outcome of Polypoid Lesion of Gallbladder 10 mm or Larger

Background and Aim. The pathogenesis and progression of pancreatic cancer involves conversion of TGFβ suppressive signaling in early lesions to an unmasked, TGFβ proliferative signaling cascade in metastatic lesions. We have previously demonstrated that TGFβ proliferative signaling downregulates the tumor suppressor PTEN and induces motility of pancreatic cancer cells after ligand-receptor binding with subsequent increase in cytoplasmic free Ca2+ concentration ([Ca2+]cyt) and activation of PKCα and NF-κB. [Ca2+]cyt homeostasis is important to maintain cell functionality dysregulation of which contributes to tumorigenesis of pancreas. Transient receptor potential ion channels (TRPC) help regulate [Ca2+]cyt, and here we evaluated the role of TRPC channels in regulating TGFβ proliferative signaling. Methods. BxPc3 pancreatic cancer cells were treated with TGFβ (10 ng/mL), and TRPC and PTEN expression were analyzed by RT-PCR and/or Western blots. [Ca2+]cyt was assessed using Fura-2 fluorescence ratio digital imaging. Pharmacological reagents (TRPC inhibitors: 2-APB, SKF96365, and LaCl3) and siRNAs to TRPCs were used to determine the role of TRPCs on TGFβ-induced PTEN regulation and cell migration. Cell motility was assessed by Boyden chamber migration assay. Results. BxPc3 cells express TRPC1, 4, and 6 isoforms. TGFβ induced an increase in [Ca2+]cyt in Ca2+-free solutions and also induced an increase in [Ca2+]cyt when extracellular Ca2+ was restored. TGFβ-induced Ca2+ entry involves the activation of TRPC, as the TRPC inhibitors 2-APB, SKF96365, and LaCl3, were able to reduce peak [Ca2+]cyt by 80%, 40%, and 29%, respectively, and reversed TGFβ-induced PTEN downregulation. The 2-APB, SKF96365, and LaCl3 inhibitors also reduced TGFβinduced cell motility. Using siRNA, inhibition of TRPC1 but not other isoforms, successfully prevented TGFβ-induced cell motility. Conclusion. TRPC1 regulates TGFβ-induced downregulation of PTEN and pancreatic cell motility. This aspect of TGFβ regulation of PTEN and pancreatic cell motility might be exploited therapeutically to control pancreatic cancer metastasis.

Abstract 1440: Downregulation of Pdcd4 leads to epithelial to mesenchymal transition and promotes metastasis in nude mice

Abstract Progression of benign tumor into invasive carcinoma is a major cause of death in cancer patients. Programmed cell death 4 (Pdcd4) is a novel tumor suppressor, which inhibits TPA-induced transformation and tumor phenotype in mouse epidermal JB6 cells. It has been reported that overexpression of pdcd4 cDNA significantly reduces invasive ability of colon carcinoma and breast carcinoma cells in Matrigel invasion assays. Conversely, knockdown of Pdcd4 leads to fibroblast-like transition, promotion of Matrigel invasion, and activation of β-catenin dependent transcription. Activation of β-catenin dependent transcription by Pdcd4 knockdown results in up-regulation of urokinase-type plasminogen activator receptor (u-PAR) and c-Myc expression. Knockdown of u-PAR or c-Myc inhibits invasion in Pdcd4 knockdown cells, suggesting that both u-PAR and c-Myc contribute to the enhanced invasion induced by Pdcd4 knockdown. However, it is unclear whether down-regulation of Pdcd4 promotes metastasis in vivo. In this study, we demonstrated that knockdown of Pdcd4 decreases the expression of epithelial markers (α-catenin and γ-catenin) and increases the protein level of mesenchymal markers (fibronectin and N-cadherin) in colon GEO and HT29 cells. These results indicate that knockdown of Pdcd4 leads to epithelial to mesenchymal transition (EMT). To delineate whether Pdcd4 knockdown promotes cell migration, wound-healing and Boyden chamber migration assays were performed. In wound-healing assay, Pdcd4 knockdown cells showed wound closure approximately 3-fold faster than control cells. In addition, the migration capacity of Pdcd4 knockdown cells is approximately 10-fold higher than that of control cells using the Boyden chamber assay. These findings indicate that knockdown of Pdcd4 promotes migration. To test whether knockdown of Pdcd4 promotes metastasis in vivo, parental, Pdcd4 knockdown, and control cells were injected into the cecal wall (orthotopic implantation) of nude mice (n = 6/group). Six weeks post-injection, samples of lung, liver, lymph node, and cecum were collected and the tumor nodules of each sample were determined. Tumors are formed on cecum in all injected mice. However, only mice injected with Pdcd4 knockdown cells developed hepatic and local lymph node metastases. No pulmonary metastasis was observed. These results indicate that Pdcd4 knockdown promotes metastasis in nude mice. Taken together, our data demonstrated that knockdown of Pdcd4 leads to EMT and promotion of migration and metastasis. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1440. doi:10.1158/1538-7445.AM2011-1440

Abstract 4814: Detection of DNAH1 fragment closely related to human colorectal cancer by RLGS and functional analysis of DNAH1 in cancer cells

Abstract [Introduction] Many cancer related genes in human cancer have been isolated by recent development of techniques in molecular biology, however, the existence of unknown genes still have been expected. Restriction landmark genomic scanning (RLGS) is one of the most powerful methods for the identification of a DNA methylation which correlate with protein expression level. This DNA analysis method is 2-dimensional gel electrophoresis of genomic DNA fragment digested with specific restriction enzyme. We have evaluated clinical samples such as breast cancer, hepatocellular carcinoma (HCC), glioma and colorectal cancer for more than 10 years. In this study, we evaluated RLGS to find genetic alterations relative to human colorectal cancer and cloned some of altered spots. We have found that one of these clone codes a part of dynein axonemal heavy chain 1 (DNAH1). Dyneins are large motor protein complexes that generate the force for the movement of eukaryotic cilia and flagella. [Materials and Methods] High molecular weight genomic DNAs were extracted from 25 colorectal cancerous tissues and corresponding normal mucosae. To analysis alteration spot, each DNA was cleaved with the restriction enzyme Not I, size fractionated by 1st-dimensional electrophoresis using Pvu II, and the DNA fragments were then cleaved by Pst I as the third enzyme in gel and separated by 2-dimensional electrophoresis. Various cell lines, such as A431, AsPC-1, TE10, MCF7, and WiDr were used for in vitro analysis. To examine gene expression of DNAH1, quantitative RT-PCR was performed using TaqMan® Gene Expression Assays. Cell migration was measured using Boyden chamber migration assay for vertical migration and EZ-TAXIScan (GE Healthcare Japan) for horizontal migration. AsPC-1 cells and AsPC-1 shRNA DNAH1 stable cells were used for xenograft model to evaluate function of DNAH1 in vivo. [Results] 19 of 25 (76.0%) colorectal cancer cases showed a spot with the same alteration, which never appeared in normal mucosae. A 410-bp DNA fragment corresponding to this spot was cloned. This sequence was found to be 98% homologous to part of dynein axonemal heavy chain 1 (DNAH1). Gene expression level of DNAH1 was correlated to cell motility in vitro migration assays. In addition, DNAH1 expression level and cell motility were altered by treatment of 5-azacytidin (a protein inhibitor of DNA methyltransferase) or siRNA of DNAH1. Histological and histopathological evaluation of xenogragt model showed that AsPC-1 is INF beta and shRNA DNAH1 stable AsPC-1 is INF alpha. [Conclusion] We have identified the genomic alteration specific in human colorectal cancer by RLGS. The expression of homologpus gene to DNAH1 is closely related to colorectal cancer. Moreover, DNAH1 might not be express only in colorectal cancer but also other cancers. Expression level of DNAH1 related to cancer cell motility and metastasis in vivo and in vitro. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4814. doi:10.1158/1538-7445.AM2011-4814

Abstract 1442: Expression of miRNA processing factor Dicer in cutaneous melanoma and its role in cell invasion

Abstract Deregulated expression of miRNAs is a hallmark of various human cancers. One potential mechanism for the aberrant expression of miRNAs in cancers is their abnormal processing due to altered expression or function of their processing factors such as Dicer and Drosha. Although altered expression of several miRNAs have been reported in melanoma, the expression profile of miRNA processing factors in this cancer is unknown. In this study we examined the expression of Dicer protein in different stages of melanocytic lesions. Using tissue microarray and immunohistochemistry, we evaluated cytoplasmic Dicer expression in 32 dysplastic nevi, 77 primary melanomas, and 48 metastatic melanomas. Our data revealed that expression of Dicer has a significant but inverse correlation with progression of melanoma (P < 0.001). Accordingly, the number of samples with moderate-strong staining for Dicer was reduced from 81.2% in dysplastic nevi to 67.5% in primary melanoma and 40.8% in metastatic melanoma. The expression of Dicer was also negatively correlated with the American Joint Committee on Cancer (AJCC) staging of the melanoma samples (p=0.019). Moreover, the reduced Dicer expression was correlated with a poorer disease-specific 5-year survival of melanoma patients (P = 0.026). Multivariate Cox regression analysis revealed that reduced nuclear Dicer expression is an independent prognostic factor to predict patient outcome (P = 0.030). We also knocked down Dicer expression and used Boyden chamber migration assay to study its role in the invasion ability of MMRU human melanoma cells. Interestingly, knockdown of Dicer enhanced the invasion ability of MMRU cells by 2-fold. Our results demonstrate the critical role of miRNA machinery in the progression of melanoma, suggesting that Dicer may be a suitable prognostic marker for human melanoma patients as well as a potential therapeutic target. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1442. doi:10.1158/1538-7445.AM2011-1442

Interaction Effect between the Receptor for Advanced Glycation End Products (RAGE) and High-Mobility Group Box-1 (HMGB-1) for the Migration of a Squamous Cell Carcinoma Cell Line

The receptor for advanced glycation end products (RAGE) is a multiligand cell surface receptor of the immunoglobulin superfamily and a newly recognized invasion-related gene. High mobility group box-1 (HMGB-1) is a 30-kD protein binding to RAGE and acting as a transcription-factor-like protein that regulates the expression of several genes. In this study, the interaction effect between RAGE and HMGB-1 on the migration of SCC7 cells was investigated along with the inhibitory effect of the drug nifedipine on this interaction effect. Ten surgical specimens from patients with squamous cell carcinoma (SCC) of the head and neck and a SCC7 cell line were stained using immunohistochemical and immunocytochemical methods. Reverse transcriptase-polymerase chain reaction (RT-PCR) was used to detect RAGE expression in SCC7 cells; Western blot analysis was used to detect HMGB-1 expression in SCC7 cells. An in vitro migration assay (Boyden chamber migration assay) was used for evaluating the interaction effect between RAGE and HMGB-1 on the migration of SCC7 cells. HMGB-1 and various concentrations of nifedipine were tested for their effect on SCC7 cell migration with in vitro migration assays. RAGE and HMGB-1 were expressed in almost all human head and neck SCC tissues and in SCC7 cells as detected by immunostaining. The migration assay showed that the interaction between RAGE and HMGB-1 increased SCC7 migration activity depending on the level of HMGB-1, and nifedipine inhibited the interaction effect between RAGE and HMGB-1 on SCC7 cells in a dose-dependent manner. The interaction between RAGE and HMGB-1 could be closely associated with metastasis of SCC of the head and neck. Nifedipine may have an inhibitory effect on tumor metastasis.

Unrestricted somatic stem cells: interaction with CD34+ cells in vitro and in vivo, expression of homing genes and exclusion of tumorigenic potential

Transplantation of allogeneic hematopoietic stem cells (HSC) within the framework of hematologic oncology or inherited diseases may be associated with complications such as engraftment failure and long-term pancytopenia. HSC engraftment can be improved, for example by co-transplantation with mesenchymal stem cells (MSC). Recently, a new multipotent MSC line from umbilical cord blood, unrestricted somatic stem cells (USSC), has been described. It was demonstrated that USSC significantly support proliferation of HSC in an in vitro feeder layer assay. A NOD/SCID mouse model was used to assess the effect of USSC on co-transplanted CD34(+) cells and look for the fate of transplanted USSC. The migration potential of USSC was studied in a Boyden chamber migration assay and in vivo. Quantitative real-time polymerase chain reaction (qRT-PCR) for CXCR4, CD44, LFA1, CD62L, VLA4, RAC2, VLA5A and RAC1 were performed. NMR1 nu/nu mice were used for a tumorigenicity test. After 4 weeks, homing of human cells (CD45(+)) to the bone marrow of NOD/SCID mice was significantly increased in mice co-transplanted with CD34(+) cells and USSC (median 30.9%, range 7-50%) compared with the CD34(+) cell-only control group (median 5.9%, range 3-10%; P = 0.004). Homing of USSC could not be shown in the bone marrow. A cell-cell contact was not required for the graft enhancing effect of USSC. An in vivo tumorigenicity assay showed no tumorigenic potential of USSC. This pre-clinical study clearly shows that USSC have an enhancing effect on engraftment of human CD34(+) cells. USSC are a safe graft adjunct.

The influence of bisphosphonates on viability, migration, and apoptosis of human oral keratinocytes—in vitro study

Bisphosphonate-associated osteonecrosis of the jaw (BP-ONJ) is one of the most often seen side effects in patients treated with bisphosphonates, presenting clinically as a non-healing wound. One theory of BP-ONJ etiology describes a negative effect on soft tissues, especially on keratinocytes, which play an important role in oral wound healing and oral soft tissue regeneration. A high cell viability of keratinocytes, which can migrate to the affected location, is essential for wound healing. The aim of this in vitro study was to investigate the effect of differently potent bisphosphonates on human oral keratinocytes (HOK).Three nitrogen-containing bisphosphonates (ibandronate, pamidronate, and zoledronate) and one non-nitrogen-containing bisphosphonate (clodronate) were compared concerning their potency on cell viability (calcein assay and MTT assay), migration ability (Boyden chamber migration assay and scratch wound proliferation assay), and apoptosis (TUNEL assay) of HOK.The nitrogen-containing bisphosphonates, particularly highly potent pamidronate and zoledronate preparations, had a strong negative influence on cell viability, migration ability, and apoptosis of HOK. The non-nitrogen-containing clodronate even increased cell viability in higher concentrations.This study demonstrates that bisphosphonates have a strong influence on HOK on different cellular levels like cell viability, migration ability, and apoptosis rate. The results support the theory that BP-ONJ is a multifactorially caused disease.Furthermore, this in vitro study confirms the theory that perioperative interruption of bisphosphonate application during dental surgical procedures might be feasible to promote better tissue regeneration and wound healing.

Thrombin-induced Migration and Matrix Metalloproteinase-9 Expression Are Regulated by MAPK and PI3K Pathways in C6 Glioma Cells

Glioblastoma multiforme is one of the most common and aggressive tumors in central nervous system. It often possesses characteristic necrotic lesions with hemorrhages, which increase the chances of exposure to thrombin. Thrombin has been known as a regulator of MMP-9 expression and cancer cell migration. However, the effects of thrombin on glioma cells have not been clearly understood. In the present study, influences of thrombin on glioma cell migration were examined using Boyden chamber migration assay and thrombin-induced changes in MMP-9 expression were measured using zymography, semi-quantitative RT-PCR, and Western blotting. Furthermore, underlying signaling pathways by which thrombin induces MMP-9 expression were examined. Thrombin-induced migration and MMP-9 expression were significantly potentiated in the presence of wortmannin, a PI3K inhibitor, whereas MAPK inhibitors suppressed thrombin-induced migration and MMP-9 expression in C6 glioma cells. The present data strongly demonstrate that MAPK and PI3K pathways evidently regulate thrombin-induced migration and MMP-9 expression of C6 glioma cells. Therefore, the control of these pathways might be a beneficial therapeutic strategy for treatment of invasive glioblastoma multiforme.

Abstract P4-05-04: Lack of the Effect of Mesenchymal Stem Cells on Breast Cancer Initiation and Growth in a Somatic Mouse Breast Cancer Model

Abstract Background: The impact of mesenchymal stem cells (MSCs) on breast cancer (BC) progression and growth remains controversial. Some studies using xenograft BC models in nude mice suggest that MSCs stimulate BC metastases, whereas other studies in syngeneic rat BC models suggest that MSCs suppress BC development and growth. In addition, whether MSCs have a role in BC initiation has not been tested. The objective of this study was to determine if bone marrow-derived MSCs affect BC initiation and progression in vitro and in clinically relevant somatic and synegeneic BC mouse models. Materials and Methods: MSCs were isolated from bone marrows of FVB wild-type mice, cultured, and characterized for their potential to differentiate into adipocytes and express flow-cytometric cell surface markers. MSC-conditioned medium (CM) was used to culture RCAS-Neu and RCAS-PyMT BC cell lines, derived from TVA-transgenic mice infected with an avian retroviral vector encoding Neu or polyoma middle T antigen (PyMT). Cell proliferation was analyzed with CellTiter 96 proliferation assay and 5-bromo-2-deoxyuridine labeling. The effect of RCAS-Neu and RCAS-PyMT cells and their CM on MSC migration was determined with Boyden chamber migration assay. In a somatic BC model, TVA-transgenic mice expressing the receptor for an avian retrovirus vector, RCAS, were infected with RCAS-PyMT vector by intraductal injection and treated with MSC (2x106cells/mouse) by i.v. injection. Mice were observed for BC development by palpation. In a syngeneic BC model, RCAS-Neu BC cells were co-implanted with MSCs (5x105/gland) at the ratios 1:0, 1:0.2, or 1:1 into the fat pad of FVB female mice. BC growth was monitored for 9 weeks. Results: CM from MSC did not significantly affect the proliferation of RCAS-Neu and RCAS-PyMT cells. However, RCAS-Neu and RCAS-PyMT cells and their CM induced morphologic changes in MSC and dramatically increased their motility. In somatic model, MSCs had no effect on BC initiation or growth with the mean tumor latency 27.5±7.5 days in MSC-treated mice and 29±5.5 days in control mice treated with PBS. In syngeneic BC model, there was no significant difference in the growth of RCAS-Neu alone or RCAS-Neu cells co-implanted with MSCs. Since BC in these models does not metastasize to distant organs, it could not be determined whether MSCs affect BC metastases. Discussion: Our results demonstrated that BC cell lines and their CM are able to induce MSCs migration and possibly differentiation. However, MSCs had no effect on BC initiation in a somatic BC model and on tumor growth in a syngeneic BC model. It is possible that the number of MSCs and schedule of MSC injection were not optimized or strong PyMT oncogene rapidly induced BC and overcame the effect of MSCs on BC formation. Experiments are ongoing to determine if multiple administrations of MSCs affect BC development induced by PyMT and Neu oncogenes. Lack of effect of MSCs in BC initiation and progression, if confirmed, will suggest that MSCs are safe for delivering novel antitumor agents for BC treatment. Citation Information: Cancer Res 2010;70(24 Suppl):Abstract nr P4-05-04.

E0194 Src family kinase SFK is essential for recruitment of bonemarrow progenitor cells to the ischaemic myocardium

BackgroundThe G protein-coupled receptor CXCR4 and its ligand stromal–cell derived factor 1 (SDF-1) play an important role in directing progenitor cells (PC) homing to ischaemic tissue. The Src family protein...

Benzyl isothiocyanate (BITC) inhibits migration and invasion of human gastric cancer AGS cells via suppressing ERK signal pathways

Metastasis suppressors and associated other regulators of cell motility play a critical initial role in tumor invasion and metastases. Benzyl isothiocyanate (BITC) is a hydrolysis compound of glucotropaeolin in dietary cruciferous vegetables. BITC has been found to exhibit prevention of cancers in laboratory animals and might also be chemoprotective in humans. Here, the purpose of this study was to investigate the effects of BITC on cell proliferation, migration, invasion and mitogen-activated protein kinase (MAPK) pathways of AGS human gastric cancer cells. Wound healing and Boyden chamber (migration and invasion) assays demonstrated that BITC exhibited an inhibitory effect on the abilities of migration and invasion in AGS cancer cells. BITC suppressed cell migration and invasion of AGS cells in a dose-dependent manner. Results from Western blotting indicated that BITC exerted an inhibitory effect on the ERK1/2, Ras, GRB2, Rho A, iNOS, COX-2 for causing the inhibitions of MMP-2, -7 and -9 then followed by the inhibitions of invasion and migration of AGS cells in vitro. BITC also promoted MKK7, MEKK3, c-jun, JNK1/2, VEGF, Sos1, phosphoinositide 3-kinase (PI3K), PKC, nuclear factor-kappaB (NF-κB) p65 in AGS cells. Results from real-time polymerized chain reaction (PCR) showed that BITC inhibited the gene expressions of MMP-2,-7 -9, FAK, ROCK1 and RhoA after BITC treatment for 24 and 48 hours in AGS cells. Taken together, the finding may provide new mechanisms and functions of BITC, which inhibit migration and invasion of human gastric cancer AGS cells.

Abstract 531: RNA interference-mediated downregulation of uPARAP decreases invasion and migration property in glioma cells through reorganization of the actin cytoskeleton

Abstract OBJECTIVE: The aim of this study was to evaluate the role of membrane protein urokinase-type plasminogen activator receptor associated protein (uPARAP), which is one of the member of serine-protease cascade, urokinase plasminogen activator system, in the motile and invasive activities of glioma cells. METHODS: In the present study, we first evaluate expression of uPARAP in various normal tissues as well as glioma patients’ samples and glioma cell lines using quantative PCR and Western blotting. To evaluate the function of uPARAP, we silence the uPARAP gene using two independent siRNAs designed for uPARAP, and evaluate its function on motile and invasive activities of glioma cells by Boyden chamber migration and invasion assays. We also evaluate the function of uPARAP on rearrangement of actin cytoskeleton by staining glioma cells with Alexa Fluor 488 conjugated Phalloidin. RESULTS: uPARAP protein was expressed four out of four (100%) glioma samples regardless of its World Health Organization grade but did not express in normal brain control. Introduction of small interfering RNA targeted uPARAP into two different glioma cell lines; KNS42 and KNS81, resulted in down-reguration of uPARAP expression, and significantly suppressed glioma cells’ migration and invasion properties in vitro. Rearrangement of actin cytoskeleton was observed at Alexa Fluor 488 conjugated Phalloidin staining. In uPARAP knocked-down glioma cells, polymeric actin became organized in stress fibers and the lamellipodia disappeared. CONCLUSION: On the basis of our findings, we conclude that RNA interference-mediated down-regulation of uPARAP decreases invasion and migration property in glioma cells. The inhibition of invasion and migration property was mediated via reorganization of the actin cytoskeleton. Downregulation of uPARAP may be a novel anti-invasion therapeutic strategy for malignant gliomas. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 531.

Lysophosphatidic acid (LPA) effects on endometrial carcinoma in vitro proliferation, invasion, and matrix metalloproteinase activity

Objectives Lysophosphatidic acid (LPA) has potent growth-regulatory effect in many cell types and has been linked to the in vivo tumor growth and metastasis in several malignancies. The goal of this study was to assess the regulation of (EC) microenvironment by LPA through the examination of its effect on cell proliferation, migration, invasion, uPA activity, and matrix metalloproteinase (MMP) secretion/activation. Methods All experiments were performed in vitro using an EC cell line, HEC-1A. Cell proliferation was determined using the Promega MTS proliferation assay following 48 h of exposures to different concentrations of LPA (0.1, 1.0 and 10.0 μM). Cell invasion was assessed using a modified Boyden chamber assay with collagen I coated on the membrane. HEC-1A motility was examined by Boyden chamber migration assay as well as the scratch wound closure assay on type I collagen. MMP secretion/activation in HEC-1A conditioned medium was detected by gelatin zymography. MMP-7 mRNA expression was determined using real-time PCR. uPA activity was measured using a coupled colorimetric assay. Results LPA, at the concentrations of 0.1 and 1.0 μM, significantly induced the proliferation of HEC-1A cells ( p < 0.01). At 10 μM, LPA- induced HEC-1A proliferation to a less extent and showed no significant effect on HEC-1A invasion and migration ( p > 0.05). Gelatin zymogram showed that HEC-1A cells secreted high levels of MMP-7, while MMP-2 and MMP-9 are barely detectable. In addition, LPA significantly enhanced uPA activity in HEC-1A conditioned medium in a concentration-dependent manner. Conclusions LPA is a potent modulator of cellular proliferation and invasion for EC cells. It also has the capacity to stimulate the secretion/activity of uPA and MMP-7. Those results suggest that LPA is a bioactive modulator of EC microenvironment and may have a distinct regulation mechanism as observed in epithelial ovarian cancer.

Reactive Astrocytes in Glial Scar Attract Olfactory Ensheathing Cells Migration by Secreted TNF-α in Spinal Cord Lesion of Rat

BackgroundAfter spinal cord injury (SCI), the formation of glial scar contributes to the failure of injured adult axons to regenerate past the lesion. Increasing evidence indicates that olfactory ensheathing cells (OECs) implanted into spinal cord are found to migrate into the lesion site and induce axons regeneration beyond glial scar and resumption of functions. However, little is known about the mechanisms of OECs migrating from injection site to glial scar/lesion site.Methods and FindingsIn the present study, we identified a link between OECs migration and reactive astrocytes in glial scar that was mediated by the tumor necrosis factor-α (TNF-α). Initially, the Boyden chamber migration assay showed that both glial scar tissue and reactive astrocyte-conditioned medium promoted OECs migration in vitro. Reactive astrocyte-derived TNF-α and its type 1 receptor TNFR1 expressed on OECs were identified to be responsible for the promoting effect on OECs migration. TNF-α-induced OECs migration was demonstrated depending on activation of the extracellular signal-regulated kinase (ERK) signaling cascades. Furthermore, TNF-α secreted by reactive astrocytes in glial scar was also showed to attract OECs migration in a spinal cord hemisection injury model of rat.ConclusionsThese findings showed that TNF-α was released by reactive astrocytes in glial scar and attracted OECs migration by interacting with TNFR1 expressed on OECs via regulation of ERK signaling. This migration-attracting effect of reactive astrocytes on OECs may suggest a mechanism for guiding OECs migration into glial scar, which is crucial for OECs-mediated axons regrowth beyond the spinal cord lesion site.

TGF-β downregulates PTEN via activation of NF-κB in pancreatic cancer cells

TGF-beta utilizes receptor-activated SMAD signaling to mediate growth suppression; however, non-SMAD signaling that modulates the TGF-beta response in epithelial cells become apparent when the SMAD signaling is abrogated, a common occurrence in pancreatic cancers. Here, we examined whether TGF-beta utilized NF-kappaB to downregulate PTEN, a gene that is rarely mutated in pancreatic cancers. SMAD4-null BxPc3 and CAPAN-1 pancreatic cancer cells were treated with TGF-beta (10 ng/ml) and lysed, and cellular proteins were analyzed by Western blots using p-IkappaB, p65, and PTEN antibodies. PTEN promoter and NF-kappaB activities were assessed by PTEN-luc and p-NF-luc constructs, respectively. Dominant negative p-IkappaB-alpha-M (NF-kappaB superrepressor) was used to block activation of NF-kappaB. Cell motility was assessed by Boyden chamber migration assay. TGF-beta induced IkappaB-alpha phosphorylation followed by NF-kappaB p65 subunit nuclear translocation and increased NF-kappaB activity. IkappaB-alpha-M blocked TGF-beta-induced NF-kappaB activity, reversed downregulated PTEN promoter activity and PTEN expression, and prevented augmentation of cell motility induced by TGF-beta. SMAD4 restoration, but not knockdown of SMAD2 and/or 3, reversed TGF-beta-induced NF-kappaB activity. Thus TGF-beta suppresses PTEN in pancreatic cancer cells through NF-kappaB activation and enhances cell motility and invasiveness in a SMAD4-independent manner that can be counteracted when TGF-beta-SMAD signaling is restored. The TGF-beta/NF-kappaB/PTEN cascade may be a critical pathway for pancreatic cancer cells to proliferate and metastasize.

Lactate promotes glioma migration by TGF-β2–dependent regulation of matrix metalloproteinase-2

Lactate dehydrogenase type A (LDH-A) is a key metabolic enzyme catalyzing pyruvate into lactate and is excessively expressed by tumor cells. Transforming growth factor-beta2 (TGF-beta2) is a key regulator of invasion in high-grade gliomas, partially by inducing a mesenchymal phenotype and by remodeling the extracellular matrix. In this study, we tested the hypothesis that lactate metabolism regulates TGF-beta2-mediated migration of glioma cells. Small interfering RNA directed against LDH-A (siLDH-A) suppresses, and lactate induces, TGF-beta2 expression, suggesting that lactate metabolism is strongly associated with TGF-beta2 in glioma cells. Here we demonstrate that TGF-beta2 enhances expression, secretion, and activation of matrix metalloproteinase-2 (MMP-2) and induces the cell surface expression of integrin alpha(v)beta(3) receptors. In spheroid and Boyden chamber migration assays, inhibition of MMP-2 activity using a specific MMP-2 inhibitor and blocking of integrin alpha(v)beta(3) abrogated glioma cell migration stimulated by TGF-beta2. Furthermore, siLDH-A inhibited MMP2 activity, leading to inhibition of glioma migration. Taken together, we define an LDH-A-induced and TGF-beta2-coordinated regulatory cascade of transcriptional regulation of MMP-2 and integrin alpha(v)beta(3). This novel interaction between lactate metabolism and TGF-beta2 might constitute a crucial mechanism for glioma migration.

Modulation of the Membrane Type 1 Matrix Metalloproteinase Cytoplasmic Tail Enhances Tumor Cell Invasion and Proliferation in Three-dimensional Collagen Matrices

Increasing evidence suggests that the cytoplasmic tail of membrane type 1 matrix metalloproteinase (MT1-MMP) is subject to phosphorylation and that this modification may influence its enzymatic activity at the cell surface. In this study, phosphorylated MT1-MMP is detected using a phospho-specific antibody recognizing a protein kinase C consensus sequence (phospho-TXR), and a MT1-MMP tail peptide is phosphorylated by exogenous protein kinase C. To characterize the potential role of cytoplasmic residue Thr(567) in these processes, mutants that mimic a state of either constitutive (T567E) or defective phosphorylation (T567A) were expressed and analyzed for their functional effects on MT1-MMP activity and cellular behavior. Phospho-mimetic mutants of Thr(567) exhibit enhanced matrix invasion as well as more extensive growth within a three-dimensional type I collagen matrix. Together, these findings suggest that MT1-MMP surface action is regulated by phosphorylation at cytoplasmic tail residue Thr(567) and that this modification plays a critical role in processes that are linked to tumor progression.

Extracellular calcium promotes the migration of breast cancer cells through the activation of the calcium sensing receptor

Breast cancer is the most frequent form of cancer in women, with the highest incidence of metastasis to the bone. The reason for the preferential destination to the bone is believed to be due to chemoattractant factors released during bone resorption, which act on the cancer cells facilitating their metastasis. One of the factors released during osteolysis that may mediate breast cancer bone localization is Ca2+. Here, we show that extracellular Ca2+ (Ca2+(o)) acting via the calcium-sensing receptor (CaSR), greatly promotes the migration of bone-preferring breast cancer cells. In Boyden Chamber and Scratch Wound migration assays, an increase in breast cancer cell migration was observed at 2.5 mM and 5 mM Ca2+(o) compared to basal levels for three of the four breast cancer cell lines tested. However, a significantly greater migratory response was observed for the highly bone metastatic MDA-MB-231 cells, compared to the MCF7 and T47D, which have a lower metastatic potential in vivo. The BT474 cells, which do not metastasize to the bone, did not respond to elevated concentrations of Ca2+(o) in the migration assays. Inhibition of either ERK1/2 MAPK or phospholipase Cbeta (PLCbeta) led to an abolition of the Ca2+(o)-induced migration, implicating these pathways in the migratory response. Knockdown of the CaSR by siRNA resulted in an inhibition of the Ca2+(o)-induced migration, demonstrating the involvement of this receptor in the effect. These results suggest that the activation of the CaSR by elevated Ca2+(o) concentrations, such as those found near resorbing bone, produces an especially strong chemoattractant effect on bone metastatic breast cancer cells toward the Ca2+-rich environment.

Identification of Gliotropic Factors That Induce Human Stem Cell Migration to Malignant Tumor

Neural stem cells are mobile, are attracted to regions of brain damage, and can migrate a considerable distance to reach a glioma site. However, the molecular basis of the progression of gliotropism to malignant gliomas remains poorly understood. With the use of clinically and histologically assessed glioma cells, we have assessed their protein and gene profiles via proteomics and microarray approaches, and have identified candidate genes from human glioma tissues. This research is expected to provide clues to the molecular mechanisms underlying the migration of neural stem cells (F3 cell) to glioma sites. The expression of 16 proteins was shown to have increased commonly in human glioma tissues. Among them, the expression of annexin A2, TIMP-1, COL11A1, bax, CD74, TNFSF8, and SPTLC2 were all increased in human glioma cells, as confirmed by Western blotting and immunohistochemical staining. In particular, annexin A2 effects an increase in migration toward F3 and glioblastoma cells (U87 cell) in a Boyden chamber migration assay. An ERK inhibitor (PD98057) and a CDK5 inhibitor (rescovitine) inhibited 50% and 90% of annexin A2-induced migration in F3 cells, respectively. A similar chemotactic migration was noted in F3 and U87 cells. These results demonstrated that 7 candidate proteins may harbor a potential glioma tropism factor relevant to the pathology of malignant glioma. These results reveal that this novel molecular approach to the monitoring of glioma may provide clinically relevant information regarding tumor malignancy, and should also prove appropriate for high-throughput clinical screening applications.

Extracellular calcium promotes the migration of breast cancer cells through the activation of the calcium sensing receptor

Breast cancer is the most frequent form of cancer in women, with the highest incidence of metastasis to the bone. The reason for the preferential destination to the bone is believed to be due to chemoattractant factors released during bone resorption, which act on the cancer cells facilitating their metastasis. One of the factors released during osteolysis that may mediate breast cancer bone localization is Ca 2+. Here, we show that extracellular Ca 2+ (Ca 2+ o) acting via the calcium-sensing receptor (CaSR), greatly promotes the migration of bone-preferring breast cancer cells. In Boyden Chamber and Scratch Wound migration assays, an increase in breast cancer cell migration was observed at 2.5 mM and 5 mM Ca 2+ o compared to basal levels for three of the four breast cancer cell lines tested. However, a significantly greater migratory response was observed for the highly bone metastatic MDA-MB-231 cells, compared to the MCF7 and T47D, which have a lower metastatic potential in vivo. The BT474 cells, which do not metastasize to the bone, did not respond to elevated concentrations of Ca 2+ o in the migration assays. Inhibition of either ERK1/2 MAPK or phospholipase Cβ (PLCβ) led to an abolition of the Ca 2+ o-induced migration, implicating these pathways in the migratory response. Knockdown of the CaSR by siRNA resulted in an inhibition of the Ca 2+ o-induced migration, demonstrating the involvement of this receptor in the effect. These results suggest that the activation of the CaSR by elevated Ca 2+ o concentrations, such as those found near resorbing bone, produces an especially strong chemoattractant effect on bone metastatic breast cancer cells toward the Ca 2+-rich environment.