Your browser doesn't support javascript.
loading
Show: 20 | 50 | 100
Results 1 - 20 de 29
Filter
Add more filters










Publication year range
1.
J Immunother Cancer ; 11(3)2023 03.
Article in English | MEDLINE | ID: mdl-36927527

ABSTRACT

BACKGROUND: The phase I first-in-human study ENGAGE-1 evaluated the humanized IgG1 OX40 agonistic monoclonal antibody GSK3174998 alone (Part 1 (P1)) or in combination with pembrolizumab (Part 2 (P2)) in patients with advanced solid tumors. METHODS: GSK3174998 (0.003-10 mg/kg) ± pembrolizumab (200 mg) was administered intravenously every 3 weeks using a continuous reassessment method for dose escalation. Primary objectives were safety and tolerability; secondary objectives included pharmacokinetics, immunogenicity, pharmacodynamics, and clinical activity. RESULTS: 138 patients were enrolled (45 (P1) and 96 (P2, including 3 crossovers)). Treatment-related adverse events occurred in 51% (P1) and 64% (P2) of patients, fatigue being the most common (11% and 24%, respectively). No dose-toxicity relationship was observed, and maximum-tolerated dose was not reached. Dose-limiting toxicities (P2) included Grade 3 (G3) pleural effusion and G1 myocarditis with G3 increased troponin. GSK3174998 ≥0.3 mg/kg demonstrated pharmacokinetic linearity and >80% receptor occupancy on circulating T cells; 0.3 mg/kg was selected for further evaluation. Limited clinical activity was observed for GSK3174998 (P1: disease control rate (DCR) ≥24 weeks 9%) and was not greater than that expected for pembrolizumab alone (P2: overall response rate 8%, DCR ≥24 weeks 28%). Multiplexed immunofluorescence data from paired biopsies suggested that increased infiltration of natural killer (NK)/natural killer T (NKT) cells and decreased regulatory T cells (Tregs) in the tumor microenvironment may contribute to clinical responses: CD16+CD56-CD134+ NK /NKT cells and CD3+CD4+FOXP3+CD134+ Tregs exhibited the largest magnitude of change on treatment, whereas CD3+CD8+granzyme B+PD-1+CD134+ cytotoxic T cells were the least variable. Tumor gene expression profiling revealed an upregulation of inflammatory responses, T-cell proliferation, and NK cell function on treatment with some inflammatory cytokines upregulated in peripheral blood. However, target engagement, evidenced by pharmacologic activity in peripheral blood and tumor tissue, did not correlate with clinical efficacy. The low number of responses precluded identifying a robust biomarker signature predictive of response. CONCLUSIONS: GSK3174998±pembrolizumab was well tolerated over the dose range tested and demonstrated target engagement. Limited clinical activity does not support further development of GSK3174998±pembrolizumab in advanced cancers. TRIAL REGISTRATION NUMBER: NCT02528357.


Subject(s)
Antineoplastic Agents , Neoplasms , Humans , Neoplasms/pathology , Antibodies, Monoclonal, Humanized/adverse effects , Antineoplastic Agents/therapeutic use , Antibodies, Monoclonal/therapeutic use , Tumor Microenvironment
2.
Expert Opin Investig Drugs ; 31(8): 781-794, 2022 Aug.
Article in English | MEDLINE | ID: mdl-35791862

ABSTRACT

INTRODUCTION: Currently, triple-negative breast cancer (TNBC) has limited therapeutic options beyond chemotherapy and has worse outcomes than other breast cancer subtypes. Initial experience with immune checkpoint blockade for the treatment of TNBC has indicated that modifying the tumor immune response represents a promising direction of investigation. Subsequent studies have led to a deeper understanding of the heterogeneity of this disease and have informed further exploration of numerous potential therapeutic approaches to intervene in the tumor microenvironment (TME). AREAS COVERED: Initial work in this arena has focused on enhanced definition of checkpoints in activation of an adaptive immune response. In this review, we discuss recent efforts that have looked into components of innate immunity to reverse immunosuppressive phenotypes and augment antitumor immune response. EXPERT OPINION: Current treatment options for TNBC have been improved with the approval of immune checkpoint inhibitors (ICI) in both advanced and early-stage disease; however, the challenge remains to expand the number of patients that will benefit from immunotherapy. Optimizing the innate immune response represents an opportunity to improve this therapeutic index, and the development of an array of novel agents is underway. Success will depend on precision characterization of the patient TME and selection of ideal combination therapy.


Subject(s)
Triple Negative Breast Neoplasms , Biomarkers, Tumor/genetics , Humans , Immunity, Innate , Immunotherapy , Triple Negative Breast Neoplasms/drug therapy , Triple Negative Breast Neoplasms/pathology , Tumor Microenvironment
3.
Mol Cancer Ther ; 20(10): 1941-1955, 2021 10.
Article in English | MEDLINE | ID: mdl-34253590

ABSTRACT

B-cell maturation antigen (BCMA) is an attractive therapeutic target highly expressed on differentiated plasma cells in multiple myeloma and other B-cell malignancies. GSK2857916 (belantamab mafodotin, BLENREP) is a BCMA-targeting antibody-drug conjugate approved for the treatment of relapsed/refractory multiple myeloma. We report that GSK2857916 induces immunogenic cell death in BCMA-expressing cancer cells and promotes dendritic cell activation in vitro and in vivo GSK2857916 treatment enhances intratumor immune cell infiltration and activation, delays tumor growth, and promotes durable complete regressions in immune-competent mice bearing EL4 lymphoma tumors expressing human BCMA (EL4-hBCMA). Responding mice are immune to rechallenge with EL4 parental and EL4-hBCMA cells, suggesting engagement of an adaptive immune response, immunologic memory, and tumor antigen spreading, which are abrogated upon depletion of endogenous CD8+ T cells. Combinations with OX40/OX86, an immune agonist antibody, significantly enhance antitumor activity and increase durable complete responses, providing a strong rationale for clinical evaluation of GSK2857916 combinations with immunotherapies targeting adaptive immune responses, including T-cell-directed checkpoint modulators.


Subject(s)
Antibodies, Monoclonal, Humanized/pharmacology , B-Cell Maturation Antigen/antagonists & inhibitors , CD8-Positive T-Lymphocytes/immunology , Immunoconjugates/pharmacology , Immunogenic Cell Death , Lymphoma/drug therapy , Multiple Myeloma/drug therapy , Animals , Antibodies, Monoclonal/chemistry , Apoptosis , B-Cell Maturation Antigen/immunology , Cell Proliferation , Female , Humans , Lymphoma/immunology , Lymphoma/metabolism , Lymphoma/pathology , Mice , Mice, Inbred C57BL , Multiple Myeloma/immunology , Multiple Myeloma/metabolism , Multiple Myeloma/pathology , Tumor Cells, Cultured , Xenograft Model Antitumor Assays
5.
J Immunother Cancer ; 7(1): 351, 2019 12 16.
Article in English | MEDLINE | ID: mdl-31843013

ABSTRACT

INTRODUCTION: OX-40 co-stimulatory signaling plays a role in mounting anti-tumor immune responses and clinical trials targeting this pathway are ongoing. However, the association of with OX-40 protein expression with clinical outcomes and pathological features in non-small cell lung cancer (NSCLC) are largely unknown. METHODS: Surgically-resected stage I-III NSCLC specimens (N = 100) were stained by immunohistochemistry (IHC) for the following immune markers: OX-40, PD-L1, PD-1, CD3, CD4, CD8, CD45RO, CD57, CD68, FOXP3, granzyme B, and ICOS. Immune-related markers mRNA expression were also assessed. We evaluated the association of OX-40 levels with major clinicopathologic variables, including molecular driver mutations. RESULTS: OX-40 IHC expression was observed in all tested tumors, predominantly localized in the membrane of the tumor immune infiltrate, and was not associated with a specific clinicopathologic or molecular subtype. High OX-40 expression levels measured by IHC median score were associated with better overall survival (OS) (p = 0.002), independent of CD3/CD8, PD-L1, and ICOS expression. High OX-40 IHC score was associated with increased expression of immune-related genes such as CD3, IFN-gamma, ICOS, CD8, CXCL9, CXCL10, CCL5, granzyme K. CONCLUSIONS: High OX-40 IHC expression in the tumor immune infiltrate is associated with favorable prognosis and increased levels of immune-related genes including IFN-gamma in patients with surgically resected stage I-III NSCLC. Its prognostic utility is independent of PD-L1 and other common markers of immune activation. High OX-40 expression potentially identifies a unique subgroup of NSCLC that may benefit from co-stimulation with OX-40 agonist antibodies and potentially enhance the efficacy of existing immune checkpoint therapies.


Subject(s)
Carcinoma, Non-Small-Cell Lung/genetics , Carcinoma, Non-Small-Cell Lung/mortality , Gene Expression , Lung Neoplasms/genetics , Lung Neoplasms/mortality , Lymphocytes, Tumor-Infiltrating/metabolism , Receptors, OX40/genetics , Adult , Aged , Aged, 80 and over , Antineoplastic Agents, Immunological/pharmacology , Biomarkers , Carcinoma, Non-Small-Cell Lung/metabolism , Carcinoma, Non-Small-Cell Lung/pathology , Cell Line, Tumor , Female , Humans , Immunohistochemistry , Lung Neoplasms/metabolism , Lung Neoplasms/pathology , Lymphocytes, Tumor-Infiltrating/immunology , Male , Middle Aged , Neoplasm Staging , Receptors, OX40/metabolism , Tumor Microenvironment
6.
Clin Cancer Res ; 25(21): 6406-6416, 2019 11 01.
Article in English | MEDLINE | ID: mdl-31371342

ABSTRACT

PURPOSE: OX40 agonist-based combinations are emerging as a novel avenue to improve the effectiveness of cancer immunotherapy. To better guide its clinical development, we characterized the role of the OX40 pathway in tumor-reactive immune cells. We also evaluated combining OX40 agonists with targeted therapy to combat resistance to cancer immunotherapy.Experimental Design: We utilized patient-derived tumor-infiltrating lymphocytes (TILs) and multiple preclinical models to determine the direct effect of anti-OX40 agonistic antibodies on tumor-reactive CD8+ T cells. We also evaluated the antitumor activity of an anti-OX40 antibody plus PI3Kß inhibition in a transgenic murine melanoma model (Braf mutant, PTEN null), which spontaneously develops immunotherapy-resistant melanomas. RESULTS: We observed elevated expression of OX40 in tumor-reactive CD8+ TILs upon encountering tumors; activation of OX40 signaling enhanced their cytotoxic function. OX40 agonist antibody improved the antitumor activity of CD8+ T cells and the generation of tumor-specific T-cell memory in vivo. Furthermore, combining anti-OX40 with GSK2636771, a PI3Kß-selective inhibitor, delayed tumor growth and extended the survival of mice with PTEN-null melanomas. This combination treatment did not increase the number of TILs, but it instead significantly enhanced proliferation of CD8+ TILs and elevated the serum levels of CCL4, CXCL10, and IFNγ, which are mainly produced by memory and/or effector T cells. CONCLUSIONS: These results highlight a critical role of OX40 activation in potentiating the effector function of tumor-reactive CD8+ T cells and suggest further evaluation of OX40 agonist-based combinations in patients with immune-resistant tumors.


Subject(s)
Antibodies, Anti-Idiotypic/pharmacology , Melanoma/drug therapy , PTEN Phosphohydrolase/genetics , Receptors, OX40/immunology , Animals , Antigens, Differentiation/genetics , Antigens, Differentiation/pharmacology , CD8-Positive T-Lymphocytes/drug effects , CD8-Positive T-Lymphocytes/immunology , Cell Line, Tumor , Female , Heterografts , Humans , Immunotherapy , Lymphocytes, Tumor-Infiltrating/drug effects , Lymphocytes, Tumor-Infiltrating/immunology , Melanoma/genetics , Melanoma/metabolism , Melanoma/pathology , Mice , Receptors, OX40/antagonists & inhibitors
7.
PLoS One ; 13(11): e0206223, 2018.
Article in English | MEDLINE | ID: mdl-30388137

ABSTRACT

Mouse syngeneic tumor models are widely used tools to demonstrate activity of novel anti-cancer immunotherapies. Despite their widespread use, a comprehensive view of their tumor-immune compositions and their relevance to human tumors has only begun to emerge. We propose each model possesses a unique tumor-immune infiltrate profile that can be probed with immunotherapies to inform on anti-tumor mechanisms and treatment strategies in human tumors with similar profiles. In support of this endeavor, we characterized the tumor microenvironment of four commonly used models and demonstrate they encompass a range of immunogenicities, from highly immune infiltrated RENCA tumors to poorly infiltrated B16F10 tumors. Tumor cell lines for each model exhibit different intrinsic factors in vitro that likely influence immune infiltration upon subcutaneous implantation. Similarly, solid tumors in vivo for each model are unique, each enriched in distinct features ranging from pathogen response elements to antigen presentation machinery. As RENCA tumors progress in size, all major T cell populations diminish while myeloid-derived suppressor cells become more enriched, possibly driving immune suppression and tumor progression. In CT26 tumors, CD8 T cells paradoxically increase in density yet are restrained as tumor volume increases. Finally, immunotherapy treatment across these different tumor-immune landscapes segregate into responders and non-responders based on features partially dependent on pre-existing immune infiltrates. Overall, these studies provide an important resource to enhance our translation of syngeneic models to human tumors. Future mechanistic studies paired with this resource will help identify responsive patient populations and improve strategies where immunotherapies are predicted to be ineffective.


Subject(s)
Neoplasms/immunology , Neoplasms/therapy , Tumor Microenvironment , Animals , CD3 Complex/metabolism , Cell Line, Tumor , Cell Proliferation , Chemokines/metabolism , Complement System Proteins/metabolism , Disease Models, Animal , Disease Progression , Female , Gene Expression Profiling , Gene Expression Regulation, Neoplastic , Immunotherapy , Ki-67 Antigen/metabolism , Mice, Inbred BALB C , Mice, Inbred C57BL , Myeloid Cells/pathology , Neoplasm Invasiveness , Neoplasms/genetics , Neoplasms/pathology , RNA, Neoplasm/genetics , RNA, Neoplasm/metabolism , Treatment Outcome
8.
Clin Cancer Res ; 24(22): 5735-5743, 2018 11 15.
Article in English | MEDLINE | ID: mdl-29784675

ABSTRACT

Purpose: Radiation is used extensively to treat localized cancer, but improved understanding of its effects on the immune system has increased interest in its potential systemic (abscopal) effects, particularly in combination with checkpoint inhibitors such as anti-PD1. The majority of patients either do not respond or develop resistance to monotherapy over time. Here, we investigated the efficacy of OX40 (CD134) stimulation as an alternative immunotherapeutic approach in combination with radiotherapy (XRT) in a murine model of anti-PD1-resistant lung tumors.Experimental Design: We established a bilateral tumor model in 129Sv/Ev mice using an anti-PD1-resistant lung tumor cell line. Primary tumors were treated with intratumoral injection of an OX40 agonist antibody, given as adjuvant therapy after XRT (36 Gy in three 12-Gy fractions), whereas secondary tumors were left untreated to investigate abscopal outcomes.Results: The combination of XRT followed by OX40 stimulation effectively inhibited local and systemic antitumor growth, limited lung metastases, and improved survival rates. This treatment regimen augmented CD4+ and CD8+ T-cell expansion. XRT induced the expression of OX40 on T cells in tumors and spleens and increased the percentages of splenic CD103+ dendritic cells.Conclusions: Our data extend the benefits of radiation to systemic disease control, especially when combined with anti-OX40 agonist to promote immunologically mediated abscopal effects. Moreover, this study provides a rational treatment approach and sequence to overcome anti-PD1-resistant poorly immunogenic tumors. Clin Cancer Res; 24(22); 5735-43. ©2018 AACR.


Subject(s)
Antineoplastic Agents, Immunological/pharmacology , Lung Neoplasms/metabolism , Programmed Cell Death 1 Receptor/antagonists & inhibitors , Receptors, OX40/metabolism , Animals , Cell Line, Tumor , Chemoradiotherapy , Disease Models, Animal , Dose-Response Relationship, Drug , Dose-Response Relationship, Radiation , Female , Humans , Lung Neoplasms/pathology , Lung Neoplasms/therapy , Lymphocytes, Tumor-Infiltrating/drug effects , Lymphocytes, Tumor-Infiltrating/immunology , Lymphocytes, Tumor-Infiltrating/metabolism , Lymphocytes, Tumor-Infiltrating/radiation effects , Mice , Receptors, OX40/antagonists & inhibitors , T-Lymphocyte Subsets/drug effects , T-Lymphocyte Subsets/immunology , T-Lymphocyte Subsets/metabolism , Xenograft Model Antitumor Assays
9.
J Pharmacol Exp Ther ; 337(3): 636-43, 2011 Jun.
Article in English | MEDLINE | ID: mdl-21378206

ABSTRACT

A major contributing factor to the high mortality rate associated with acute myeloid leukemia and multiple myeloma is the development of resistance to chemotherapy. We have shown that the combination of tipifarnib, a nonpeptidomimetic farnesyltransferase inhibitor (FTI), with bortezomib, a proteosome inhibitor, promotes synergistic death and overcomes de novo drug resistance in acute myeloid leukemia cell lines. Experiments were undertaken to identify the molecular mechanisms by which tipifarnib produces cell death in acute myeloid leukemia and multiple myeloma cell lines (U937 and 8226, respectively). Tipifarnib, but not other FTIs tested [N-[4-[2(R)-amino-3-mercaptopropyl]amino-2-phenylbenzoyl]methionine methyl ester trifluoroacetate salt (FTI-277) and 2'-methyl-5-((((1-trityl-1H-imidazol-4-yl)methyl)amino)methyl)-[1,1'-biphenyl]-2-carboxylic acid (FTI-2153), promotes elevations in intracellular free-calcium concentrations ([Ca(2+)](i)) in both cell lines. These elevations in [Ca(2+)](i) were accompanied by highly dynamic plasmalemmal blebbing and frequently resulted in membrane lysis. The tipifarnib-induced elevations in [Ca(2+)](i) were not blocked by thapsigargin or ruthenium red, but were inhibited by application of Ca(2+)-free extracellular solution and by the Ca(2+) channel blockers Gd(3+) and La(3+). Conversely, 2-aminoethoxydiphenyl borate (2-APB) potentiated the tipifarnib-evoked [Ca(2+)](i) overload. Preventing Ca(2+) influx diminished tipifarnib-evoked cell death, whereas 2-APB potentiated this effect, demonstrating a link between tipifarnib-induced Ca(2+) influx and apoptosis. These data suggest that tipifarnib exerts its effects by acting on a membrane channel with pharmacological properties consistent with store-operated channels containing the Orai3 subunit. It is noteworthy that Orai3 transcripts were found to be expressed at lower levels in tipifarnib-resistant 8226/R5 cells. Our results indicate tipifarnib causes cell death via a novel mechanism involving activation of a plasma membrane Ca(2+) channel and intracellular Ca(2+) overload.


Subject(s)
Antineoplastic Agents/pharmacology , Apoptosis/drug effects , Calcium Channels/metabolism , Calcium/metabolism , Cell Membrane/metabolism , Farnesyltranstransferase/antagonists & inhibitors , Quinolones/pharmacology , Cell Membrane/drug effects , Endoplasmic Reticulum/drug effects , Humans , Leukemia, Myeloid, Acute/drug therapy , Leukemia, Myeloid, Acute/metabolism , Leukemia, Myeloid, Acute/pathology , Multiple Myeloma/drug therapy , Multiple Myeloma/metabolism , Multiple Myeloma/pathology , Poly (ADP-Ribose) Polymerase-1 , Poly(ADP-ribose) Polymerases/metabolism , Tumor Cells, Cultured , U937 Cells
10.
Cancer Res ; 69(4): 1517-26, 2009 Feb 15.
Article in English | MEDLINE | ID: mdl-19208838

ABSTRACT

Inhibition of specific matrix metalloproteinases (MMP) is an attractive noncytotoxic approach to cancer therapy. MMP-14, a membrane-bound zinc endopeptidase, has been proposed to play a central role in tumor growth, invasion, and neovascularization. Besides cleaving matrix proteins, MMP-14 activates proMMP-2 leading to an amplification of pericellular proteolytic activity. To examine the contribution of MMP-14 to tumor growth and angiogenesis, we used DX-2400, a highly selective fully human MMP-14 inhibitory antibody discovered using phage display technology. DX-2400 blocked proMMP-2 processing on tumor and endothelial cells, inhibited angiogenesis, and slowed tumor progression and formation of metastatic lesions. The combination of potency, selectivity, and robust in vivo activity shows the potential of a selective MMP-14 inhibitor for the treatment of solid tumors.


Subject(s)
Antibodies, Monoclonal , Antineoplastic Agents/therapeutic use , Cell Division/drug effects , Enzyme Inhibitors/therapeutic use , Matrix Metalloproteinase Inhibitors , Neovascularization, Pathologic/prevention & control , Animals , Antibodies, Monoclonal, Humanized , Breast Neoplasms/pathology , Cell Line, Tumor , Endothelium, Vascular/cytology , Endothelium, Vascular/drug effects , Female , Genes, Reporter , Humans , Immunohistochemistry , Mice , Neoplasm Invasiveness/pathology , Transfection , Transplantation, Heterologous , Umbilical Veins/cytology , Umbilical Veins/drug effects
11.
Clin Cancer Res ; 12(2): 591-9, 2006 Jan 15.
Article in English | MEDLINE | ID: mdl-16428505

ABSTRACT

It has been established in preclinical models of multiple myeloma and acute myeloid leukemia (AML) that the bone marrow microenvironment provides protection from chemotherapy- and death receptor-mediated apoptosis. This form of resistance, termed de novo drug resistance, occurs independent of chronic exposure to cancer-related therapies and likely promotes the development of multidrug resistance. Consequently, it is of major interest to identify compounds or drug combinations that can overcome environment-mediated resistance. In this study, we investigated the activity of tipifarnib (Zarnestra, formerly R115777) combined with bortezomib (Velcade, formerly PS-341) in microenvironment models of multiple myeloma and AML. The combination proved to be synergistic in multiple myeloma and AML cell lines treated in suspension culture. Even in tumor cells relatively resistant to tipifarnib, combined activity was maintained. Tipifarnib and bortezomib were also effective when multiple myeloma and AML cells were adhered to fibronectin, providing evidence that the combination overcomes cell adhesion-mediated drug resistance (CAM-DR). Of importance, activation of the endoplasmic reticulum stress response was enhanced and correlated with apoptosis and reversal of CAM-DR. Multiple myeloma and AML cells cocultured with bone marrow stromal cells also remained sensitive, although stromal-adhered tumor cells were partially protected (relative to cells in suspension or fibronectin adhered). Evaluation of the combination using a transwell apparatus revealed that stromal cells produce a protective soluble factor. Investigations are under way to identify the cytokines and/or growth factors involved. In summary, our study provides the preclinical rationale for trials testing the tipifarnib and bortezomib combination in patients with multiple myeloma and AML.


Subject(s)
Antineoplastic Agents/pharmacology , Boronic Acids/pharmacology , Cell Adhesion/drug effects , Drug Resistance, Neoplasm , Leukemia, Myeloid, Acute/drug therapy , Multiple Myeloma/drug therapy , Pyrazines/pharmacology , Quinolones/pharmacology , Bone Marrow/drug effects , Bone Marrow/metabolism , Bortezomib , Drug Synergism , Drug Therapy, Combination , Fibronectins/metabolism , Humans , Leukemia, Myeloid, Acute/metabolism , Multiple Myeloma/metabolism , Stromal Cells/cytology , Stromal Cells/drug effects , Stromal Cells/metabolism , Tumor Cells, Cultured
12.
J Cell Biochem ; 97(1): 198-206, 2006 Jan 01.
Article in English | MEDLINE | ID: mdl-16200638

ABSTRACT

Vitamin D3 analogs are potential anti-cancer agents with theoretically wide therapeutic index, but there have been limited studies directed towards human neuroblastoma. The antiproliferative ability of the novel vitamin D3 hybrid analog QW-1624F2-2 (QW, 1-hydroxymethyl-16-ene-24, 24-F2-26, 27-bishomo-25-hydroxyvitamin D3) was examined in two human neuroblastoma-derived cell-lines. Analog QW inhibited cell-cycle progression of IMR5 cells with accumulation in G1 phase. QW induced the differentiation of CHP134 as evidenced by increased neurite length. These effects were accompanied by decreased expression of MYCN in both the cell-lines treated with QW. Furthermore, QW inhibited the migration of CHP134 cells in matrigel invasion assays, indicating its anti-invasive ability. In athymic nude mice, we found that QW was less calcemic than EB1089 (1alpha, 25-dihydroxy-22, 24-diene-24, 26,27-trishomovitamin D3). Systemic administration of QW in a mouse xenotransplantation model revealed that it is more effective than EB1089 in suppressing the growth of CHP134 flank tumors. In summary, the low-calcemic hybrid analog QW showed significant anti-tumor activity in vivo and thus exhibits potential as a novel cancer therapeutic.


Subject(s)
Antineoplastic Agents/pharmacology , Calcitriol/analogs & derivatives , Calcitriol/pharmacology , Neuroblastoma/pathology , Animals , Blotting, Western , Cell Cycle/drug effects , Cell Division/drug effects , Cell Line, Tumor , Cell Survival/drug effects , Down-Regulation/drug effects , Genes, myc , Humans , Mice , Mice, Nude , Neoplasm Transplantation
13.
Int J Cancer ; 115(6): 998-1005, 2005 Jul 20.
Article in English | MEDLINE | ID: mdl-15723303

ABSTRACT

Recombinant adeno-associated viruses (rAAV) have become the vector of choice for many gene therapy protocols. rAAVs have a number of attractive features including long-term transgene expression and the ability to transduce both dividing and non-dividing cells. We have shown previously the anti-cancer role of tissue factor pathway inhibitor-2 (TFPI-2), a matrix-associated serine protease inhibitor, in human glioblastomas. As a result of our present study, in which 0.8-kb fragment of human TFPI-2 was cloned into the adeno-associated viral vectors (rAAA-TFPI-2), rAAV-TFPI-2 infection of SNB19 cells significantly increased TFPI-2 as determined by Western blotting. As assessed by spheroid and Matrigel assays, infection of SNB19 cells with rAAV-TFPI-2 significantly reduced migration and invasion in a dose-dependent manner. Tumor spheroids infected with rAAV-TFPI-2 and co-cultured with fetal rat brain aggregates did not invade rat brain aggregates, whereas 90-95% of the mock and AAV-CMV infected cells invaded rat brain aggregates. In vitro angiogenesis studies (tumor cells co-cultured with endothelial cells or endothelial cells seeded on matrigel) showed reduction of capillary-like structure formation in rAAV-TFPI-2-treated cells as compared to parental and mock-transfected cells. In in vivo angiogenesis results demonstrated the formation of microvessels in SNB19 parental cells and this formation was inhibited when the SNB19 cells were infected with rAAV-TFPI-2. Further, we observed a large reduction of tumor growth in SNB19 cells treated with rAAV-TFPI-2 virus injected intracerebrally when compared to controls. Our study demonstrates that rAAV-TFPI-2-mediated gene therapy offers a novel tool for the treatment of brain tumors.


Subject(s)
Dependovirus/genetics , Glycoproteins/physiology , Neovascularization, Pathologic/genetics , Animals , Cell Movement , Cell Proliferation , Genetic Vectors , Glioblastoma , Glycoproteins/genetics , Humans , Mice , Mice, Nude , Neoplasm Invasiveness , Neoplasm Transplantation , Tumor Cells, Cultured
14.
Cancer Res ; 64(12): 4069-77, 2004 Jun 15.
Article in English | MEDLINE | ID: mdl-15205313

ABSTRACT

We have shown previously that urokinase plasminogen activator receptor (uPAR) and cathepsin B are overexpressed during glioma progression, particularly at the leading edge of the tumor. In the present study, we simultaneously down-regulated uPAR and cathepsin B in SNB19 glioma cell monolayer or SNB19 spheroids using an adenoviral vector carrying antisense uPAR and antisense cathepsin B and a combination of these genes as determined by Western blot analysis. The Ad-uPAR-Cath B-infected cells revealed a marked reduction in tumor growth and invasiveness as compared with the parental and vector controls. In vitro and in vivo angiogenic assays demonstrated inhibition of capillary-like structure formation and microvessel formation after Ad-uPAR-Cath B infection of SNB19 cells when compared with Ad-cytomegalovirus (CMV)-infected or mock-infected controls. Furthermore, using a near infrared fluorescence probe, in vivo imaging for cathepsin B indicated low/undetectable levels of fluorescence after injection of the Ad-uPAR-Cath B construct into pre-established s.c. tumors as compared with Ad-CMV-treated and untreated tumors. The effect with bicistronic construct (Ad-uPAR-Cath B) was much higher than with single (Ad-uPAR/Ad-Cath B) constructs. These results indicate that the down-regulation of cathepsin B and uPAR plays a significant role in inhibiting tumor growth, invasion, and angiogenesis. Hence, the targeting of these two proteases may be a potential therapy for brain tumors and other cancers.


Subject(s)
Cathepsin B/antagonists & inhibitors , DNA, Antisense/genetics , Glioblastoma/genetics , Glioblastoma/therapy , Neovascularization, Pathologic/genetics , Neovascularization, Pathologic/therapy , Receptors, Cell Surface/antagonists & inhibitors , Adenoviridae/genetics , Animals , Cathepsin B/genetics , Cell Division/genetics , Cell Movement/genetics , DNA, Antisense/administration & dosage , Female , Genetic Therapy/methods , Glioblastoma/blood supply , Glioblastoma/virology , Humans , Male , Mice , Mice, Nude , Neoplasm Invasiveness , Neovascularization, Pathologic/pathology , Receptors, Cell Surface/genetics , Receptors, Urokinase Plasminogen Activator , Spheroids, Cellular , Xenograft Model Antitumor Assays
15.
Oncogene ; 23(27): 4681-9, 2004 Jun 10.
Article in English | MEDLINE | ID: mdl-15122332

ABSTRACT

Extracellular proteases have been shown to cooperatively influence matrix degradation and tumor cell invasion through proteolytic cascades, with individual proteases having distinct roles in tumor growth, invasion, migration and angiogenesis. Matrix metalloproteases (MMP)-9 and cathepsin B have been shown to participate in the processes of tumor growth, vascularization and invasion of gliomas. In the present study, we used a cytomegalovirus promoter-driven DNA template approach to induce hairpin RNA (hpRNA)-triggered RNA interference (RNAi) to block MMP-9 and cathepsin B gene expression with a single construct. Transfection of a plasmid vector-expressing double-stranded RNA (dsRNA) for MMP-9 and cathepsin B significantly inhibited MMP-9 and cathepsin B expression and reduced the invasive behavior of SNB19, glioblastoma cell line in Matrigel and spheroid invasion models. Downregulation of MMP-9 and cathepsin B using RNAi in SNB19 cells reduced cell-cell interaction of human microvascular endothelial cells, resulting in the disruption of capillary network formation in both in vitro and in vivo models. Direct intratumoral injections of plasmid DNA expressing hpRNA for MMP-9 and cathepsin B significantly inhibited established glioma tumor growth and invasion in intracranial tumors in vivo. Further intraperitoneal (i.p.) injections of plasmid DNA expressing hpRNA for MMP-9 and cathepsin B completely regressed pre-established tumors for a long time (4 months) without any indication of these tumor cells. For the first time, these observations demonstrate that the simultaneous RNAi-mediated targeting of MMP-9 and cathepsin B has potential application for the treatment of human gliomas.


Subject(s)
Cathepsin B/antagonists & inhibitors , Gene Expression Regulation, Neoplastic , Glioblastoma/metabolism , Matrix Metalloproteinase 9/metabolism , RNA Interference , Animals , Blotting, Western , Cathepsin B/administration & dosage , Cell Division/genetics , Cell Line, Tumor , Cell Movement/genetics , Collagen/metabolism , Down-Regulation , Drug Combinations , Glioblastoma/blood supply , Glioblastoma/genetics , Glioblastoma/pathology , Humans , Injections, Intraperitoneal , Injections, Intraventricular , Laminin/metabolism , Matrix Metalloproteinase 9/administration & dosage , Mice , Mice, Nude , Models, Biological , Neoplasm Invasiveness , Neoplasm Transplantation , Neovascularization, Pathologic/genetics , Proteoglycans/metabolism , Spheroids, Cellular , Transplantation, Heterologous
16.
Oncogene ; 23(13): 2339-46, 2004 Mar 25.
Article in English | MEDLINE | ID: mdl-14767475

ABSTRACT

Glioblastoma multiforme, the most common brain tumor, typically exhibits markedly increased angiogenesis, which is crucial for tumor growth and invasion. Antiangiogenic strategies based on disruption of the tumor microvasculature have proven effective for the treatment of experimental brain tumors. Here, we have overexpressed human caspase-9 by stable transfection in the SNB19 glioblastoma cell line, which normally expresses low levels of caspase-9. Our studies revealed that overexpression of caspase-9 coupled with radiation has a synergistic effect on the inhibition of glioma invasion as demonstrated by Matrigel assay (> 65%). Furthermore, sense caspase stable clones cocultured with fetal rat brain aggregates along with radiation showed complete inhibition as compared to the parental and vector controls. During in vitro angiogenesis, SNB19 cells cocultured with human microvascular endothelial cells (HMEC) showed vascular network formation after 48-72 h. In contrast, these capillary-like structures were inhibited when HMEC cells were cocultured with sense caspase stable SNB19 cells. This effect was further enhanced by radiation (5 Gy). Signaling mechanisms revealed that apoptosis is induced by cleavage of caspase-9 by radiation, loss of mitochondrial membrane potential and activation of caspase-3. These results demonstrate that activation of caspase-9 disrupts glioma cell invasion and angiogenesis in vitro. Hence, overexpression of proapoptotic molecules such as caspase-9 may be an important determinant of the therapeutic effect of radiation in cancer therapy.


Subject(s)
Caspases/radiation effects , Glioma/radiotherapy , Neoplasm Invasiveness , Neovascularization, Pathologic/radiotherapy , Animals , Apoptosis/radiation effects , Capillaries/enzymology , Capillaries/growth & development , Caspase 9 , Caspases/metabolism , Cytochromes c/biosynthesis , Cytochromes c/genetics , Humans , Membrane Potentials/physiology , Mice , Mitochondria/metabolism , Proto-Oncogene Proteins c-bcl-2/biosynthesis , Proto-Oncogene Proteins c-bcl-2/genetics
17.
Oncogene ; 23(12): 2224-30, 2004 Mar 18.
Article in English | MEDLINE | ID: mdl-14730346

ABSTRACT

The cysteine proteinase cathepsin B has been implicated in tumor progression by virtue of its increased mRNA and protein levels, as well as its localization at the invading front of the tumor. In this study, we examined whether blocking cathepsin B expression in human glioblastoma SNB19 cells affects angiogenesis. Stable transfectants of human glioblastoma cells with a plasmid containing antisense cathepsin B cDNA showed decreased migration rates in wound- and spheroid-migration assays. Analysis showed a reduction in VEGF protein and MMP-9 activity in the cathepsin B antisense cDNA-transfected cells. Regarding angiogenesis in vitro, we found that the conditioned medium of glioblastoma cells with downregulated cathepsin B expression reduced cell-cell interaction of human microvascular endothelial cells, resulting in the disruption of capillary-like network formation. Furthermore, a marked reduction in microvasculature development was seen in an in vivo dorsal air sac assay of glioblastoma cells with downregulated cathepsin B expression. Taken together, these results provide evidence that inhibition of cathepsin B expression can suppress glioblastoma-induced neovascularization.


Subject(s)
Angiogenesis Inhibitors/pharmacology , Brain Neoplasms/metabolism , Cathepsin B/antagonists & inhibitors , Glioblastoma/metabolism , Neoplasms/blood supply , Neovascularization, Pathologic , Animals , Biological Assay , Brain Neoplasms/genetics , Brain Neoplasms/pathology , Cell Line, Tumor , Cell Movement , Chromones/pharmacology , Culture Media, Conditioned , Culture Media, Serum-Free , Endothelium, Vascular/cytology , Enzyme Inhibitors/pharmacology , Flavonoids/pharmacology , Glioblastoma/genetics , Glioblastoma/pathology , Humans , Matrix Metalloproteinase Inhibitors , Mice , Mice, Nude , Morpholines/pharmacology , Tetradecanoylphorbol Acetate/pharmacology , Transfection , Vascular Endothelial Growth Factor A/metabolism
18.
Oncogene ; 22(38): 5967-75, 2003 Sep 04.
Article in English | MEDLINE | ID: mdl-12955075

ABSTRACT

Urokinase-type plasminogen activator (uPA) and its receptor (uPAR) play an important role in the invasiveness of gliomas and other infiltrative tumors. In glioma cell lines and tumors, high grade correlates with increased expression of uPAR and uPA. We report here the downregulation of uPAR and uPA by delivery of antisense sequences of uPAR and uPA in a single adenoviral vector, Ad-uPAR-uPA (Ad, adenovirus). The bicistronic construct (Ad-uPAR-uPA) infected glioblastoma cell line had significantly reduced levels of uPAR, uPA enzymatic activity and immunoreactivity for these proteins when compared to controls. The Ad-uPAR-uPA infected cells showed a markedly lower level of invasion in the Matrigel invasion assays, and their spheroids failed to invade the fetal rat brain aggregates in the coculture system. Intracranial injection of SNB19 cells with the Ad-uPAR-uPA antisense bicistronic construct showed inhibited invasiveness and tumorigenicity. Subcutaneous injections of bicistronic antisense constructs into established tumors (U87 MG) caused regression of those tumors. Our results support the therapeutic potential of targeting the individual components of the uPAR-uPA system by using a single adenovirus construct for the treatment of glioma and other invasive cancers.


Subject(s)
Central Nervous System Neoplasms/genetics , Central Nervous System Neoplasms/therapy , Glioma/genetics , Glioma/therapy , Neovascularization, Pathologic/therapy , Receptors, Cell Surface/genetics , Urokinase-Type Plasminogen Activator/genetics , Adenoviridae/genetics , Animals , Carcinogenicity Tests , Cell Movement , Central Nervous System Neoplasms/blood supply , Central Nervous System Neoplasms/pathology , DNA, Antisense/administration & dosage , DNA, Antisense/genetics , Gene Expression , Genetic Therapy/methods , Genetic Vectors/genetics , Glioma/blood supply , Glioma/pathology , Humans , Mice , Mice, Nude , Neoplasm Invasiveness , Neovascularization, Pathologic/genetics , Receptors, Cell Surface/immunology , Receptors, Cell Surface/metabolism , Receptors, Urokinase Plasminogen Activator , Tumor Cells, Cultured , Urokinase-Type Plasminogen Activator/immunology , Urokinase-Type Plasminogen Activator/metabolism
19.
Clin Exp Metastasis ; 20(4): 375-83, 2003.
Article in English | MEDLINE | ID: mdl-12856725

ABSTRACT

In recent years there has been an increasing interest in compounds present in foods that may prevent or slow the progression of chronic illnesses, such as cardiovascular disease, osteoporosis and cancer. Saponins have been reported to have important time-dependent anti-cancer properties. We have used a highly purified and characterized saponin fraction containing the soyasapogenol B glycosides (the 'B group' saponins) from soybeans (Glycine max L.) to demonstrate a reduction in SNB 19 human glioblastoma cell invasion (45% decrease compared to untreated cells) in vitro in a Matrigel invasion assay. We have also demonstrated that triterpenoid saponin induces apopotosis and affects mictochondiral function. Dose-dependent loss of mitochondrial trans-membrane potential in SNB 19 cells occurred with treatment, along with release of cytochrome c, processing of caspase-9, and -3 and specific cleavage of poly ADP-ribose polymerase (PARP), a substrate of caspase-3. The results suggest that the saponin fraction induces apoptosis in SNB19 human glioblastoma cells by stimulating cytochrome-c release and subsequent activation of a caspase cascade. Our observations clearly demonstrate the pro-apoptotic and anti-invasive activities of the soyasapogenol B glycosides from soybeans.


Subject(s)
Apoptosis , Brain Neoplasms/metabolism , Caspases/physiology , Glioblastoma/metabolism , Neoplasm Invasiveness , Saponins/pharmacology , Annexin A5/metabolism , Brain Neoplasms/pathology , Cell Cycle/drug effects , Cell Division/drug effects , Collagen/chemistry , Cytochrome c Group/metabolism , Down-Regulation , Drug Combinations , Enzyme Activation , Glioblastoma/pathology , Humans , Immunoblotting , Immunoenzyme Techniques , Laminin/chemistry , Membrane Potentials/drug effects , Mitochondria/drug effects , Mitochondria/metabolism , Mitochondria/ultrastructure , Poly(ADP-ribose) Polymerases/metabolism , Proteoglycans/chemistry , Glycine max/chemistry , Tumor Cells, Cultured/drug effects , Tumor Cells, Cultured/metabolism , Tumor Cells, Cultured/ultrastructure
20.
Oncogene ; 22(29): 4509-16, 2003 Jul 17.
Article in English | MEDLINE | ID: mdl-12881707

ABSTRACT

We have shown previously that the tissue factor pathway inhibitor-2 (TFPI-2), a broad range proteinase inhibitor, is highly expressed in low-grade gliomas, but, minimally expressed or undetectable in glioblastomas, and that enforced expression of this gene reduces the invasive properties of brain tumor cells. Here, we examined the role of promoter methylation as a mechanism of TFPI-2 gene silencing. In SNB19 glioblastoma cells, which have no detectable TFPI-2 expression, 5-aza-2'-deoxycytidine (5aC), an inhibitor of DNA methyltransferase, induced TFPI-2 mRNA in a dose-dependent manner. Trichostatin A (TSA), the histone deacetylase (HDAC) inhibitor, by itself, was more efficient than 5aC in inducing TFPI-2 transcripts, and the 5aC+TSA combination resulted in highly synergistic reactivation of the gene, both at the transcript and protein levels. In Hs683 glioma cells, which express the TFPI-2 gene at high levels, transfection of the in vitro methylated TFPI-2 promoter constructs resulted in a drastic decrease of promoter activity compared to the unmethylated promoter. Further, the methylation-specific PCR in SNB19 and Hs683 cells showed that TFPI-2 gene repression was closely linked with methylation of the CpG islands in the promoter. Finally, the chromatin immunoprecipitation assays in SNB19 cells showed that the methylated and repressed TFPI-2 promoter was associated with the methyl-CpG binding protein 2 (MeCP2), and that gene reactivation resulted in the loss of MeCP2 from this site. These studies establish that TFPI-2 is transcriptionally silenced through promoter methylation in SNB19 cells.


Subject(s)
Brain Neoplasms/metabolism , Chromosomal Proteins, Non-Histone , DNA Methylation , Gene Silencing , Glioblastoma/metabolism , Glioma/metabolism , Glycoproteins/genetics , Promoter Regions, Genetic , Repressor Proteins , Azacitidine/pharmacology , Brain Neoplasms/drug therapy , Brain Neoplasms/genetics , Chromatin/metabolism , CpG Islands , DNA (Cytosine-5-)-Methyltransferases/antagonists & inhibitors , DNA (Cytosine-5-)-Methyltransferases/metabolism , DNA-Binding Proteins/drug effects , DNA-Binding Proteins/metabolism , Enzyme Inhibitors/pharmacology , Glioblastoma/drug therapy , Glioblastoma/genetics , Glioma/drug therapy , Glioma/genetics , Glycoproteins/metabolism , Histone Deacetylase Inhibitors , Histone Deacetylases/metabolism , Humans , Hydroxamic Acids/pharmacology , Methyl-CpG-Binding Protein 2 , Polymerase Chain Reaction/methods
SELECTION OF CITATIONS
SEARCH DETAIL
...