ABSTRACT
BACKGROUND: Generalized anxiety disorder (GAD) is a prevalent anxiety disorder characterized by uncontrollable worry, trouble sleeping, muscle tension, and irritability. Cognitive behavioural therapy (CBT) is one of the first-line treatments that has demonstrated high efficacy in reducing symptoms of anxiety. Electronically delivered CBT (e-CBT) has been a promising adaptation of in-person treatment, showing comparable efficacy with increased accessibility and scalability. Finding further scalable interventions that can offer benefits to patients requiring less intensive interventions can allow for better resource allocation. Some studies have indicated that weekly check-ins can also lead to improvements in GAD symptoms. However, there is a lack of research exploring the potential benefits of online check-ins for patients with GAD. OBJECTIVE: This study aims to investigate the effects of weekly online asynchronous check-ins on patients diagnosed with GAD and compare it with a group receiving e-CBT. METHODS: Participants (n e-CBT = 45; n check-in = 51) with GAD were randomized into either an e-CBT or a mental health check-in program for 12 weeks. Participants in the e-CBT program completed pre-designed modules and homework assignments through a secure online delivery platform where they received personalized feedback from a trained care provider. Participants in the mental health check-in condition had weekly asynchronous messaging communication with a care provider where they were asked structured questions with a different weekly theme to encourage conversation. RESULTS: Both treatments demonstrated statistically significant reductions in GAD-7-item questionnaire (GAD-7) scores over time, but when comparing the groups there was no significant difference between the treatments. The number of participants who dropped out and baseline scores on all questionnaires were comparable for both groups. CONCLUSIONS: The findings support the effectiveness of e-CBT and mental health check-ins for the treatment of GAD.
Comparing the Effectiveness of Electronically Delivered Therapy (e-CBT) to Weekly Online Mental Health Check-ins for Generalized Anxiety DisorderA Randomized Controlled TrialPlain Language SummaryGeneralized anxiety disorder (GAD) is a prevalent psychiatric condition that leads to symptoms like uncontrollable worry, trouble sleeping, muscle tension, and irritability. Cognitive behavioural therapy (CBT) is a common psychotherapy used for GAD since it has been shown to reduce symptoms. However, traditional CBT that is in person can have barriers such as being inaccessible and costly, and therefore electronically delivered CBT (e-CBT) is a viable alternative since previous studies have shown its efficacy in reducing symptoms and being similar compared to face-to-face CBT. Previous studies have also shown reductions in GAD symptomology through the use of checking in on people and their mental health. Therefore, this study aimed to compare e-CBT to a check-in condition and had a total of 45 individuals in e-CBT and 51 participants in the check-in condition. Participants in the e-CBT condition completed 12 weeks of predesigned e-CBT modules, homework and received personalized feedback from a care provider. In contrast, individuals in the check-in condition completed 12 weeks of unstructured asynchronous messaging with a care provider. Results from the study showed that both the e-CBT and check-in condition demonstrated statistically significant improvements in GAD-7 across time, but when comparing the groups there was no significant difference. The results show the efficacy of e-CBT and checking in on people's mental health to reduce GAD and future research should examine the 2 conditions combined.
ABSTRACT
BACKGROUND: Lockdowns and social distancing resulting from the COVID-19 pandemic have worsened the population's mental health and made it more difficult for individuals to receive care. Electronic cognitive behavioral therapy (e-CBT) is a cost-effective and evidence-based treatment for anxiety and depression and can be accessed remotely. OBJECTIVE: The objective of the study was to investigate the efficacy of online psychotherapy tailored to depression and anxiety symptoms during the pandemic. METHODS: The pilot study used a pre-post design to evaluate the efficacy of a 9-week e-CBT program designed for individuals with depression and anxiety affected by the pandemic. Participants were adults (N=59) diagnosed with major depressive disorder and generalized anxiety disorder, whose mental health symptoms initiated or worsened during the COVID-19 pandemic. The online psychotherapy program focused on teaching coping, mindfulness, and problem-solving skills. Symptoms of anxiety and depression, resilience, and quality of life were assessed. RESULTS: Participants demonstrated significant improvements in symptoms of anxiety (P=.02) and depression (P=.03) after the intervention. Similar trends were observed in the intention-to-treat analysis. No significant differences were observed in resilience and quality-of-life measures. The sample comprised mostly females, making it challenging to discern the benefits of the intervention in males. Although a pre-post design is less rigorous than a controlled trial, this design was selected to observe changes in scores during a critical period. CONCLUSIONS: e-CBT for COVID-19 is an effective and accessible treatment option. Improvements in clinical symptoms of anxiety and depression can be observed in individuals whose mental health is affected by the COVID-19 pandemic. TRIAL REGISTRATION: ClinicalTrials.gov NCT04476667; https://clinicaltrials.gov/study/NCT04476667. INTERNATIONAL REGISTERED REPORT IDENTIFIER (IRRID): RR2-10.2196/24913.
Subject(s)
COVID-19 , Cognitive Behavioral Therapy , Depressive Disorder, Major , Mindfulness , Adult , Female , Humans , Male , Anxiety/therapy , Anxiety Disorders/therapy , Cognitive Behavioral Therapy/methods , Communicable Disease Control , COVID-19/epidemiology , Depression/therapy , Depressive Disorder, Major/psychology , Pandemics , Pilot Projects , Quality of LifeABSTRACT
BACKGROUND: A subset of men with metastatic prostate cancer (mPC) responds to immune checkpoint inhibitors, and there is an unmet need to predict those most likely to benefit. We characterized circulating tumor cells (CTCs) for expression of immune checkpoint ligands in men with mPC as a non-invasive biomarker of immune evasion and immunotherapy benefit. METHODS: Three cohorts of patients were enrolled: 1) men with mCRPC starting abiraterone acetate/prednisone or enzalutamide (pre-ARSI), 2) men with mCRPC who were progressing on enzalutamide or abiraterone acetate/prednisone (post-ARSI), and 3) men with newly diagnosed metastatic hormone sensitive prostate cancer (mHSPC) starting androgen deprivation therapy. CTCs were captured using the CellSearch® system and stained for PD-L1, PD-L2, B7-H3, and CTLA-4 at baseline, on treatment, and disease progression. Summary statistics on mean CTCs per cohort, as well as rates of ligand positivity were used to analyze CTCs by cohort and by timepoint. RESULTS: Men in all cohorts and timepoints had prevalent CTC B7-H3 expression (> 80%). We found evidence for CTC PD-L1 expression across disease states, in which > 1 positive CTC or > 50% of CTCs were positive for PD-L1 in 40 and 30% of men with mHSPC, respectively, 60 and 20% of men with mCRPC pre-ARSI, and 70 and 30% of men with mCRPC post-ARSI. CTC PD-L2 expression was present in 20-40% of men in each disease state, while CTC CTLA-4 expression was rare, present in 20% of men with mCRPC pre-ARSI and 10% of men with mCRPC post-ARSI or with mHSPC. CTC immune checkpoint expression was heterogeneous within/between men and across disease states. CONCLUSIONS: We have identified that CTCs from men with mPC heterogeneously express immune checkpoints B7-H3, PD-L1, PD-L2, and CTLA-4, and the detection of these immune checkpoints may enable monitoring on immunotherapy.
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BACKGROUND: Osteosarcoma is a rare but aggressive bone cancer that occurs primarily in children. Like other rare cancers, treatment advances for osteosarcoma have stagnated, with little improvement in survival for the past several decades. Developing new treatments has been hampered by extensive genomic heterogeneity and limited access to patient samples to study the biology of this complex disease. METHODS: To overcome these barriers, we combined the power of comparative oncology with patient-derived models of cancer and high-throughput chemical screens in a cross-species drug discovery pipeline. RESULTS: Coupling in vitro high-throughput drug screens on low-passage and established cell lines with in vivo validation in patient-derived xenografts we identify the proteasome and CRM1 nuclear export pathways as therapeutic sensitivities in osteosarcoma, with dual inhibition of these pathways inducing synergistic cytotoxicity. CONCLUSIONS: These collective efforts provide an experimental framework and set of new tools for osteosarcoma and other rare cancers to identify and study new therapeutic vulnerabilities.
ABSTRACT
Multiple studies evaluated the outcomes and complications rate of Takotsubo Syndrome (TTS) in patients with and without advanced chronic kidney disease (CKD), revealed conflicting results. This study aims to assess the clinical outcomes and impact of advanced CKD on patients hospitalized with Takotsubo Syndrome. Patients who presented with Takotsubo cardiomyopathy between 2010 and 2014 were identified in the National Inpatient Sample (NIS) database using the International Classification of Diseases, 9th Revision, Clinical Modification (ICD-9-CM), and subsequently were divided into two groups, with advanced CKD and without advanced CKD. NIS is the largest all-payer inpatient stays database in the United States. The primary outcome was the effect of advanced CKD on inpatient mortality in comparison to the non-advanced CKD group. Secondary outcomes were the impact of CKD on TTS in-hospital complications. We also evaluated the length of hospital stay and the cost of hospitalization. Propensity score-matched analysis was performed to address potential confounding. The advanced CKD group had no significant increase in the risk of In-hospital mortality (OR 0.99; 95% CI 0.75-1.31, Pâ¯=â¯0.269). However, advanced CKD patients were more likely to develop acute kidney injury (AKI) requiring dialysis (OR: 5.12, 95% CI: 3.16-8.30, Pâ¯=â¯<0.0001), and were more likely to stay longer at the hospital (OR 1.12; 95% CI 1.03 to 1.22, P 0.010). In conclusion, advanced chronic kidney disease does not increase immediate in-hospital mortality, neither most of the TTS in-hospital complications, apart from AKI and hospital length of stay, in comparison to the patients with non-advanced CKD.
Subject(s)
Hospital Mortality/trends , Length of Stay/trends , Propensity Score , Renal Insufficiency, Chronic/mortality , Takotsubo Cardiomyopathy/mortality , Aged , Aged, 80 and over , Female , Hospitalization/trends , Humans , Male , Middle Aged , Renal Insufficiency, Chronic/diagnosis , Renal Insufficiency, Chronic/physiopathology , Takotsubo Cardiomyopathy/diagnosis , Takotsubo Cardiomyopathy/physiopathology , Treatment OutcomeABSTRACT
While inhibitors of BCL-2 family proteins (BH3 mimetics) have shown promise as anti-cancer agents, the various dependencies or co-dependencies of diverse cancers on BCL-2 genes remain poorly understood. Here we develop a drug screening approach to define the sensitivity of cancer cells from ten tissue types to all possible combinations of selective BCL-2, BCL-XL, and MCL-1 inhibitors and discover that most cell lines depend on at least one combination for survival. We demonstrate that expression levels of BCL-2 genes predict single mimetic sensitivity, whereas EMT status predicts synergistic dependence on BCL-XL+MCL-1. Lastly, we use a CRISPR/Cas9 screen to discover that BFL-1 and BCL-w promote resistance to all tested combinations of BCL-2, BCL-XL, and MCL-1 inhibitors. Together, these results provide a roadmap for rationally targeting BCL-2 family dependencies in diverse human cancers and motivate the development of selective BFL-1 and BCL-w inhibitors to overcome intrinsic resistance to BH3 mimetics.
Subject(s)
Neoplasms/metabolism , Proto-Oncogene Proteins c-bcl-2/metabolism , Animals , Antineoplastic Agents, Phytogenic/pharmacology , Bridged Bicyclo Compounds, Heterocyclic/pharmacology , Cell Line, Tumor , Dose-Response Relationship, Drug , Male , Mice , RNA, Messenger/metabolism , Sulfonamides/pharmacology , bcl-X Protein/metabolismABSTRACT
Most patients with ovarian cancer are diagnosed late in progression and often experience tumor recurrence and relapses due to drug resistance. Surface expression of matrix metalloprotease (MMP)-14 on ovarian cancer cells stimulates a tumor-stromal signaling pathway that promotes angiogenesis and tumor growth. In a cohort of 92 patients, we found that MMP-14 was increased in the serum of women with malignant ovarian tumors. Therefore, we investigated the preclinical efficacy of a MMP-14 monoclonal antibody that could inhibit the migratory and invasive properties of aggressive ovarian cancer cells in vitro. MMP-14 antibody disrupted ovarian tumor-stromal communication and was equivalent to Avastin in suppressing blood vessel growth in mice harboring Matrigel plugs. These effects on angiogenesis correlated with downregulation of several important angiogenic factors. Furthermore, mice with ovarian cancer tumors treated with anti-MMP-14 monotherapy showed a marked and sustained regression in tumor growth with decreased angiogenesis compared with immunoglobulin G (IgG)-treated controls. In a model of advanced peritoneal ovarian cancer, MMP-14-dependent invasion and metastasis was effectively inhibited by intraperitoneal administration of monoclonal MMP-14 antibody. Together, these studies provide a preclinical proof-of-concept for MMP-14 targeting as an adjuvant treatment strategy for advanced ovarian cancer.
Subject(s)
Antibodies, Monoclonal/pharmacology , Matrix Metalloproteinase 14/metabolism , Matrix Metalloproteinase Inhibitors/pharmacology , Neovascularization, Pathologic/metabolism , Ovarian Neoplasms/metabolism , Ovarian Neoplasms/pathology , Tumor Burden/drug effects , Animals , Antibodies, Monoclonal/administration & dosage , Antineoplastic Agents/pharmacology , Biomarkers, Tumor/blood , Cell Line, Tumor , Cell Movement/drug effects , Disease Models, Animal , Docetaxel , Female , Human Umbilical Vein Endothelial Cells/drug effects , Human Umbilical Vein Endothelial Cells/metabolism , Humans , Matrix Metalloproteinase 14/blood , Matrix Metalloproteinase 14/genetics , Matrix Metalloproteinase Inhibitors/administration & dosage , Mice , Mice, Nude , Neoplasm Invasiveness , Neoplasm Metastasis , Neovascularization, Pathologic/genetics , Ovarian Neoplasms/genetics , Taxoids/pharmacology , Tumor Microenvironment/drug effects , Tumor Microenvironment/genetics , Xenograft Model Antitumor AssaysABSTRACT
Protease-activated receptors (PARs) are G-protein-coupled receptors that are activated by proteolytic cleavage and generation of a tethered ligand. High PAR1 expression has been documented in a variety of invasive cancers of epithelial origin. In the present study, we investigated the contribution of the four PAR family members to motility of lung carcinomas and primary tumor samples from patients. We found that of the four PARs, only PAR1 expression was highly increased in the lung cancer cell lines. Primary lung cancer cells isolated from patient lung tumors migrated at a 10- to 40-fold higher rate than epithelial cells isolated from nonmalignant lung tissue. Cell-penetrating pepducin inhibitors were generated against the first (i1) and third (i3) intracellular loops of PAR1 and tested for their ability to inhibit PAR1-driven migration and extracellular regulated kinase (ERK)1/2 activity. The PAR1 pepducins showed significant inhibition of cell migration in both primary and established cell lines similar to silencing of PAR1 expression with short hairpin RNA (shRNA). Unlike i1 pepducins, the i3 loop pepducins were effective inhibitors of PAR1-mediated ERK activation and tumor growth. Comparable in efficacy with Bevacizumab, monotherapy with the PAR1 i3 loop pepducin P1pal-7 provided significant 75% inhibition of lung tumor growth in nude mice. We identify the PAR1-ERK1/2 pathway as a feasible target for therapy in lung cancer.
Subject(s)
Adenocarcinoma/drug therapy , Lipopeptides/pharmacology , Lung Neoplasms/drug therapy , Receptor, PAR-1/antagonists & inhibitors , Receptor, PAR-1/metabolism , Adenocarcinoma/metabolism , Adenocarcinoma/pathology , Animals , Apoptosis , Blotting, Western , Cell Line, Tumor , Cell Movement , Cell Proliferation , Cytoplasm/metabolism , Epithelial Cells/drug effects , Epithelial Cells/metabolism , Female , Humans , Immunoenzyme Techniques , Lipopeptides/pharmacokinetics , Lung/cytology , Lung/drug effects , Lung/metabolism , Lung Neoplasms/metabolism , Lung Neoplasms/pathology , Mice , Mice, Nude , Mitogen-Activated Protein Kinase 1/genetics , Mitogen-Activated Protein Kinase 1/metabolism , Mitogen-Activated Protein Kinase 3/genetics , Mitogen-Activated Protein Kinase 3/metabolism , RNA, Messenger/genetics , RNA, Small Interfering/genetics , Receptor, PAR-1/genetics , Receptors, G-Protein-Coupled/metabolism , Reverse Transcriptase Polymerase Chain Reaction , Signal Transduction , Tissue DistributionABSTRACT
At the 2nd Pepducin Science Symposium held in Cambridge, Massachusetts, on November 4-5, 2010, investigators working in G protein-coupled receptor (GPCR) research convened to discuss progress since last year's inaugural conference. This year's symposium focused on increasing knowledge of the structure and function of this ubiquitous superfamily of membrane receptors and their potential modulation for disease treatment. Presentations also focused on how GPCR mechanisms might be exploited to treat diseases with pepducins, novel synthetic lipopeptide pharmacophores that modulate heptahelical GPCR activity. While the multiple roles of GPCRs in physiological and pathophysiological processes offer significant opportunities for novel drug development, the global nature of their activity challenges drug-specific and validated target identification. This year's conference highlighted advances in understanding of GPCR agonist and antagonist ligand-binding motifs, their ligand-independent functions, structure-activity relationships (SARs), and evolving unique methods to probe GPCR structure and function. Study results summarized at the meeting also provided evidence for evolving views of how signaling mechanisms work through these receptors.
Subject(s)
Congresses as Topic , Lipopeptides/metabolism , Receptors, G-Protein-Coupled/metabolism , Signal Transduction/physiology , Drug Design , Humans , Lipopeptides/chemistry , Neoplasms/physiopathology , Neoplasms/therapy , Receptors, G-Protein-Coupled/chemistry , Structure-Activity RelationshipABSTRACT
Sepsis is a deadly disease characterized by the inability to regulate the inflammatory-coagulation response in which the endothelium plays a key role. The cause of this perturbation remains poorly understood and has hampered the development of effective therapeutics. Matrix metalloproteases (MMPs) are involved in the host response to pathogens, but can also cause uncontrolled tissue damage and contribute to mortality. We found that human sepsis patients had markedly elevated plasma proMMP-1 and active MMP-1 levels, which correlated with death at 7 and 28 days after diagnosis. Likewise, septic mice had increased plasma levels of the MMP-1 ortholog, MMP-1a. We identified mouse MMP-1a as an agonist of protease-activated receptor-1 (PAR1) on endothelial cells. MMP-1a was released from endothelial cells in septic mice. Blockade of MMP-1 activity suppressed endothelial barrier disruption, disseminated intravascular coagulation (DIC), lung vascular permeability as well as the cytokine storm and improved survival, which was lost in PAR1-deficient mice. Infusion of human MMP-1 increased lung vascular permeability in normal wild-type mice but not in PAR1-deficient mice. These findings implicate MMP-1 as an important activator of PAR1 in sepsis and suggest that therapeutics that target MMP1-PAR1 may prove beneficial in the treatment of sepsis.
Subject(s)
Capillary Permeability/physiology , Inflammation/metabolism , Matrix Metalloproteinase 1/blood , Receptor, PAR-1/metabolism , Sepsis/metabolism , Sepsis/mortality , Animals , Endothelial Cells/cytology , Endothelial Cells/physiology , Humans , Matrix Metalloproteinase Inhibitors , Mice , Mice, Inbred C57BL , Mice, Knockout , Receptor, PAR-1/genetics , Signal Transduction/physiology , rho GTP-Binding Proteins/metabolismABSTRACT
Ovarian cancer is a lethal gynecologic malignancy that may benefit from new therapies that block key paracrine pathways involved in tumor-stromal interactions and tumor vascularity. It was recently shown that matrix metalloprotease-1 (MMP1) activation of the G protein-coupled receptor protease-activated receptor-1 (PAR1) is an important stimulator of angiogenesis and metastasis in peritoneal mouse models of ovarian cancer. In the present study, we tested the hypothesis that MMP1-PAR1 promotes angiogenesis through its paracrine control of angiogenic chemokine receptors. We found that MMP1-PAR1 activation induces the secretion of several angiogenic factors from ovarian carcinoma cells, most prominently interleukin (IL)-8, growth-regulated oncogene-alpha (GRO-alpha), and monocyte chemoattractant protein-1. The secreted IL-8 and GRO-alpha acts on endothelial CXCR1/2 receptors in a paracrine manner to cause robust endothelial cell proliferation, tube formation, and migration. A cell-penetrating pepducin, X1/2pal-i3, which targets the conserved third intracellular loop of both CXCR1 and CXCR2 receptors, significantly inhibited endothelial cell proliferation, tube formation, angiogenesis, and ovarian tumor growth in mice. Matrigel plugs mixed with MMP1-stimulated, OVCAR-4-conditioned media showed a dramatic 33-fold increase in blood vessel formation in mice. The X1/2pal-i3 pepducin completely inhibited MMP1-dependent angiogenesis compared with a negative control pepducin or vehicle. Conversely, a vascular endothelial growth factor-directed antibody, Avastin, suppressed angiogenesis in mice but, as expected, was unable to inhibit IL-8 and GRO-alpha-dependent endothelial tube formation in vitro. These studies identify a critical MMP1-PAR1-CXCR1/2 paracrine pathway that might be therapeutically targeted for ovarian cancer treatment.
Subject(s)
Angiogenesis Inhibitors/pharmacology , Matrix Metalloproteinase 1/metabolism , Ovarian Neoplasms/metabolism , Receptor, PAR-1/metabolism , Amino Acid Sequence , Animals , Cell Communication/physiology , Cell Line, Tumor , Chemokine CXCL1/metabolism , Endothelial Cells/drug effects , Endothelial Cells/metabolism , Endothelial Cells/pathology , Female , Humans , Interleukin-8/metabolism , Mice , Mice, Nude , Molecular Sequence Data , Neovascularization, Pathologic/drug therapy , Neovascularization, Pathologic/metabolism , Neovascularization, Pathologic/pathology , Ovarian Neoplasms/blood supply , Ovarian Neoplasms/drug therapy , Ovarian Neoplasms/pathology , Peptide Fragments/pharmacology , Receptors, Interleukin-8A/metabolism , Receptors, Interleukin-8B/metabolism , Xenograft Model Antitumor AssaysABSTRACT
The inaugural Pepducin Science Symposium convened in Cambridge, Massachusetts on March 8-9, 2009 provided the opportunity for an international group of distinguished scientists to present and discuss research regarding G protein-coupled receptor-related research. G protein-coupled receptors (GPCRs) are, arguably, one of the most important molecular targets in drug discovery and pharmaceutical development today. This superfamily of membrane receptors is central to nearly every signaling pathway in the human body and has been the focus of intense research for decades. However, as scientists discover additional properties of GPCRs, it has become clear that much is yet to be understood about how these receptors function. Everyone agrees, however, that tremendous potential remains if specific GPCR signaling pathways can be modulated to correct pathological states. One exciting new approach to this challenge involves pepducins: novel, synthetic lipopeptide pharmacophores that modulate heptahelical GPCR activity from inside the cell membrane.
Subject(s)
Drug Delivery Systems/methods , Pharmaceutical Preparations , Receptors, G-Protein-Coupled/agonists , Receptors, G-Protein-Coupled/antagonists & inhibitors , Allosteric Regulation/drug effects , Allosteric Regulation/physiology , Animals , Computational Biology , Drug Evaluation , Female , Humans , Ligands , Models, Biological , Models, Molecular , Morphine Dependence/etiology , Morphine Dependence/metabolism , Ovarian Neoplasms/drug therapy , Pharmaceutical Preparations/isolation & purification , Receptors, G-Protein-Coupled/chemistry , Receptors, G-Protein-Coupled/metabolism , Sepsis/drug therapy , Synaptic Transmission/drug effectsABSTRACT
Protease-activated receptor 1 (PAR1) is a G protein-coupled receptor that is not expressed in normal breast epithelia but is up-regulated in invasive breast carcinomas. In the present study, we found that matrix metalloprotease-1 (MMP-1) robustly activates the PAR1-Akt survival pathway in breast carcinoma cells. This process is blocked by a cell-penetrating lipopeptide "pepducin," P1pal-7, which is a potent inhibitor of cell viability in breast carcinoma cells expressing PAR1. Both a MMP-1 inhibitor and P1pal-7 significantly promote apoptosis in breast tumor xenografts and inhibit metastasis to the lungs by up to 88%. Dual therapy with P1pal-7 and Taxotere inhibits the growth of MDA-MB-231 xenografts by 95%. Consistently, biochemical analysis of xenograft tumors treated with P1pal-7 or MMP-1 inhibitor showed attenuated Akt activity. Ectopic expression of constitutively active Akt rescues breast cancer cells from the synergistic cytotoxicity of P1pal-7 and Taxotere, suggesting that Akt is a critical component of PAR1-dependent cancer cell viability. Together, these findings indicate that blockade of MMP1-PAR1 signaling may provide a benefit beyond treatment with Taxotere alone in advanced, metastatic breast cancer.
Subject(s)
Breast Neoplasms/drug therapy , Breast Neoplasms/metabolism , Peptides/pharmacology , Proto-Oncogene Proteins c-akt/antagonists & inhibitors , Receptor, PAR-1/antagonists & inhibitors , Animals , Antineoplastic Combined Chemotherapy Protocols/administration & dosage , Apoptosis/drug effects , Breast Neoplasms/enzymology , Breast Neoplasms/pathology , Caspases/metabolism , Cell Line, Tumor , Cell Survival/drug effects , Cell Survival/physiology , Docetaxel , Drug Synergism , Enzyme Activation , Female , Humans , Lung Neoplasms/metabolism , Lung Neoplasms/secondary , Matrix Metalloproteinase 1/metabolism , Matrix Metalloproteinase Inhibitors , Mice , Mice, Nude , Peptides/administration & dosage , Peptides/pharmacokinetics , Proto-Oncogene Proteins c-akt/metabolism , RNA, Small Interfering/genetics , Receptor, PAR-1/genetics , Receptor, PAR-1/metabolism , Signal Transduction/drug effects , Taxoids/administration & dosage , Xenograft Model Antitumor AssaysABSTRACT
Gene chip and proteomic analyses of tumors and stromal tissue has led to the identification of dozens of candidate tumor and host components potentially involved in tumor-stromal interactions, angiogenesis, and progression of invasive disease. In particular, matrix metalloproteases (MMP) have emerged as important biomarkers and prognostic factors for invasive and metastatic cancers. From an initial screen of benign versus malignant patient fluids, we delineated a metalloprotease cascade comprising MMP-14, MMP-9, and MMP-1 that culminates in activation of PAR1, a G protein-coupled protease-activated receptor up-regulated in diverse cancers. In xenograft models of advanced peritoneal ovarian cancer, PAR1-dependent angiogenesis, ascites formation, and metastasis were effectively inhibited by i.p. administration of cell-penetrating pepducins based on the intracellular loops of PAR1. These data provide an in vivo proof-of-concept that targeting the metalloprotease-PAR1 signaling system may be a novel therapeutic approach in the treatment of ovarian cancer.
Subject(s)
Ascites/enzymology , Metalloproteases/antagonists & inhibitors , Neovascularization, Pathologic/enzymology , Oligopeptides/pharmacology , Ovarian Neoplasms/pathology , Receptor, PAR-1/antagonists & inhibitors , Signal Transduction/drug effects , Animals , Antineoplastic Combined Chemotherapy Protocols/pharmacology , Body Fluids/enzymology , Cell Line, Tumor , Cell Movement/drug effects , Disease Progression , Docetaxel , Female , Fibroblasts/drug effects , Fibroblasts/enzymology , Humans , Mice , Mice, Nude , NIH 3T3 Cells , Neoplasm Invasiveness , Oligopeptides/administration & dosage , Oligopeptides/therapeutic use , Ovarian Neoplasms/drug therapy , Ovarian Neoplasms/enzymology , Peritoneum/enzymology , Peritoneum/pathology , Taxoids/administration & dosage , Taxoids/pharmacology , Xenograft Model Antitumor AssaysABSTRACT
Sepsis is a deadly disease characterized by considerable derangement of the proinflammatory, anti-inflammatory and coagulation responses. Protease-activated receptor 1 (PAR1), an important regulator of endothelial barrier function and blood coagulation, has been proposed to be involved in the lethal sequelae of sepsis, but it is unknown whether activation of PAR1 is beneficial or harmful. Using a cell-penetrating peptide (pepducin) approach, we provide evidence that PAR1 switched from being a vascular-disruptive receptor to a vascular-protective receptor during the progression of sepsis in mice. Unexpectedly, we found that the protective effects of PAR1 required transactivation of PAR2 signaling pathways. Our results suggest therapeutics that selectively activate PAR1-PAR2 complexes may be beneficial in the treatment of sepsis.
Subject(s)
Endothelial Cells/physiology , Receptor, PAR-1/physiology , Receptor, PAR-2/physiology , Sepsis/metabolism , Signal Transduction/physiology , Animals , Capillary Permeability , Cell Communication , Cell Line , Mice , Receptor, PAR-1/metabolism , Receptor, PAR-2/agonists , Receptor, PAR-2/metabolism , Sepsis/physiopathology , Vascular Diseases/etiologyABSTRACT
We describe a new therapeutic approach for the treatment of lethal sepsis using cell-penetrating lipopeptides-termed pepducins-that target either individual or multiple chemokine receptors. Interleukin-8 (IL-8), a ligand for the CXCR1 and CXCR2 receptors, is the most potent endogenous proinflammatory chemokine in sepsis. IL-8 levels rise in blood and lung fluids to activate neutrophils and other cells, and correlate with shock, lung injury and high mortality. We show that pepducins derived from either the i1 or i3 intracellular loops of CXCR1 and CXCR2 prevent the IL-8 response of both receptors and reverse the lethal sequelae of sepsis, including disseminated intravascular coagulation and multi-organ failure in mice. Conversely, pepducins selective for CXCR4 cause a massive leukocytosis that does not affect survival. CXCR1 and CXCR2 pepducins conferred nearly 100% survival even when treatment was postponed, suggesting that our approach might be beneficial in the setting of advanced disease.
Subject(s)
Peptides/pharmacology , Receptors, CXCR4/antagonists & inhibitors , Receptors, Interleukin-8A/antagonists & inhibitors , Receptors, Interleukin-8B/antagonists & inhibitors , Systemic Inflammatory Response Syndrome/drug therapy , Systemic Inflammatory Response Syndrome/physiopathology , Amino Acid Sequence , Animals , Cells, Cultured , Female , Humans , Interleukin-8/physiology , Mice , Mice, Inbred Strains , Molecular Sequence Data , Neutrophils/drug effects , Protein Conformation , Protein Subunits , Receptors, CXCR4/physiology , Receptors, Interleukin-8A/chemistry , Receptors, Interleukin-8A/physiology , Receptors, Interleukin-8B/chemistry , Receptors, Interleukin-8B/physiologyABSTRACT
The physiologic response to stress is highly dependent on the activation of corticotropin-releasing hormone (CRH) neurons by various neurotransmitters. A particularly rich innervation of hypophysiotropic CRH neurons has been detected by nerve fibers containing the neuropeptide PACAP, a potent activator of the cAMP-protein kinase A (PKA) system. Intracerebroventricular (icv) injections of PACAP also elevate steady-state CRH mRNA levels in the paraventricular nucleus (PVN), but it is not known whether PACAP effects can be associated with acute stress responses. Likewise, in cell culture studies, pharmacologic activation of the PKA system has stimulated CRH gene promoter activity through an identified cAMP response element (CRE); however, a direct link between PACAP and CRH promoter activity has not been established. In our present study, icv injection of 150 or 300 pmol PACAP resulted in robust phosphorylation of the transcription factor CREB in the majority of PVN CRH neurons at 15 to 30 min post-injection and induced nuclear Fos labeling at 90 min. Simultaneously, plasma corticosterone concentrations were elevated in PACAP-injected animals, and significant increases were observed in face washing, body grooming, rearing and wet-dog shakes behaviors. We investigated the effect of PACAP on human CRH promoter activity in alphaT3-1 cells, a PACAP-receptor expressing cell line. Cells were transiently transfected with a chloramphenicol acetyltransferase (CAT) reporter vector containing region - 663/+124 of the human CRH gene promoter then treated for with PACAP (100 nM) or with the adenylate cyclase activating agent, forskolin (2.5 muM). Both PACAP and forskolin significantly increased wild-type hCRH promoter activity relative to vehicle controls. The PACAP response was abolished in the CRE-mutant construct. Pretreatment of transfected cells with the PKA blocker, H-89, completely prevented both PACAP- and forskolin-induced increases in CRH promoter activity. Furthermore, CREB overexpression strongly enhanced PACAP-mediated stimulation of hCRH promoter activity, an effect which was also lost with mutation of the CRE. Thus, we demonstrate that icv PACAP administration to rats under non-stressed handling conditions leads to cellular, hormonal and behavioral responses recapitulating manifestations of the acute stress response. Both in vivo and in vitro data point to the importance of PACAP-mediated activation of the cAMP/PKA signaling pathway for stimulation of CRH gene transcription, likely via the CRE.
Subject(s)
Corticotropin-Releasing Hormone/metabolism , Cyclic AMP-Dependent Protein Kinases/metabolism , Gene Expression Regulation/drug effects , Nerve Growth Factors/pharmacology , Neuropeptides/pharmacology , Neurotransmitter Agents/pharmacology , Paraventricular Hypothalamic Nucleus/drug effects , Stress, Physiological/physiopathology , Analysis of Variance , Animals , Behavior, Animal/drug effects , Cell Line , Corticosterone/blood , Corticotropin-Releasing Hormone/genetics , Dose-Response Relationship, Drug , Drug Interactions , Enzyme Inhibitors/pharmacology , Immunohistochemistry/methods , Injections, Intraventricular/methods , Male , Mice , Oncogene Proteins v-fos/metabolism , Paraventricular Hypothalamic Nucleus/cytology , Pituitary Adenylate Cyclase-Activating Polypeptide , RNA, Messenger/metabolism , Random Allocation , Rats , Rats, Sprague-Dawley , Reverse Transcriptase Polymerase Chain Reaction/methods , Time Factors , Transfection/methodsABSTRACT
Protease-activated receptors (PARs) are a unique class of G protein-coupled receptors that play critical roles in thrombosis, inflammation, and vascular biology. PAR1 is proposed to be involved in the invasive and metastatic processes of various cancers. However, the protease responsible for activating the proinvasive functions of PAR1 remains to be identified. Here, we show that expression of PAR1 is both required and sufficient to promote growth and invasion of breast carcinoma cells in a xenograft model. Further, we show that the matrix metalloprotease, MMP-1, functions as a protease agonist of PAR1 cleaving the receptor at the proper site to generate PAR1-dependent Ca2+ signals and migration. MMP-1 activity is derived from fibroblasts and is absent from the breast cancer cells. These results demonstrate that MMP-1 in the stromal-tumor microenvironment can alter the behavior of cancer cells through PAR1 to promote cell migration and invasion.
Subject(s)
Breast Neoplasms/metabolism , Carcinoma/metabolism , Cell Transformation, Neoplastic/metabolism , Matrix Metalloproteinase 1/metabolism , Receptor, PAR-1/metabolism , Animals , Binding Sites/physiology , Breast Neoplasms/physiopathology , Calcium Signaling/physiology , Carcinoma/physiopathology , Cell Line, Tumor , Cell Movement/physiology , Disease Models, Animal , Extracellular Matrix/metabolism , Female , Humans , Mice , Mice, Nude , NIH 3T3 Cells , Neoplasm Invasiveness , Transplantation, HeterologousABSTRACT
Oviduct-specific glycoprotein (OGP) is a high molecular weight glycoprotein belonging to the chitinase protein family. OGPs are found to associate with the zona pellucida and plasma membrane of the oocyte and developing embryo. Recent studies have shown that OGP plays an important role in pre-fertilization reproductive events (i.e. sperm capacitation, sperm-zona binding and zona penetration). To have a better understanding of human OGP (HuOGP) gene expression, a 3.3 kb DNA fragment containing the 5'-flanking and the intron I region of the HuOGP gene was isolated. DNA sequence analysis of the HuOGP putative promoter revealed little homology with its hamster and mouse ogp gene counterparts. One transcription initiation site was found 12 nucleotides upstream of the first ATG codon of the HuOGP gene. The HuOGP gene promoter lacked typical CAAT or GC boxes, but contained eight half estrogen-responsive elements (ERE) and an imperfect ERE (iERE; 5'-GGTCANNNTGACT-3') site. Deletion mutants of the 3.3 kb DNA fragment were generated and fused to a promoterless beta-galactosidase (beta-Gal) gene. Transfection studies revealed that in the presence of 100 nmol/l estradiol-17 beta (E(2)), a minimal 0.3 kb promoter construct (pH-298/+25 beta Gal) mediated a high level of beta-Gal expression in immortalized human oviductal epithelial OE-E6/E7 cells, but not in MCF-7 and CHO-K1 cells. By electromobility shift assay and specific estrogen receptor antibodies, we demonstrated that estrogen receptor beta present in the OE-E6/E7 cells binds to the iERE. These findings allow a better understanding of the regulation of OGP gene expression in the human oviduct.