ABSTRACT
BACKGROUND: Treatment of renal cancer has significantly improved with the arrival to the clinic of kinase inhibitors and immunotherapies. However, the disease is still incurable in advanced stages. The fact that several approved inhibitors for kidney cancer target receptor tyrosine kinases (RTKs) suggests that these proteins play a critical role in the pathophysiology of the disease. Based on these precedents, we decided to explore whether RTKs other than those targeted by approved drugs, contribute to the development of kidney cancer. METHODS: The activation status of 49 RTKs in 44 paired samples of normal and tumor kidney tissue was explored using antibody arrays, with validation by western blotting. Genetic and pharmacologic approaches were followed to study the biological implications of targeting the epidermal growth factor receptor (EGFR) and its ligand Transforming Growth Factor-α (TGFα). RESULTS: Activation of the EGFR was found in a substantial number of tumors. Moreover, kidney tumors expressed elevated levels of TGFα. Down-regulation of EGFR or TGFα using RNAi or their pharmacological targeting with blocking antibodies resulted in inhibition of the proliferation of in vitro cellular models of renal cancer. Importantly, differences in the molecular forms of TGFα expressed by tumors and normal tissues were found. In fact, tumor TGFα was membrane anchored, while that expressed by normal kidney tissue was proteolytically processed. CONCLUSIONS: The EGFR-TGFα axis plays a relevant role in the pathophysiology of kidney cancer. This study unveils a distinctive feature in renal cell carcinomas, which is the presence of membrane-anchored TGFα. That characteristic could be exploited therapeutically to act on tumors expressing transmembrane TGFα, for example, with antibody drug conjugates that could recognize the extracellular region of that protein.
Subject(s)
Biomarkers, Tumor , Carcinoma, Renal Cell/metabolism , Protein Precursors/metabolism , Transforming Growth Factor alpha/metabolism , Carcinoma, Renal Cell/drug therapy , Carcinoma, Renal Cell/etiology , Carcinoma, Renal Cell/pathology , Case-Control Studies , Cell Line, Tumor , Clinical Decision-Making , Disease Management , Disease Susceptibility , Humans , Immunohistochemistry , Ligands , Molecular Targeted Therapy , Protein Kinase Inhibitors/pharmacology , Protein Precursors/antagonists & inhibitors , Protein Precursors/genetics , Receptor Protein-Tyrosine Kinases/antagonists & inhibitors , Receptor Protein-Tyrosine Kinases/genetics , Receptor Protein-Tyrosine Kinases/metabolism , Signal Transduction/drug effects , Transforming Growth Factor alpha/antagonists & inhibitors , Transforming Growth Factor alpha/geneticsABSTRACT
Background. Xuanwei City in Yunnan province has been one of the towns with highest lung cancer mortality in China. The high content of amorphous silica in the bituminous coal from Xuanwei of Yunnan is mainly present as irregular and spherical silica nanoparticles (SiNPs). It has been reported that silica nanoparticles in bituminous coal correlated with the high incidence of lung cancer in Xuanwei. To explore the role and mechanism of SiNPs in the tumorigenesis of lung cancer in Xuanwei, human mononuclear cells (THP-1) and human bronchial epithelial cells (BEAS-2B) were cocultured in a transwell chamber. Combined with Benzo[a]pyrene-7, 8-dihydrodiol-9, and 10-epoxide (BPDE), SiNPs could significantly promote the proliferation and Epithelial-Mesenchymal Transition (EMT) and inhibit apoptosis of BEAS-2B cells and induce the release of TGF-α from THP-1 cells. After neutralizing TGF-α with antibody, the proliferation and EMT were decreased and enhanced apoptosis of BEAS-2B cells. Furthermore, the results showed that TGF-α in the sera of patients with lung adenocarcinoma in Xuanwei were significantly higher than in patients with benign pulmonary lesions in Xuanwei and those with lung adenocarcinoma in outside of Xuanwei of Yunnan. Taken together, our study found that SiNPs promoted the proliferation and EMT of BEAS-2B cells by inducing the release of TGF-α from THP-1 cells.
Subject(s)
Epithelial-Mesenchymal Transition/genetics , Lung Neoplasms/drug therapy , Lung/drug effects , Transforming Growth Factor alpha/genetics , Antibodies, Neutralizing/pharmacology , Apoptosis/drug effects , Cell Proliferation/drug effects , Coculture Techniques , Epithelial Cells/drug effects , Epithelial-Mesenchymal Transition/drug effects , Humans , Leukocytes, Mononuclear/drug effects , Lung/pathology , Lung Neoplasms/genetics , Lung Neoplasms/pathology , Nanoparticles/administration & dosage , Nanoparticles/chemistry , Silicon Dioxide/chemistry , Silicon Dioxide/pharmacology , Transforming Growth Factor alpha/antagonists & inhibitorsABSTRACT
INTRODUCTION: Adult T-cell leukemia (ATL) is an aggressive form of malignancy caused by human T- cell lymphotropic virus 1 (HTLV-1). Currently, there is no effective treatment for ATL. Thymoquinone has been reported to have anti-cancer properties. OBJECTIVE: The aim of this study is to investigatthe effects of TQ on proliferation, apoptosis induction and the underlying mechanism of action in both HTLV-1 positive (C91-PL and HuT-102) and HTLV-1 negative (CEM and Jurkat) malignant T-lymphocytes. MATERIALS AND METHODS: Cells were incubated with different thymoquinone concentrations for 24h. Cell cytotoxicity was assayed using the CytoTox 96® Non-Radioactive Cytotoxicity Assay Kit. Cell proliferation was determined using CellTiter 96® Non-Radioactive Cell Proliferation. Cell cycle analysis was performed by staining with propidium iodide. Apoptosis was assessed using cell death ELISA kit. The effect of TQ on p53, p21, Bcl-2 protein expression was determined using Western blot analysis while TGF mRNA expression was determined by RT-PCR. RESULTS: At non-cytotoxic concentrations of TQ, it resulted in the inhibition of proliferation in a dose dependent manner. Flow cytometric analysis revealed a shift in the cell cycle distribution to the PreG1 phase which is a marker of apoptosis. Also TQ increase DNA fragmentation. TQ mediated its anti-proliferative effect and apoptosis induction by an up-regulation of TGFß1, p53 and p21 and a down-regulation of TGF-α and Bcl-2α. CONCLUSION: Thymoquinone presents antiproliferative and proapoptotic effects in ATL cells. For this reason, further research is required to investigate its possible application in the treatment of ATL.
Subject(s)
Apoptosis/drug effects , Benzoquinones/pharmacology , Cyclin-Dependent Kinase Inhibitor p21/metabolism , Leukemia-Lymphoma, Adult T-Cell/drug therapy , Proto-Oncogene Proteins c-bcl-2/antagonists & inhibitors , Transforming Growth Factor alpha/antagonists & inhibitors , Tumor Suppressor Protein p53/metabolism , Benzoquinones/chemistry , Cell Line, Tumor , Cell Proliferation/drug effects , Cyclin-Dependent Kinase Inhibitor p21/genetics , Dose-Response Relationship, Drug , Drug Screening Assays, Antitumor , Humans , Leukemia-Lymphoma, Adult T-Cell/metabolism , Leukemia-Lymphoma, Adult T-Cell/pathology , Molecular Structure , Proto-Oncogene Proteins c-bcl-2/genetics , Proto-Oncogene Proteins c-bcl-2/metabolism , Structure-Activity Relationship , Transforming Growth Factor alpha/genetics , Transforming Growth Factor alpha/metabolism , Tumor Suppressor Protein p53/geneticsABSTRACT
Transforming growth factor-alpha (TGFA) has been shown to play a role in experimental chronic kidney disease associated with nephron reduction, while its role in diabetic kidney disease (DKD) is unknown. We show here that intrarenal TGFA mRNA expression, as well as urine and serum TGFA, are increased in human DKD. We used a TGFA neutralizing antibody to determine the role of TGFA in two models of renal disease, the remnant surgical reduction model and the uninephrectomized (uniNx) db/db DKD model. In addition, the contribution of TGFA to DKD progression was examined using an adeno-associated virus approach to increase circulating TGFA in experimental DKD. In vivo blockade of TGFA attenuated kidney disease progression in both nondiabetic 129S6 nephron reduction and Type 2 diabetic uniNx db/db models, whereas overexpression of TGFA in uniNx db/db model accelerated renal disease. Therapeutic activity of the TGFA antibody was enhanced with renin angiotensin system inhibition with further improvement in renal parameters. These findings suggest a pathologic contribution of TGFA in DKD and support the possibility that therapeutic administration of neutralizing antibodies could provide a novel treatment for the disease.
Subject(s)
Diabetic Nephropathies/metabolism , Kidney/metabolism , Transforming Growth Factor alpha/metabolism , Aged , Angiotensin-Converting Enzyme Inhibitors/pharmacology , Animals , Antibodies, Monoclonal/pharmacology , Antibodies, Neutralizing/pharmacology , Blood Pressure , Cells, Cultured , Dependovirus/genetics , Diabetes Mellitus, Type 2/complications , Diabetic Nephropathies/drug therapy , Diabetic Nephropathies/genetics , Diabetic Nephropathies/physiopathology , Disease Models, Animal , Disease Progression , ErbB Receptors/metabolism , Extracellular Matrix/metabolism , Female , Gene Transfer Techniques , Genetic Vectors , Glomerular Filtration Rate , Humans , Hypertension/complications , Hypertension/physiopathology , Kidney/drug effects , Kidney/physiopathology , Kidney/surgery , Male , Mice, 129 Strain , Mice, Knockout , Middle Aged , Nephrectomy , Phosphorylation , Renin-Angiotensin System , Signal Transduction , Time Factors , Transforming Growth Factor alpha/antagonists & inhibitors , Transforming Growth Factor alpha/deficiency , Transforming Growth Factor alpha/geneticsABSTRACT
Recent studies have implicated a role of the epidermal growth factor receptor (EGFR) pathway in kidney disease. Skin toxicity associated with therapeutics which completely block the EGFR pathway precludes their use in chronic dosing. Therefore, we developed antibodies which specifically neutralize the EGFR ligands TGFα (transforming growth factor-alpha) and epiregulin but not EGF (epidermal growth factor), amphiregulin, betacellulin, HB-EGF (heparin-binding epidermal growth factor), or epigen. The epitope of one such neutralizing antibody, LY3016859, was characterized in detail to elucidate the structural basis for ligand specificity. Here we report a crystal structure of the LY3016859 Fab fragment in complex with soluble human TGFα. Our data demonstrate a conformational epitope located primarily within the C-terminal subdomain of the ligand. In addition, point mutagenesis experiments were used to highlight specific amino acids which are critical for both antigen binding and neutralization, most notably Ala41 , Glu44 , and His45 . These results illustrate the structural basis for the ligand specificity/selectivity of LY3016859 and could also provide insight into further engineering to alter specificity and/or affinity of LY3016859.
Subject(s)
Antibodies, Neutralizing/chemistry , Antibody Specificity , Epiregulin/chemistry , Epitopes/chemistry , Immunoglobulin Fab Fragments/chemistry , Transforming Growth Factor alpha , Animals , Humans , Mice , Transforming Growth Factor alpha/antagonists & inhibitors , Transforming Growth Factor alpha/chemistryABSTRACT
OBJECTIVE: Transforming growth factor α (TGFα) is increased in osteoarthritic (OA) cartilage in rats and humans and modifies chondrocyte phenotype. CCL2 is increased in OA cartilage and stimulates proteoglycan loss. This study was undertaken to test whether TGFα and CCL2 cooperate to promote cartilage degradation and whether inhibiting either reduces disease progression in a rat model of posttraumatic OA. METHODS: Microarray analysis was used to profile expression of messenger RNA (mRNA) for Tgfa, Ccl2, and related genes in a rat model of posttraumatic OA. Rat primary chondrocytes and articular cartilage explants were treated with TGFα in the presence or absence of MEK-1/2, p38, phosphatidylinositol 3-kinase, Rho-associated protein kinase, or CCR2 inhibitors and immunostained for markers of cartilage degradation. The rat model was used to administer pharmacologic inhibitors of TGFα (AG1478) and CCL2 (RS504393) signaling for up to 10 weeks and assess histopathology and serum biomarkers of cartilage synthesis (C-propeptide of type II collagen [CPII]) and breakdown (C2C). RESULTS: Tgfa and Ccl2 mRNA were simultaneously up-regulated in articular cartilage in the rat model of posttraumatic OA. TGFα induced expression of CCL2, Mmp3, and Tnf in primary chondrocytes. Cleavage of type II collagen and aggrecan (by matrix metalloproteinases and ADAMTS-4/5, respectively) induced by TGFα was blocked by pharmacologic inhibition of CCL2 in cartilage explants. In vivo pharmacologic inhibition of TGFα or CCL2 signaling reduced Osteoarthritis Research Society International cartilage histopathology scores and increased serum CPII levels, but only TGFα inhibition reduced C2C levels intreated versus untreated rat OA cartilage. CONCLUSION: TGFα signaling stimulates cartilage degradation via a CCL2-dependent mechanism, but pharmacologic inhibition of the TGFα-CCL2 axis reduces experimental posttraumatic OA progression in vivo.
Subject(s)
Chemokine CCL2/antagonists & inhibitors , Disease Progression , Osteoarthritis/prevention & control , Osteoarthritis/physiopathology , Signal Transduction/physiology , Wounds and Injuries/complications , Animals , Benzoxazines/pharmacology , Cartilage, Articular/pathology , Cartilage, Articular/physiopathology , Chemokine CCL2/drug effects , Chemokine CCL2/physiology , Disease Models, Animal , Male , Osteoarthritis/etiology , Quinazolines/pharmacology , Rats , Rats, Sprague-Dawley , Signal Transduction/drug effects , Spiro Compounds/pharmacology , Transforming Growth Factor alpha/antagonists & inhibitors , Transforming Growth Factor alpha/drug effects , Transforming Growth Factor alpha/physiology , Tyrphostins/pharmacology , Up-Regulation/physiologyABSTRACT
We studied the effects of interleukin (IL)-1ß on DNA synthesis and cell proliferation in primary cultures of adult rat hepatocytes in order to elucidate the mechanisms of its action. Hepatocyte parenchymal cells maintained in a serum-free, defined medium synthesized DNA and proliferated in the presence of IL-1ß (3-30 ng/ml), but not IL-1α (0.1-30 ng/ml) in a time- and dose-dependent manner. Specific inhibitors of growth-related signal transducers, such as AG1478, LY294002, PD98059, and rapamycin, completely abolished IL-1ß-stimulated hepatocyte DNA synthesis and proliferation. Western blot analysis showed that IL-1ß significantly stimulated mitogen-activated protein (MAP) kinase activation within 10 min. Addition of a monoclonal antibody against transforming growth factor (TGF)-α, but not a monoclonal antibody against insulin-like growth factor-I, to the culture dose-dependently inhibited IL-1ß-induced hepatocyte mitogenesis. Culture medium TGF-α levels increased significantly within 3 min in response to IL-1ß from baseline levels. Peak TGF-α levels (33 pg/ml) were reached at 10 min after IL-1ß stimulation. These results indicate that the proliferative mechanism of action of IL-1ß is mediated through an increase in autocrine secretion of TGF-α from primary cultured hepatocytes. Secreted TGF-α, in turn, acts as a complete mitogen to induce hepatocyte mitogenesis through the receptor tyrosine kinase/phosphatidylinositol 3-kinase/MAP kinase/mammalian target of rapamycin pathway.
Subject(s)
Hepatocytes/drug effects , Hepatocytes/metabolism , Interleukin-1beta/pharmacology , Transforming Growth Factor alpha/metabolism , Animals , Cell Cycle/drug effects , Cell Proliferation/drug effects , Cell Proliferation/physiology , Cells, Cultured , Chromones/pharmacology , DNA/biosynthesis , Dose-Response Relationship, Drug , Flavonoids/pharmacology , Hepatocytes/cytology , Interleukin 1 Receptor Antagonist Protein/metabolism , Interleukin 1 Receptor Antagonist Protein/pharmacology , Interleukin-1alpha/administration & dosage , Interleukin-1alpha/metabolism , Interleukin-1alpha/pharmacology , Interleukin-1beta/administration & dosage , Interleukin-1beta/metabolism , MAP Kinase Signaling System/drug effects , Male , Morpholines/pharmacology , Quinazolines/pharmacology , Rats , Rats, Wistar , Signal Transduction/drug effects , Sirolimus/pharmacology , Transforming Growth Factor alpha/antagonists & inhibitors , Tyrphostins/pharmacologyABSTRACT
Osteosarcoma is the most common primary malignancy of bone and is characterized by a high malignant and metastatic potential. Transforming growth factor alpha (TGF-α) is classified as the EGF (epidermal growth factor)-like family, which is involved in cancer cellular activities such as proliferation, motility, migration, adhesion and invasion abilities. However, the effect of TGF-α on human osteosarcoma is largely unknown. We found that TGF-α increased the cell migration and expression of intercellular adhesion molecule-1 (ICAM-1) in human osteosarcoma cells. Transfection of cells with ICAM-1 siRNA reduced TGF-α-mediated cell migration. We also found that the phosphatidylinositol 3'-kinase (PI3K)/Akt/NF-κB pathway was activated after TGF-α treatment, and TGF-α-induced expression of ICAM-1 and cell migration was inhibited by the specific inhibitors and siRNAs of PI3K, Akt, and NF-κB cascades. In addition, knockdown of TGF-α expression markedly decreased cell metastasis in vitro and in vivo. Our results indicate that TGF-α/EGFR interaction elicits PI3K and Akt activation, which in turn activates NF-κB, resulting in the expression of ICAM-1 and contributing the migration of human osteosarcoma cells.
Subject(s)
Bone Neoplasms/metabolism , ErbB Receptors/agonists , Intercellular Adhesion Molecule-1/metabolism , Osteosarcoma/secondary , Phosphatidylinositol 3-Kinase/metabolism , Second Messenger Systems , Transforming Growth Factor alpha/metabolism , Animals , Antineoplastic Agents/pharmacology , Antineoplastic Agents/therapeutic use , Bone Neoplasms/drug therapy , Bone Neoplasms/pathology , Cell Line, Tumor , Cell Movement/drug effects , ErbB Receptors/antagonists & inhibitors , ErbB Receptors/metabolism , Humans , Intercellular Adhesion Molecule-1/chemistry , Intercellular Adhesion Molecule-1/genetics , Lung Neoplasms/metabolism , Lung Neoplasms/pathology , Lung Neoplasms/prevention & control , Lung Neoplasms/secondary , Male , Mice , Mice, SCID , NF-kappa B/antagonists & inhibitors , NF-kappa B/genetics , NF-kappa B/metabolism , Osteosarcoma/drug therapy , Osteosarcoma/metabolism , Osteosarcoma/pathology , Phosphatidylinositol 3-Kinase/chemistry , Phosphatidylinositol 3-Kinase/genetics , Phosphoinositide-3 Kinase Inhibitors , Proto-Oncogene Proteins c-akt/agonists , Proto-Oncogene Proteins c-akt/antagonists & inhibitors , Proto-Oncogene Proteins c-akt/genetics , Proto-Oncogene Proteins c-akt/metabolism , RNA Interference , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Second Messenger Systems/drug effects , Transforming Growth Factor alpha/antagonists & inhibitors , Transforming Growth Factor alpha/genetics , Tumor Burden/drug effects , Up-Regulation/drug effects , Xenograft Model Antitumor AssaysABSTRACT
Sorafenib, the first-line systemic drug for advanced hepatocellular carcinoma (HCC), has demonstrated limited benefits with very low response rates. Thus it is essential to investigate the underlying mechanisms for the resistance to sorafenib and seek potential strategy to enhance its efficacy. Hypoxic cells inside solid tumors are extremely resistant to therapies as their survival ability is increased due to the cellular adaptive response to hypoxia, which is controlled by hypoxia-inducible factor (HIF)-1 and HIF-2. Sorafenib inhibits HIF-1α synthesis, making the hypoxic response switch from HIF-1α- to HIF-2α-dependent pathways and providing a mechanism for more aggressive growth of tumors. The present study has demonstrated that upregulation of HIF-2α induced by sorafenib contributes to the resistance of hypoxic HCC cells by activating the transforming growth factor (TGF)-α/epidermal growth factor receptor (EGFR) pathway. Blocking the TGF-α/EGFR pathway by gefitinib, a specific EGFR inhibitor, reduced the activation of STAT (signal transducer and activator of transcription) 3, AKT and ERK (extracellular signal-regulated kinase), and synergized with sorafenib to inhibit proliferation and induce apoptosis of hypoxic HCC cells. Transfection of HIF-2α siRNA into HCC cells downregulated the expression of VEGF (vascular endothelial growth factor), cyclin D1, HIF-2α and TGF-α, and inhibited the activation of EGFR. HIF-2α siRNA inhibited the proliferation and promoted the apoptosis of HCC cells in vitro, and synergized with sorafenib to suppress the growth of HCC tumors in vivo. The results indicate that targeting HIF-2α-mediated activation of the TGF-α/EGFR pathway warrants further investigation as a potential strategy to enhance the efficacy of sorafenib for treating HCC.
Subject(s)
Antineoplastic Agents/pharmacology , Basic Helix-Loop-Helix Transcription Factors/metabolism , ErbB Receptors/metabolism , Niacinamide/analogs & derivatives , Phenylurea Compounds/pharmacology , Transforming Growth Factor alpha/metabolism , Up-Regulation/drug effects , Animals , Basic Helix-Loop-Helix Transcription Factors/antagonists & inhibitors , Basic Helix-Loop-Helix Transcription Factors/genetics , Carcinoma, Hepatocellular/metabolism , Carcinoma, Hepatocellular/pathology , Cell Line, Tumor , Drug Resistance, Neoplasm/genetics , ErbB Receptors/antagonists & inhibitors , ErbB Receptors/genetics , Extracellular Signal-Regulated MAP Kinases/metabolism , Gefitinib , Gene Expression Regulation, Neoplastic , Humans , Liver Neoplasms/metabolism , Liver Neoplasms/pathology , Male , Mice , Mice, Inbred BALB C , Mice, Nude , Niacinamide/pharmacology , Quinazolines/pharmacology , Signal Transduction/drug effects , Sorafenib , Transforming Growth Factor alpha/antagonists & inhibitors , Transforming Growth Factor alpha/geneticsABSTRACT
Dietary isoprenic derivatives such as ß-ionone (ßI) are a promising class of chemopreventive agents. In this study, cellular aspects of ßI protective activities during early hepatocarcinogenesis were evaluated. Male Wistar rats were submitted to "resistant hepatocyte" model and then received daily 16 mg/100 g body weight (b.w.) of ßI (ßI group) or only 0.25 mL/100 g b.w. of corn oil (vehicle, control group [CO]) during 4 wk, specifically during early promotion phase. Compared to controls, ßI inhibited (P < 0.05) the development of persistent preneoplastic lesions (pPNL), considered to be potential hepatocellular carcinoma (HCC) progression sites, and increased remodeling PNL (rPNL) (P < 0.05) that tend to regress to a normal phenotype. Increased ßI hepatic levels (P < 0.05), in the ßI group, were associated with its chemopreventive actions. Compared to control rats, ßI reduced the frequency of both pPNL and rPNL positive for tumor growth factor (TGF)-α (P < 0.05), reduced the frequency of pPNL stained for p65 (nuclear factor-kappaB; NF-κB) (P < 0.05), and reduced the frequency of pPNL positive for cytoplasmic p53 (P < 0.05). Our data demonstrated that ßI targets TGF-α, NF-κB, and p53 in initial phases of hepatocarcinogenesis and specifically inhibits PNL with increased probability to progress to HCC. This isoprenoid may represent a chemopreventive agent of choice for HCC control.
Subject(s)
Anticarcinogenic Agents/pharmacology , Carcinoma, Hepatocellular/drug therapy , Liver Neoplasms, Experimental/drug therapy , Norisoprenoids/pharmacology , Animals , Chemoprevention , Liver/drug effects , Liver/metabolism , Male , NF-kappa B/antagonists & inhibitors , NF-kappa B/metabolism , Rats , Rats, Wistar , Transforming Growth Factor alpha/antagonists & inhibitors , Transforming Growth Factor alpha/metabolism , Tumor Suppressor Protein p53/antagonists & inhibitors , Tumor Suppressor Protein p53/metabolismABSTRACT
Recent advances in the field of cancer therapeutics come from the development of drugs that specifically recognize validated oncogenic or pro-metastatic targets. The latter may be mutated proteins with altered function, such as kinases that become constitutively active, or critical components of growth factor signaling pathways, whose deregulation leads to aberrant malignant cell proliferation and dissemination to metastatic sites. We herein focus on the description of the overlapping activities of two important developmental pathways often exacerbated in cancer, namely Transforming Growth Factor-ß (TGF-ß) and Hedgehog (HH) signaling, with a special emphasis on the unifying oncogenic role played by GLI1/2 transcription factors. The latter are the main effectors of the canonical HH pathway, yet are direct target genes of TGF-ß/SMAD signal transduction. While tumor-suppressor in healthy and pre-malignant tissues, TGF-ß is often expressed at high levels in tumors and contributes to tumor growth, escape from immune surveillance, invasion and metastasis. HH signaling regulates cell proliferation, differentiation and apoptosis, and aberrant HH signaling is found in a variety of cancers. We discuss the current knowledge on HH and TGF-ß implication in cancer including cancer stem cell biology, as well as the current state, both successes and failures, of targeted therapeutics aimed at blocking either of these pathways in the pre-clinical and clinical settings.
Subject(s)
Antineoplastic Agents/therapeutic use , Hedgehog Proteins/antagonists & inhibitors , Neoplasms/drug therapy , Signal Transduction/drug effects , Transforming Growth Factor alpha/antagonists & inhibitors , Animals , Antibodies, Monoclonal/administration & dosage , Antibodies, Monoclonal/pharmacology , Antibodies, Monoclonal/therapeutic use , Antineoplastic Agents/administration & dosage , Antineoplastic Agents/pharmacology , Clinical Trials as Topic , Hedgehog Proteins/genetics , Humans , Neoplasms/metabolism , Oligonucleotides, Antisense/administration & dosage , Oligonucleotides, Antisense/pharmacology , Oligonucleotides, Antisense/therapeutic use , Small Molecule Libraries/administration & dosage , Small Molecule Libraries/pharmacology , Small Molecule Libraries/therapeutic use , Transforming Growth Factor alpha/geneticsABSTRACT
Renal fibrosis is a major hallmark of chronic kidney disease, regardless of the initial causes, and prominent renal fibrosis predicts poor prognosis for renal insufficiency. Transforming growth factor (TGF)-ß plays a pivotal role in the progression of renal fibrosis, and therapeutic interventions targeting TGF-ß have been successful and well tolerated in animal models. However, these interventions might have adverse effects by inducing systemic inflammation due to the strong bifunctional role of TGF-ß (pro-fibrotic and anti-inflammatory). This review of the current literature focuses on the inhibitors/antagonists of TGF-ß, and discusses possible therapeutic approaches targeting them, describing the effectiveness of orally active bone morphogenetic protein 7 mimetics in reversing established fibrosis. It will conclude with a brief discussion of possible future directions for research.
Subject(s)
Kidney/pathology , Renal Insufficiency, Chronic/drug therapy , Transforming Growth Factor alpha/antagonists & inhibitors , Animals , Bone Morphogenetic Protein 7/agonists , Bone Morphogenetic Protein 7/antagonists & inhibitors , Bone Morphogenetic Protein 7/physiology , Fibrosis , Humans , Kidney/drug effects , Kidney/physiopathology , Renal Insufficiency, Chronic/physiopathology , Signal Transduction , Transforming Growth Factor alpha/physiologyABSTRACT
AIM: To investigate the relation between neutrophil elastase (NE) and proliferation of breast cancer cells and whether the NE inhibitor sivelestat could both contribute and be applied to therapy for anti-epithelial growth factor receptor 2 (HER2)-positive breast cancers. MATERIALS AND METHODS: The proliferation or inhibition of breast cancer cell line SKBR-3 by each agent was evaluated by methylthiazole tetrazolium (MTT) assay. Signal transduction and expression of signaling molecules were evaluated by Western blot analysis. RESULTS: The auto tumor progression mechanism initiated by NE through tumor growth factor-α (TGF-α) was present in breast cancer cells, and this mechanism was intensively suppressed by sivelestat. The effect of trastuzumab was suppressed, and trastuzumab-induced HER2 down-regulation was impaired by TGF-α. TGF-α not only promoted cell proliferation as a ligand but also enhanced resistance to trastuzumab by impairing HER2 down-regulation. Furthermore, combined use of trastuzumab and sivelestat suppressed cell proliferation more intensively than either drug alone and did not provoke impairment by TGF-α of HER2-induced down-regulation. CONCLUSION: Combinatorial use of sivelestat and trastuzumab might be a novel therapeutic strategy for HER2-positive breast cancer.
Subject(s)
Breast Neoplasms/drug therapy , Cell Proliferation/drug effects , Glycine/analogs & derivatives , Leukocyte Elastase/antagonists & inhibitors , Receptor, ErbB-2/antagonists & inhibitors , Serine Proteinase Inhibitors/pharmacology , Sulfonamides/pharmacology , Antibodies, Monoclonal, Humanized/pharmacology , Antineoplastic Agents/pharmacology , Antineoplastic Combined Chemotherapy Protocols , Blotting, Western , Breast Neoplasms/metabolism , Breast Neoplasms/pathology , Cell Line, Tumor , Drug Resistance, Neoplasm/drug effects , Drug Synergism , Female , Gene Expression Regulation, Neoplastic , Glycine/pharmacology , Humans , Leukocyte Elastase/metabolism , Receptor, ErbB-2/metabolism , Signal Transduction , Transforming Growth Factor alpha/antagonists & inhibitors , Transforming Growth Factor alpha/genetics , Transforming Growth Factor alpha/metabolism , TrastuzumabABSTRACT
The peroxisome proliferator-activated receptors (PPARs), PPARα, PPARδ, and PPARγ, are nuclear ligand-activated transcription factors that are best known as regulators of glucose and lipid metabolism. PPARα agonists have been shown to have profound anti-inflammatory and antiproliferative effects on human keratinocytes, and are important for maintaining homeostasis of the skin. To better characterize the mechanisms underlying these phenomena, we analysed the effects of PPARα agonists on transforming growth factor (TGF)-α-induced expression of matrix metalloproteinase (MMP)-9. MMP-9 expression in keratinocytes has previously been linked to inflammatory skin diseases, regenerative skin mechanisms, and tumour development and metastasis. We found that PPARα agonists effectively inhibited TGF-α-induced MMP-9 expression in human keratinocytes via a post-transcriptional mechanism, revealing a novel and important aspect of the anti-inflammatory and anticarcinogenic action of these compounds.
Subject(s)
Enzyme Inhibitors/pharmacology , Keratinocytes/enzymology , Matrix Metalloproteinase 9/metabolism , PPAR alpha/agonists , Transforming Growth Factor alpha/antagonists & inhibitors , Enzyme Activation/drug effects , Enzyme-Linked Immunosorbent Assay , Humans , Keratinocytes/drug effects , Polymerase Chain ReactionSubject(s)
Anti-Inflammatory Agents/therapeutic use , Antibodies, Monoclonal/therapeutic use , Crohn Disease/drug therapy , Tissue Inhibitor of Metalloproteinase-1/metabolism , Apoptosis/drug effects , Crohn Disease/metabolism , Humans , Infliximab , Transforming Growth Factor alpha/antagonists & inhibitors , Transforming Growth Factor alpha/metabolismABSTRACT
Insulin-like growth factor 1 (IGF1) is involved in the proliferation of mouse and rat endometrial cells in a paracrine or autocrine manner. Insulin-like growth factor binding protein-3 (IGFBP3) modulates actions of IGFs directly or indirectly. The present study aimed to determine whether IGFBP3 is involved in the regulation of proliferation of mouse endometrial cells. Mouse endometrial epithelial cells and stromal cells were isolated, and cultured in a serum free medium. IGF1 stimulated DNA synthesis by endometrial epithelial and stromal cells, and IGFBP3 inhibited IGF1-induced DNA synthesis. Estradiol-17beta (E2) decreased the Igfbp3 mRNA level in endometrial stromal cells, whereas it increased the Igf1 mRNA level. Transforming growth factor alpha (TGFalpha) significantly decreased IGFBP3 expression at both the mRNA and secreted protein levels in endometrial stromal cells. Progesterone (P4) did not affect the E2-induced down-regulation of Igfbp3 mRNA expression in endometrial stromal cells, although P4 alone increased Igfbp3 mRNA levels. The present findings suggest that in mouse endometrial stromal cells E2 enhances IGF1 action through enhancement of IGF1 synthesis and reduction of IGFBP3 synthesis, and that TGFalpha affects IGF1 actions through modulation of IGFBP3 levels.
Subject(s)
Endometrium/metabolism , Estradiol/pharmacology , Insulin-Like Growth Factor Binding Protein 3/biosynthesis , Progesterone/pharmacology , Transforming Growth Factor alpha/pharmacology , Animals , Blotting, Western , DNA/biosynthesis , Endometrium/cytology , Endometrium/drug effects , Enzyme Inhibitors/pharmacology , Epithelial Cells/drug effects , Epithelial Cells/metabolism , Female , Insulin-Like Growth Factor Binding Protein 3/antagonists & inhibitors , Insulin-Like Growth Factor Binding Protein 3/genetics , Insulin-Like Growth Factor I/biosynthesis , Insulin-Like Growth Factor I/genetics , Mice , Mice, Inbred ICR , Quinazolines , RNA, Messenger/biosynthesis , RNA, Messenger/genetics , Receptor, IGF Type 1/biosynthesis , Receptor, IGF Type 1/genetics , Reverse Transcriptase Polymerase Chain Reaction , Stromal Cells/drug effects , Stromal Cells/metabolism , Transforming Growth Factor alpha/antagonists & inhibitors , Tyrphostins/pharmacologyABSTRACT
Previous studies have demonstrated that monospecific antisense oligonucleotides (oligos) directed against mRNA encoding proteins associated with tumor growth, death, and survival are efficacious against breast and prostate tumors. Targeted proteins, associated with different signal transduction pathways, have included transforming growth factor-alpha [TGF-alpha (MR(1))], its binding site the epidermal growth factor receptor [EGFR (MR(2))] sharing sequence homology to the breast cancer prognostic marker Her-2/neu, an apoptosis inhibiting protein [bcl-2 (MR(4))], and the androgen receptor [AR (MR(5))]. In attempts to enhance antisense therapy, recent reports describe how two of the binding sites mentioned above can be sequentially placed within a single complementary (bispecific) strand and administered either in the presence or absence of additional therapeutic agents. When tested against breast and prostate tumor cell lines specific differences were noted: MCF-7 breast cancer cells were more receptive to the inhibitory effects of monospecific oligos, whereas PC-3 and LNCaP prostate cells were particularly responsive to bispecifics. In an effort to identify agents which enhance the activity of oligos and which possess less toxicity than traditionally employed chemotherapeutics, Rapamycin, an immunosuppressive agent known to regulate tumor growth and signal transduction mediated by the mTOR receptor, is compared to paclitaxel in combination therapy employing monospecific or bispecific oligos. Bispecifics were constructed recognizing the binding sites for TGF-alpha and EGFR mRNA [TGF-alpha/EGFR (MR(12)) and EGFR/TGF-alpha (MR(21))]; another pair recognized binding sites for EGFR and bcl-2 [EGFR/bcl-2 (MR(24)) and bcl-2/EGFR (MR(42))]; while a third pair employed only against the LNCaP prostate cell line recognized bcl-2 and the androgen receptor [bcl-2/AR (MR4(45)) and AR/bcl-2 (MR(54))]. Oligo pairs differ in their 5'-3' linear binding site orientations, and were tested in vitro against MCF-7 breast and PC-3 and LNCaP prostate tumor cell lines. Following cell attachment, incubations were done for 2 days with the agents followed by 2 days in their absence. Five experiments evaluated the effect of monospecific or bispecific antisense oligos in combination with an LD(50) dosage of either Rapamycin or paclitaxel and led to the conclusion that although these agents act via different mechanisms, they are comparable in effectiveness.
Subject(s)
Antibiotics, Antineoplastic/therapeutic use , Antineoplastic Agents, Phytogenic/therapeutic use , Breast Neoplasms/therapy , Oligonucleotides, Antisense , Paclitaxel/therapeutic use , Prostatic Neoplasms/therapy , Sirolimus/therapeutic use , Androgen Receptor Antagonists , Apoptosis Regulatory Proteins/antagonists & inhibitors , Apoptosis Regulatory Proteins/genetics , Breast Neoplasms/drug therapy , Breast Neoplasms/metabolism , Cell Line, Tumor , Combined Modality Therapy , ErbB Receptors/antagonists & inhibitors , ErbB Receptors/genetics , Female , Gene Targeting/methods , Humans , Male , Prostatic Neoplasms/drug therapy , Prostatic Neoplasms/metabolism , Receptors, Androgen/genetics , Signal Transduction/drug effects , Transforming Growth Factor alpha/antagonists & inhibitors , Transforming Growth Factor alpha/geneticsABSTRACT
Up to now clinical experiences focusing EGF receptor, an attractive target for cancer therapy, have been limited to passive therapies, suggesting that therapeutic cancer vaccines inducing anti-epidermal growth factor receptor (EGFR) antibodies could also work. Here, the humoral immune response induced in mice with a vaccine formulation containing the human EGFR-extracellular domain and very small-sized proteoliposomes (VSSP), a novel nanoparticulated adjuvant was assessed. In vaccinated mice sera average of the specific polyclonal antibodies (PAb) titers was 10(-5). Anti-EGFR PAb were able to bind EGFR+ tumor cell lines, expressing different levels of the molecule. Noteworthy, the presence of Cetuximab only partially inhibited the vaccine-induced antibodies binding to H125 cells. Anti-EGFR PAb abrogated ligands-dependent EGFR phosphorylation, provoking tumor cells apoptosis. The described EGFR-based vaccine might be a superior therapeutic approach for patients with EGFR+ tumors.
Subject(s)
Antibodies/immunology , Apoptosis/immunology , Cancer Vaccines/immunology , ErbB Receptors/immunology , Animals , Antibody Formation/drug effects , Caco-2 Cells , Cell Line , Cell Line, Tumor , Cell Proliferation , Epidermal Growth Factor/antagonists & inhibitors , Epidermal Growth Factor/metabolism , ErbB Receptors/metabolism , Female , Humans , Mice , Mice, Inbred BALB C , Mice, Inbred C57BL , Protein Binding/immunology , Proteolipids , Transforming Growth Factor alpha/antagonists & inhibitors , Transforming Growth Factor alpha/metabolismABSTRACT
We determined whether phosphorylated epidermal growth factor receptor (EGFR) expressed on tumor-associated endothelial cells is a primary target for therapy with EGFR tyrosine kinase inhibitors (TKIs). Human colon cancer cells SW620CE2 (parental) that do not express EGFR or human epidermal growth factor receptor 2 (HER2) but express transforming growth factor alpha (TGF-alpha) were transduced with a lentivirus carrying nontargeting small hairpin RNA (shRNA) or TGF-alpha shRNA. The cell lines were implanted into the cecum of nude mice. Two weeks later, treatment began with saline, 4-[R]-phenethylamino-6-[hydroxyl] phenyl-7H-pyrrolo [2,3-D]-pyrimidine (PKI166), or irinotecan. Endothelial cells in parental and nontargeting shRNA tumors expressed phosphorylated EGFR. Therapy with PKI166 alone or with irinotecan produced apoptosis of these endothelial cells and necrosis of the EGFR-negative tumors. Endothelial cells in tumors that did not express TGF-alpha did not express EGFR, and these tumors were resistant to treatment with PKI166. The response of neoplasms to EGFR antagonists has been correlated with EGFR mutations, HER2 expression, Akt activation, and EGFR gene copy number. Our present data using colon cancer cells that do not express EGFR or HER2 suggest that the expression of TGF-alpha by tumor cells leading to the activation of EGFR in tumor-associated endothelial cells is a major determinant for the susceptibility of neoplasms to therapy by specific EGFR-TKI.
Subject(s)
Colonic Neoplasms/drug therapy , ErbB Receptors/metabolism , Protein Kinase Inhibitors/pharmacology , Pyrimidines/pharmacology , Pyrroles/pharmacology , Animals , Apoptosis/drug effects , Blotting, Western , Camptothecin/analogs & derivatives , Camptothecin/pharmacology , Cecum/metabolism , Cecum/pathology , Cell Proliferation , Colonic Neoplasms/enzymology , Colonic Neoplasms/pathology , Drug Therapy, Combination , Endothelial Cells/drug effects , Endothelial Cells/metabolism , Enzyme Inhibitors/pharmacology , ErbB Receptors/antagonists & inhibitors , ErbB Receptors/genetics , Humans , Immunoenzyme Techniques , In Situ Nick-End Labeling , Irinotecan , Lymphatic Metastasis , Male , Mice , Mice, Nude , Necrosis , Phosphorylation/drug effects , Proto-Oncogene Proteins c-akt/genetics , Proto-Oncogene Proteins c-akt/metabolism , RNA, Small Interfering/pharmacology , Receptor, ErbB-2/genetics , Receptor, ErbB-2/metabolism , Topoisomerase I Inhibitors , Transforming Growth Factor alpha/antagonists & inhibitors , Transforming Growth Factor alpha/genetics , Transforming Growth Factor alpha/metabolismABSTRACT
Numerous epidemiological studies have shown an inverse correlation between helminth infections and the manifestation of atopic diseases, yet the immunological mechanisms governing this phenomenon are indistinct. We therefore investigated the effects of infection with the filarial parasite Litomosoides sigmodontis on allergen-induced immune reactions and airway disease in a murine model of asthma. Infection with L. sigmodontis suppressed all aspects of the asthmatic phenotype: Ag-specific Ig production, airway reactivity to inhaled methacholine, and pulmonary eosinophilia. Similarly, Ag-specific recall proliferation and overall Th2 cytokine (IL-4, IL-5, and IL-3) production were significantly reduced after L. sigmodontis infection. Analysis of splenic mononuclear cells and mediastinal lymph nodes revealed a significant increase in the numbers of T cells with a regulatory phenotype in infected and sensitized mice compared with sensitized controls. Additionally, surface and intracellular staining for TGF-beta on splenic CD4(+) T cells as well as Ag-specific TGF-beta secretion by splenic mononuclear cells was increased in infected and sensitized animals. Administration of Abs blocking TGF-beta or depleting regulatory T cells in infected animals before allergen sensitization and challenges reversed the suppressive effect with regard to airway hyperreactivity, but did not affect airway inflammation. Despite the dissociate results of the blocking experiments, these data point toward an induction of regulatory T cells and enhanced secretion of the immunomodulatory cytokine TGF-beta as one principle mechanism. In conclusion, our data support the epidemiological evidence and enhance the immunological understanding concerning the impact of helminth infections on atopic diseases thus providing new insights for the development of future studies.