A recombinant decoy comprising EGFR and ErbB-4 inhibits tumor growth and metastasis.

Epidermal growth factor (EGF)-like growth factors control tumor progression as well as evasion from the toxic effects of chemotherapy. Accordingly, antibodies targeting the cognate receptors, such as EGFR/ErbB-1 and the co-receptor HER2/ErbB-2, are widely used to treat cancer patients, but agents that target the EGF-like growth factors are not available. To circumvent the existence of 11 distinct ErbB ligands, we constructed a soluble fusion protein (hereinafter: TRAP-Fc) comprising truncated extracellular domains of EGFR/ErbB-1 and ErbB-4. The recombinant TRAP-Fc retained high-affinity ligand binding to EGF-like growth factors and partially inhibited growth of a variety of cultured tumor cells. Consistently, TRAP-Fc displayed an inhibitory effect in xenograft models of human cancer, as well as synergy with chemotherapy. Additionally, TRAP-Fc inhibited invasive growth of mammary tumor cells and reduced their metastatic seeding in the lungs of animals. Taken together, the activities displayed by TRAP-Fc reinforce critical roles of EGF-like growth factors in tumor progression, and they warrant further tests of TRAP-Fc in preclinical models.

Modeling ductal carcinoma in situ: a HER2-Notch3 collaboration enables luminal filling.

A large fraction of ductal carcinoma in situ (DCIS), a non-invasive precursor lesion of invasive breast cancer, overexpresses the HER2/neu oncogene. The ducts of DCIS are abnormally filled with cells that evade apoptosis, but the underlying mechanisms remain incompletely understood. We overexpressed HER2 in mammary epithelial cells and observed growth factor-independent proliferation. When grown in extracellular matrix as three-dimensional spheroids, control cells developed a hollow lumen, but HER2-overexpressing cells populated the lumen by evading apoptosis. We demonstrate that HER2 overexpression in this cellular model of DCIS drives transcriptional upregulation of multiple components of the Notch survival pathway. Importantly, luminal filling required upregulation of a signaling pathway comprising Notch3, its cleaved intracellular domain and the transcriptional regulator HES1, resulting in elevated levels of c-MYC and cyclin D1. In line with HER2-Notch3 collaboration, drugs intercepting either arm reverted the DCIS-like phenotype. In addition, we report upregulation of Notch3 in hyperplastic lesions of HER2 transgenic animals, as well as an association between HER2 levels and expression levels of components of the Notch pathway in tumor specimens of breast cancer patients. Therefore, it is conceivable that the integration of the Notch and HER2 signaling pathways contributes to the pathophysiology of DCIS.

Modeling invasive breast cancer: growth factors propel progression of HER2-positive premalignant lesions.

The HER2/neu oncogene encodes a receptor-like tyrosine kinase whose overexpression in breast cancer predicts poor prognosis and resistance to conventional therapies. However, the mechanisms underlying aggressiveness of HER2 (human epidermal growth factor receptor 2)-overexpressing tumors remain incompletely understood. Because it assists epidermal growth factor (EGF) and neuregulin receptors, we overexpressed HER2 in MCF10A mammary cells and applied growth factors. HER2-overexpressing cells grown in extracellular matrix formed filled spheroids, which protruded outgrowths upon growth factor stimulation. Our transcriptome analyses imply a two-hit model for invasive growth: HER2-induced proliferation and evasion from anoikis generate filled structures, which are morphologically and transcriptionally analogous to preinvasive patients' lesions. In the second hit, EGF escalates signaling and transcriptional responses leading to invasive growth. Consistent with clinical relevance, a gene expression signature based on the HER2/EGF-activated transcriptional program can predict poorer prognosis of a subgroup of HER2-overexpressing patients. In conclusion, the integration of a three-dimensional cellular model and clinical data attributes progression of HER2-overexpressing lesions to EGF-like growth factors acting in the context of the tumor's microenvironment.

Combining epitope-distinct antibodies to HER2: cooperative inhibitory effects on invasive growth.

Monoclonal antibodies (mAbs) to HER2 are currently used to treat breast cancer, but low clinical efficacy, along with primary and acquired resistance to therapy, commonly limit clinical applications. We previously reported that combinations of antibodies directed at non-overlapping epitopes of HER2 are endowed with enhanced antitumor effects, probably due to accelerated receptor degradation. Here, we extend these observations to three-dimensional mammary cell models, and compare the effects of single mAbs with the effects of antibody combinations. Collectively, our in vitro assays and computational image analyses indicate that combining mAbs against different epitopes of HER2 better inhibits invasive growth. Importantly, while growth factors are able to reduce intraluminal apoptosis and induce an invasive phenotype, combinations of mAbs better than single mAbs can reverse the growth factor-induced phenotypes of HER2-overexpressing spheroids. In conclusion, our studies propose that mAb combinations negate the biological effects of growth factors on invasive growth of HER2-overexpressing cells. Hence, combining mAbs offers a therapeutic strategy, potentially able to enhance clinical efficacy of existing antireceptor immunotherapeutics.

13 Cis-retinoic acid mediates apoptosis in Dalton's lymphoma ascites cells by regulating gene expression.

Apoptosis is a form of regulated cell death that plays an important role in the maintenance of tissue homeostasis. Treatment of Dalton's lymphoma ascites cells (DLA)-induced experimental solid tumor tumor cells with 13 cis-retinoic acid, at concentrations of 25 microg/mL and 50 microg/mL, produced apoptotic morphologic changes, such as nuclear condensation and DNA-ladder formation after 48h incubation at 37 degrees C. in addition, the compound upregulated caspase-3 expression and downregulated bcl-2 gene expression. In vivo treatment with 13 cis-retinoic acid (200 micromoles/kg body weight, ip) inhibited solid tumor development induced with DLA cells in Swiss albino mice. In conclusion, the results suggest that the anti-tumor activity of 13 cis-retinoic acid in DLA cells is due to apoptosis mediated by the regulation of bcl-2 and caspase-3 gene expression.

Expression of vascular endothelial growth factor (VEGF) and VEGF receptors in tumor angiogenesis and malignancies.

Angiogenesis is a process by which new blood vessels are formed from preexisting vessels. New blood vessel formation by angiogenesis involves the degradation of extra-cellular matrix combined with sprouting and migration of endothelial cells from preexisting capillaries. Solid tumors consist of several components, including normal and stromal cells, extracellular matrix, and vasculature. To grow and metastasize, tumors must stimulate the development of new vasculature through angiogenesis. Vascular endothelial growth factor (VEGF) is a potent angiogenic peptide with biologic effects that include regulation of hematopoietic stem cell development, extracellular matrix remodeling, and inflammatory cytokine regeneration. VEGF is both a vascular growth factor and a vascular permeability factor. Its expression can upregulate several proangiogenic and prometa-static molecules. As a central mediator of angiogenesis, VEGF has emerged as an important target for antiangiogenic therapy. In this review, the authors describe the essential characteristics of VEGF and the VEGF family of ligands and their receptors. They also provide an overview of the central role of VEGF in physiologic and pathologic angiogenesis, directly or indirectly. This review sheds light on the importance of VEGF-targeted antiangiogenic therapy based on the monoclonal antibodies against VEGF, small interfering RNA, and therapy directed against VEGF-VEGFR kinase. It also gives a brief overview of the natural products or dietary compounds that could be used as antiangiogenic agents. Therapeutic inhibition of vessel formation could be best suited to preventive strategies aimed at the suppression of angiogenesis in primary tumors in subjects at risk or of micrometastases after surgical removal of primary tumor.

Piperine is a potent inhibitor of nuclear factor-kappaB (NF-kappaB), c-Fos, CREB, ATF-2 and proinflammatory cytokine gene expression in B16F-10 melanoma cells.

Immune regulation, induction of various inflammatory and growth regulatory genes such as IL-1beta, IL-6, TNF-alpha and GM-CSF require activation of transcription factors such as nuclear factor-kappaB (NF-kappaB), activated transcription factor (ATF-2), c-Fos and cAMP response element-binding protein (CREB). Untreated B16F-10 cells produce very high amount of proinflammatory cytokines such as IL-1beta, IL-6, TNF-alpha and GM-CSF. Piperine treatment significantly reduced the above proinflammatory cytokines. We also found that piperine could reduce the expression of IL-1beta, IL-6, TNF-alpha, GM-CSF and IL-12p40 genes. Piperine at a concentration of 2.5, 5 and 10 microg/ml inhibited the collagen matrix invasion of B16F-10 melanoma cells in a dose-dependent manner. Piperine could inhibit the matrix metalloproteinase production which was demonstrated by zymographic analysis. We found that the nuclear translocation of p65, p50, c-Rel subunits of NF-kappaB and other transcription factors such as ATF-2, c-Fos and CREB were inhibited by the treatment of piperine.

Effect of beta-carotene on the inhibition of lung metastasis in mice.

Effect of beta-carotene on the inhibition of lung metastasis induced by B16F-10 melanoma cells was studied in C57BL/6 mice. Simultaneous administration of the compound after tumor induction produced a significant reduction (71.36%) in tumor nodule formation. Increased lung collagen hydroxyproline (22.37 microg/mg protein) in the metastasized lungs of control animals compared to the normal animals (0.95 microg/mg protein) was significantly reduced (4.19 microg/mg protein) in the beta-carotene treated animals. High amount of uronic acid (355.83 microg/100mg tissue ) in the metastasized control animals was significantly reduced (87.87 microg/100 mg tissue) in the animals treated with beta-carotene. Lung hexosamine content also was inhibited significantly in the beta-carotene treated animals (1.58 mg/100 mg lyophilized tissue) compared to the untreated control animals (4.2 mg/100 mg lyophilized tissue). The elevated levels of serum sialic acid and serum gamma glutamyl transpeptidase activity in the untreated control animals was significantly reduced in the animals treated with beta-carotene. Beta carotene treated animals were survived up to 69 days. Histopathology of the lung tissue also correlated with the above parameters and life span of the drug treated animals. Our results reveal the antimetastatic activity of beta-carotene which are abundantly present in green plants, vegetables and fruits.

Effect of piperine on the inhibition of nitric oxide (NO) and TNF-alpha production.

Effect of piperine which is an alkaloid present in plants such as Piper nigrum and Piper longum on the production of nitric oxide (NO) and tumor necrosis factor-alpha (TNF-alpha) level was analyzed using in vitro as well as in vivo systems. The level of nitrite in the LPS stimulated Balb/C mice (95.3 microM) was reduced in the piperine treated animals (25 microM) significantly. Nitrite level in the Concanavalin-A (Con-A) treated control animals (83.1 microM) was also significantly reduced to 18.5 microM in the piperine treated mice. The drastically elevated levels of TNF-alpha in the lipopolysaccharide (LPS) stimulated animals (625.8 pg/mL) was lowered in the piperine treated animals (105.8 pg/mL). Piperine also inhibited the Con-A induced TNF-alpha production. Piperine could inhibit the nitrite production by in vitro activated macrophages (116.25 microM) to the normal level (15.67 microM) at concentration of 5 microg/mL. In vitro L929 bioassay also revealed the inhibition of TNF-alpha production by the piperine treatment.

Effect of piperine on the inhibition of lung metastasis induced B16F-10 melanoma cells in mice.

The effect of piperine on the inhibition of lung metastasis induced by B16F-10 melanoma cells was studied in C57BL/6 mice. Simultaneous administration of the compound with tumor induction produced a significant reduction (95.2%) in tumor nodule formation. Increased lung collagen hydroxyproline (22.37 microg/mg protein) in the metastasized lungs of the control animals compared to normal animals (0.95 microg/mg protein) was significantly reduced (2.59 microg/mg protein) in the piperine-treated animals. The high amount of uronic acid (355.83 microg/100 mg tissue) in the metastasized control animals was significantly reduced (65 microg/100 mg tissue) in the animals treated with piperine. Lung hexosamine content was also significantly reduced in the piperine-treated animals (0.98 mg/100 mg lyophilized tissue) compared to the untreated tumor-bearing animals (4.2 mg/100 mg lyophilized tissue). The elevated levels of serum sialic acid and serum gamma glutamyl transpeptidase activity in the untreated control animals was significantly reduced in the animals treated with piperine. The piperine-treated animals even survived the experiment (90 days). Histopathology of the lung tissue also correlated with the lifespan of the drug-treated animals. Our results demonstrate the antimetastatic activity of piperine, an alkaloid present in plants such as Piper nigrum and Piper longum.