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1.  Enzastaurin, an inhibitor of PKCβ, Enhances Antiangiogenic Effects and Cytotoxicity of Radiation against Endothelial Cells1,2 
Translational Oncology  2008;1(4):195-201.
PURPOSE: Angiogenesis plays an important role in pancreas cancer pathobiology. Pancreatic tumor cells secrete vascular endothelial growth factor (VEGF), activating endothelial cell protein kinase C beta (PKCβ) that phosphorylates GSK3β to suppress apoptosis and promote endothelial cell proliferation and microvessel formation. We used Enzastaurin (Enz) to test the hypothesis that inhibition of PKCβ results in radiosensitization of endothelial cells in culture and in vivo. MATERIALS/METHODS: We measured PKCβ phosphorylation, VEGF pathway signaling, colony formation, and capillary sprout formation in primary human dermal microvessel endothelial cells (HDMECs) after Enz or radiation (RT) treatment. Microvessel density and tumor volume of human pancreatic cancer xenografts in nude mice were measured after treatment with Enz, RT, or both. RESULTS: Enz inhibited PKCβ and radiosensitized HDMEC with an enhancement ratio of 1.31 ± 0.05. Enz combined with RT reduced HDMEC capillary sprouting to a greater extent than either agent alone. Enz prevented radiation-induced GSK3β phosphorylation of serine 9 while having no direct effect on VEGFR phosphorylation. Treatment of xenografts with Enz and radiation produced greater reductions in microvessel density than either treatment alone. The reduction in microvessel density corresponded with increased tumor growth delay. CONCLUSIONS: Enz-induced PKCβ inhibition radiosensitizes human endothelial cells and enhances the antiangiogenic effects of RT. The combination of Enz and RT reduced microvessel density and resulted in increased growth delay in pancreatic cancer xenografts, without increase in toxicity. These results provide the rationale for combining PKCβ inhibition with radiation and further investigating such regimens in pancreatic cancer.
PMCID: PMC2582168  PMID: 19043530
2.  Metronomic small molecule inhibitor of Bcl-2 (TW-37) is anti-angiogenic and potentiates the anti-tumor effect of ionizing radiation 
Purpose
To investigate the effect of a metronomic (low dose, high frequency) small molecule inhibitor of Bcl-2 (TW-37) in combination with radiotherapy on microvascular endothelial cells in vitro and in tumor angiogenesis in vivo.
Methods and materials
Primary human dermal microvascular endothelial cells (HDMEC) were exposed to ionizing radiation and/or TW-37, and colony formation as well as capillary sprouting in 3-D collagen matrices, was evaluated. Xenografts vascularized with human blood vessels were engineered by co-transplantation of human squamous cell carcinoma cells (OSCC3) and HDMEC seeded in highly porous biodegradable scaffolds into the subcutaneous space of immunodeficient mice. Mice were treated with metronomic TW-37 and/or radiation, and tumor growth was evaluated.
Results
Low dose TW-37 sensitized primary endothelial cells to radiation-induced inhibition of colony formation. Low dose TW-37 or radiation partially inhibited endothelial cell sprout formation, while in combination these therapies abrogated new sprouting. Combination of metronomic TW-37 and low dose radiation inhibited tumor growth and resulted in significant increase in time to failure as compared to controls, whereas single agents did not. Notably, histopathological analysis revealed that tumors treated with TW-37 (with or without radiation) are more differentiated and showed more cohesive invasive fronts, which is consistent with less aggressive phenotype.
Conclusions
These results demonstrate that metronomic TW-37 potentiates the anti-tumor effects of radiotherapy, and suggest that patients with head and neck cancer might benefit from the combination of small molecule inhibitor of Bcl-2 and radiation therapy.
doi:10.1016/j.ijrobp.2010.04.024
PMCID: PMC2946486  PMID: 20675079
Developmental therapeutics; Radiotherapy; Head and Neck Cancer; Apoptosis; Neovascularization
3.  Role of α5β1 Integrin Up-regulation in Radiation-Induced Invasion by Human Pancreatic Cancer Cells1 
Translational Oncology  2011;4(5):282-292.
Radiotherapy is used in the management of pancreatic cancer because of its high propensity for locoregional relapse: one third of patients succumb to localized disease. Thus, strategies to improve the efficacy of radiotherapy in pancreatic cancer are important to pursue. We used naturally serum-free, selectively permeable basement membranes and confocal microscopy of fluorescent antibody-stained human Panc-1, MiaPaCa-2, and BxPC-3 pancreatic cancer cell lines to investigate the effects of ionizing radiation on α5β1 integrin fibronectin receptor expression and on α5β1-mediated invasion. We report that radiation rapidly induces pancreatic cancer cell invasion, and that radiation-induced invasion is caused by up-regulation of α5β1 integrin fibronectin receptors by transcriptional and/or postendocytic recycling mechanisms. We also report that radiation causes α5β1 up-regulation in Panc-1, MiaPaCa-2, and BxPC-3 tumor xenografts and that upregulated α5β1 colocalizes with upregulated early or late endosomes in Panc-1 or BxPC-3 tumors, respectively, although it may colocalize significantly with both endosome types in MiaPaCa-2 tumors. Our results suggest that systemic inhibition of α5β1-mediated invasion might be an effective way to reduce radiation-induced pancreatic cancer cell invasion, thereby improving the efficacy of radiotherapy.
PMCID: PMC3162303  PMID: 21966545
4.  Ablation of Breast Cancer Stem Cells with Radiation1 
Translational Oncology  2011;4(4):227-233.
Tumor radioresistance leads to recurrence after radiation therapy. The radioresistant phenotype has been hypothesized to reside in the cancer stem cell (CSC) component of breast and other tumors and is considered to be an inherent property of CSC. In this study, we assessed the radiation resistance of breast CSCs using early passaged, patient-derived xenografts from two separate patients. We found a patient-derived tumor in which the CSC population was rapidly depleted 2 weeks after treatment with radiation, based on CD44+ CD24- lin- phenotype and aldehyde dehydrogenase 1 immunofluorescence, suggesting sensitivity to radiotherapy. The reduction in CSCs according to phenotypic markers was accompanied by a decrease in functional CSC activity measured by tumor sphere frequency and the ability to form tumors in mice. In contrast, another patient tumor sample displayed enrichment of CSC after irradiation, signifying radioresistance, in agreement with others. CSC response to radiation did not correlate with the level of reactive oxygen species in CSC versus non-CSC. These findings demonstrate that not all breast tumor CSCs are radioresistant and suggest a mechanism for the observed variability in breast cancer local recurrence.
PMCID: PMC3140010  PMID: 21804918
5.  Loss of Tumor-Initiating Cell Activity in Cyclophosphamide-Treated Breast Xenografts1 
Translational Oncology  2010;3(3):149-152.
Cancer stem cells (CSCs) are a subpopulation of tumor cells with preferential tumor-initiating capacity and have been purported to be resistant to chemotherapy. It has been shown that breast CSC are, on average, enriched in patient tumors after combination neoadjuvant chemotherapy including docetaxel, doxorubicin, and cyclophosphamide (CPA). Here, we investigate the resistance of breast CSC to CPA alone in a xenograft model. CPA treatment led to a 48% reduction in tumor volume during a 2-week period. Cells bearing the CD44+ CD24- phenotype were reduced by 90% (2.5% to 0.24%) in CPA-treated tumors, whereas cells with aldehyde dehydrogenase activity were reduced by 64% (4.7% to 1.7%). A subsequent functional analysis showed that CPA-treated tumors were impaired in their ability to form tumors, indicating loss of functional tumor-initiating activity. These results are consistent with a CSC phenotype that is sensitive to CPA and indicate that some patient CSC may not display the expected resistance to therapy. Deciphering the mechanism for this difference may lead to therapies to counteract resistance.
PMCID: PMC2887643  PMID: 20563255
6.  In Vivo Bioluminescent Imaging of Irradiated Orthotopic Pancreatic Cancer Xenografts in Nonobese Diabetic-Severe Combined Immunodeficient Mice: A Novel Method for Targeting and Assaying Efficacy of Ionizing Radiation 
Translational Oncology  2010;3(3):153-159.
Adenocarcinoma of the pancreas is a lethal malignancy, and better models to study tumor behavior in vivo are needed for the development ofmore effective therapeutics. Ionizing radiation is a treatment modality that is commonly used in the clinical setting, in particular, for locally confined disease; however, good model systems to study the effect of ionizing radiation in orthotopic tumors have not been established. In an attempt to create clinically relevant models for studying treatments directed against pancreatic cancer, we have defined a methodology to measure the effect of varying doses of radiation in established human pancreatic cancer orthotopic xenografts using two different pancreatic cancer cell lines (Panc-1 and BXPC3) infected with a lentiviral vector expressing CMV promoter-driven luciferase to allow bioluminescence imaging of live animals in real time. Quantifiable photon emission from luciferase signaling in vivo correlated well with actual tumor growth. Bioluminescence imaging of the established pancreatic xenografts was used to direct delivery of radiation to the orthotopic tumors and minimize off-target adverse effects. Growth delay was observed with schedules in the range of 7.5 Gy in five fractions to 10 Gy in four fractions, whereas doses 3 Gy or higher produced toxic adverse effects. In conclusion, we describe a model in which the effects of ionizing radiation, alone or in combination with other therapeutics, in orthotopic xenografts, can be studied.
PMCID: PMC2887644  PMID: 20563256
7.  GSK3β and β-Catenin Modulate Radiation Cytotoxicity in Pancreatic Cancer1 
Neoplasia (New York, N.Y.)  2010;12(5):357-365.
Background
Knowledge of factors and mechanisms contributing to the inherent radioresistance of pancreatic cancer may improve cancer treatment. Irradiation inhibits glycogen synthase kinase 3β (GSK3β) by phosphorylation at serine 9. In turn, release of cytosolic membrane β-catenin with subsequent nuclear translocation promotes survival. Both GSK3β and β-catenin have been implicated in cancer cell proliferation and resistance to death.
Methods
We investigated pancreatic cancer cell survival after radiation in vitro and in vivo, with a particular focus on the role of the function of the GSK3β/β-catenin axis.
Results
Lithium chloride, RNAi-medicated silencing of GSK3β, or the expression of a kinase dead mutant GSK3β resulted in radioresistance of Panc1 and BxPC3 pancreatic cancer cells. Conversely, ectopic expression of a constitutively active form of GSK3β resulted in radiosensitization of Panc1 cells. GSK3β silencing increased radiation-induced β-catenin target gene expression asmeasured by studies of AXIN2 and LEF1 transcript levels. Western blot analysis of total and phosphorylated levels of GSK3β and β-catenin showed that GSK3β inhibition resulted in stabilization of β-catenin. Xenografts of both BxPC3 and Panc1 with targeted silencing of GSK3β exhibited radioresistance in vivo. Silencing of β-catenin resulted in radiosensitization, whereas a nondegradable β-catenin construct induced radioresistance.
Conclusions
These data support the hypothesis that GSK3β modulates the cellular response to radiation in a β-catenin-dependent mechanism. Further understanding of this pathway may enhance the development of clinical trials combining drugs inhibiting β-catenin activation with radiation and chemotherapy in locally advanced pancreatic cancer.
PMCID: PMC2864473  PMID: 20454507
8.  TW-37, a small molecule inhibitor of Bcl-2, mediates S phase cell cycle arrest and suppresses head and neck tumor angiogenesis 
Molecular cancer therapeutics  2009;8(4):893-903.
Members of the Bcl-2 family play a major role in the pathobiology of head and neck cancer. We have shown that Bcl-2 orchestrates a crosstalk between tumor cells and endothelial cells that have a direct impact on the progression of head and neck squamous cell carcinoma (HNSCC). Notably, Bcl-2 is significantly upregulated in the tumor associated endothelial cells as compared to the endothelial cells of normal oral mucosa in patients with HNSCC. Here, we evaluated the effect of TW-37, a small molecule inhibitor of Bcl-2, on the cell cycle and survival of endothelial cells and HNSCC and on the progression of xenografted tumors. TW-37 has an IC50 of 1.1 µM for primary human endothelial cells and averaged 0.3 µM for head and neck cancer cells (OSCC3, UM-SCC-1, UM-SCC-74A). Combination of TW-37 and cisplatin showed enhanced cytotoxic effects for endothelial cells and HNSCC in vitro, as compared with single drug treatment . Notably, while cisplatin led to an expected G2/M cell cycle arrest, TW-37 mediated an “S” phase cell cycle arrest in endothelial cells and in HNSCC. In vivo, TW-37 inhibited tumor angiogenesis and induced tumor apoptosis without significant systemic toxicities. Combination of TW-37 and cisplatin enhanced the time to tumor failure (i.e. 4-fold increase in tumor volume), as compared to either drug given separately. Collectively, these data reveal that therapeutic inhibition of Bcl-2 function with TW-37 is sufficient to arrest endothelial cells and HNSCC in the “S” phase of cell cycle, and to inhibit head and neck tumor angiogenesis.
doi:10.1158/1535-7163.MCT-08-1078
PMCID: PMC2709836  PMID: 19372562
apoptosis; neovascularization; cisplatin; cancer
9.  α5β1 Integrin Ligand PHSRN Induces Invasion and α5 mRNA in Endothelial Cells to Stimulate Angiogenesis1 
Translational Oncology  2009;2(1):8-20.
Angiogenesis requires endothelial cell invasion and is crucial for wound healing and for tumor growth and metastasis. Invasion of native collagen is mediated by the α5β1 integrin fibronectin receptor. Thus, α5β1 up-regulation on the surfaces of endothelial cells may induce endothelial cell invasion to stimulate angiogenesis. We report that the interaction of α5β1 with its PHSRN peptide ligand induces human microvascular endothelial cell invasion and that PHSRN-induced endothelial cell invasion is regulated by α4β1 integrin and requires matrix metalloproteinase 1 (MMP-1). Moreover, our results show that exposure to PHSRN causes rapid, specific up-regulation of surface levels of α5β1 integrin and significantly increases α5 integrin mRNA in microvascular endothelial cells. Consistent with these results, α5 small interfering RNA abrogates PHSRN-induced surface α5 and MMP-1 up-regulation, as well as blocking invasion induction. We also observed dose-dependent, PHSRN-induced α5β1 integrin up-regulation on endothelial cells in vivo in Matrigel plugs. We further report that the PHSCN peptide, an α5β1-targeted invasion inhibitor, blocks PHSRN-induced invasion, α5β1 up-regulation, α5 mRNA induction, and MMP-1 secretion in microvascular endothelial cells and that systemic PHSCN administration prevents PHSRN-induced α5β1 up-regulation and angiogenesis in Matrigel plugs. These results demonstrate a critical role for α5β1 integrin and MMP-1 in mediating the endothelial cell invasion and angiogenesis and suggest that PHSRN-induced α5 transcription and α5β1 up-regulation may form an important feed-forward mechanism for stimulating angiogenesis.
PMCID: PMC2647698  PMID: 19252747
10.  Endothelial Cells Enhance Tumor Cell Invasion through a Crosstalk Mediated by CXC Chemokine Signaling1 
Neoplasia (New York, N.Y.)  2008;10(2):131-139.
Field cancerization involves the lateral spread of premalignant or malignant disease and contributes to the recurrence of head and neck tumors. The overall hypothesis underlying this work is that endothelial cells actively participate in tumor cell invasion by secreting chemokines and creating a chemotactic gradient for tumor cells. Here we demonstrate that conditioned medium from head and neck tumor cells enhance Bcl-2 expression in neovascular endothelial cells. Oral squamous cell carcinoma-3 (OSCC3) and Kaposi's sarcoma (SLK) show enhanced invasiveness when cocultured with pools of human dermal microvascular endothelial cells stably expressing Bcl-2 (HDMEC-Bcl-2), compared to cocultures with empty vector controls (HDMEC-LXSN). Xenografted OSCC3 tumors vascularized with HDMEC-Bcl-2 presented higher local invasion than OSCC3 tumors vascularized with control HDMEC-LXSN. CXCL1 and CXCL8 were upregulated in primary endothelial cells exposed to vascular endothelial growth factor (VEGF), as well as in HDMEC-Bcl-2. Notably, blockade of CXCR2 signaling, but not CXCR1, inhibited OSCC3 and SLK invasion toward endothelial cells. These data demonstrate that CXC chemokines secreted by endothelial cells induce tumor cell invasion and suggest that the process of lateral spread of tumor cells observed in field cancerization is guided by chemotactic signals that originated from endothelial cells.
PMCID: PMC2244688  PMID: 18283335
11.  Increased Expression of Death Receptors 4 and 5 Synergizes the Apoptosis Response to Combined Treatment with Etoposide and TRAIL 
Molecular and Cellular Biology  2000;20(1):205-212.
Chemotherapeutic genotoxins induce apoptosis in epithelial-cell-derived cancer cells. The death receptor ligand TRAIL also induces apoptosis in epithelial-cell-derived cancer cells but generally fails to induce apoptosis in nontransformed cells. We show here that the treatment of four different epithelial cell lines with the topoisomerase II inhibitor etoposide in combination with TRAIL (tumor necrosis factor [TNF]-related apoptosis-inducing ligand) induces a synergistic apoptotic response. The mechanism of the synergistic effect results from the etoposide-mediated increase in the expression of the death receptors 4 (DR4) and 5 (DR5). Inhibition of NF-κB activation by expression of kinase-inactive MEK kinase 1(MEKK1) or dominant-negative IκB (ΔIκB) blocked the increase in DR4 and DR5 expression following etoposide treatment. Addition of a soluble decoy DR4 fusion protein (DR4:Fc) to cell cultures reduced the amount of etoposide-induced apoptosis in a dose-dependent manner. The addition of a soluble TNF decoy receptor (TNFR:Fc) was without effect, demonstrating the specificity of DR4 binding ligands in the etoposide-induced apoptosis response. Thus, genotoxin treatment in combination with TRAIL is an effective inducer of epithelial-cell-derived tumor cell apoptosis relative to either treatment alone.
PMCID: PMC85076  PMID: 10594023

Results 1-11 (11)