Benjamin David Zeitlin

Bcl-2 Acts in a Proangiogenic Signaling Pathway through Nuclear Factor-κB and CXC Chemokines

Vascular endothelial growth factor (VEGF) induces expression of Bcl-2 in tumor-associated microvascular endothelial cells. We have previously reported that up-regulated Bcl-2 expression in microvascular endothelial cells is sufficient to enhance intratumoral angiogenesis and to accelerate tumor growth. We initially attributed these results to Bcl-2-mediated endothelial cell survival. However, in recent experiments, we observed that conditioned medium from Bcl-2-transduced human dermal microvascular endothelial cells (HDMEC-Bcl-2) is sufficient to induce potent neovascularization in the rat corneal assay, whereas conditioned medium from empty vector controls (HDMEC-LXSN) does not induce angiogenesis. These results cannot be attributed to the role of Bcl-2 in cell survival. To understand this unexpected observation, we did gene expression arrays that revealed that the expression of the proangiogenic chemokines interleukin-8 (CXCL8) and growth-related oncogene-alpha (CXCL1) is significantly higher in HDMEC exposed to VEGF and in HDMEC-Bcl-2 than in controls. Inhibition of Bcl-2 expression with small interfering RNA-Bcl-2, or the inhibition of Bcl-2 function with small molecule inhibitor BL-193, down-regulated CXCL8 and CXCL1 expression and caused marked decrease in the angiogenic potential of endothelial cells without affecting cell viability. Nuclear factor-kappaB (NF-kappaB) is highly activated in HDMEC exposed to VEGF and HDMEC-Bcl-2 cells, and genetic and chemical approaches to block the activity of NF-kappaB down-regulated CXCL8 and CXCL1 expression levels. These results reveal a novel function for Bcl-2 as a proangiogenic signaling molecule and suggest a role for this pathway in tumor angiogenesis.

Bcl-2 orchestrates a cross-talk between endothelial and tumor cells that promotes tumor growth.

The current understanding of the interaction between the endothelium and cancer cells is fundamentally based on the concept that endothelial cells are responsive to differentiation and survival signals originating from the tumor cells. Whereas the effect of tumor cell-secreted factors on angiogenesis is well established, little is known about the effect of factors secreted by endothelial cells on tumor cell gene expression and tumor progression. Here, we show that bcl-2 gene expression is significantly higher in the tumor-associated endothelial cells of patients with head and neck squamous cell carcinomas (HNSCC) as compared with endothelial cells from the normal oral mucosa. Bcl-2 induces vascular endothelial growth factor (VEGF) expression in neovascular endothelial cells through a signal transducer and activator of transcription 3 (STAT3)-mediated pathway. Endothelial cell-derived VEGF signals through VEGFR1 and induces expression of Bcl-2 and the proangiogenic chemokines CXCL1 and CXCL8 in HNSCC cells. Notably, inhibition of Bcl-2 expression in neovascular endothelial cells with RNA interference down-regulates expression of Bcl-2, CXCL8, and CXCL1 in HNSCC cells, and is sufficient to inhibit growth and decrease the microvessel density of xenografted HNSCC in immunodeficient mice. Together, these results show that Bcl-2 is the orchestrator of a cross-talk between neovascular endothelial cells and tumor cells, which has a direct effect on tumor growth. This work identifies a new function for Bcl-2 in cancer biology that is beyond its classic role in cell survival.

Effects of Morphogen and Scaffold Porogen on the Differentiation of Dental Pulp Stem Cells

INTRODUCTION Dental pulp tissue engineering is an emerging field that can potentially have a major impact on oral health. However, the source of morphogens required for stem cell differentiation into odontoblasts and the scaffold characteristics that are more conducive to odontoblastic differentiation are still unclear. This study investigated the effect of dentin and scaffold porogen on the differentiation of human dental pulp stem cells (DPSCs) into odontoblasts. METHODS Poly-L-lactic acid (PLLA) scaffolds were prepared in pulp chambers of extracted human third molars using salt crystals or gelatin spheres as porogen. DPSCs seeded in tooth slice/scaffolds or control scaffolds (without tooth slice) were either cultured in vitro or implanted subcutaneously in immunodefficient mice. RESULTS DPSCs seeded in tooth slice/scaffolds but not in control scaffolds expressed putative odontoblastic markers (DMP-1, DSPP, and MEPE) in vitro and in vivo. DPSCs seeded in tooth/slice scaffolds presented lower proliferation rates than in control scaffolds between 7 and 21 days (p < 0.05). DPSCs seeded in tooth slice/scaffolds and transplanted into mice generated a tissue with morphological characteristics similar to those of human dental pulps. Scaffolds generated with gelatin or salt porogen resulted in similar DPSC proliferation. The porogen type had a relatively modest impact on the expression of the markers of odontoblastic differentiation. CONCLUSIONS Collectively, this work shows that dentin-related morphogens are important for the differentiation of DPSC into odontoblasts and for the engineering of dental pulp-like tissues and suggest that environmental cues influence DPSC behavior and differentiation potential.

A hydrogel scaffold that maintains viability and supports differentiation of dental pulp stem cells

OBJECTIVES The clinical translation of stem cell-based Regenerative Endodontics demands further development of suitable injectable scaffolds. Puramatrix™ is a defined, self-assembling peptide hydrogel which instantaneously polymerizes under normal physiological conditions. Here, we assessed the compatibility of Puramatrix™ with dental pulp stem cell (DPSC) growth and differentiation. METHODS DPSC cells were grown in 0.05-0.25% Puramatrix™. Cell viability was measured colorimetrically using the WST-1 assay. Cell morphology was observed in 3D modeling using confocal microscopy. In addition, we used the human tooth slice model with Puramatrix™ to verify DPSC differentiation into odontoblast-like cells, as measured by expression of DSPP and DMP-1. RESULTS DPSC survived and proliferated in Puramatrix™ for at least three weeks in culture. Confocal microscopy revealed that cells seeded in Puramatrix™ presented morphological features of healthy cells, and some cells exhibited cytoplasmic elongations. Notably, after 21 days in tooth slices containing Puramatrix™, DPSC cells expressed DMP-1 and DSPP, putative markers of odontoblastic differentiation. SIGNIFICANCE Collectively, these data suggest that self-assembling peptide hydrogels might be useful injectable scaffolds for stem cell-based Regenerative Endodontics.

Antiangiogenic Effect of TW37, a Small-Molecule Inhibitor of Bcl-2

Bcl-2 is an antiapoptotic protein that is up-regulated in several tumor types, and its expression levels have strong correlation to development of resistance to therapy and poor prognosis. We have shown recently that Bcl-2 also functions as a proangiogenic signaling molecule that activates a nuclear factor-kappaB-mediated pathway resulting in up-regulation of the angiogenic chemokines CXCL1 and CXCL8 by neovascular endothelial cells. Here, we evaluate the antiangiogenic effect of the novel small-molecule inhibitor of Bcl-2 (TW37) developed using a structure-based design strategy. We observed that TW37 has an IC(50) of 1.8 mumol/L for endothelial cells but showed no cytotoxic effects for fibroblasts at concentrations up to 50 mumol/L. The mechanism of TW37-induced endothelial cell death was apoptosis, in a process mediated by mitochondrial depolarization and activation of caspase-9 and caspase-3. The effect of TW37 on endothelial cell apoptosis was not prevented by coexposure to the growth factor milieu secreted by tumor cells. Inhibition of the angiogenic potential of endothelial cells (i.e., migration and capillary sprouting assays) and expression of the angiogenic chemokines CXCL1 and CXCL8 were accomplished at subapoptotic TW37 concentrations (0.005-0.05 micromol/L). Notably, administration of TW37 i.v. resulted in a decrease in the density of functional human microvessels in the severe combined immunodeficient mouse model of human angiogenesis. In conclusion, we describe functionally separate proapoptotic and antiangiogenic mechanisms for a small-molecule inhibitor of Bcl-2 and show the potential for Bcl-2 inhibition as a target for antiangiogenic therapy.

From combinatorial peptide selection to drug prototype (I): Targeting the vascular endothelial growth factor receptor pathway

Inhibition of blood vessel formation is a viable therapeutic approach in angiogenesis-dependent diseases. We previously used a combinatorial screening on vascular endothelial growth factor (VEGF)-activated endothelial cells to select the sequence CPQPRPLC and showed that the motif Arg-Pro-Leu targets VEGF receptor-1 and neuropilin-1. Here, we evaluated and validated D(LPR), a derivative molecule with strong antiangiogenesis attributes. This prototype drug markedly inhibits neovascularization in three mouse models: Matrigel-based assay, functional human/murine blood vessel formation, and retinopathy of prematurity. In addition to its systemic activity, D(LPR) also inhibits retinal angiogenesis when administered in an eye-drop formulation. Finally, in preliminary studies, we have showed targeted drug activity in an experimental tumor-bearing mouse model. These results show that drugs targeting extracellular domains of VEGF receptors are active, affect signal transduction, and have potential for clinical application. On a larger context, this study illustrates the power of ligand-directed selection plus retro-inversion for rapid drug discovery and development.

TW-37, a small-molecule inhibitor of Bcl-2, mediates S-phase cell cycle arrest and suppresses head and neck tumor angiogenesis.

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 cross talk between tumor cells and endothelial cells that have a direct effect on the progression of head and neck squamous cell carcinoma (HNSCC). Notably, Bcl-2 is significantly up-regulated in the tumor-associated endothelial cells compared with 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 μmol/L for primary human endothelial cells and averaged 0.3 μmol/L for head and neck cancer cells (OSCC3, UM-SCC-1, and UM-SCC-74A). The combination of TW-37 and cisplatin showed enhanced cytotoxic effects for endothelial cells and HNSCC in vitro, compared with single drug treatment. Notably, whereas 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), compared with 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 the cell cycle and to inhibit head and neck tumor angiogenesis.[Mol Cancer Ther 2009;8(4):893–903]

RAIN-Droplet: a novel 3D in vitro angiogenesis model

Angiogenesis is fundamentally required for the initialization, development and metastatic spread of cancer. A rapidly expanding number of new experimental, chemical modulators of endothelial cell function have been described for the therapeutic inhibition of angiogenesis in cancer. Despite this expansion, there has been very limited parallel growth of in vitro angiogenesis models or experimental tools. Here we present the Responsive Angiogenic Implanted Network (RAIN)-Droplet model and novel angiogenesis assay using an endothelial cell culture model of microvascular endothelial cells encapsulated in a spontaneously self-assembling, toroidal hydrogel droplet uniquely yielding discrete, pre-formed, angiogenic networks that may be embedded in 3D matrices. On embedding, radial growth of capillary-like sprouts and cell invasion was observed. The sprouts formed not only as outgrowths from endothelial cells on the surface of the droplets, but also, uniquely, from the pre-formed network structures within the droplet. We demonstrate proof of principle for the utility of the model showing significant inhibition of sprout formation (P<0.001) in the presence of bevacizumab, an anti-angiogenic antibody. Using the RAIN-Droplet assay, we also demonstrate a novel dose-dependent pro-angiogenic function for the characteristically anti-angiogenic multi-kinase inhibitor sorafenib. Exposure of endothelial cells in 3D culture to low, non-lethal doses (<1 μM) of sorafenib after initiation of sprouting resulted in the formation of significantly (P<0.05) more endothelial sprouts compared with controls over a 48-h period. Higher doses of sorafenib (5 μM) resulted in a significant (P<0.05) reduction of sprouting over the same time period. The RAIN-Droplet model is a highly versatile and simply constructed 3D focal sprouting approach well suited for the study of vascular morphogenesis and for preclinical testing of drugs. Furthermore, the RAIN-Droplet model has facilitated the discovery of a novel pro-angiogenic capacity for sorafenib, which may impact the clinical application and dosing regimen of that drug.

Metronomic Dosing of BH3 Mimetic Small Molecule Yields Robust Antiangiogenic and Antitumor Effects

Bcl-2 is an antiapoptotic protein that has also been found to function as a proangiogenic signaling molecule. Improvements in antiangiogenic therapy can be engendered by metronomic dosing. Thus, we hypothesized that BH3-mimetic drugs that antagonize Bcl-2 family proteins may exert a greater efficacy when dosed metronomically. To examine this hypothesis, we employed AT101, an orally available and well-tolerated BH3-mimetic drug that has been established as effective. In a mouse xenograft model of human squamous cell carcinomas (SCC) that includes a humanized vasculature, we explored the effects of docetaxel in combination with either daily (metronomic) or weekly (bolus) doses of AT101. In addition, we explored the effect of single or combination therapy on angiogenesis and survival of endothelial or SCC cells in vitro. Metronomic AT101 therapy increased mouse survival, decreased tumor mitotic index, and decreased tumor microvessel density, compared with bolus therapy. Therapeutic potentiation was achieved by similar overall drug exposure and without altering systemic toxicities. Combinations of AT101 and docetaxel produced additive toxicity in both endothelial and SCC tumor cells. Notably, subapoptotic concentrations of AT101 potently inhibited the angiogenic potential of endothelial cells. Taken together, our findings unveil the efficacious benefits that can be achieved by metronomic delivery of BH3-mimetic drugs, in particular suggesting that SCC patients with might benefit from low-dose continuous administration of these drugs.

Metronomic small molecule inhibitor of Bcl-2 (TW-37) is antiangiogenic and potentiates the antitumor 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 were exposed to ionizing radiation and/or TW-37 and colony formation, as well as capillary sprouting in three-dimensional collagen matrices, was evaluated. Xenografts vascularized with human blood vessels were engineered by cotransplantation of human squamous cell carcinoma cells (OSCC3) and human dermal microvascular endothelial cells 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, and 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 compared with controls, whereas single agents did not. Notably, histopathologic 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 antitumor 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.