Endothelial interleukin-6 defines the tumorigenic potential of primary human cancer stem cells.

Head and neck squamous cell carcinomas (HNSCC) contain a small subpopulation of stem cells endowed with unique capacity to generate tumors. These cancer stem cells (CSC) are localized in perivascular niches and rely on crosstalk with endothelial cells for survival and self-renewal, but the mechanisms involved are unknown. Here, we report that stromal interleukin (IL)-6 defines the tumorigenic capacity of CSC sorted from primary human HNSCC and transplanted into mice. In search for the cellular source of Interleukin-6 (IL-6), we observed a direct correlation between IL-6 levels in tumor-associated endothelial cells and the tumorigenicity of CSC. In vitro, endothelial cell-IL-6 enhanced orosphere formation, p-STAT3 activation, survival, and self-renewal of human CSC. Notably, a humanized anti-IL-6R antibody (tocilizumab) inhibited primary human CSC-mediated tumor initiation. Collectively, these data demonstrate that endothelial cell-secreted IL-6 defines the tumorigenic potential of CSC, and suggest that HNSCC patients might benefit from therapeutic inhibition of IL-6/IL-6R signaling.

Establishment and characterization of cMYB-expressing human salivary adenoid cystic carcinoma cell lines (UM-HACC-14, UM-HACC-6) and matching patient-derived xenograft model (UM-PDX-HACC-14).

OBJECTIVE: Limited availability of authentic human adenoid cystic carcinoma (ACC) cell lines has hindered progress in understanding mechanisms underpinning the biology of this disease and the development of safe and effective therapies. STUDY DESIGN: Surgical human ACC specimens (UM-HACC-6, UM-HACC-14) were dissociated into single cell suspensions and cultured in fibronectin-coated flasks. Alternatively, tumor fragments were transplanted subcutaneously into female immunodeficient (SCID) mice to establish patient-derived xenograft tumors (PDX; UM-PDX-HACC-14). RESULTS: Both ACC cell lines showed continuous growth in monolayers for over 100 passages. Total RNA-Seq, RT-PCR, and FISH analysis revealed that both are MYB-NFIB fusion negative. Western blots revealed passage-dependent expression of E-Cadherin, PCNA, p63, phospho-c-MYB, and NFIB. Both, UM-HACC-14 and UM-HACC-6 cells exhibited tumorigenic potential when injected orthotopically into mouse submandibular glands. CONCLUSION: UM-HACC-14, patient-matching UM-PDX-HACC-14, and the UM-HACC-6 cell line are new, authenticated preclinical models of ACC that are well suited for mechanistic and developmental therapeutics studies.

Crosstalk between tumor and endothelial cells promotes tumor angiogenesis by MAPK activation of Notch signaling.

While significant progress has been made in understanding the induction of tumor vasculature by secreted angiogenic factors, little is known regarding contact-dependent signals that promote tumor angiogenesis. Here, we report that the Notch ligand Jagged1 induced by growth factors via mitogen-activating protein kinase (MAPK) in head and neck squamous cell carcinoma (HNSCC) cells triggered Notch activation in neighboring endothelial cells (ECs) and promoted capillary-like sprout formation. Jagged1-expressing HNSCC cells significantly enhanced neovascularization and tumor growth in vivo. Moreover, the level of Jagged1 was significantly correlated with tumor blood vessel content and associated with HNSCC development. Our results elucidate a novel mechanism by which the direct interplay between tumor cells and ECs promotes angiogenesis through MAPK and Notch signaling pathways.

A curriculum for the new dental practitioner: preparing dentists for a prospective oral health care environment.

The emerging concept of prospective health care would shift the focus of health care from disease management to disease prevention and health management. Dentistry has a unique opportunity to embrace this model of prospective and collaborative care and focus on the management of oral health. Academic dentistry must better prepare future dentists to succeed in this new health care environment by providing them with the scientific and technical knowledge required to understand and assess risk and practice disease prevention. Dental schools must consider creating career pathways for enabling future graduates to assume important leadership roles that will advance a prospective oral health care system.

Endothelial cell Bcl-2 and lymph node metastasis in patients with oral squamous cell carcinoma.

BACKGROUND: Loco-regional spread of disease causes high morbidity and is associated with the poor prognosis of malignant oral tumors. Better understanding of mechanisms underlying the establishment of lymph node metastasis is necessary for the development of more effective therapies for patients with oral cancer. The aims of this work were to evaluate a possible correlation between endothelial cell Bcl-2 and lymph node metastasis in patients with oral squamous cell carcinoma (OSCC), and to study signaling pathways that regulate Bcl-2 expression in lymphatic endothelial cells. METHODS: Endothelial cells were selectively retrieved from paraffin-embedded tissue sections of primary human OSCC from patients with or without lymph node metastasis by laser capture microdissection. RT-PCR was used to evaluate Bcl-2 expression in tumor-associated endothelial cells and in tumor cells. In vitro, mechanistic studies were performed to examine the effect of vascular endothelial growth factor (VEGF)-C on the expression of Bcl-2 in primary human lymphatic endothelial cells. RESULTS: We observed that Bcl-2 expression is upregulated in the endothelial cells of human oral tumors with lymph node metastasis as compared to endothelial cells from stage-matched tumors without metastasis. VEGF-C induced Bcl-2 expression in lymphatic endothelial cells via VEGFR-3 and PI3k/Akt signaling. Notably, OSCC cells express VEGF-C and induce Bcl-2 in lymphatic endothelial cells. CONCLUSIONS: Collectively, this work unveiled a mechanism for the induction of Bcl-2 in lymphatic endothelial cells and suggested that endothelial cell Bcl-2 contributes to lymph node metastasis in patients with oral squamous cell carcinoma.

Crosstalk between cancer stem cells and the tumor microenvironment drives progression of premalignant oral epithelium.

Cancer stem cells (CSC) are a subpopulation of cancer cells that exhibit properties of self-renewal and differentiation and have been implicated in metastasis and treatment failures. There is mounting evidence that carcinogen-initiated mucosal epithelial stem cells acquire the CSC phenotype following exposure to environmental or infectious mutagens and are responsible for promoting the malignant transformation of premalignant (dysplastic) epithelium. CSC further contribute to the progression of dysplasia by activating signaling pathways through crosstalk with various cell populations in the tumor microenvironment. Two cell types, tumor-associated macrophages (TAM) and vascular endothelial cells (EC) nurture CSC development, support CSC stemness, and contribute to tumor progression. Despite mounting evidence implicating CSC in the initiation and progression of dysplastic oral epithelium to squamous cell carcinoma (SCC), the molecular mechanisms underlying these synergistic biological processes remain unclear. This review will examine the mechanisms that underlie the transformation of normal epithelial stem cells into CSC and the mechanistic link between CSC, TAM, and EC in the growth and the malignant conversation of dysplastic oral epithelium.

The IL-6R and Bmi-1 axis controls self-renewal and chemoresistance of head and neck cancer stem cells.

Despite major progress in elucidating the pathobiology of head and neck squamous cell carcinoma (HNSCC), the high frequency of disease relapse correlates with unacceptably deficient patient survival. We previously showed that cancer stem-like cells (CSCs) drive tumorigenesis and progression of HNSCC. Although CSCs constitute only 2-5% of total tumor cells, CSCs contribute to tumor progression by virtue of their high tumorigenic potential and their resistance to chemo-, radio-, and immunotherapy. Not only are CSCs resistant to therapy, but cytotoxic agents actually enhance cancer stemness by activating transcription of pluripotency factors and by inducing expression of Bmi-1, a master regulator of stem cell self-renewal. We hypothesized therapeutic inhibition of interleukin-6 receptor (IL-6R) suppresses Bmi-1 to overcome intrinsic chemoresistance of CSCs. We observed that high Bmi-1 expression correlates with decreased (p = 0.04) recurrence-free survival time in HNSCC patients (n = 216). Blockade of IL-6R by lentiviral knockdown or pharmacologic inhibition with a humanized monoclonal antibody (Tocilizumab) is sufficient to inhibit Bmi-1 expression, secondary sphere formation, and to decrease the CSC fraction even in Cisplatin-resistant HNSCC cells. IL-6R inhibition with Tocilizumab abrogates Cisplatin-mediated increase in CSC fraction and induction of Bmi-1 in patient-derived xenograft (PDX) models of HNSCC. Notably, Tocilizumab inhibits Bmi-1 and suppresses growth of xenograft tumors generated with Cisplatin-resistant HNSCC cells. Altogether, these studies demonstrate that therapeutic blockade of IL-6R suppresses Bmi-1 function and inhibits cancer stemness. These results suggest therapeutic inhibition of IL-6R might be a viable strategy to overcome the CSC-mediated chemoresistance typically observed in HNSCC patients.

Active Smoking Induces Aberrations in Digestive Tract Microbiota of Rats.

Cigarette smoking could have certain effects on gut microbiota. Some pioneering studies have investigated effects of active smoking on the microbiome in local segments of the digestive tract, while active smoking-induced microbiome alterations in the whole digestive tract have not been fully investigated. Here, we developed a rat model of active smoking and characterized the effects of active smoking on the microbiota within multiple regions along the digestive tract. Blood glucose and some metabolic factors levels, the microbial diversity and composition, relative abundances of taxa, bacterial network correlations and predictive functional profiles were compared between the control group and active smoking group. We found that active smoking induced hyperglycemia and significant reductions in serum insulin and leptin levels. Active smoking induced region-specific shifts in microbiota structure, composition, network correlation and metabolism function along the digestive tract. Our results demonstrated that active smoking resulted in a reduced abundance of some potentially beneficial genera (i.e. Clostridium, Turicibacter) and increased abundance of potentially harmful genera (i.e. Desulfovibrio, Bilophila). Functional prediction suggested that amino acid, lipid, propanoate metabolism function could be impaired and antioxidant activity may be triggered. Active smoking may be an overlooked risk to health through its potential effects on the digestive tract microbiota, which is involved in the cause and severity of an array of chronic diseases.

HPV16 drives cancer immune escape via NLRX1-mediated degradation of STING.

The incidence of human papillomavirus-positive (HPV+) head and neck squamous cell carcinoma (HNSCC) has surpassed that of cervical cancer and is projected to increase rapidly until 2060. The coevolution of HPV with transforming epithelial cells leads to the shutdown of host immune detection. Targeting proximal viral nucleic acid-sensing machinery is an evolutionarily conserved strategy among viruses to enable immune evasion. However, E7 from the dominant HPV subtype 16 in HNSCC shares low homology with HPV18 E7, which was shown to inhibit the STING DNA-sensing pathway. The mechanisms by which HPV16 suppresses STING remain unknown. Recently, we characterized the role of the STING/type I interferon (IFN-I) pathway in maintaining immunogenicity of HNSCC in mouse models. Here we extended those findings into the clinical domain using tissue microarrays and machine learning-enhanced profiling of STING signatures with immune subsets. We additionally showed that HPV16 E7 uses mechanisms distinct from those used by HPV18 E7 to antagonize the STING pathway. We identified NLRX1 as a critical intermediary partner to facilitate HPV16 E7-potentiated STING turnover. The depletion of NLRX1 resulted in significantly improved IFN-I-dependent T cell infiltration profiles and tumor control. Overall, we discovered a unique HPV16 viral strategy to thwart host innate immune detection that can be further exploited to restore cancer immunogenicity.

Bcl-2 protects endothelial cells against gamma-radiation via a Raf-MEK-ERK-survivin signaling pathway that is independent of cytochrome c release.

The Bcl-2 oncoprotein is a potent inhibitor of apoptosis and is overexpressed in a wide variety of malignancies. Until recently, it was generally accepted that Bcl-2 primarily mediates its antiapoptotic function by regulating cytochrome c release from mitochondria. However, more recent studies have shown that Bcl-2 is present on several intracellular membranes and mitochondria may not be the only site where Bcl-2 exercises its survival function. In this study, we investigated if Bcl-2 can protect endothelial cells against gamma-radiation by a cytochrome c-independent signaling pathway. Human dermal microvascular endothelial cells (HDMEC), when exposed to gamma-radiation, exhibited a time-dependent activation of caspase-3 that was associated with increased cytochrome c release from mitochondria. Bcl-2 expression in endothelial cells (HDMEC-Bcl-2) significantly inhibited irradiation-induced caspase-3 activation. However, Bcl-2-mediated inhibition of caspase-3 was significantly reversed by inhibition of the Raf-mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase (ERK) kinase (MEK)-ERK pathway. Interestingly, caspase-3 activation in HDMEC-Bcl-2 cells was not associated with cytochrome c release. We also observed that endothelial cell Bcl-2 expression significantly increased the expression of survivin and murine double minute-2 (Mdm2) via the Raf-MEK-ERK pathway. Endothelial cells expressing Bcl-2 also inhibited gamma-radiation-induced activation of p38 MAPK and p53 accumulation. Inhibition of p53 accumulation in HDMEC-Bcl-2 could be due to the enhanced expression of Mdm2 in these cells. Taken together, these results show three mechanisms by which Bcl-2 may mediate endothelial cell cytoprotection independently of cytochrome c release: (a) increased survivin expression, (b) inhibition of p53 accumulation, and (c) inhibition of p38 MAPK.

Arsenic trioxide enhances the therapeutic efficacy of radiation treatment of oral squamous carcinoma while protecting bone.

Therapeutic radiation is commonly used in the treatment of squamous cell carcinoma of the oral cavity and pharynx. Despite the proven efficacy of this form of anticancer therapy, high-dose radiation treatment is invariably associated with numerous unwanted side effects. This is particularly true for bone, in which radiation treatment often leads to osteoradionecrosis. The aim of this study was to investigate if treatment with arsenic trioxide (As(2)O(3)) could enhance the antitumor effect of radiotherapy whereas minimizing the destructive effects of radiation on bone. As(2)O(3) treatment induced a dose-dependent (1-20 mumol/L) inhibition of endothelial and tumor cell (OSCC-3 and UM-SCC-74A) survival and significantly enhanced radiation-induced endothelial cell and tumor cell death. In contrast, As(2)O(3) treatment (0.5-7.5 mumol/L) induced the proliferation of osteoblasts and also protected osteoblasts against radiation-induced cell death. Furthermore, As(2)O(3) treatment was able to significantly enhance radiation-induced inhibition of endothelial cell tube formation and tumor cell colony formation. To test the effectiveness of As(2)O(3) and radiation treatment in vivo, we used a severe combined immunodeficiency mouse model that has a bone ossicle and tumor growing side by side subcutaneously. Animals treated with As(2)O(3) and radiation showed a significant inhibition of tumor growth, tumor angiogenesis, and tumor metastasis to the lungs as compared with As(2)O(3) treatment or radiation treatment alone. In contrast, As(2)O(3) treatment protected bone ossicles from radiation-induced bone loss. These results suggest a novel strategy to enhance the therapeutic efficacy of radiation treatment while protecting bone from the adverse effects of therapeutic radiation.

Endothelial cells expressing Bcl-2 promotes tumor metastasis by enhancing tumor angiogenesis, blood vessel leakiness and tumor invasion.

Metastatic spread of tumor cells to vital organs is the major cause of mortality in cancer patients. Bcl-2, a key antiapoptotic protein, is expressed at high levels in a number of human tumors. We have recently shown that Bcl-2 is also overexpressed in tumor-associated blood vessels in head-and-neck cancer patients. Interestingly, enhanced Bcl-2 expression in tumor blood vessels is directly correlated with metastatic status of these cancer patients. In addition, endothelial cells (ECs) expressing Bcl-2 showed increased production of interleukin-8 (IL-8) resulting in significantly enhanced tumor cell proliferation and tumor cell invasion. Therefore, we hypothesized that Bcl-2 expression in tumor-associated ECs may promote tumor metastasis by enhancing tumor cell invasiveness and release in the circulation. To test our hypothesis, we coimplanted tumor cells along with ECs expressing Bcl-2 (EC-Bcl-2) in the flanks of SCID mice. Our results demonstrate that incorporation of EC-Bcl-2 in primary tumors significantly enhanced tumor cell metastasis to lungs and this EC-Bcl-2-mediated tumor metastasis was independent of primary tumor size. In addition, Bcl-2-mediated tumor metastasis directly correlated with increased tumor angiogenesis. Bcl-2 expression in ECs also promoted transendothelial cell permeability, blood vessel leakiness and tumor cell invasion. EC-Bcl-2-mediated tumor cell proliferation and tumor cell invasion were significantly mediated by IL-8. These results suggest that Bcl-2, when expressed at higher levels in tumor-associated ECs, may promote tumor metastasis by enhancing tumor angiogenesis, blood vessel leakiness and tumor cell invasiveness.

Integrative and collaborative care models between pediatric oral health and primary care providers: a scoping review of the literature.

OBJECTIVES: Collaborative and/or integrative care between oral health and primary care providers can increase access to care to a more expansive population, helping to mitigate oral health related disease. The objective of this review was to present and evaluate different types of care models that exist between oral health and primary care providers in pediatric settings. METHODS: A literature search was conducted using five databases: MEDLINE/PubMed, ISI Web of Science, Dentistry and Oral Sciences Source, Cochrane Database, and EMBASE, to identify literature from January 1990 to January 2016. Combinations of controlled terms were utilized. Eligible sources targeted pediatric populations ages 1-17 and provided descriptions of existing collaborative and/or integrative models. RESULTS: Data related to the practice model, oral care provided, level of integration/collaboration and workflow were extracted. Sixteen articles were included that discussed 24 models of collaboration. These models provided ranges of services, but each offered a minimum of oral health risk assessment, oral health instruction, topical fluoride application and assessment for further treatment. These models included different levels of collaboration based off a ranking system created by the authors with 16.6 percent (4) classified as low, 54.2 percent (13) as medium and 29.2 percent (7) as high. CONCLUSIONS: Existing care models offered varying services and levels of integration and/or collaboration, but each offered a baseline of oral care. Most of these collaborations were based within Federally Qualified Health Centers and aimed to ease access to care issues.

Advancing Dental Education in the 21st Century: Phase 2 Report on Strategic Analysis and Recommendations.

In Phase 1 of the "Advancing Dental Education in the 21st Century" project, research was conducted and published on a number of serious challenges facing dental and allied dental education, both presently and projected to 2040. Those findings informed the strategic analysis and recommendations developed in Phase 2 of the project. This report provides an overview of the Phase 2 conclusions and presents recommendations to address the challenges identified. The recommendations propose ways to educate a workforce prepared to meet the oral health needs of the population; develop a sustainable economic model that allows schools to meet their education, research, and service missions; make dental and allied dental education and practice an integral part of the larger health education and delivery systems; and keep dentistry advancing as a "learned" profession. This report begins with an Executive Summary and then presents the strategic analysis of challenges facing dental schools and allied dental programs and provides a brief explanation of the rationale for each recommendation. Two appendices are included with the report: the first summarizes discussions held at the national conference to consider the recommendations; and the second provides additional documentation of calculations used to estimate the number of new dental graduates needed in 2040.

UM-HACC-2A: MYB-NFIB fusion-positive human adenoid cystic carcinoma cell line.

OBJECTIVES: Limited availability of validated human adenoid cystic carcinoma (ACC) cell lines has hindered the mechanistic understanding of the pathobiology of this malignancy and the development of effective therapies. The purpose of this work was to generate and characterize a human ACC cell line. MATERIAL AND METHODS: Immediately after surgery, a tumor fragment from a minor salivary gland from the tongue of a female Caucasian was minced, dissociated, and a single cell suspension was plated in fibronectin-coated flasks. A culture medium containing bovine brain extract and rhEGF was optimized for these cells. Whole exome sequencing was used to evaluate the presence of MYB-NFIB translocation. RESULTS: The University of Michigan-Human Adenoid Cystic Carcinoma (UM-HACC)-2A cells showed continuous growth in monolayers for at least 180 in vitro passages while maintaining epithelial morphology. Short-tandem repeat (STR) profiling confirmed a 100% match to patient DNA. Whole exome sequencing revealed the presence of the MYB-NFIB fusion in UM-HACC-2A cells, which was confirmed by PCR analysis. Western blots revealed high expression of epithelial markers (e.g. E-cadherin, EGFR, pan-cytokeratin) and proteins associated with ACC (e.g. c-Myb, p63). Developmental therapeutic studies showed that UM-HACC-2A cells were resistant to cisplatin (IC50 = 44.7 µM) while more responsive to paclitaxel (IC50 = 0.0006 µM). In a pilot study, we observed that UM-HACC-2A cells survived orthotopic transplantation into the submandibular gland. Notably, one of the mice injected with UM-HACC-2A cells exhibited lung metastasis after 6 months. CONCLUSION: UM-HACC-2A is a MYB-NFIB fusion-positive ACC cell line that is suitable for mechanistic and developmental therapeutics studies.

Mitigating SOX2-potentiated Immune Escape of Head and Neck Squamous Cell Carcinoma with a STING-inducing Nanosatellite Vaccine.

Purpose: The response rates of Head and Neck Squamous Cell Carcinoma (HNSCC) to checkpoint blockade are below 20%. We aim to develop a mechanism-based vaccine to prevent HNSCC immune escape.Experimental Design: We performed RNA-Seq of sensitive and resistant HNSCC cells to discover central pathways promoting resistance to immune killing. Using biochemistry, animal models, HNSCC microarray, and immune cell deconvolution, we assessed the role of SOX2 in inhibiting STING-type I interferon (IFN-I) signaling-mediated antitumor immunity. To bypass SOX2-potentiated STING suppression, we engineered a novel tumor antigen-targeted nanosatellite vehicle to enhance the efficacy of STING agonist and sensitize SOX2-expressing HNSCC to checkpoint blockade.Results: The DNA-sensing defense response is the most suppressed pathway in immune-resistant HNSCC cells. We identified SOX2 as a novel inhibitor of STING. SOX2 facilitates autophagy-dependent degradation of STING and inhibits IFN-I signaling. SOX2 potentiates an immunosuppressive microenvironment and promotes HNSCC growth in vivo in an IFN-I-dependent fashion. Our unique nanosatellite vehicle significantly enhances the efficacy of STING agonist. We show that the E6/E7-targeted nanosatellite vaccine expands the tumor-specific CD8+ T cells by over 12-fold in the tumor microenvironment and reduces tumor burden. A combination of nanosatellite vaccine with anti-PD-L1 significantly expands tumor-specific CTLs and limits the populations expressing markers for exhaustion, resulting in more effective tumor control and improved survival.Conclusions: SOX2 dampens the immunogenicity of HNSCC by targeting the STING pathway for degradation. The nanosatellite vaccine offers a novel and effective approach to enhance the adjuvant potential of STING agonist and break cancer tolerance to immunotherapy. Clin Cancer Res; 24(17); 4242-55. ©2018 AACR.

Quantification of human angiogenesis in immunodeficient mice using a photon counting-based method.

Testing new antiangiogenic drugs for cancer treatment requires the use of animal models, since stromal cells and extracellular matrices mediate signals to endothelial cells that cannot be fully reproduced in vitro. Most methods used for analysis of antiangiogenic drugs in vivo utilized histologic examination of tissue specimens, which often requires large sample sizes to obtain reliable quantitative data. Furthermore, these assays rely on the analysis of murine vasculature that may not be correlated with the responses of human endothelial cells. Here, we engineered human blood vessels in immunodeficient mice with human endothelial cells expressing luciferase, demonstrated that these cells line functional blood vessels, and quantified angiogenesis over time using a photon counting-based method. In a proof-of-principle experiment with PTK/ZK, a small molecule inhibitor of vascular endothelial growth factor (VEGF) tyrosine kinase receptors, a strong correlation was observed between the decrease in bioluminescence (9.12-fold) in treated mice and the actual decrease in microvessel density (9.16-fold) measured after retrieval of the scaffolds and immunohistochemical staining of endothelial cells. The method described here allows for quantitative and noninvasive investigation into the effects of anti-cancer drugs on human angiogenesis in a murine host.

Bcl-2 acts in a proangiogenic signaling pathway through nuclear factor-kappaB 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.