Suppression of beta 2 adrenergic receptor actions prevent UVB mediated cutaneous squamous cell tumorigenesis through inhibition of VEGF-A induced angiogenesis.

Although beta 2 adrenergic receptors (ß2 ADR) are present in the keratinocytes, their role in cutaneous squamous cell tumorigenesis needs to be ascertained. For the first time, we report here that selective ß2 ADR antagonists by inhibiting ß2 ADR actions significantly retarded the progression of ultraviolet B (UVB) induced premalignant cutaneous squamous cell lesions. These antagonists acted by inhibiting vascular endothelial growth factor-A (VEGF) mediated angiogenesis to prevent UVB radiation-induced squamous cell carcinoma of the skin.

Positron Emission Tomographic Imaging Elucidates the Complex Relationship Between Glucose Uptake and Tissue Blood Flow Mechanism in Squamous Cell Oral Cancer Patients.

BACKGROUND Through the clinical use of positron emission tomography, we aimed to elucidate the complex relationship between glucose uptake and squamous cell oral cancer (ScOC) growth, along with its mechanism with respect to tissue blood flow (tBF). MATERIAL AND METHODS We retrospectively reviewed a total of 69 newly diagnosed ScOC patients by Fluorine-18 fluorodeoxyglucose (18F-FDG) positron emission tomography (PET). Maximum and mean standard uptake values (SUV↑ and SUV) were recorded to assess glucose uptake. Multi-shot spin-echo echo-planar imaging-based pseudo-continuous arterial spin labeling (pcASL) technique at 3.0 T MRI was used to obtain tBF values in ScOC (tBF-ScOC). Patients were divided according to T-stage and location. Pearson's correlation coefficients were calculated between both SUV and tBF-ScOC for significant correlations. RESULTS Forty-one (59.4%) patients had oropharynx and the other 28 (40.6%) patients had laryngopharynx. Significant positive correlations were detected between SUV↑, SUV, tBF-ScOC and non-advanced T-stage (T1a, T1b, T2 and T3), while a negative correlation was observed in the advanced T-stage (T4a and T4b). CONCLUSIONS Using PET imaging, we established the relationship between glucose uptake and ScOC growth on the basis of the division of T-stage and tumor location of ScOC, thereby elucidating the underlying mechanism. Our findings provide insights important to the diagnosis, treatment, and care of ScOC patients.

Blueberry inhibits invasion and angiogenesis in 7,12-dimethylbenz[a]anthracene (DMBA)-induced oral squamous cell carcinogenesis in hamsters via suppression of TGF-ß and NF-κB signaling pathways.

Aberrant activation of oncogenic signaling pathways plays a pivotal role in tumor initiation and progression. The purpose of the present study was to investigate the chemopreventive and therapeutic efficacy of blueberry in the hamster buccal pouch (HBP) carcinogenesis model based on its ability to target TGF-ß, PI3K/Akt, MAPK and NF-κB signaling and its impact on invasion and angiogenesis. Squamous cell carcinomas were induced in the HBP by 7,12-dimethylbenz[a]anthracene (DMBA). The effect of blueberry on the oncogenic signaling pathways and downstream events was analyzed by quantitative real-time PCR and immunoblotting. Experiments with the ECV304 cell line were performed to explore the mechanism by which blueberry regulates angiogenesis. Blueberry supplementation inhibited the development and progression of HBP carcinomas by abrogating TGF-ß and PI3K/Akt pathways. Although blueberry failed to influence MAPK, it suppressed NF-κB activation by preventing nuclear translocation of NF-κB p65. Blueberry also modulated the expression of the oncomiR miR-21 and the tumor suppressor let-7. Collectively, these changes induced a shift to an anti-invasive and anti-angiogenic phenotype as evidenced by downregulating matrix metalloproteinases and vascular endothelial growth factor. Blueberry also inhibited angiogenesis in ECV304 cells by suppressing migration and tube formation. The results of the present study suggest that targeting oncogenic signaling pathways that influence acquisition of cancer hallmarks is an effective strategy for chemointervention. Identification of modulatory effects on phosphorylation, intracellular localization of oncogenic transcription factors and microRNAs unraveled by the present study as key mechanisms of action of blueberry is critical from a therapeutic perspective.

In Vivo Correlation of Glucose Metabolism, Cell Density and Microcirculatory Parameters in Patients with Head and Neck Cancer: Initial Results Using Simultaneous PET/MRI.

OBJECTIVE: To demonstrate the feasibility of simultaneous acquisition of 18F-FDG-PET, diffusion-weighted imaging (DWI) and T1-weighted dynamic contrast-enhanced MRI (T1w-DCE) in an integrated simultaneous PET/MRI in patients with head and neck squamous cell cancer (HNSCC) and to investigate possible correlations between these parameters. METHODS: 17 patients that had given informed consent (15 male, 2 female) with biopsy-proven HNSCC underwent simultaneous 18F-FDG-PET/MRI including DWI and T1w-DCE. SUVmax, SUVmean, ADCmean, ADCmin and Ktrans, kep and ve were measured for each tumour and correlated using Spearman's ρ. RESULTS: Significant correlations were observed between SUVmean and Ktrans (ρ = 0.43; p ≤ 0.05); SUVmean and kep (ρ = 0.44; p ≤ 0.05); Ktrans and kep (ρ = 0.53; p ≤ 0.05); and between kep and ve (ρ = -0.74; p ≤ 0.01). There was a trend towards statistical significance when correlating SUVmax and ADCmin (ρ = -0.35; p = 0.08); SUVmax and Ktrans (ρ = 0.37; p = 0.07); SUVmax and kep (ρ = 0.39; p = 0.06); and ADCmean and ve (ρ = 0.4; p = 0.06). CONCLUSION: Simultaneous 18F-FDG-PET/MRI including DWI and T1w-DCE in patients with HNSCC is feasible and allows depiction of complex interactions between glucose metabolism, microcirculatory parameters and cellular density.

Monitoring microcirculatory alterations in oral squamous cell carcinoma following photodynamic therapy.

BACKGROUND: One of the mechanisms through which photodynamic therapy (PDT) is thought to elicit tumour destruction is by producing microvascular damage and obstruction of nutritive blood flow. The aim of this study was to directly monitor and quantify microcirculatory changes following tissue illumination by PDT for oral squamous cell carcinoma. METHODS: Ten consecutive patients receiving PDT for a carcinoma in situ, a T1 or T2 tumour in the oral cavity without evidence of lymph node metastasis were selected for this study. Tumour and marginal healthy mucosa total capillary density (TCD) and functional capillary density (FCD) inside the field of illumination were measured and compared using sidestream dark-field (SDF) imaging prior to tissue illumination, immediately after PDT, and again after 15min. RESULTS: Baseline mean tumour TCD was 21.2±5capillaries per square millimetres (cpll/mm²) and 24.9±19cpll/mm² in the surrounding marginal healthy tissue; there were no significant differences between tumour and healthy tissue or time points. Comparisons between baseline and post-illumination time points revealed significant differences in both tumour and healthy tissue FCD (P<0.05). No significant differences in FCD were observed between the two tissues. CONCLUSIONS: Our findings using SDF imaging demonstrate that PDT significantly attenuates tumour and marginal healthy tissue perfusion by directly disrupting the functionality of the microcirculation.

Apelin expression in human non-small cell lung cancer: role in angiogenesis and prognosis.

INTRODUCTION: The recently discovered bioactive peptide, apelin, has been demonstrated to stimulate angiogenesis in various experimental systems. However, its clinical significance and role in tumor vascularization have not yet been investigated in a human malignancy. Therefore, our aim was to study whether apelin expression is associated with angiogenesis and/or tumor growth/behavior in human non-small cell lung cancer (NSCLC). METHODS: A total of 94 patients with stage I-IIIA NSCLC and complete follow-up information were included. Apelin expression in human NSCLC samples and cell lines was measured by quantitative reverse-transcriptase polymerase chain reaction, enzyme-linked immunosorbent assay, and immunohistochemistry. Effects of exogenous apelin and apelin transfection were studied on NSCLC cell lines in vitro. In vivo growth of tumors expressing apelin or control vectors were also assessed. Morphometric variables of human and mouse tumor capillaries were determined by anti-CD31 labeling. RESULTS: Apelin was expressed in all of the six investigated NSCLC cell lines both at the mRNA and protein levels. Although apelin overexpression or apelin treatments did not increase NSCLC cell proliferation in vitro, increasing apelin levels by gene transfer to NSCLC cells significantly stimulated tumor growth and microvessel densities and perimeters in vivo. Apelin mRNA levels were significantly increased in human NSCLC samples compared with normal lung tissue, and high apelin protein levels were associated with elevated microvessel densities and poor overall survival. CONCLUSIONS: This study reveals apelin as a novel angiogenic factor in human NSCLC. Moreover, it also provides the first evidence for a direct association of apelin expression with clinical outcome in a human cancer.

VEGF and VEGFRs expression in oral squamous cell carcinoma.

Oral cancer is an important cause of worldwide morbidity and mortality, with substantial economic, physiological, and psychosocial impacts due to its treatment modality and a great risk for recurrences and second primary oral squamous cell carcinomas (OSCC) development. Therefore, it is very important to understand the underlying cell biology of such tumors. It is now a well-accepted fact that angiogenesis is essential for the growth and metastasis of solid tumors, including head and neck squamous cell carcinoma. The main factor responsible for angiogenesis is VEGF and its receptors. It has been demonstrated that VEGFRs are also present on tumor cells themselves and other cells from the tumor microenvironment, in addition to tumoral endothelial cells (ECs). Therefore between these cells take place numerous and different interactions mediated via paracrine/autocrine pathways that promote angiogenesis, uncontrolled tumor proliferation and metastasation. In consequence, estimation of VEGF expression and its receptors became a reliable prognostic tool in OSCCS, predicting the poor disease-free survival, poor overall survival, and metastatic disease. Understanding the distribution and role of VEGF and its receptors in the progression of OSCC will be essential to the development and design of new therapeutic strategies.

RhoC expression and head and neck cancer metastasis.

RhoC protein, a known marker of metastases in aggressive breast cancers and melanoma, has also been found to be overexpressed in certain head and neck cancers, thus we investigated the correlation between RhoC expression and the metastatic behavior of head and neck squamous cell carcinoma. Selective inhibition of RhoC expression was achieved using lentiviral small hairpin RNA (shRNA) transduced and tracked with green fluorescent protein to achieve 70% to 80% RhoC inhibition. Fluorescence microscopy of the RhoC knockdown stable clones showed strong green fluorescence in the majority of cells, signifying a high efficiency of transduction. Importantly, quantitative real-time PCR showed no significant decrease in the mRNA expression levels of other members of the Ras superfamily. Cell motility and invasion were markedly diminished in RhoC-depleted cell lines as compared with control transduced lines. H&E staining of lung tissue obtained from severe combined immunodeficiency mice, which had been implanted with RhoC knockdown cells, showed a marked decrease in lung metastasis and inflammation of the blood vessels. The cultured lung tissue showed a significant decrease in cell growth in mice implanted with RhoC-depleted cell lines as compared with shRNA-scrambled sequence control lines. Microscopic studies of CD31 expression revealed substantial quantitative and qualitative differences in the primary tumor microvessel density as compared with parental and shRNA-scrambled controls. This study is the first of its kind to establish the involvement of RhoC specifically in head and neck metastasis. These findings suggest that RhoC warrants further investigation to delineate its robustness as a novel potentially therapeutic target.

Pilot study on confocal endomicroscopy for determination of the depth of squamous cell esophageal cancer in vivo.

BACKGROUND AND AIM: Confocal endomicroscopy is ultra-high-magnification endoscopy with histological observation during ongoing endoscopy. We planned a pilot study of the diagnosis of the depth of esophageal cancer using confocal endomicroscopy for treatment strategies. METHODS: Patients had 14 superficial esophageal cancers and one dysplasia. The depth of neoplasms in 15 lesions was confirmed by endoscopic mucosal resection or surgery. We examined the rate of delineation and compared results of confocal imaging with histological findings. We classified two cellular and three microvascular patterns on confocal endomicroscopic images: CP-N for normal squamous mucosa and CP-Ca for cancerous lesion; VP-type A for normal squamous mucosa; VP-type B for T1a-EP and T1a-LPM cancers; and VP-type C for T1a-MM or a more invasive cancer pattern. We measured diameters of microvessels for the three patterns of confocal endomicroscopic images and histological specimens. RESULTS: The rate of delineation was 73.3% (11/15) for esophageal cancer. The results of confocal imaging coincided well with microvessel distribution on horizontal histology. Two endoscopists blindly diagnosed the two types by cellular pattern and the three types by vascular pattern: their overall accuracies were 96% and 89% for the cellular pattern and 85% and 85% for the vascular pattern, respectively. The k value of the cellular pattern and the vascular pattern diagnosis was 0.84 and 0.75, respectively. CONCLUSION: Scoring and quantification of confocal endomicroscopic images may be useful for the differential diagnosis and diagnosis of superficial invasion by squamous cell carcinoma.

VEGF-C and VEGF-D expression is correlated with lymphatic vessel density and lymph node metastasis in oral squamous cell carcinoma: Implications for use as a prognostic marker.

The prognosis of patients with oral squamous cell carcinoma (SCC) is influenced by the presence of lymph node metastasis. In this study, we analyzed the relationship between lymphangiogenesis and the expression of VEGF-C and VEGF-D in association with lymph node metastasis in oral SCC. Oral SCC biopsy specimens (160 cases) were examined for lymphatic vessel density (LVD) and the expression of VEGF-C and VEGF-D immunohistochemically. The levels of VEGF-C and VEGF-D expression and LVD were significantly associated with lymph node metastasis (p<0.001). The expression of VEGF-C and VEGF-D increased the LVD significantly (p<0.001). Multivariate analysis showed that VEGF-C expression and LVD were significantly associated with lymph node metastasis (p<0.001). This study presents clinical evidence for the important roles of VEGF-C and VEGF-D in lymphangiogenesis and lymphatic metastasis of oral SCC, and suggests that VEGF-C or LVD can effectively predict lymphatic metastasis of oral SCC.

Tetra-arsenic oxide (Tetras) enhances radiation sensitivity of solid tumors by anti-vascular effect.

Tetras (tetra-arsenic oxide, As(4)O(6)) is a derivative of arsenic used in Korean traditional medicine for the treatment of cancer, but its mechanism remains largely undefined. Recently, a similar arsenic derivative, diarsenic trioxide (As(2)O(3), ATO), has been shown to mediate anti-tumor activity, therefore reigniting interest in the therapeutic effect of arsenic compounds. Here we report that Tetras can effectively mediate an anti-vascular effect on tumors, leading to delay in tumor growth and increased survival. Our study demonstrates for the first time the potential use of Tetras as a radiation therapy enhancement agent for solid tumors. These findings reveal an unappreciated role of Tetras in cancer therapy and its potential application to radiotherapy in achieving local tumor control.

A Phase II study of SU5416 in patients with advanced or recurrent head and neck cancers.

BACKGROUND: SU5416 (semaxanib) is a synthetic small molecule inhibitor of the tyrosine kinase domain of vascular endothelial growth factor receptor 2 (VEGFR2). This Phase II study was conducted to determine the safety and efficacy of SU5416 in patients with recurrent or metastatic head and neck cancers. PATIENTS AND METHODS: This was an open label, single arm, Phase 2 study for patients who had received no more than 2 cytotoxic regimens. Thirty-five patients received intravenous SU5416 (145 mg/m(2)) twice per week by intravenous catheter. Radiologic imaging for response assessment was planned at the conclusion of each 8 week cycle. Serum VEGF levels and power Doppler ultrasound tumor imaging were explored as potential surrogate markers for SU5416 activity. RESULTS: Thirty-two patients had received prior radiotherapy, including 18 patients who received prior concurrent chemoradiotherapy. Twelve patients had received prior chemotherapy in the recurrent disease setting. Thirty-one patients were evaluable for response. There was one partial response and one minor response. The median number of 8-week cycles received was 1 (range 1-4). Median overall survival was 6.25 months. The most common > or = grade 3 toxicity was headache (31%). There was one fatal carotid artery hemorrhage. Fatigue, nausea, and vomiting were common grade 1-2 adverse events. Power Doppler ultrasound demonstrated decreased tumor vascularity in 5 of 7 patients. CONCLUSIONS: Treatment with SU5416 in patients with head and neck cancers is feasible, but objective responses are rare. Studies evaluating more potent anti-angiogenic agents in this disease are of interest.

IFN-alpha induced adenosine production on the endothelium: a mechanism mediated by CD73 (ecto-5'-nucleotidase) up-regulation.

CD73 (ecto-5'-nucleotidase; EC 3.1.3.5) participates in lymphocyte binding to endothelial cells and converts extracellular AMP into a potent anti-inflammatory substance adenosine. However, the regulation of expression and function of CD73 has remained largely unknown. In this study, we show that IFN-alpha produces a time- and dose-dependent long-term up-regulation of CD73 on endothelial cells, but not on lymphocytes both at protein and RNA levels. Moreover, CD73-mediated production of adenosine is increased after IFN-alpha treatment on endothelial cells, resulting in a decrease in the permeability of these cells. Subsequent to induction with PMA, FMLP, dibutyryl cAMP, thrombin, histamine, IL-1beta, TNF-alpha, and LPS, no marked changes in the level of CD73 expression on endothelial cells are observed. We also show that CD73 is up-regulated in vivo on the vasculature after intravesical treatment of urinary bladder cancers with IFN-alpha. In conclusion, distinct behavior of lymphocyte and endothelial CD73 subsequent to cytokine treatment further emphasizes the existence of cell type-specific mechanisms in the regulation of CD73 expression and function. Overall, these results suggest that IFN-alpha is a relevant in vivo regulator of CD73 in the endothelial-leukocyte microenvironment in infections/inflammations, and thus has a fundamental role in controlling the extent of inflammation via CD73-dependent adenosine production.

Studying the growth of cervical carcinoma nests and angiogenesis by immunostaining, quantitation and three-dimensional structural analysis.

OBJECTIVE: To analyze the relationship and mutual effect of the growth of cervical carcinoma nests and angiogenesis. STUDY DESIGN: Serial paraffin sections of cervical squamous carcinoma were stained in repeated order with hematoxylin-eosin (HE), immunostain for factor VIII-related antigen, type IV collagen and proliferating cell nuclear antigen (PCNA). Three-dimensional reconstructions were made, and the volumes of carcinoma nests, necrosis and microvessels were measured. RESULTS: Two types of cervical carcinoma nests were distinguished on the basis of their growth characteristics and vascularity (groups I and II). Group I nests: The carcinoma cells were proliferating actively, as determined by their morphology and PCNA staining. There were abundant microvessels. Some endothelial sprouts and cords penetrated the nests and then formed networks and new vessels. The volume ratio of microvessels, including sprouts and cords, to the nests was 0.6282:1. Group II nests: The center of these nests underwent necrosis. The peripheral cells were rather small, with no mitosis. The PCNA index was rather low; these nests grew very slowly. There were only a few surrounding microvessels with no endothelial sprouts or cords. The volume ratio of vessels to nest was 0.0657:1. CONCLUSION: Two types of cervical carcinoma nests show a close relationship and mutual effect of the growth of carcinoma nest and angiogenesis. Group I nests grow and develop well, with abundant microvessels. Thus, many tumor cells may be angiogenic and induce angiogenesis; growth of the nests seemed dependent on adequate neovascularization. Group II nests grow slowly, with a few microvessels; the center of the nests undergoes necrosis. The inadequate blood supply must be one of the important causes of necrosis. Considering that there must have been abundant neovascularization during their growth in the past, most of the microvessels must have been obliterated and then reabsorbed to make the remaining vessels so few.

Cross-species comparison of angiogenesis during the premalignant stages of squamous carcinogenesis in the human cervix and K14-HPV16 transgenic mice.

Infection of the human cervix with certain papillomavirus subtypes is associated with the development of neoplastic squamous lesions that can progress to overt cervical malignancies. Recently, multistage squamous carcinogenesis has been achieved in K14-HPV16 transgenic mice, wherein expression of the human papillomavirus (HPV) type 16 early genes is targeted to basal squamous epithelial cells by regulatory elements of the human keratin-14 (K14) promoter. Immunostaining of the endothelial marker vWf revealed a parallel upregulation of angiogenesis during the early neoplastic stages in both human cervix and the epidermis of K14-HPV16 transgenic mice. Moreover, high-grade premalignant lesions and cancers in humans and transgenic mice were characterized by an additional increment in the number of new capillaries and close apposition of the microvasculature to the overlying neoplastic epithelium. Expression of the potent angiogenic factor VEGF was progressively up-regulated during carcinogenesis in both species, correlating with the increased density and altered distribution of the microvasculature. Thus, angiogenesis occurs during the premalignant stages of squamous carcinogenesis in both human cervical disease and a relevant transgenic model and may be controlled by similar molecular mechanisms in both species. These results validate the use of the transgenic model to elucidate the role of angiogenesis during HPV-associated neoplastic progression.