Expression of SIRT2 and SIRT6 in retinoblastoma.

PURPOSE: SIRT2 and SIRT6 are members of the sirtuin family and are associated with cancer development and progression in certain tumours, but their expression in retinoblastoma has not been studied. The primary objective of our study was to determine the expression of SIRT2 and SIRT6 in human retinoblastoma cases. METHODS: Eighteen formalin-fixed paraffin-embedded blocks of retinoblastoma cases from the Ocular Pathology Registry at the Henry C. Witelson Ocular Pathology Laboratory were obtained, classified and immunostained for SIRT2 and SIRT6 using mouse monoclonal antibodies. RESULTS: Sixteen cases were poorly differentiated retinoblastoma cases. SIRT2 and SIRT6 were expressed in all cases of retinoblastoma although differences in the staining intensity were found between cases. SIRT2 and SIRT6 expression was also observed in various normal structures of the remaining ocular tissue. CONCLUSIONS: SIRT2 and SIRT6 are expressed in retinoblastoma, as well as in some normal ocular structures. While precise roles of these proteins must still be determined in retinoblastoma, their expression profiles suggest that further functional studies of both SIRT2 and SIRT6 should be pursued in this cancer.

Expression of the metastasis suppressor BRMS1 in uveal melanoma.

AIMS: To determine the expression of breast metastasis suppressor 1 (BRMS1) in human uveal melanoma (UM) tissues and cell lines. In addition, we intend to establish a possible association between BRMS1 expression and the presence of metastatic disease. METHODS: Thirty-one formalin-fixed paraffin-embedded tissues from enucleated eyes of patients with UM were immunostained. Clinical-pathological data were obtained, including age, tumour location, largest dimension, cell type, and occurrence of metastasis. The expression of BRMS1 mRNA in four human UM cell lines was determined by real-time reverse transcriptase polymerase chain reaction, and protein expression was assessed by immunocytochemistry and western blot. The association between BRMS1 immunostaining and location, largest tumour dimension, and tumour cell type was determined using the correlation coefficient test. The association between BRMS1 immunostaining and the incidence of metastasis was assessed using Kaplan-Meier analysis. RESULTS: Of the 31 cases of UM, 24 (77.42%) stained positive and seven (22.58%) negative for BRMS1. From the positively stained tumours, 21 (87.50%) showed cytoplasmatic staining. Macrophages were usually positive when present in the tumour and staining intensity was generally higher than in UM cells. BRMS1 mRNA was present in all four human UM cell lines, as well as cytoplasmatic immunoexpression of BRMS1. Immunoblotting showed variable BRMS1 protein levels between the different cell lines. No statistically significant correlation was found between BRMS1 protein expression and survival (P = 0.69), tumour cell type (P = 0.68), largest tumour dimension (P = 0.75), and tumour location (P = 0.11). CONCLUSIONS: BRMS1 is expressed in UM both at the mRNA and protein level; however, neither was associated with any of the prognosticor outcome parameters that we tested.

Rapid growth of an orbital hemangiopericytoma with atypical histopathological findings.

Hemangiopericytoma is a rare vascular tumor that originates from pericytes. The orbit is a rare location for this particular tumor, and corresponds to 0.8% to 3% of all primary orbital tumors. We report a case of a hemangiopericytoma in a 45-year-old man that had an unusual presentation, as a rapidly growing mass in the anterior right inferior orbit. Given that there are no clinical or radiological signs pathognomonic of this tumor, a careful histopathological examination is necessary to confirm the diagnosis. In our case, it presented also with unusual histopathological findings. The clinical features, radiological findings, differential diagnosis and treatment of this challenging entity are reviewed in this case report.

Expression of the Metastasis Suppressor KAI1 in Uveal Melanoma.

Introduction. Uveal melanoma (UM) is an intraocular tumor that leads to metastatic disease in approximately 50% of afflicted patients. There is no efficacious treatment for metastatic disease in this cancer. Identification of markers that can offer prognostic and therapeutic value is a major focus in this field at present. KAI1 is a metastasis suppressor gene that has been reported to play a role in various human malignancies, although it has not previously been evaluated in UM. Purpose. To investigate the expression of KAI1 in UM and its potential value as a prognostic marker. Materials and Methods. 18 cases of human primary UM were collected and immunostained for KAI1 expression. A pathologist evaluated staining intensity and distribution semiquantitatively. Each case was categorized as group 1 (low staining) or group 2 (high staining). Results. In group 2, two of the 12 cases presented with metastasis. Conversely, in group 1, five out of 6 cases had metastasis. The mean follow-up of patients who did not develop metastasis was 81.81 months (median: 75 months) versus 42.14 months (median: 44 months) for patients with metastasis. Conclusions. KAI1 is a promising candidate marker that may offer prognostic value in UM; it may also represent a therapeutic target in metastatic disease.

Expression of SIRT1 in choroidal neovascular membranes.

PURPOSE: SIRT1 is a deacetylase that has been shown to be instrumental in embryonic and pathologic vascular formation. The purpose of this study was to evaluate the potential role of SIRT1 in the pathogenesis of choroidal neovascularization in age-related macular degeneration. METHODS: The expression of SIRT1 was assessed via immunohistochemistry in nine excised human choroidal neovascularization membranes and seven non-age-related macular degeneration donor eyes. Enzyme-linked immunosorbent assay-based angiogenesis arrays were used to assess the potential of an SIRT1 inhibitor, nicotinamide, to reduce secretion of 10 unique proangiogenic cytokines from retinal pigment epithelial cells. RESULTS: SIRT1 was expressed more frequently in choroidal neovascularization membranes than donor eyes about vascular endothelial cells (78 vs. 29% positive cases) and retinal pigment epithelial cells (57 vs. 14% positive cases). SIRT1 inhibition in retinal pigment epithelial cells correlated with significantly decreased secretion of three potent proangiogenic cytokines: angiogenin, platelet-derived growth factor BB, and vascular endothelial growth factor A. CONCLUSION: SIRT1 levels appear elevated in human choroidal neovascularization membranes compared with control eyes. Moreover, inhibition of SIRT1 activity is correlated with decreased secretion of potent proangiogenic cytokines. Collectively, these data support a potential role for SIRT1 in the pathogenesis of neovascular age-related macular degeneration.

Expression of SIRT1 and DBC1 in Developing and Adult Retinas.

Sirtuin 1 (SIRT1) is a deacetylase that can regulate various biological processes via repression of transcription. Its activity has been linked to the differentiation of neural progenitor cells, although little is known about its function during retinal development. The study described herein was undertaken to evaluate the expression of SIRT1 and its innate inhibitor, DBC1, in retinal tissues and progenitor cells. We found both SIRT1 and DBC1 to be widely expressed in mouse and human retinas, with subtle differences in subcellular distribution of each protein. We further demonstrate that nuclear-localized SIRT1 is only seen in human-derived retinal progenitor cells and not in adult retinas, suggesting that this nuclear localization may be important in retinal development. Moreover, we observed cytoplasmic DBC1 in a subset of progenitor cells as well as in mature ganglion cells, indicating that the progenitor cell subset, which was comprised predominantly of small cells, may represent a population of ganglion cell precursors. Collectively, the data presented in this study provide support for SIRT1 and DBC1 as regulators of retinal development and normal retinal physiology.

Choroidal neovascular membranes express toll-like receptor 3.

BACKGROUND: Recent evidence has suggested a role for toll-like receptor 3 (TLR3) in experimental models of age-related macular degeneration (AMD). To date, however, few data exist about TLR3 in human AMD. The purpose of this study was to investigate the expression of TLR3 in human choroidal neovascular (CNV) membranes. METHODS: Immunostaining for TLR3 was performed on sections of CNV membranes from 8 AMD patients and eyes from 4 donors without CNV. RESULTS: All CNV membranes expressed TLR3 in retinal pigment epithelial (RPE) cells. One was classified as having strong intensity, 5 as having moderate intensity and 2 as having weak intensity. All cases had ≥30% of the RPE cells staining for TLR3, ranging from 30 to 90%. No expression of TLR3 was observed in vascular endothelial cells or fibroblasts in any CNV membrane. In the donor eyes, the RPE cells near the ora serrata stained stronger than those at the posterior pole, where no staining was observed in 3 out of 4 cases. CONCLUSION: TLR3 was found in all CNV membranes and was expressed exclusively in RPE cells. The observed difference in RPE staining for TLR3 in donor eyes and CNV membranes suggests a possible role for this receptor in human neovascular AMD.

SPARC is expressed in human uveal melanoma and its abrogation reduces tumor cell proliferation.

BACKGROUND: Secreted protein acidic and rich in cysteine (SPARC) has been shown to play an integral role in the progression of numerous malignancies. The aim of this study was to investigate the expression of SPARC in uveal melanoma (UM). MATERIALS AND METHODS: SPARC expression was assessed in UM cell lines using RT-PCR and immunocytochemistry. Small interfering RNA directed against SPARC was used to transfect each of the cell lines, which were subsequently run in proliferation assays. SPARC expression was further investigated in 19 cases of human UM and 11 primary and 8 metastatic tumors from a rabbit xenograft model. RESULTS: The cell lines transfected with SPARC siRNA showed a significant decrease in proliferation compared to controls. All cases of human uveal melanoma demonstrated positive staining for SPARC as did all primary and metastatic tumors from the xenograft model. CONCLUSION: SPARC may represent a novel target to inhibit growth of UM.

Expression of cyclooxygenase-2 in choroidal neovascular membranes from age-related macular degeneration patients.

PURPOSE: The objective of this study was to investigate the expression of cyclooxygenase (COX)-2 in human choroidal neovascular membranes. METHODS: Paraffin-embedded sections of choroidal neovascular membranes excised from 16 patients with wet age-related macular degeneration were used for this study. Sections were subjected to immunohistochemistry using a monoclonal mouse antihuman COX-2 antibody. Staining was classified as either negative or positive in retinal pigment epithelial cells, vascular endothelial cells, and fibroblasts. Serial sections were stained for vimentin expression to confirm tissue antigenicity. RESULTS: Eleven of 16 (69%) choroidal neovascular membranes stained positive for COX-2 in retinal pigment epithelial cells, with 6 (38%) of these also expressing COX-2 in vascular endothelial cells and 6 (38%) in fibroblasts. None of the sections that were negative for COX-2 in the retinal pigment epithelial cells showed COX-2 expression in the other cell types assessed. There was a statistically significant difference (P = 0.0097) in the mean ages between the COX-2 positive group (65.6 years) and COX-2 negative group (76.8 years) as determined by a two-tailed, unpaired Student's t-test. CONCLUSION: The expression of COX-2 in human choroidal neovascular membranes suggests a possible role for this modulator in age-related macular degeneration pathogenesis. The age-dependent expression observed is novel and warrants further investigation.

Single-cell tumor dormancy model of uveal melanoma.

BACKGROUND: Ocular melanoma is easily treated by the removal of the eye or through plaque radiotherapy. However, after removal or control of the primary tumor, patients can develop fatal liver metastases up to 20 years later. It has been reported that difficulties in imaging single cells and the propensity for tumor cells to replicate rapidly in animal models account for the deficit of single-cell tumor dormancy models. METHODS: In this paper, we performed two animal experiments using green fluorescent-labeled uveal melanoma cells in nude mice. Cells were injected via tail-vein and the experiments ran 20 and 42 days, respectively. Labeled cells were imaged in vivo via skin-flap and epifluorescent microscopy. RESULTS: The first experiment exemplified the feasibility of a single-cell tumor dormancy model; cells were present in multiple organs post-injection, but persisted solely in the liver for the duration of the experiment. The second experiment, demonstrating the presence and viability of these single, metastatic seeds 6 weeks after injection. CONCLUSION: Due to the inherent difficulties in establishing single-celled tumor dormancy models, few exist. In this paper, we have successfully developed a single-cell dormancy model of uveal melanoma, a disease that, in patients, epitomizes tumor dormancy. This model has the potential to reveal the mechanisms behind dormancy, identify patients at high risk for metastatic development, and develop new serum biomarkers for micrometastasis detection.

The use of a cyclooxygenase-2 inhibitor (Nepafenac) in an ocular and metastatic animal model of uveal melanoma.

The expression of cyclooxygenase-2 (COX-2) has been reported as an indicator of poor prognosis in a wide variety of human tumors, including colon, breast and uveal melanoma (UM). COX-2 inhibitors have shown promise in controlling the malignancy of several types of tumors. Previous studies have demonstrated the efficacy of a COX-2 inhibitor on the proliferation rates of human UM cells. The goal of this experiment was to investigate the efficiency of Nepafenac, a topically administered COX-2 inhibitor, in a rabbit model of UM. The animals were divided into two groups of 14 animals for the duration of the 12-week experiment. One animal per group was killed each week to evaluate disease progression and for histopathological studies. The experimental group received drops containing 0.3% Nepafenac solution. Intraocular tumor growth was evaluated weekly by fundoscopic examination and each animal was weighed prior to examination. Blood samples were taken weekly from all rabbits to detect circulating malignant cells (CMCs) throughout the experiment. After the second week of inoculation, the experimental group weighed significantly more than the control group. The control group developed more intraocular tumors and presented with metastases and higher detectable levels of CMCs before the treated group. These results indicate that the topical administration of a COX-2 inhibitor delayed the progression of this malignancy in our animal model. A clinical trail using an anti-COX-2 inhibitor for patients with UM should be considered.

Expression and migratory analysis of 5 human uveal melanoma cell lines for CXCL12, CXCL8, CXCL1, and HGF.

BACKGROUND: The aim of this study was to characterize the presence and roles of CXCL12, CXCL8, CXCL1, and HGF in five human uveal melanoma cell lines, using different methods, in order to ascertain their significance in this disease. METHODS: Five human uveal melanoma cell lines (92.1, SP6.5, MKT-BR, OCM-1, and UW-1) of known proliferative, invasive, and metastatic potential were used in this experiment. A migration assay was used in order to assess the responsiveness of each cell line towards the four chosen chemotactic factors. Immunohistochemistry was then performed for all five cell lines (cytospins) using antibodies directed toward CXCL1, CXCL8 and their receptors CXCR2 and CXCR1 respectively. Quantitative real-time PCR was then performed on all five cell lines in order to establish the presence of these four chemotactic factors. RESULTS: All five human uveal melanoma cell lines migrated towards the four chosen chemotactic factors at a level greater than that of the negative control. Chemokines CXCL1 and CXCL8 resulted in the greatest number of migrating cells in all five of our cell lines. Immunohistochemistry confirmed the expression of CXCL1, CXCL8, and their receptors CXCR2 and CXCR1 in all five of the cell lines. Quantitative real-time PCR results established expression of CXCL8, CXCL1, and HGF in all 5 cell lines tested. CXCL1 and CXCL8 are highly expressed in SP6.5 and UW-1. None of the five cell lines expressed any detectable levels of CXCL12. CONCLUSION: The migratory ability of the 5 human uveal melanoma cell lines was positively influenced by the four chemotactic factors tested, namely CXCL12, CXCL8, CXCL1, and HGF. Self-expression of chemotactic factors CXCL8, CXCL1, and HGF may indicate an autocrine system, which perhaps contributes to the cells' metastatic ability in vivo.