Differential Effects of Benzalkonium Chloride on Human Trabecular Meshwork Cells Not Treated or Treated with Transforming Growth Factor-ß2 or Dexamethasone.

Purpose: The objective of the present study was to evaluate the effects of low concentrations of benzalkonium chloride (BAC) (10-7%, 10-6%, or 10-5%) on healthy and glaucomatous human trabecular meshwork (HTM) cells. For this purpose, we used in vitro models replicating a healthy HTM and HTM with primary open-angle glaucoma (POAG) or steroid-induced glaucoma (SG) using two-dimensional (2D) cultures of HTM cells not treated or treated with a 5 ng/mL solution of transforming growth factor-ß2 or 250 nM dexamethasone (DEX). Methods: Analyses were carried out for (1) the intercellular affinity function of 2D HTM monolayers, as determined by transepithelial electrical resistance (TEER) measurements; (2) cell viability; (3) cellular metabolism by using a Seahorse bioanalyzer; and (4) expression of extracellular matrix (ECM) molecules, an ECM modulator, and cell junction-related molecules. Results: In the absence and presence of BAC (10-7% or 10-5%), intercellular affinity function determined by TEER and cellular metabolic activities were significantly and dose dependently affected in both healthy and glaucomatous HTM cells despite the fact that there was no significant decrease in cell viabilities. However, the effects based on TEER values were significantly greater in the healthy HTM. The mRNA expression of several molecules that were tested was not substantially modulated by these concentrations of BAC. Conclusions: The findings reported herein suggest that low concentrations of BAC may have unfavorable adverse effects on cellular metabolic capacity by inducing increases in the intercellular affinity properties of the HTM, but those effects of BAC were different in healthy and glaucomatous HTM cells.

Significant and Various Effects of ML329-Induced MITF Suppression in the Melanoma Cell Line.

To study the inhibitory effects on microphthalmia-associated transcription factor (MITF)-related biological aspects in malignant melanomas (MMs) in the presence or absence of the low-molecular MITF specific inhibitor ML329, cell viability, cellular metabolic functions, and three-dimensional (3D) spheroid formation efficacy were compared among MM cell lines including SK-mel-24, A375, dabrafenib- and trametinib-resistant A375 (A375DT), and WM266-4. Upon exposure to 2 or 10 µM of ML329, cell viability was significantly decreased in WM266-4, SK-mel-24, and A375DT cells, but not A375 cells, in a dose-dependent manner, and these toxic effects of ML329 were most evident in WM266-4 cells. Extracellular flux assays conducted using a Seahorse bioanalyzer revealed that treatment with ML329 increased basal respiration, ATP-linked respiration, proton leakage, and non-mitochondrial respiration in WM266-4 cells and decreased glycolytic function in SK-mel-24 cells, whereas there were no marked effects of ML329 on A375 and A375DT cells. A glycolytic stress assay under conditions of high glucose concentrations also demonstrated that the inhibitory effect of ML329 on the glycolytic function of WM266-4 cells was dose-dependent. In addition, ML329 significantly decreased 3D-spheroid-forming ability, though the effects of ML329 were variable among the MM cell lines. Furthermore, the mRNA expression levels of selected genes, including STAT3 as a possible regulator of 3D spheroid formation, KRAS and SOX2 as oncogenic-signaling-related factors, PCG1a as the main regulator of mitochondrial biogenesis, and HIF1a as a major hypoxia transcriptional regulator, fluctuated among the MM cell lines, possibly supporting the diverse ML329 effects mentioned above. The findings of diverse ML329 effects on various MM cell lines suggest that MITF-associated biological activities are different among various types of MM.

Physical Properties and Cellular Metabolic Characteristics of 3D Spheroids Are Possible Definitive Indices for the Biological Nature of Cancer-Associated Fibroblasts.

The current study's objective was to elucidate some currently unknown biological indicators to evaluate the biological nature of cancer-associated fibroblasts (CAFs). For this purpose, four different CAFs, CAFS1, CAFS2, SCC17F and MO-1000, were established using surgical specimens from oral squamous cell carcinomas (OSCC) with different clinical malignant stages (CAFS1 and CAFS2, T2N0M0, stage II; SCC17F and MO-1000, T4aN2bM0, stage IVA). Fibroblasts unrelated to cancer (non-CAFs) were also prepared and used as controls. Initially, confirmation that these four fibroblasts were indeed CAFs was obtained by their mRNA expression using positive and negative markers for the CAF or fibroblasts. To elucidate possible unknown biological indicators, these fibroblasts were subjected to a cellular metabolic analysis by a Seahorse bioanalyzer, in conjugation with 3D spheroid cultures of the cells and co-cultures with a pancreas ductal carcinoma cell line, MIA PaCa-2. The mitochondrial and glycolytic functions of human orbital fibroblasts (HOF) were nearly identical to those of Graves'-disease-related HOF (GOF). In contrast, the characteristics of the metabolic functions of these four CAFs were different from those of human conjunctival fibroblasts (HconF), a representative non-CAF. It is particularly noteworthy that CAFS1 and CAFS2 showed markedly reduced ratios for the rate of oxygen consumption to the extracellular acidification rate, suggesting that glycolysis was enhanced compared to mitochondrial respiration. Similarly, the physical aspects, their appearance and stiffness, of their 3D spheroids and fibroblasts that were induced effects based on the cellular metabolic functions of MIA PaCa-2 were also different between CAFs and non-CAFs, and their levels for CAFS1 or SCC17F were similar to those for CAFS2 or MO-1000 cells, respectively. The findings reported herein indicate that cellular metabolic functions and the physical characteristics of these types of 3D spheroids may be valuable and useful indicators for estimating potential biological diversity among various CAFs.

Three-Dimensional Spheroid Configurations and Cellular Metabolic Properties of Oral Squamous Carcinomas Are Possible Pharmacological and Pathological Indicators.

The objective of the current study was to elucidate the clinicopathological significance and appearance of in vitro three-dimension (3D) spheroid models of oral malignant tumors that were prepared from four pathologically different squamous cell carcinoma (OSCC; low-grade; SSYP and MO-1000, intermediate-grade; LEM2) and oral adenosquamous carcinoma (OASC; high-grade; Mesimo) obtained from patients with different malignant stages. To characterize the biological significance of these cell lines themselves, two-dimensional (2D) cultured cells were subjected to cellular metabolic analysis by a Seahorse bioanalyzer alongside the measurement of the cytotoxicity of cisplatin (CDDP). The appearance of their 3D spheroids was then observed by phase contrast microscopy, and both 2D and 3D cultured cells were subject to trypsin digestion and qPCR analysis of factors related to oncogenic signaling and other related analyses. ATP-linked respiration and proton leaking were significantly different among the four cell lines, and the malignant stages of these cultures were significantly associated with increased ATP-linked respiration and decreased proton leakage. Alternatively, the appearances of these 3D spheroids were also significantly diverse among them, and their differences increased in the order of LEM2, MO-1000, SSYP, and Mesimo. Interestingly, these orders were exactly the same in that the efficacies of CDDP-induced cytotoxicity increased in the same order. qPCR analysis indicated that the levels of expression of oncogenic signaling-related factors varied among these four cell lines, and the values for fibronectin and a key regulator of mitochondrial biogenesis, PGC-1α, were prominently elevated in cultures of the worst malignant Mesimo cells. In addition, although 0.25% trypsin-induced destruction was comparable among all four 2D cultured cells, the values for the 3D spheroids were also substantially varied among these cultures. The findings reported herein indicate that cellular metabolic functions and 3D spheroid architectures may be valuable and useful indicators for estimating the pathological and drug-sensitive aspects of OSCC and OASC malignancies.

Prevention of murine experimental corneal trauma by epigenetic events regulating claudin 6 and claudin 9.

PURPOSE: To study the effects of epigenetic events on corneal barrier functions, we examined tight junctions (TJs), the most important factor in the barrier function of the cornea, by using in vitro and in vivo corneal trauma models. METHODS: We examined alteration of TJ-associated gene expression and corneal barrier function in human corneal cells by methylating and demethylating promoter cytosine (CpG) islands, with and without epigenetic regulators such as trichostatin A, (TSA), 5-azacytidine (5-aza), and dimethyl sulfoxide (DMSO). We also investigated the clinical relevance of epigenetic regulators by applying these agents to murine experimental corneal trauma. RESULTS: Treatment with epigenetic regulators such as TSA, 5-aza, and DMSO significantly enhanced the expression of TJ-associated genes such as claudin 6 and claudin 9 in corneal cells, changing transcriptional signals by demethylating CpG islands. In addition, the epigenetic regulators increased transendothelial electrical resistance and suppressed fluxes of corneal cells, thus enhancing the corneal barrier function. These epigenetic regulators mediated TJ-associated gene enhancement, and the corneal barrier function enhancement was sufficient to limit the corneal fluxes in murine experimental corneal trauma. CONCLUSIONS: Epigenetic regulators enhance corneal barrier impairment by modulating TJs, so epigenetic regulation is a possible therapeutic method for corneal trauma.

An intractable case of Pseudomonas aeruginosa infection after scleral buckling for rhegmatogenous retinal detachment.

BACKGROUND: Scleral buckling is still a common procedure to repair rhematogenous retinal detachment, and acute or chronic infection of the scleral explant is rare. We report an intractable case of acute scleral explant infection by Pseudomonas aeruginosa. CASE: A 36-year-old man suffered from acute scleral explant infection by P. aeruginosa forty-eight hours after scleral buckling for rhegmatogenous retinal detachment. The infection was treated by intravenous administration of various appropriate antibiotics for eighteen days and washing the scleral explant with appropriate antibiotics, and appeared to be resolved. However, three months after the initial surgery, we had to remove the scleral explant because of recurrent infection. OBSERVATIONS: We encountered an intractable case of acute scleral explant infection by P. aeruginosa, that recurred and forced the removal of the scleral explant. CONCLUSIONS: We found that recurrence of infection necessitated removal of the scleral explant, even though the organism was sensitive to the antibiotics used to treat the infection, and there was an appropriate duration of treatment. Early diagnosis and countermeasures, first considering conservative management, which may have a role in delaying buckle removal, and thus reduce the risk of retinal redetachment, and help prolong the time until surgical treatment such as removing the scleral explant is required.

Glial cell-derived cytokines attenuate the breakdown of vascular integrity in diabetic retinopathy.

The blood-retinal barrier (BRB) is a biological unit comprised of specialized capillary endothelial cells firmly connected by intercellular tight junctions and endothelium-surrounding glial cells. The BRB is essential for maintaining the retinal microenvironment and low permeability and is compromised in an early phase during the progression of diabetic retinopathy. Here, we demonstrate that retinoic acid receptor (RAR)alpha stimulants preferentially act on glial cells rather than endothelial cells, resulting in the enhanced expression of glial cell line-derived neurotrophic factor (GDNF) through recruitment of the RARalpha-driven trans-acting coactivator to the 5'-flanking region of the gene promoter. Conversely, RARalpha decreases expression of vascular endothelial growth factor (VEGF)/vascular permeability factor. These gene expression alterations causally limit vascular permeability by modulating the tight junction function of capillary endothelium in a paracrine manner in vitro. The phenotypic transformation of glial cells mediated by RARalpha is sufficient for significant reductions of vascular leakage in the diabetic retina, suggesting that RARalpha antagonizes the loss of tight junction integrity induced by diabetes. These findings reveal that glial cell-derived cytokines such as GDNF and VEGF regulate BRB function, implying that the glial cell can be a possible therapeutic target in diabetic retinopathy.

Occludin-mediated premature senescence is a fail-safe mechanism against tumorigenesis in breast carcinoma cells.

We have previously demonstrated that epigenetic silencing of occludin, a tight junction-associated membrane protein, results in the acquisition of apoptotic resistance to various apoptogenic stimuli, causally contributing to the enhanced tumorigenicity of cancer cells. However, it remains to be examined whether occludin expression in transformed cells has an alternative impact that is important for cancer progression. Here we show that forced expression of occludin induces anoikis and promotes oxidative stress-induced premature senescence in breast carcinoma cells, which is accompanied by upregulation of negative cell cycle regulators such as p16(INK4A), p21(Waf1/Cip1) and p27(Kip1) but not p53. The senescent phenotype is reversed by specific inhibition of mitogen-activated protein kinase. Endogenous reexpression of occludin mediated by a synergistic effect with a demethylator and histone deacetylase inhibitor or retinoids that stimulate retinoic acid receptor alpha is also sufficient for provoking the senescent phenotype. In addition, tumors that developed from occludin-expressing cells in mice showed a feature of cellular senescence that has not been described as a consequence of occludin signaling. These findings suggest that the loss of occludin expression is at least partially involved in the senescence-escape program during mammary tumorigenesis.

Epigenetic silencing of occludin promotes tumorigenic and metastatic properties of cancer cells via modulations of unique sets of apoptosis-associated genes.

Occludin is the first identified integral protein for the tight junction (TJ), and its long COOH-terminal domain is considered to have functions in receiving and transmitting cell survival signals. Loss of TJ-associated molecules, such as occludin, has been correlated with tumor progression in carcinogenesis; however, the precise molecular mechanisms explaining its loss of expression and whether occludin expression has any effects on cancer phenotypes remain to be clarified. Here, we show that forced expression of occludin in cancer cells exhibits enhanced sensitivity to differently acting apoptogenic factors, and thus inhibits the tumorigenicity of transformed cells, via modulation of unique sets of apoptosis-associated genes. In addition, studies using deletion mutants of occludin constructs show that 44 amino acids at the COOH-terminal end play a critical role in modifying the cellular phenotypes. Interestingly, occludin decreases cellular invasiveness and motility, thereby abrogating metastatic potencies of cancer cells. We also found that occludin expression is silenced by CpG island hypermethylation on its promoter region. Synergy with a demethylator and histone deacetylase inhibitor or retinoids that stimulate retinoic acid receptor alpha induces endogenous occludin, which is sufficient for apoptotic sensitization. Our results show the functional diversity of occludin and suggest that methylator phenotype of occludin provides enhanced tumorigenic, invasive, and metastatic properties of cancer cells, identifying occludin as a likely candidate for a tumor-suppressor gene in certain types of cancer.

Glyceraldehyde-derived advanced glycation end-products preferentially induce VEGF expression and reduce GDNF expression in human astrocytes.

The blood-brain barrier (BBB) is a biological unit composed of capillary endothelial cells and astrocytes. Here we examined the effects of various types of advanced glycation end-products (AGEs) on astrocytes and BBB-forming endothelial cells. While no type of AGE we examined changed the permeability of endothelial sheets, glyceraldehyde-derived AGE induced VEGF expression most significantly in astrocytes. The expression of glial cell line-derived neurotrophic factor (GDNF), which reduces the vascular permeability, was decreased in the astrocytes by treatment with glyceraldehyde-derived AGE. These results indicate that glyceraldehyde-derived AGE is the biologically active substance for astrocytes by regulating the VEGF and GDNF expression, which is causally contributing to an increase in the permeability of the BBB.