Biology subjects

Kennedy, B. N.

Publications and source records attributed to Kennedy, B. N..

9 recordsLinked to original sources

bioRxiv2021

Inhibition of Metastatic Uveal Melanoma Cell Proliferation by the Histone Deacetylase 6 Inhibitor, Ricolinostat, is Linked to Altered Microphthalmia-associated Transcription Factor and Phospho-ERK Expression

Metastatic uveal melanoma (MUM) is characterized by poor patient survival. Unfortunately, current treatment options demonstrate limited benefits. In this study, we evaluate the efficacy of ACY-1215, a histone deacetylase 6 inhibitor (HDAC6i), to attenuate MUM cell growth in vitro and in vivo, and elucidate the underlying molecular mechanisms. Treatment of OMM2.5 MUM cells with ACY-1215 resulted in a significant (p = 0.0001), dose-dependent reduction in cell survival and proliferation in vitro, and in vivo regression of primary OMM2.5 xenografts in zebrafish larvae. Furthermore, flow cytometry analysis revealed that ACY-1215 significantly arrested the OMM2.5 cell cycle in S phase (p = 0.0006) following 24 hours of treatment and significant apoptosis was triggered in a time- and dose-dependent manner (p = <0.0001). Additionally, ACY-1215 treatment resulted in a significant reduction in OMM2.5 p-ERK expression levels. Through proteome-profiling, attenuation of the microphthalmia-associated transcription factor (MITF) signaling pathway was linked to the observed anti-cancer effects of ACY-1215. In agreement, pharmacological inhibition of MITF signaling with ML329, significantly reduced OMM2.5 cell survival and viability in vitro (p = 0.0001) and in vivo (p = 0.0006). Our findings provide evidence that ACY-1215 and ML329 are efficacious against growth and survival of MUM cells and are potential therapeutic options for MUM. Simple SummaryUveal melanoma (UM) is the most common adult eye cancer. UM originates in the iris, ciliary body, or choroid (collectively known as the uvea), in the middle layer of the eye. This first or primary UM is treated by targeting the cancer cells using ocular radiation implants or by surgical removal of the eye. However, when UM spreads to the liver and other parts of the body, patients have a poor survival prognosis. Unfortunately, there are no effective treatment options for UM that has spread. Our aim is to help identify effective treatments for UM cancer that has spread. In our study, we identified that the drug ACY-1215 prevents the growth of UM cells from the liver. Our study has found a promising treatment approach for advanced UM.

cancer biology↗

bioRxiv2021

Affordable and Effective Optokinetic Response Methods to Assess Visual Acuity and Contrast Sensitivity in Larval to Juvenile Zebrafish

BackgroundThe optokinetic response (OKR) is an effective behavioural assay to investigate functional vision in zebrafish. The rapid and widespread use of gene editing, drug screening and environmental modulation technologies have resulted in a broader need for visual neuroscience researchers to access affordable and more sensitive OKR, contrast sensitivity (CS) and visual acuity (VA) assays. Here, we demonstrate how 2D- and 3D-printed, striped patterns or drums coupled with a motorised base and microscope provide a simple, cost-effective but efficient means to assay OKR, CS and VA in larval-juvenile zebrafish. ResultsIn wild-type, 5 days post-fertilisation (dpf) zebrafish, the 2D or 3D drums printed with the standard OKR stimulus of 0.02 cycles per degree (cpd), 100% black-white contrast evoked equivalent responses of 24.2 or 21.8 saccades per minute, respectively. Furthermore, although the OKR number was significantly reduced compared to the 0.02 cpd drum (p<0.0001), the 2D and 3D drums evoked respectively equivalent responses with the 0.06 and 0.2 cpd drums. Notably, standard OKR responses varied with time of day; peak responses of 29.8 saccades per minute occurred in the early afternoon with significantly reduced responses occurring in the early morning or late afternoon, (18.5 and 18.4 saccades per minute, respectively). A customised series of 2D printed drums enabled analysis of visual acuity and contrast sensitivity in 5-21 dpf zebrafish. The saccadic frequency in visual acuity and contrast sensitivity assays, was inversely proportional to age, spatial frequency and contrast of the stimulus. ConclusionsOKR, VA and CS of zebrafish larvae can be efficiently measured using 2D- or 3D-printed striped drums. For data consistency the luminance of the OKR light source, the time of day when the analysis performed, and the order of presentation of VA and CS drums must be considered. These simple methods allow effective and more sensitive analysis of functional vision in zebrafish.

neuroscience↗

bioRxiv2020

High Cysteinyl Leukotriene Receptor 1 Expression Correlates with Poor Survival of Uveal Melanoma Patients and Cognate Antagonist Drugs Modulate the Growth, Cancer Secretome, and Metabolism of Uveal Melanoma Cells.

Uveal melanoma (UM) is a rare, but often lethal, form of ocular cancer arising from melanocytes within the uveal tract. UM has a high propensity to spread hematogenously to the liver, with up to 50% of patients developing liver metastases. Unfortunately, once liver metastasis occurs, patient prognosis is extremely poor with as few as 8% of patients surviving beyond two years. There are no standard-of-care therapies available for the treatment of metastatic uveal melanoma, hence it is a clinical area of urgent unmet need. Here, the clinical relevance and therapeutic potential of cysteinyl leukotriene receptors (CysLT1 and CysLT2) in UM was evaluated. High expression of CYSLTR1 or CYSLTR2 transcripts is significantly associated with poor disease-free survival and poor overall survival in UM patients. Digital pathology analysis identified high expression of CysLT1 in primary UM is associated with reduced disease-specific survival (p = 0.012) and overall survival (p = 0.011). High CysLT1 expression shows a statistically significant (p = 0.041) correlation with ciliary body involvement, a poor prognostic indicator in UM. Small molecule drugs targeting CysLT1 were vastly superior at exerting anti-cancer phenotypes in UM cell lines and zebrafish xenografts than drugs targeting CysLT2. Quininib, a selective CysLT1 antagonist, significantly inhibits survival (p < 0.0001), long-term proliferation (p < 0.0001), and oxidative phosphorylation (p < 0.001), but not glycolysis, in primary and metastatic UM cell lines. Quininib exerts opposing effects on the secretion of inflammatory markers in primary versus metastatic UM cell lines. Quininib significantly downregulated IL-2 and IL-6 in Mel285 cells (p < 0.05), but significantly upregulated IL-10, IL-1{beta}, IL-2 (p < 0.0001), IL-13, IL-8 (p < 0.001), IL-12p70 and IL-6 (p < 0.05) in OMM2.5 cells. Finally, quininib significantly inhibits tumour growth in orthotopic zebrafish xenograft models of UM. These preclinical data suggest that antagonism of CysLT1, but not CysLT2, may be of therapeutic interest in the treatment of UM.

cancer biology↗

bioRxiv2020

Non-photopic and photopic visual cycles differentially regulate immediate, early and late-phases of cone photoreceptor-mediated vision

Cone photoreceptors in the retina enable vision over a wide range of light intensities. However, the processes enabling cone vision in bright light (i.e. photopic vision) are not adequately understood. Chromophore regeneration of cone photopigments may require the retinal pigment epithelium (RPE) and/or retinal Muller glia. In the RPE, isomerization of all-trans-retinyl esters (atRE) to 11-cis-retinol (11cROL) is mediated by the retinoid isomerohydrolase Rpe65. An alternative retinoid isomerase, dihydroceramide desaturase-1 (DES1), is expressed in RPE and Muller cells. The retinol-isomerase activities of Rpe65 and Des1 are inhibited by emixustat and fenretinide, respectively. Here, we tested the effects of these visual cycle inhibitors on immediate, early and late phases of cone photopic vision. In zebrafish larvae raised under cyclic light conditions, fenretinide impaired late cone photopic vision, whereas emixustat-treated zebrafish unexpectedly had normal vision. In contrast, emixustat-treated larvae raised under extensive dark-adaption displayed significantly attenuated immediate photopic vision concomitantly with significantly reduced 11-cis-retinaldehyde (11cRAL). Following 30 minutes of light, early photopic vision recovered, despite 11cRAL levels remaining significantly reduced. Defects in immediate cone photopic vision were rescued in emixustat- or fenretinide-treated larvae following exogenous 9-cis-retinaldehyde (9cRAL) supplementation. Genetic knockout of degs1 or retinaldehyde-binding protein 1b (rlbp1b) revealed that neither are required for photopic vision in zebrafish. Our findings define the molecular and temporal requirements of the non-photopic and photopic visual cycles for mediating vision in bright light.

biochemistry↗

bioRxiv2019

Rab28 is localised to photoreceptor outer segments, regulates outer segment shedding but is not linked to retinal degeneration in zebrafish

The photoreceptor outer segment is the canonical example of a modified and highly specialised cilium, with an expanded membrane surface area in the form of discs or lamellae for efficient light detection. Many ciliary proteins are essential for normal photoreceptor function and cilium dysfunction often results in retinal degeneration leading to impaired vision. Herein, we investigate the function and localisation of the ciliary G-protein RAB28 in zebrafish cone photoreceptors. CRISPR-Cas9 generated rab28 mutant zebrafish display a reduction in shed outer segment material in the RPE at 1 month post fertilisation (mpf), but otherwise normal retinal structure and visual function up to 12 mpf. Cone photoreceptor-specific transgenic reporter lines show Rab28 localises almost exclusively to outer segments, independently of nucleotide binding. Co-immunoprecipitation analysis demonstrates tagged Rab28 interacts with components of the phototransduction cascade, including opsins, Phosphodiesterase 6C and Guanylate Cyclase 2D. Our data shed light on RAB28 function in cones and provide a model for RAB28-associated cone-rod dystrophy.

cell biology↗

bioRxiv2019

A ciliary BBSome-ARL-6-PDE6D pathway trafficks RAB-28, a negative regulator of extracellular vesicle biogenesis

Cilia both receive and send information, the latter in the form of extracellular vesicles (EVs). EVs are nano-communication devices that cells shed to influence cell, tissue, and organism behavior. Mechanisms driving ciliary EV biogenesis and environment release are almost entirely unknown. Here, we show that the ciliary G-protein RAB28, associated with human autosomal recessive cone-rod dystrophy, negatively regulates EV levels in the sensory organs of Caenorhabditis elegans. We also find that sequential targeting of lipidated RAB28 to periciliary and ciliary membranes is highly dependent on the BBSome and PDE6D, respectively, and that BBSome loss causes excessive and ectopic EV production. Our data indicate that RAB28 and the BBSome are key in vivo regulators of EV production at the periciliary membrane. Our findings also suggest that EVs control sensory organ homeostasis by mediating communication between ciliated neurons and glia, and that defects in ciliary EV biogenesis may contribute to human ciliopathies.

cell biology↗

bioRxiv2019

Orthogonal Drug Pooling Enhances Phenotype-based Discovery of Ocular Anti-angiogenic Drugs in Zebrafish Larvae

Unbiased screening of large randomized chemical libraries in vivo is a powerful tool to find new drugs and targets. However, forward chemical screens in zebrafish can be time consuming and usually >99% of test compounds have no significant effect on the desired phenotype. Here, we sought to find bioactive drugs more efficiently and to comply with the 3R principles of replacement, reduction and refinement of animals in research. We investigated if pooling of drugs to simultaneously test 8-10 compounds in zebrafish larvae can increase the screening efficiency of an established assay that identifies drugs inhibiting developmental angiogenesis in the eye. In a phenotype-based screen, we tested 1760 small molecule compounds from the ChemBridge DIVERSet chemical library for their ability to inhibit the formation of distinct primary hyaloid vessels in the eye. Applying orthogonal pooling of the chemical library, we treated zebrafish embryos from 3 to 5 days post fertilization with pools of 8 or 10 compounds at 10 M each. This reduced the number of tests from 1760 to 396. In 63% of cases, treatment showed sub-threshold effects of <40% reduction of primary hyaloid vessels. From 18 pool hits, we identified 8 compounds that reduce hyaloid vessels in the larval zebrafish eye by at least 40%. Compound 4-[4-(1H-benzimidazol-2-yl)phenoxy]aniline ranked as the most promising candidate with reproducible and dose-dependent effects. To our knowledge, this is the first report of a self-deconvoluting matrix strategy applied to drug screening in zebrafish. We conclude that the orthogonal drug pooling strategy is a cost-effective, time-saving and unbiased approach to discover novel inhibitors of developmental angiogenesis in the eye. Ultimately, this approach may identify new drugs or targets to mitigate disease caused by pathological angiogenesis in the eye e.g. diabetic retinopathy or age-related macular degeneration wherein blood vessel growth and leaky vessels lead to vision impairment or clinical blindness.

pharmacology and toxicology↗

bioRxiv2018

1,4-dihydroxy Quininib Attenuates Growth of Colorectal Cancer Cells and Xenografts and Regulates the TIE-2 Signaling Pathway in Patient Tumours

Colorectal cancer (CRC) is the second leading cause of cancer associated deaths in developed countries. Cancer progression and metastatic spread is reliant on new blood vasculature, or angiogenesis. Tumour-related angiogenesis is regulated by pro- and anti-angiogenic factors secreted from malignant tissue in a stepwise process. Previously we structurally modified the small anti-angiogenic molecule quininib and discovered a more potent anti-angiogenic compound 1, 4 dihydroxy quininib (Q8), an antagonist of cysteinyl leukotriene receptor-1 with VEGF-independent bioactivity. Here, Q8, quininib (Q1) and five structural analogues were assayed for anti-tumorigenic effects in pre-clinical cancer models. Q8 reduced clone formation of the human colorectal cancer cell line HT29-Luc2. Gene silencing of CysLT1 in HT29-Luc2 cells significantly reduced expression of calpain-2. In human ex vivo colorectal cancer tumour explants, Q8 significantly decreased the secretion of both TIE-2 and VCAM-1 expression. In vivo Q8 was well tolerated up to 50 mg/kg by Balb/C mice and significantly more effective at reducing tumour volume in colorectal tumour xenografts compared to the parent drug quininib. In tumour xenografts, Q8 significantly reduced expression of the angiogenic marker calpain-2 in. In summary, we propose Q8 may act on the TIE-2-Angiopoietin signalling pathway to significantly inhibit the process of tumour angiogenesis in colorectal cancer.

cancer biology↗

bioRxiv2018

Pharmacological Restoration of Visual Function in a Zebrafish Model of von-Hippel Lindau Disease

Von Hippel-Lindau (VHL) syndrome is rare, autosomal dominant disorder, characterized by hypervascularised tumour formation in multiple organ systems. Vision loss associated with retinal capillary hemangioblastomas remains one of the earliest complications of VHL disease. The mortality of Vhl-/- mice in utero restricted modelling of VHL disease in this mammalian model. Zebrafish harbouring a recessive germline mutation in the vhl gene represent a viable, alterative vertebrate model to investigate associated ocular loss-of-function phenotypes. Previous studies reported neovascularization of the brain, eye and trunk together with odema in the vhl-/- zebrafish eye. In this study, we demonstrate vhl-/- zebrafish almost entirely lack visual function. Furthermore, hyaloid vasculature networks in the vhl-/- eye are improperly formed and this phenotype is concomitant with development of an ectopic intraretinal vasculature. Sunitinib, a multi tyrosine kinase inhibitor, market authorised for cancer, reversed the ocular behavioural and morphological phenotypes observed in vhl-/- zebrafish. We conclude that the zebrafish vhl gene contributes to the endogenous molecular barrier that prevents development of intraretinal vasculature and that sunitinib can improve visual function and hyaloid vessel patterning while reducing abnormally formed ectopic intraretinal and choroidal vessels in vhl-/- zebrafish.

pharmacology and toxicology↗