Bioenergetic modulation with the mitochondria uncouplers SR4 and niclosamide prevents proliferation and growth of treatment-naïve and vemurafenib-resistant melanomas.

BRAF mutations are detected in >50% of all melanomas. These mutations impair the LKB1-AMPK signaling, an important metabolic pathway associated with cell growth, proliferation and survival. Melanoma patients with BRAF mutations are usually treated with BRAF inhibitors such as vemurafenib, but responses are short-lived as drug resistant tumors metabolically switch to mitochondrial oxidative phosphorylation (OXPHOS) to escape metabolic stress-induced BRAF inhibition. Additionally, a large subset of melanoma utilizes OXPHOS in their metabolism, which can confer de novo resistance to BRAF inhibitors. Therefore, uncoupling of OXPHOS to perturb energy homeostasis and to indirectly stimulate AMPK could be a novel treatment for melanoma and to overcome intrinsic and acquired resistance to BRAF inhibitors. Here, we investigated the effects of SR4 and niclosamide, two small molecule mitochondria uncouplers, on the growth and proliferation of treatment-naïve and vemurafenib-resistant melanomas in vitro and in vivo. SR4 and niclosamide inhibited melanoma proliferation irrespective of BRAF/NRAS status. Melanomas with greater OXPHOS phenotype (higher OCR/ECAR), with LKB1 mutation, or with acquired resistance to vemurafenib displayed greater sensitivity to both uncouplers. More importantly, SR4 and niclosamide inhibited tumor growth in both treatment-naïve and vemurafenib-resistant xenograft mice models. Mechanistic studies indicate both uncouplers induced energetic stress, modulated the AMPK-mTOR pathway, and promoted apoptosis without affecting MEK-ERK MAPK signaling. These results suggest that uncouplers such as SR4 and niclosamide may be useful as first line treatment against melanoma regardless of BRAF/NRAS status, and as an adjuvant therapy for patients failing MAPK inhibitors.

Effect of brimonidine on retinal and choroidal neovascularization in a mouse model of retinopathy of prematurity and laser-treated rats.

PURPOSE: To determine whether chronic treatment with brimonidine (BRI) attenuates retinal vascular leakage and neovascularization in neonatal mice after exposure to high oxygen in a mouse model of retinopathy of prematurity (ROP), and choroidal neovascularization (CNV) in rats after laser treatment. METHODS: Experimental CNV was induced by laser treatment in Brown Norway (BN) rats. BRI or vehicle (VEH) was administered by osmotic minipumps, and CNV formation was measured 11 days after laser treatment. Oxygen-induced retinopathy was generated in neonatal mice by exposure to 75% oxygen from postnatal day (P)7 to P12. BRI or VEH was administered by gavage, and vitreoretinal vascular endothelial growth factor (VEGF) concentrations and retinal vascular leakage, neovascularization, and vaso-obliteration were measured on P17. Experimental CNV was induced in rabbits by subretinal lipopolysaccharide/fibroblast growth factor-2 injection. RESULTS: Systemic BRI treatment significantly attenuated laser-induced CNV formation in BN rats when initiated 3 days before or within 1 hour after laser treatment. BRI treatment initiated during exposure to high oxygen significantly attenuated vitreoretinal VEGF concentrations, retinal vascular leakage, and retinal neovascularization in P17 mice subjected to oxygen-induced retinopathy. Intravitreal treatment with BRI had no effect on CNV formation in a rabbit model of nonischemic angiogenesis. CONCLUSIONS: BRI treatment significantly attenuated vitreoretinal VEGF concentrations, retinal vascular leakage, and retinal and choroidal neovascularization in animal models of ROP and CNV. BRI may inhibit underlying event(s) of ischemia responsible for upregulation of vitreoretinal VEGF and thus reduce vascular leakage and retinal-choroidal neovascularization.

Inhibition of vitreoretinal VEGF elevation and blood-retinal barrier breakdown in streptozotocin-induced diabetic rats by brimonidine.

PURPOSE: To determine whether long-term brimonidine (BRI) treatment prevents the hyperglycemia-induced increase in vitreoretinal vascular endothelial growth factor (VEGF) expression and breakdown of the blood-retinal barrier (BRB) in streptozotocin (STZ)-induced diabetic rats. METHODS: Brown Norway/Long-Evans rats were divided into three groups with similar distributions of blood glucose and body weight. Two groups received a single intravenous injection of STZ (65 mg/kg) and the remaining control group received vehicle. Drug treatment administered via miniosmotic pumps was initiated 1 or 6 weeks later. The STZ-induced diabetic rats were treated with BRI (1 mg/kg/d) or vehicle (VEH) and control nondiabetic rats were treated with VEH for 4 weeks. Vitreoretinal VEGF protein, vitreal glutamate, and BRB breakdown were then measured. RESULTS: At 5 weeks after STZ treatment, STZ-treated diabetic rats demonstrated significantly elevated vitreoretinal VEGF expression, vitreal glutamate concentrations, and BRB leakage compared with nondiabetic control rats. Chronic BRI treatment had no effect on vitreal glutamate concentrations in diabetic animals but significantly decreased vitreoretinal VEGF expression and BRB breakdown to levels similar to those observed in control rats. BRI also significantly reduced BRB breakdown in aged diabetic rats at 10 weeks after STZ treatment. CONCLUSIONS: BRI produced marked decreases in vitreoretinal VEGF and inhibition of BRB breakdown in diabetic rats. The mechanism for these effects may involve attenuation of retinal NMDA receptor activity by BRI. The results suggest that BRI would be useful for treatment of ocular diseases associated with BRB leakage, such as diabetic macular edema and retinopathy.

Effect of memantine on neuroretinal function and retinal vascular changes of streptozotocin-induced diabetic rats.

PURPOSE: To test whether chronic memantine (MEM) treatment improves retinal function and prevents neurodegeneration and vascular changes in the retinas of streptozotocin (STZ)-induced diabetic rats. METHODS: Based on basal body weight and blood glucose, Brown Norway (BN) rats were divided into three groups. One group of rats was treated with vehicle (VEH), and the other two groups were treated with 65 mg/kg STZ. After 7 days, VEH-treated rats were treated further with a second VEH, and STZ-treated diabetic rats were treated either with the second VEH or with MEM (10 mg/kg daily) for another 3 to 4 weeks using mini-osmotic pumps. At end of the study, electroretinogram findings, retinal ganglion cell (RGC) count, vitreoretinal vascular endothelial growth factor (VEGF) protein levels, and blood-retinal barrier (BRB) breakdown of the animals were measured. RESULTS: Within 4 to 5 weeks of STZ treatment, the diabetic rats demonstrated significantly less retinal function and fewer RGCs than VEH-treated nondiabetic rats. The diabetic animals also had significantly elevated VEGF protein levels in retina and vitreous fluid and BRB breakdown compared with control nondiabetic rats. Chronic MEM treatment significantly improved retinal function and protected RGC loss in STZ-induced diabetic rats. MEM treatment also significantly decreased elevated vitreoretinal VEGF protein levels and retinal BRB leakage in the diabetic rats. This effect of MEM was not seen in nondiabetic rats. CONCLUSIONS: These results indicate that MEM could be useful for the treatment of ocular diseases, including diabetic retinopathy with neurodegeneration, elevated vitreoretinal VEGF protein levels, and increased BRB breakdown. In addition to the neuroprotective effect of this compound, MEM can reduce vascular changes seen in diabetic retinas. These data are the first to identify the vasculoprotective effect of MEM.