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The main objective of this study was to determine the inhibition of pro-inflammatory cytokines and their associated signaling molecules by δ-opioid receptor activation by a selective ligand, SNC-121 in chronic rat glaucoma model. Intraocular pressure was raised in rat eyes by injecting 2 M hypertonic saline into the limbal veins. SNC-121 (1 mg/kg; i. p) or Stattic (5 mg/kg; i. p) was administered in Brown Norway rats daily for 7 days. The mRNA expression of IL-1ß, TNF-α, Fas, IL-6, leukemia inhibitory factor, and IFN-γ was increased significantly in the retina of ocular hypertensive animals at day 7, post injury. Administration of SNC-121 (1 mg/kg; i. p. injection) for 7 days (once a day) completely inhibited the increase in the mRNA and protein expression of pro-inflammatory cytokines. Mechanistically, we provide data showing a significant increase in the phosphorylation of STAT3 at tyrosine 705 whereas a moderate but significant increase in the total STAT3 protein expression was also seen in the retina of ocular hypertensive animals. Data illustrated that SNC-121 administration completely abrogated ocular hypertension-induced increase in STAT3Y705 phosphorylation. Interestingly, acetylation of STAT3 at lysine 685 (AcK685) was reduced in ocular hypertensive animals and subsequently increased significantly by SNC-121 treatment. Stattic, a selective STAT3 inhibitor, administration resulted in a complete attenuation in the production of IL-1ß and IL-6 in ocular hypertensive animals. In conclusion, δ-opioid receptor activation suppressed the phosphorylation of STAT3 at tyrosine 705 and increased acetylation at lysine 686 and these posttranslational modifications can regulate the production of some but not all pro-inflammatory cytokines in response to glaucomatous injury.
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Purpose: This study determines if δ-opioid receptor agonist (i.e. SNC-121)-induced epigenetic changes via regulation of histone deacetylases (HDACs) for retinal ganglion cell (RGC) neuroprotection in glaucoma model. Methods: Intraocular pressure was raised in rat eyes by injecting 2M hypertonic saline into the limbal veins. SNC-121 (1 mg/kg; i.p.) was administered to the animals for 7 days. Retinas were collected at days 7 and 42, post-injury followed by measurement of HDAC activities, mRNA, and protein expression by enzyme assay, quantitative real-time PCR (qRT-PCR), Western blotting, and immunohistochemistry. Results: The visual acuity, contrast sensitivity, and pattern electroretinograms (ERGs) were declined in ocular hypertensive animals, which were significantly improved by SNC-121 treatment. Class I and IIb HDACs activities were significantly increased at days 7 and 42 in ocular hypertensive animals. The mRNA and protein expression of HDAC 1 was increased by 1.33 ± 0.07-fold and 20.2 ± 2.7%, HDAC 2 by 1.4 ± 0.05-fold and 17.0 ± 2.4%, HDAC 3 by 1.4 ± 0.06-fold and 17.4 ± 3.4%, and HDAC 6 by 1.5 ± 0.09-fold and 15.1 ± 3.3% at day 7, post-injury. Both the mRNA and protein expression of HDACs were potentiated further at day 42 in ocular hypertensive animals. HDAC activities, mRNA, and protein expression were blocked by SNC-121 treatment at days 7 and 42 in ocular hypertensive animals. Conclusions: Data suggests that class I and IIb HDACs are activated and upregulated during early stages of glaucoma. Early intervention with δ-opioid receptor activation resulted in the prolonged suppression of class I and IIb HDACs activities and expression, which may, in part, play a crucial role in RGC neuroprotection.
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Epigénesis Genética/efectos de los fármacos , Glaucoma/metabolismo , Histona Desacetilasas/metabolismo , Receptores Opioides delta/agonistas , Animales , Western Blotting , Modelos Animales de Enfermedad , Electrorretinografía , Femenino , Glaucoma/enzimología , Histona Desacetilasas/efectos de los fármacos , Presión Intraocular , Masculino , Ratas , Reacción en Cadena en Tiempo Real de la Polimerasa , Células Ganglionares de la Retina/efectos de los fármacos , Células Ganglionares de la Retina/metabolismoRESUMEN
Purpose: We determined whether δ-opioid receptor agonist (SNC-121) regulates acetylation homeostasis via controlling histone deacetylases (HDACs) activity and expression in optic nerve head (ONH) astrocytes. Methods: ONH astrocytes were treated with SNC-121 (1 µM) for 24 hours. The HDAC activity was measured using HDAC-specific fluorophore-conjugated synthetic substrates, Boc-Lys(Ac)-AMC and (Boc-Lys(Tfa)-AMC). Protein and mRNA expression of each HDAC was determined by Western blotting and quantitative real-time PCR. IOP in rats was elevated by injecting 2.0 M hypertonic saline into the limbal veins. Results: Delta opioid receptor agonist, SNC-121 (1 µM), treatment increased acetylation of histone H3, H2B, and H4 by 128 ± 3%, 45 ± 1%, and 68 ± 2%, respectively. The addition of Garcinol, a histone-acetyltransferase inhibitor, fully blocked SNC-121-induced histone H3 acetylation. SNC-121 reduced the activities of class I and IIb HDACs activities significantly (17 ± 3%) and this decrease in HDACs activities was fully blocked by a selective δ-opioid receptors antagonist, naltrindole. SNC-121 also decrease the mRNA expression of HDAC-3 and HDAC-6 by 19% and 18%, respectively. Furthermore, protein expression of HDAC 1, 2, 3, and 6 was significantly (P < 0.05) decreased by SNC-121 treatment. SNC-121 treatment also reduced lipopolysaccharide-induced TNF-α production from ONH astrocytes and glial fibrillary acidic protein immunostaining in the optic nerve of ocular hypertensive animals. Conclusions: We provided evidence that δ-opioid receptor agonist activation increased histone acetylation, decrease HDACs class I and class IIb activities, mRNA, and protein expression, lipopolysaccharide-induced TNF-α production in ONH astrocytes. Our data also demonstrate that SNC-121 treatment decrease glial fibrillary acidic protein immunostaining in the optic nerves of animals with ocular hypertension.
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Astrocitos/efectos de los fármacos , Benzamidas/farmacología , Histona Desacetilasas/metabolismo , Disco Óptico/efectos de los fármacos , Piperazinas/farmacología , Células Ganglionares de la Retina/efectos de los fármacos , Acetilación , Anciano , Anciano de 80 o más Años , Animales , Astrocitos/metabolismo , Astrocitos/patología , Western Blotting , Cadáver , Células Cultivadas , Electrorretinografía , Femenino , Humanos , Masculino , Persona de Mediana Edad , Disco Óptico/metabolismo , Disco Óptico/patología , Ratas , Ratas Endogámicas BN , Receptores Opioides delta/agonistas , Células Ganglionares de la Retina/metabolismo , Células Ganglionares de la Retina/patologíaRESUMEN
The outcome of patients with metastatic osteosarcoma has not improved since the introduction of chemotherapy in the 1970s. Development of therapies targeting the metastatic cascade is a tremendous unmet medical need. The Wnt signaling pathway has been the focus of intense investigation in osteosarcoma because of its role in normal bone development. Although the role of Wnt signaling in the pathogenesis of osteosarcoma is controversial, there are several reports of dickkopf-1 (DKK-1), a Wnt signaling antagonist, possibly playing a pro-tumorigenic role. In this work we investigated the effect of anti-DKK-1 antibodies on the growth and metastasis of patient-derived osteosarcoma xenografts. We were able to detect human DKK-1 in the blood of tumor-bearing mice and found a correlation between DKK-1 level and tumor proliferation. Treatment with the anti-DKK-1 antibody, BHQ880, slowed the growth of orthotopically implanted patient-derived osteosarcoma xenografts and inhibited metastasis. This effect was correlated with increased nuclear beta-catenin staining and increased expression of the bone differentiation marker osteopontin. These findings suggest that Wnt signaling is anti-tumorigenic in osteosarcoma, and support the targeting of DKK-1 as an anti-metastatic strategy for patients with osteosarcoma.