Notch Signaling Suppresses Melanoma Tumor Development in BRAF/Pten Mice.

Both oncogenic and tumor suppressor roles have been assigned to Notch signaling in melanoma. In clinical trials, Notch inhibitors proved to be ineffective for melanoma treatment. Notch signaling has also been implicated in melanoma transdifferentiation, a prognostic feature in primary melanoma. In this study, we investigated the role of Notch signaling in melanoma tumor development and growth using the genetic model of mouse melanoma by crossing BRAFCA/+/Pten+/+/Tyr-CreER+ (B) and BRAFCA/+/Pten-/-/Tyr-CreER + (BP) mice with Notch1 or Notch2 floxed allele mice. The topical application of tamoxifen induced tumors in BP mice but not in B mice with or without the deletion of either Notch1 or Notch2. These data show that the loss of either Notch1 nor Notch2 can substitute the tumor suppressor function of Pten in BRAFV600E-induced melanomagenesis. However, in Pten-null background, the loss of either Notch1 or Notch2 appeared to accelerate BRAFV600E-induced tumor development, suggesting a tumor suppressor role for Notch1 and Notch2 in BRAFV600E/Pten-null driven melanomagenesis. Quantitative immunochemical analysis of a human cutaneous melanoma tissue microarray that consists of >100 primary tumors with complete clinical history showed a weak to moderate correlation between NOTCH protein levels and clinical and pathological parameters. Our data show that Notch signaling is involved during melanomagenesis and suggest that the identification of genes and signaling pathways downstream of Notch could help devise strategies for melanoma prevention.

Microfluidic Model to Evaluate Astrocyte Activation in Penumbral Region following Ischemic Stroke.

Stroke is one of the main causes of death in the US and post-stroke treatment options remain limited. Ischemic stroke is caused by a blood clot that compromises blood supply to the brain, rapidly leading to tissue death at the core of the infarcted area surrounded by a hypoxic and nutrient-starved region known as the penumbra. Recent evidence suggests that astrocytes in the penumbral region play a dual role in stroke response, promoting further neural and tissue damage or improving tissue repair depending on the microenvironment. Thus, astrocyte response in the hypoxic penumbra could promote tissue repair after stroke, salvaging neurons in the affected area and contributing to cognitive recovery. However, the complex microenvironment of ischemic stroke, characterized by gradients of hypoxia and nutrients, poses a unique challenge for traditional in vitro models, which in turn hinders the development of novel therapies. To address this challenge, we have developed a novel, polystyrene-based microfluidic device to model the necrotic and penumbral region induced by an ischemic stroke. We demonstrated that when subjected to hypoxia, and nutrient starvation, astrocytes within the penumbral region generated in the microdevice exhibited long-lasting, significantly altered signaling capacity including calcium signaling impairment.

Up-regulation of Sirtuin-1/autophagy signaling in human cerebral ischemia: possible role in caspase-3 mediated apoptosis.

Aim: Autophagy is a catabolic process, which plays a pivotal role in neuronal homeostases. Sirtuin-1 (Sirt1, Silent information regulator family protein 1) is a protein deacetylase that is activated by nicotinamide adenine dinucleotide (NAD+), is also influenced by starvation and stress response similar to autophagy. Sirt1 is necessary for the induction of autophagy and is critical for neuronal survival in neurodegeneration. The present study investigates the role of Sirt1/autophagy signaling and its possible involvement in neuronal cell death in the brains of cerebral ischemia (CI) patients. Patients and methods: Autopsied brain tissues from three healthy subjects and ten CI patients were obtained from National Institute of Mental Health and Neurosciences (NIMHANS); Bangalore, India. Western blotting and immunostaining were performed to assess the expression changes in Sirt1, autophagy mediators including Beclin-1, autophagy-related (Atg) proteins-3, 5, 7, 12-5, microtubule-associated protein-1A light chain3 (Lc3-I/II), and caspase-3 in stroke patients. Results: Our study showed that, in stroke patients, expression of Sirt1, Beclin-1, Atg-3, 5, 7, 12-5, and Lc3-II/I were upregulated. Further, our immunohistochemistry results show increased immunoreactivity of Sirt1, Beclin-1, Atg-7, Lc3-I/II, and cleaved caspase-3 in stroke brains. Conclusion: The present data suggesting a role for Sirt1/autophagy signaling in regulating neuronal cell survival in CI.

Differential role of SIRT1/MAPK pathway during cerebral ischemia in rats and humans.

Cerebral ischemia (CI) is a severe cause of neurological dysfunction and mortality. Sirtuin-1 (Silent information regulator family protein 1, SIRT1), an oxidized nicotinamide adenine dinucleotide (NAD+)-dependent protein deacetylase, plays an important role in protection against several neurodegenerative disorders. The present study aims to investigate the protective role of SIRT1 after CI in experimental young and aged rats and humans. Also, the study examines the possible regulatory mechanisms of neuronal death in CI settings. Immunoblotting and immunohistochemistry were used to evaluate changes in the expression of SIRT1, JNK/ERK/MAPK/AKT signaling, and pro-apoptotic caspase-3 in experimental rats and CI patients. The study findings demonstrated that, in aged experimental rats, SIRT1 activation positively influenced JNK and ERK phosphorylation and modulated neuronal survival in AKT-dependent manner. Further, the protection conferred by SIRT1 was effectively reversed by JNK inhibition and increased pro-apoptotic caspase-3 expression. In young experimental rats, SIRT1 activation decreased the phosphorylation of stress-induced JNK, ERK, caspase-3, and increased the phosphorylation of AKT after CI. Inhibition of SIRT1 reversed the protective effect of resveratrol. More importantly, in human patients, SIRT1 expression, phosphorylation of JNK/ERK/MAPK/AKT signaling and caspase-3 were up-regulated. In conclusion, SIRT1 could possibly be involved in the modulation of JNK/ERK/MAPK/AKT signaling pathway in experimental rats and humans after CI.

Up-regulation of Sirt1/miR-149-5p signaling may play a role in resveratrol induced protection against ischemia via p53 in rat brain.

Micro-RNA(miRNA) are well studied small noncoding RNA, which plays a diverse role in the regulation of vital elements in cell survival and apoptosis. However, the functional significance of miRNAs after the pathogenesis of ischemic stroke remains unclear. The present study is designed to investigate the regulatory role of miR-149-5p on Sirtuin-1/p53 axis during ischemic-reperfusion-induced injury. Middle cerebral artery occlusion (MCAO) was performed by nylon monofilament for 60 min. Resveratrol was administered via intraperitoneal (IP) route, 30 min before the MCAO. Our study demonstrated that the miR-149-5p levels were markedly decreased at 24 h after ischemic-reperfusion (I/R) injury. Further, we observed decreased p53 protein expression and increased miR-149-5p activity on sirtuin1 (Sirt1) activation with resveratrol after 24 h following MCAO. Moreover, immunohistochemistry studies found that resveratrol treatment significantly decreased the immunoreactivity of p53 and caspase-3 on activation of Sirt1/miR149-5p axis. In conclusion, our findings suggest that miR-149-5p could play a regulatory role in neuronal cell death via Sirt1/p53 axis, which offers a new target for novel therapeutic interventions during acute ischemic stroke.