Asialo-rhuEPO as a Potential Neuroprotectant for Ischemic Stroke Treatment.

Neuroprotective drugs to protect the brain against cerebral ischemia and reperfusion (I/R) injury are urgently needed. Mammalian cell-produced recombinant human erythropoietin (rhuEPOM) has been demonstrated to have excellent neuroprotective functions in preclinical studies, but its neuroprotective properties could not be consistently translated in clinical trials. The clinical failure of rhuEPOM was thought to be mainly due to its erythropoietic activity-associated side effects. To exploit its tissue-protective property, various EPO derivatives with tissue-protective function only have been developed. Among them, asialo-rhuEPO, lacking terminal sialic acid residues, was shown to be neuroprotective but non-erythropoietic. Asialo-rhuEPO can be prepared by enzymatic removal of sialic acid residues from rhuEPOM (asialo-rhuEPOE) or by expressing human EPO gene in glycoengineered transgenic plants (asialo-rhuEPOP). Both types of asialo-rhuEPO, like rhuEPOM, displayed excellent neuroprotective effects by regulating multiple cellular pathways in cerebral I/R animal models. In this review, we describe the structure and properties of EPO and asialo-rhuEPO, summarize the progress on neuroprotective studies of asialo-rhuEPO and rhuEPOM, discuss potential reasons for the clinical failure of rhuEPOM with acute ischemic stroke patients, and advocate future studies needed to develop asialo-rhuEPO as a multimodal neuroprotectant for ischemic stroke treatment.

Downregulation of TRIM33 Promotes Survival and Epithelial-Mesenchymal Transition in Gastric Cancer.

Among all malignancies worldwide, gastric cancer is the fifth most common cancer with the third highest mortality rate. One of the main reasons for the low survival rate is the recurrence and metastasis that occurs in many patients after surgery. Numerous studies have shown that abnormal TRIM33 expression is associated with the progression of malignant tumors. TRIM33 can function either as a tumor suppressor or tumor promoter in different cancers. Our data showed that TRIM33 was highly expressed in stomach cancer, and in human gastric cancer tissues, low expression of TRIM33 was associated with poor prognosis in patients with gastric cancer. To clarify the function of TRIM33 in survival and epithelial-mesenchymal transition in gastric cancer cells, we investigated the effect of TRIM33 knockdown in several gastric cancer cell lines. Downregulation of TRIM33 in BGC-823 and SGC-7901 cells enhanced the proliferation, colony formation, and migratory ability of these gastric cancer cells. It also promoted epithelial-mesenchymal transition; transfection of cells with siRNA targeting TRIM33 led to the upregulation of vimentin and N-Cadherin expression, and downregulation of E-Cadherin expression. Meanwhile, the transforming growth factor beta pathway was activated: levels of transforming growth factor beta were elevated and the expressions of p-Smad2, Smad2, Smad3, and Smad4 were activated. To confirm the role of TRIM33 in vivo, a xenograft model was established in nude mice. Immunohistochemical analysis identified that the protein levels of TRIM33, p-Smad2, Smad2, Smad3, Smad4, vimentin, and N-Cadherin were increased, and E-Cadherin levels were decreased, in xenograft tumors from the si-TRIM33 group. Taken together, these results suggest that TRIM33 may be a potential marker for the diagnosis and prognosis of gastric cancer. Furthermore, it may also serve as a novel target for gastric cancer treatment.

Rethinking the necessity of low glucose intervention for cerebral ischemia/reperfusion injury.

Glucose is the essential and almost exclusive metabolic fuel for the brain. Ischemic stroke caused by a blockage in one or more cerebral arteries quickly leads to a lack of regional cerebral blood supply resulting in severe glucose deprivation with subsequent induction of cellular homeostasis disturbance and eventual neuronal death. To make up ischemia-mediated adenosine 5'-triphosphate depletion, glucose in the ischemic penumbra area rapidly enters anaerobic metabolism to produce glycolytic adenosine 5'-triphosphate for cell survival. It appears that an increase in glucose in the ischemic brain would exert favorable effects. This notion is supported by in vitro studies, but generally denied by most in vivo studies. Clinical studies to manage increased blood glucose levels after stroke also failed to show any benefits or even brought out harmful effects while elevated admission blood glucose concentrations frequently correlated with poor outcomes. Surprisingly, strict glycaemic control in clinical practice also failed to yield any beneficial outcome. These controversial results from glucose management studies during the past three decades remain a challenging question of whether glucose intervention is needed for ischemic stroke care. This review provides a brief overview of the roles of cerebral glucose under normal and ischemic conditions and the results of managing glucose levels in non-diabetic patients. Moreover, the relationship between blood glucose and cerebral glucose during the ischemia/reperfusion processes and the potential benefits of low glucose supplements for non-diabetic patients are discussed.

MicroRNA-205-5p inhibits skin cancer cell proliferation and increase drug sensitivity by targeting TNFAIP8.

Tumor necrosis factor-α-induced protein 8 (TNFAIP8) is a member of the TIPE/TNFAIP8 family which regulates tumor growth and survival. Our goal is to delineate the detailed oncogenic role of TNFAIP8 in skin cancer development and progression. Here we demonstrated that higher expression of TNFAIP8 is associated with basal cell carcinoma (BCC), squamous cell carcinoma (SCC), and melanoma development in patient tissues. Induction of TNFAIP8 expression by TNFα or by ectopic expression of TNFAIP8 in SCC or melanoma cell lines resulted in increased cell growth/proliferation. Conversely, silencing of TNFAIP8 decreased cell survival/cell migration in skin cancer cells. We also showed that miR-205-5p targets the 3'UTR of TNFAIP8 and inhibits TNFAIP8 expression. Moreover, miR-205-5p downregulates TNFAIP8 mediated cellular autophagy, increased sensitivity towards the B-RAFV600E mutant kinase inhibitor vemurafenib, and induced cell apoptosis in melanoma cells. Collectively our data indicate that miR-205-5p acts as a tumor suppressor in skin cancer by targeting TNFAIP8.

Mitochondrial dysfunction contributes to Rapamycin-induced apoptosis of Human Glioblastoma Cells - A synergistic effect with Temozolomide.

Mammalian target of rapamycin (mTOR) is upregulated in a high percentage of glioblastomas. While a well-known mTOR inhibitor, rapamycin, has been shown to reduce glioblastoma survival, the role of mitochondria in achieving this therapeutic effect is less well known. Here, we examined mitochondrial dysfunction mechanisms that occur with the suppression of mTOR signaling. We found that, along with increased apoptosis, and a reduction in transformative potential, rapamycin treatment significantly affected mitochondrial health. Specifically, increased production of reactive oxygen species (ROS), depolarization of the mitochondrial membrane potential (MMP), and altered mitochondrial dynamics were observed. Furthermore, we verified the therapeutic potential of rapamycin-induced mitochondrial dysfunction through co-treatment with temzolomide (TMZ), the current standard of care for glioblastoma. Together these results demonstrate that the mitochondria remain a promising target for therapeutic intervention against human glioblastoma and that TMZ and rapamycin have a synergistic effect in suppressing glioblastoma viability, enhancing ROS production, and depolarizing MMP.

Activation of Sirtuin1 by lyceum barbarum polysaccharides in protection against diabetic cataract.

ETHNOPHARMACOLOGICAL RELEVANCE: Lycium barbarum polysaccharide (LBP) extracted from the Lycium barbarum L. has been widely used to improve diabetes and its relative complications. However, the mechanisms have not fully understood. A recent study has demonstrated that LBP upregulates suituin 1 (SIRT1). OBJECTIVE: This study was to define the role of Sirt1 and its downstream signaling pathways in diabetic cataract using in vitro and in vivo models. MATERIALS AND METHODS: Human lens epithelial cell line SRA01/04 cells were cultured under high glucose (HG) medium with treatment of LBP or vehicle. Cell viability, apoptosis, protein and/or mRNA levels of Sirt1, BAX, Bcl-2, active-caspase-3, FOXO1, p27 and acetylated p53 were measured. SIRT1 upregulated- and knocked-down cells were generated and tested in high glucose culture. Diabetes mellitus was induced in rats by streptozotocin injection. Body weight, blood glucose levels, lens transparency and retinal function were assessed and SIRT1, as well as the aforementioned biomarkers were measured using Western blotting and qPCR in the animal lens samples. RESULTS: The results showed that HG decreased cell viability and LBP prevented the decrease. The reduced viability in HG cultured SRA01/04 cells was associated with increased levels of BAX, active caspase 3, FOXO1, p27, and p53 and decreased levels of SIRT1 and Bcl-2. Further experiments using sirt1 gene modulated cells showed that upregulation of Sirt1 improved viability, increase cell division as reflected by an increased proportion of S phase in the cell cycle, reduced the number of apoptotic cell death and suppressed p53 acetylation and caspase 3 activation. Opposite results were observed in SIRT1 knock-down cells. Treating diabetic animals with LBP reduced body weight loss and blood glucose content in diabetic animals. Similarly, LBP hindered the development of cataract in lenses and improved retinal function. The beneficial effect of LBP on diabetic cataract was associated with the supression of p53, caspase 3, FOXO1, BAX, p27 and elevation of SIRT1 and Bcl-2, which were consistent with the in vitro findings. CONCLUSION: Our findings showed that diabetes caused cataract is associated with suppression of SIRT1 and Bcl-2 and activation of other cell death related genes. LBP prevented diabetic cataract in animals by upregulating Sirt1 and Bcl-2 and suppressing cell death related genes.

The Immp2l Mutation Causes Ovarian Aging Through ROS-Wnt/ß-Catenin-Estrogen Pathway: Preventive Effect of Melatonin.

Mitochondria play important roles in ovarian follicle development. Mitochondrial dysfunction, including mitochondrial gene deficiency, impairs ovarian development. Here, we explored the role and mechanism of mitochondrial inner membrane gene Immp2l in ovarian follicle growth and development. Our results revealed that female Immp2l-/- mice were infertile, whereas Immp2l+/- mice were normal. Body and ovarian weights were reduced in the female Immp2l-/- mice, ovarian follicle growth and development were stunted in the secondary follicle stage. Although a few ovarian follicles were ovulated, the oocytes were not fertilized because of mitochondrial dysfunction. Increased oxidative stress, decreased estrogen levels, and altered genes expression of Wnt/ß-catenin and steroid hormone synthesis pathways were observed in 28-day-old Immp2l-/- mice. The Immp2l mutation accelerated ovarian aging process, as no ovarian follicles were detected by age 5 months in Immp2l-/- mice. All the aforementioned changes in the Immp2l-/- mice were reversed by administration of antioxidant melatonin to the Immp2l-/- mice. Furthermore, our in vitro study using Immp2l knockdown granulosa cells confirmed that the Immp2l downregulation induced granulosa cell aging by enhancing reactive oxygen species (ROS) levels, suppressing Wnt16, increasing ß-catenin, and decreasing steroid hormone synthesis gene cyp19a1 and estrogen levels, accompanied by an increase in the aging phenotype of granulosa cells. Melatonin treatment delayed granulosa cell aging progression. Taken together, Immp2l causes ovarian aging through the ROS-Wnt/ß-catenin-estrogen (cyp19a1) pathway, which can be reversed by melatonin treatment.

Polycomb-like 2 regulates PRC2 components to affect proliferation in glioma cells.

INTRODUCTION: The Polycomb group (PcG) is an important family of transcriptional regulators that controls growth and tumorigenesis. The PcG mainly consists of two complexes, PRC1 and Polycomb Repressive Complex 2 (PRC2). Polycomb-like 2 (PCL2) is known to interact with the PRC2 protein. The role of PCL2 in the development and progression of glioma is unclear. METHODS: We use The Cancer Genome Atlas (TCGA) database to detect the expression of PCL2 in various tumors. 117 cases of clinical glioma (WHOI-IV) were collected, and PCL2 expression and localization were detected by immunohistochemical staining. Glioma cells U87/U251 were infected with overexpressed and interfered PCL2. CCK8 assay, colony formation assay, EdU method, cell cycle and apoptosis were used to detect cell proliferation and apoptosis. Western blot was used to detect the expression of PRC2-related core proteins. After DZNeP intervention, PRC2 protein expression was again measured to discuss the mechanism of PCL2 action. RESULTS: TCGA database results and immunohistochemical staining results suggest that PCL2 is highly expressed in gliomas. We found that the PCL2 gene promoted tumor cell proliferation, enhanced the colony formation ability, and increased S phase in the cell cycle. The overexpression of PCL2 upregulated the expression levels of EZH2 and EED (two core members of PRC2), decreased the expression of SUZ12, increased the level of H3K27 trimethylation (H3K27me3), H3K4 dimethylation (H3K4me2), and decreased H3K9 dimethylation (H3K9me2). The result after interfering with PCL2 was the opposite. CONCLUSIONS: As an important accessory protein of PRC2, PCL2 can not only change the expression of PRC2 components, but also affect the expression level of Histone methylation. Therefore, PCL2 may be an important hub for regulating the synergy among PRC2 members. This study revealed PCL2 as a new target for tumor research and open up a new avenue for future research in glioma.

Silver Nanoparticle Exposure Causes Pulmonary Structural Damage and Mitochondrial Dynamic Imbalance in the Rat: Protective Effects of Sodium Selenite.

BACKGROUND: With the increased application of Silver nanoparticles (AgNP), its potential concerns to the health of human beings remain to be defined. This study aims to explore the harmful effects of AgNP on lung tissue in animals and to examine the mechanisms of protection achieved by sodium selenite. METHODS: Sprague-Dawley(SD) rats were exposed to AgNP (200 µL,1mg/mL) through a single intratracheal instillation. Sodium selenite (0.2mg/kg) was i.p. injected. Malondialdehyde (MDA) and glutathione (GSH) were measured using a spectrophotometer. Histological outcomes and ultrastructural changes were assessed by hematoxylin and eosin (HE) staining and electronic microscopy. Caspases and mitochondrial fission and fusion markers were measured by Western blotting. RESULTS: The histopathologic findings showed that AgNP significantly increased the thickness of alveolar septa, accumulation of macrophage, and the formation of pulmonary bullae and pulmonary consolidation. Ultrastructural studies showed localization of AgNP inside the mitochondria, hyperplasia and vacuolation of type I and type II alveolar cells, lysis of osmiophilic lamellar bodies, and swollen of the mitochondria. AgNP elevated MDA and reduced GSH levels. AgNP activated caspases-3, increased mitochondrial fission markers Dynamin-related protein 1 (Drp1) and phospho-Drp1(p-Drp1), and decreased fusion proteins optic atrophy 1 (Opa1) and mitofusins 2 (Mfn2). Treatment with sodium selenite for 7 days corrected the AgNP-caused alterations in morphological, ultrastructural, oxidative stress, caspase-3 activation and mitochondrial dynamic imbalance. CONCLUSION: We conclude that the exposure of AgNP causes lung tissue damage by enhances oxidative stress, activates caspases-3, and triggers mitochondrial dynamic imbalance towards fission. Sodium selenite effectively detoxifies the AgNP-induced damage to the lung tissue by preventing the above alterations.

Coenzyme Q10 Protects Astrocytes from Ultraviolet B-Induced Damage Through Inhibition of ERK 1/2 Pathway Overexpression.

Overexpression of extracellular signal-regulated kinase ½ (ERK ½) signaling pathway leads to overproduction of reactive oxygen species (ROS) which induces oxidative stress. Coenzyme Q10 (CoQ10) scavenges ROS and protects cells against oxidative stress. The present study was designed to examine whether the protection of Coenzyme Q10 against oxidative damage in astrocytes is through regulating ERK 1/2 pathway. Ultraviolet B (UVB) irradiation was chosen as a tool to induce oxidative stress. Murine astrocytes were treated with 10 µg/ml and 25 µg/ml of CoQ10 for 24 h prior to UVB and maintained during UVB and 24 h post-UVB. Cell viability was evaluated by counting viable cells and MTT conversion assay. ROS production was measured using fluorescent probes. Levels of p-ERK 1/2, ERK 1/2, p-PKA, PKA were detected using immunocytochemistry and/or Western blotting. The results showed that UVB irradiation decreased the number of viable cells. This damaging effect was associated with accumulation of ROS and elevations of p-ERK 1/2 and p-PKA. Treatment with CoQ10 at 25 µg/ml significantly increased the number of viable cells and prevented the UVB-induced increases of ROS, p-ERK 1/2, and p-PKA. It is concluded that suppression of the PKA-ERK 1/2 signaling pathway may be one of the important mechanisms by which CoQ10 protects astrocytes from UVB-induced oxidative damage.

Rapamycin treatment increases hippocampal cell viability in an mTOR-independent manner during exposure to hypoxia mimetic, cobalt chloride.

BACKGROUND: Cobalt chloride (CoCl2) induces chemical hypoxia through activation of hypoxia-inducible factor-1 alpha (HIF-1α). Mammalian target of rapamycin (mTOR) is a multifaceted protein capable of regulating cell growth, angiogenesis, metabolism, proliferation, and survival. In this study, we tested the efficacy of a well-known mTOR inhibitor, rapamycin, in reducing oxidative damage and increasing cell viability in the mouse hippocampal cell line, HT22, during a CoCl2-simulated hypoxic insult. RESULTS: CoCl2 caused cell death in a dose-dependent manner and increased protein levels of cleaved caspase-9 and caspase-3. Rapamycin increased viability of HT22 cells exposed to CoCl2 and reduced activation of caspases-9 and -3. Cells exposed to CoCl2 displayed increased reactive oxygen species (ROS) production and hyperpolarization of the mitochondrial membrane, both of which rapamycin successfully blocked. mTOR protein itself, along with its downstream signaling target, phospho-S6 ribosomal protein (pS6), were significantly inhibited with CoCl2 and rapamycin addition did not significantly lower expression further. Rapamycin promoted protein expression of Beclin-1 and increased conversion of microtubule-associated protein light chain 3 (LC3)-I into LC3-II, suggesting an increase in autophagy. Pro-apoptotic protein, Bcl-2 associated × (Bax), exhibited a slight, but significant decrease with rapamycin treatment, while its anti-apoptotic counterpart, B cell lymphoma-2 (Bcl-2), was to a similar degree upregulated. Finally, the protein expression ratio of phosphorylated mitogen-activated protein kinase (pMAPK) to its unphosphorylated form (MAPK) was dramatically increased in rapamycin and CoCl2 co-treated cells. CONCLUSIONS: Our results indicate that rapamycin confers protection against CoCl2-simulated hypoxic insults to neuronal cells. This occurs, as suggested by our results, independent of mTOR modification, and rather through stabilization of the mitochondrial membrane with concomitant decreases in ROS production. Additionally, inhibition of caspase-9 and -3 activation and stimulation of protective autophagy reduces cell death, while a decrease in the Bax/Bcl-2 ratio and an increase in pMAPK promotes cell survival during CoCl2 exposure. Together these results demonstrate the therapeutic potential of rapamycin against hypoxic injury and highlight potential pathways mediating the protective effects of rapamycin treatment.

Inhibition of MAPK signaling pathways enhances cell death induced by 5-Aminolevulinic acid-photodynamic therapy in skin squamous carcinoma cells.

Combination of a photosensitizer, 5-aminolevulinic acid (ALA), with photodynamic therapy (PDT) has been widely used to treat skin squamous cell carcinoma (SCC). However, a portion of SCC patients do not respond well to PDT. The molecular reason for this resistance is not clear. We hypothesize that mitogen-activated phosphorylation kinase (MAPK) plays a key role in mediating SCC resistance to PDT. To determine whether inhibition of MAPK signaling enhances the anti-tumor effect of ALA-PDT in SCC. The human squamous carcinoma cell line, SCL-1, was either untreated or treated with various combinations of ALA, PDT light source and inhibitors of MAPK signaling components. ALA-PDT treatment significantly decreased cell viability, increased the percentage of annexin-V positive cells and resulted in formation of apoptotic bodies. ALA-PDT treated cells showed increased levels of p-MEK, p-ERK1/2, p-p38, p-Elk-1, p-JNK and p-c-Jun. Addition of inhibitors for ERK1/2 (PD98059), p38 (SB203580) and JNK (SP60125) reversed the changes and led to a more dramatic decrease in SCL-1 cell viability than seen with ALA-PDT alone. Inhibition of the MAPK pathway enhances the cytotoxic effect of ALA-PDT on SCL-1.

Overexpression of human selenoprotein H in neuronal cells ameliorates ultraviolet irradiation-induced damage by modulating cell signaling pathways.

Selenoprotein H (SelH) is one of the 25 so far identified selenoproteins. Selenoproteins may function as antioxidants, heavy metal antidotes, and neural survival factors. Previous studies have shown that overexpression of SelH in HT22 cells protected the cells from UVB irradiation-induced death by reducing superoxide formation. The objective of this study was to determine the effects of SelH on cell signaling pathways after UVB irradiation. We exposed both human SelH- and vector-transfected HT22 cells to UVB irradiation and collected samples at 5 and 17 h of recovery. Cell viability was assessed, as well as protein levels of caspase-3, -8, -9, apoptosis-inducing factor (AIF), P53, nuclear respiratory factor-1 (NRF-1) and heat shock protein 40 (HSP40). Mitochondrial membrane potential was determined by flow cytometry. Overexpression of SelH protected cells against UVB-induced injury by blockade of the mitochondria-initiated cell death pathway, prevention of mitochondrial membrane depolarization, and suppression of the increase of p53. Furthermore, overexpression of SelH increased levels of NRF-1, an antioxidant, and HSP40, a protein chaperone that repairs denatured protein. We conclude that SelH protects neurons against UVB-induced damage by inhibiting apoptotic cell death pathways, by preventing mitochondrial depolarization, and by promoting cell survival pathways.

Hyperglycemia-enhanced ischemic brain damage in mutant manganese SOD mice is associated with suppression of HIF-1alpha.

Both preischemic hyperglycemia and reduction of manganese superoxide dismutase activity are known to enhance neuronal death induced by transient cerebral ischemia. Transcriptional factor hypoxia-inducible factor 1 (HIF-1) regulates multiple downstream genes that modulate cell metabolism, survival, death, angiogenesis, hematopoiesis, and other functions. The objectives of this study were to determine (i) whether hyperglycemia is able to increase ischemic brain damage in mutant manganese superoxide dismutase (SOD2) mice and (ii) whether the reduction of SOD2 activity has a profound effect on HIF-1 protein expression under hyperglycemic ischemic condition. Both wild type and mutant SOD deficient (SOD2(-/+)) mice were induced to hyperglycemia 30min before induction of a 30-min transient middle cerebral artery occlusion (tMCAO). Brains were extracted after 5 and 24h of reperfusion for immunohistochemistry and Western blot analyses. The results showed that preischemic hyperglycemia significantly increased infarct volume in SOD2(-/+)mice and that HIF-1alpha protein levels were significantly reduced in ischemic core area at 5- and 24-h of reperfusion in hyperglycemic SOD2(-/+) mice. However, the HIF-1alpha protein levels were not significantly decreased in hyperglycemic wild type animals subjected to stroke. The results suggest that the increased brain damage observed in hyperglycemic SOD2(-/+) mice is associated with HIF-1alpha suppression, while hyperglycemia per se does not seem to exert its detrimental effects on ischemic brain via modulating HIF-1 pathway.