Geraniol attenuates oxygen-glucose deprivation/reoxygenation-induced ROS-dependent apoptosis and permeability of human brain microvascular endothelial cells by activating the Nrf-2/HO-1 pathway.

Blood-brain barrier breakdown and ROS overproduction are important events during the progression of ischemic stroke aggravating brain damage. Geraniol, a natural monoterpenoid, possesses anti-apoptotic, cytoprotective, anti-oxidant, and anti-inflammatory activities. Our study aimed to investigate the effect and underlying mechanisms of geraniol in oxygen-glucose deprivation/reoxygenation (OGD/R)-induced human brain microvascular endothelial cells (HBMECs). Apoptosis, caspase-3 activity, and cytotoxicity of HBMECs were evaluated using TUNEL, caspase-3 activity, and CCK-8 assays, respectively. The permeability of HBMECs was examined using FITC-dextran assay. Reactive oxygen species (ROS) production was measured using the fluorescent probe DCFH-DA. The protein levels of zonula occludens-1 (ZO-1), occludin, claudin-5, ß-catenin, nuclear factor erythroid 2-related factor 2 (Nrf2), and heme oxygenase-1 (HO-1) were determined by western blotting. Geraniol showed no cytotoxicity in HBMECs. Geraniol and ROS scavenger N-acetylcysteine (NAC) both attenuated OGD/R-induced apoptosis and increase of caspase-3 activity and the permeability to FITC-dextran in HBMECs. Geraniol relieved OGD/R-induced ROS accumulation and decrease of expression of ZO-1, occludin, claudin-5, and ß-catenin in HBMECs. Furthermore, we found that geraniol activated Nrf2/HO-1 pathway to inhibit ROS in HBMECs. In conclusion, geraniol attenuated OGD/R-induced ROS-dependent apoptosis and permeability in HBMECs through activating the Nrf2/HO-1 pathway.

Hederagenin Upregulates PTPN1 Expression in Aß-Stimulated Neuronal Cells, Exerting Anti-Oxidative Stress and Anti-Apoptotic Activities.

Alzheimer's disease (AD) is a prevalently neurodegenerative disease characterized by neuronal damage which is associated with amyloid-ß (Aß) accumulation. Hederagenin is a triterpenoid saponin, exerting anti-apoptotic, anti-oxidative, anti-inflammatory, anti-tumoral, and neuroprotective activities. However, its role in AD progression is still obscure. The aim of this study was to explore the influences of hederagenin on Aß-caused neuronal injury in vitro. Neuronal cells were treated with Aß25-35 (Aß) to establish a cellular model of AD. Cell viability was assessed using cell counting kit-8 (CCK-8). Oxidative stress was evaluated by detecting reactive oxygen species (ROS) generation and superoxide dismutase (SOD) activity. Apoptosis was investigated using TUNEL staining and caspase-3 activity assays. Protein tyrosine phosphatase nonreceptor type 1 (PTPN1) was screened by bioinformatics analysis. Protein levels of PTPN1 and protein kinase B (Akt) were measured by western blotting. Hederagenin (2.5, 5, and 10 µM) alone did not affect viability of neuronal cells, but relieved Aß-induced viability reduction. Hederagenin mitigated Aß-induced increase in ROS accumulation and decrease in SOD activity. Hederagenin attenuated Aß-induced increase in apoptotic rate and caspase-3 activity. PTPN1 was screened as a target of hederagenin against AD by bioinformatics analysis. Hederagenin treatment resisted Aß-induced decrease in PTPN1 mRNA and protein levels in neuronal cells. PTPN1 silencing attenuated the suppressive functions of hederagenin in Aß-stimulated oxidative stress and apoptosis. Hederagenin mitigated Aß-induced Akt signaling inactivation by upregulating PTPN1 expression. In conclusion, hederagenin attenuates oxidative stress and apoptosis in neuronal cells stimulated with Aß by promoting PTPN1/Akt signaling activation.

Targeting MAGI2-AS3-modulated Akt-dependent ATP-binding cassette transporters as a possible strategy to reverse temozolomide resistance in temozolomide-resistant glioblastoma cells.

Drug resistance is a major impediment to the successful treatment of glioma. This study aimed to elucidate the effects and mechanisms of the long noncoding RNA membrane-associated guanylate kinase inverted-2 antisense RNA 3 (MAGI2-AS3) on temozolomide (TMZ) resistance in glioma cells. MAGI2-AS3 expression in TMZ-resistant glioblastoma (GBM) cells was analyzed using the Gene Expression Omnibus data set GSE113510 and quantitative real-time PCR (qRT-PCR). Cell viability and TMZ half-maximal inhibitory concentration values were determined using the MTT assay. Apoptosis and cell cycle distribution were evaluated using flow cytometry. The expression of multidrug resistance 1 (MDR1), ATP-binding cassette superfamily G member 2 (ABCG2), protein kinase B (Akt), and phosphorylated Akt was detected using qRT-PCR and/or western blot analysis. MAGI2-AS3 was expressed at low levels in TMZ-resistant GBM cells relative to that in their parental cells. MAGI2-AS3 re-expression alleviated TMZ resistance in TMZ-resistant GBM cells. MAGI2-AS3 overexpression also accelerated TMZ-induced apoptosis and G2/M phase arrest. Mechanistically, MAGI2-AS3 overexpression reduced MDR1 and ABCG2 expression and inhibited the Akt pathway, whereas Akt overexpression abrogated the reduction in MDR1 and ABCG2 expression induced by MAGI2-AS3. Moreover, activation of the Akt pathway inhibited the effects of MAGI2-AS3 on TMZ resistance. MAGI2-AS3 inhibited tumor growth and enhanced the suppressive effect of TMZ on glioma tumorigenesis in vivo. In conclusion, MAGI2-AS3 reverses TMZ resistance in glioma cells by inactivating the Akt pathway.

Rutaecarpine attenuates high glucose-induced damage in AC16 cardiomyocytes by suppressing the MAPK pathway.

Diabetic cardiomyopathy is a common diabetic complication, resulting in heart failure. Rutaecarpine is an active compound with cardiovascular protective effects. However, the function of rutaecarpine in diabetic cardiomyopathy is largely unknown. The aim of this research was to study the effect and action mechanism of rutaecarpine in high glucose (HG)-induced cardiomyocyte damage. The overlapping genes of diabetic cardiomyopathy and rutaecarpine were analyzed according to GeneCards, DisGeNet, and SwissTargetPrediction. Cell damage was investigated by determining apoptosis, oxidative stress, and inflammatory response in HG-stimulated AC16 cells. The expression of proteins involved in the mitogen-activated protein kinase (MAPK) signaling was measured using Western blotting. Totally seven overlapping genes of diabetic cardiomyopathy and rutaecarpine were screened out and predicted to be associated with the MAPK signaling. Rutaecarpine protected against HG-induced cardiomyocyte damage by enhancing cell viability and reducing cell apoptosis, caspase-3 activity, and lactate dehydrogenase (LDH) release. Rutaecarpine mitigated HG-induced oxidative stress in cardiomyocytes through decreasing reactive oxygen species (ROS) formation and malondialdehyde (MDA) level and elevating superoxide dismutase (SOD) activity and glutathione peroxidase (GSH-Px) level. Rutaecarpine alleviated HG-induced inflammatory response via reducing the level of interleukin (IL)-1ß, IL-6, tumor necrosis factor (TNF)-α, and IL-8. Moreover, rutaecarpine inhibited HG-induced activation of the MAPK pathway. Treatment with MAPK signaling agonist reversed the suppressive effect of rutaecarpine on HG-induced damage. In conclusion, rutaecarpine alleviated HG-induced cardiomyocyte damage through decreasing apoptosis, oxidative stress, and inflammatory response by inactivating the MAPK pathway.

Cardamonin attenuates cerebral ischemia/reperfusion injury by activating the HIF-1α/VEGFA pathway.

Cardamonin is a chalcone with neuroprotective activity. The aim of our study was to explore the functions and mechanism of action of cardamonin in ischemic stroke. Oxygen-glucose deprivation and reperfusion (OGD/R)-induced human brain microvascular endothelial cells (HBMECs) and middle cerebral artery occlusion (MCAO) mouse model were utilized to mimic ischemic stroke. Cell viability was analyzed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide. Permeability was investigated via fluorescein isothiocyanate-dextran assay. Apoptosis was detected by TdT-Mediated dUTP Nick End Labeling staining. Hypoxia-inducible factor (HIF)-1α and vascular endothelial growth factor A (VEGFA) protein levels were measured using Western blotting. Brain injury was evaluated by 2,3,5-triphenyltetrazolium chloride staining, neurological score and brain water content. The 37 overlapping targets of ischemic stroke and cardamonin were predicted to be associated with the HIF-1/VEGFA signaling. Cardamonin alleviated OGD/R-induced viability reduction and increase of permeability and apoptosis in HBMECs. Cardamonin increased OGD/R-induced activation of the HIF-1α/VEGFA pathway. Inhibition of the HIF-1α/VEGFA signaling using inhibitor relieved the effect of cardamonin on cell viability, permeability and apoptosis in HBMECs under OGD/R. Cardamonin mitigated brain injury and promoted activation of the HIF-1α/VEGFA signaling in MCAO-treated mice. Overall, cardamonin protected against OGD/R-induced HBMEC damage and MACO-induced brain injury through activating the HIF-1α/VEGFA pathway.

WDR34 affects PI3K/Akt and Wnt/ß-catenin pathways to regulates malignant biological behaviors of glioma cells.

PURPOSE: Glioma is the most prevalent primary intracranial tumor globally. WDR34, a member of the WDR superfamily with five WD40 repeats, is involved in the pathogenesis of several tumors. However, the role of WDR34 in glioma progression is unknown. METHODS: The expression and prognostic significance of WDR34 in glioma patients were analyzed using GEPIA. WDR34 expression was detected by qRT-PCR. Western blot was employed to determine the expression of Ki67, proliferating cell nuclear antigen (PCNA), matrix metallopeptidase (MMP)2, MMP9, phosphatase and tensin homolog, protein kinase B (Akt), phosphorylated Akt, ß-catenin, and c-Myc. CCK-8, BrdU incorporation assay, Transwell invasion assay, flow cytometry analysis, and measurement of caspase-3 and caspase-9 activities were conducted to examine the effects of WDR34 knockdown on glioma cells. RESULTS: WDR34 was upregulated in glioma, which predicted a poor prognosis in glioma patients. WDR34 knockdown inhibited cell proliferation and reduced the expression of Ki67 and PCNA in glioma cells. WDR34 knockdown repressed the invasive ability of glioma cells by decreasing MMP-2 and MMP-9 expression. WDR34 knockdown increased the apoptotic rate and caspase-3 and caspase-9 activities in glioma cells. The PI3K/Akt and Wnt/ß-catenin pathways were inhibited after WDR34 knockdown in glioma cells. Moreover, overexpression of Akt or ß-catenin reversed the function of WDR34 knockdown on proliferation, invasion, and apoptosis. WDR34 knockdown reduced tumor growth in vivo. CONCLUSIONS: WDR34 knockdown inhibited malignant biological behaviors of glioma cells by inactivating the PI3K/Akt and Wnt/ß-catenin signaling cascades.

SRY-Box 21 Antisense RNA 1 Knockdown Diminishes Amyloid Beta25-35-Induced Neuronal Damage by miR-132/PI3K/AKT Pathway.

Our study aimed to explore the function and mechanism of action of long noncoding RNA (lncRNA) SRY-Box 21 antisense RNA 1 (SOX21-AS1) in amyloid beta25-35 (Aß25-35)-induced neuronal damage. To induce neuronal damage, neuronal cells and differentiated IMR-32 neuroblastoma cells were challenged by Aß25-35. SOX21-AS1 and miR-132 quantities were detected by quantitative reverse transcription polymerase chain reaction. Cell damage was evaluated by detecting the changes of cell viability, apoptosis, and oxidative stress. Cell viability was measured using cell counting kit-8. Cell apoptosis was evaluated by flow cytometry and caspase-3 activity. The oxidative stress was analyzed by reactive oxygen species level. The expression of proteins associated with the phosphoinositide 3-kinase (PI3K)/protein kinase B (AKT) pathway was examined by western blot. SOX21-AS1 abundance was up-regulated in Aß25-35-challenged neuronal cells. Silencing of SOX21-AS1 attenuated Aß25-35-induced viability reduction and promotion of apoptosis and oxidative stress, suggesting that silencing of SOX21-AS1 repressed Aß25-35-induced neuronal damage. miR-132 quantity was reduced in Aß25-35-challenged neuronal cells, and negatively controlled by SOX21-AS1. miR-132 knockdown abolished the effect of SOX21-AS1 silencing on Aß25-35-induced neuronal damage, indicating that SOX21-AS1 controls Aß25-35-induced neuronal damage via regulating miR-132. The PI3K/AKT signaling was repressed in Aß25-35-challenged cells, but this effect was counteracted upon overexpression of miR-132. In conclusion, SOX21-AS1 knockdown mitigated Aß25-35-dependent neuronal cell damage by promoting miR-132/PI3K/AKT pathway.

Pseudogene ANXA2P2 knockdown shows tumor-suppressive function by inhibition of the PI3K/PKB pathway in glioblastoma cells.

The pseudogene annexin A2 pseudogene 2 (ANXA2P2) is highly expressed in glioblastoma (GBM). However, its role and mechanism involved in the progression of GBM remain poorly understood. ANXA2P2 messenger RNA expression was measured by quantitative reverse transcription-polymerase chain reaction. The protein levels were detected by Western blot. Cell viability was evaluated by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide and lactate dehydrogenase (LDH) release assays. Cell invasive ability was investigated by the transwell assay and by epithelial-mesenchymal transition (EMT). Cell apoptosis was examined by flow cytometry. The results showed that ANXA2P2 expression was increased in GBM tissues and cells. Silencing of ANXA2P2 inhibited the activation of the phosphoinositide 3-kinase (PI3K)/protein kinase B (PKB) pathway in GBM cells. Knockdown of ANXA2P2 decreased cell viability, promoted LDH release, suppressed cell invasive ability, and EMT, and induced cell apoptosis in GBM cells. The addition of the PI3K/PKB activator 740Y-P abrogated the effects of ANXA2P2 knockdown on cell viability, LDH release, invasive ability, and apoptosis. In conclusion, knockdown of ANXA2P2 inhibited cell viability and invasion but promoted the apoptotic rate by suppressing the PI3K/PKB pathway in GBM cells. ANXA2P2 may represent a new target for the treatment of GBM.

The nuclear transporter importin-11 regulates the Wnt/ß-catenin pathway and acts as a tumor promoter in glioma.

Karyopherins mediate the macromolecular transport between the cytoplasm and the nucleus and participate in cancer progression. However, the role and mechanism of importin-11 (IPO11), a member of the karyopherin family, in glioma progression remain undefined. Effects of IPO11 on glioma progression were detected using CCK-8, colony formation assay, flow cytometry analysis, caspase-3 activity assay, and Transwell invasion assay. Western blot analysis was used to detect the expression of active caspase-3, active caspase-7, active caspase-9, N-cadherin, Vimentin, E-cadherin, ß-catenin, and c-Myc. The activity of Wnt/ß-catenin pathway was evaluated by the T-cell factor/lymphoid enhancer factor (TCF/LEF) transcription factor reporter assay. Results showed that IPO11 knockdown inhibited proliferation and reduced colony number in glioma cells. IPO11 silencing promoted the apoptotic rate, increased expression levels of active caspase-3, caspase-7, and caspase-9, and enhanced caspase-3 activity. Moreover, IPO11 silencing inhibited glioma cell invasion by suppressing epithelial-to-mesenchymal transition (EMT). Mechanistically, IPO11 knockdown inactivated the Wnt/ß-catenin pathway. ß-Catenin overexpression abolished the effects of IPO11 silencing on the proliferation, apoptosis, and invasion in glioma cells. Furthermore, IPO11 silencing blocked the malignant phenotypes and repressed the Wnt/ß-catenin pathway in vivo. In conclusion, IPO11 knockdown suppressed the malignant phenotypes of glioma cells by inactivating the Wnt/ß-catenin pathway.

LncRNA SAMMSON Knockdown Inhibits the Malignancy of Glioblastoma Cells by Inactivation of the PI3K/Akt Pathway.

Dysregulated lncRNAs are proposed to be tightly associated with the progression of various tumors including glioblastoma (GBM). LncRNA Survival Associated Mitochondrial Melanoma-Specific Oncogenic Non-Coding RNA (SAMMSON) has been reported to be an oncogenic lncRNA in several tumors. Nevertheless, the specific role and molecular mechanism of SAMMSON in GBM progression remain unknown. Expression of SAMMSON in GBM tissues and cells was detected by qRT-PCR. CCK-8 and LDH release assays were applied to evaluate cellular viability. Invasion effect was assessed by Transwell invasion assay and western blot analysis of E-cadherin and N-cadherin expression. Apoptosis was detected using flow cytometry analysis and caspase-3 activity assay. The protein levels of phosphatidylinositol-3-kinase (PI3K), phosphorylated (p)-PI3K, protein kinase B (Akt) and p-Akt were estimated by western blot. We found that SAMMSON was highly expressed in GBM tissues and cells. SAMMSON knockdown suppressed cell viability and increased LDH release in GBM cells. Moreover, SAMMSON silencing impeded the invasive ability of GBM cells by regulating epithelial-to-mesenchymal transition (EMT). Furthermore, SAMMSON downregulation increased the apoptotic rate and caspase-3 activity in GBM cells. Additionally, it was demonstrated that the PI3K/Akt pathway was inhibited following SAMMSON silencing in GBM cells. Rescue assays revealed that activation of the PI3K/Akt pathway by 740Y-P abolished SAMMSON knockdown-induced viability reduction, invasion suppression and apoptosis in GBM cells. Taken together, lncRNA SAMMSON knockdown inhibited the malignancy of GBM cells by inactivation of the PI3K/Akt pathway.

SMAGP knockdown inhibits the malignant phenotypes of glioblastoma cells by inactivating the PI3K/Akt pathway.

Small trans-membrane and glycosylated protein (SMAGP), a novel small trans-membrane glycoprotein, is reported to be upregulated in multiple cancers and involved in tumor development. However, little is known about its role in the development of glioblastoma (GBM). GEPIA database was used to analyze SMAGP expression and evaluate the prognostic value of SMAGP in GBM. GO and KEGG pathway enrichment analyses were used to predict the biological functions and pathways of SMAGP and 948 SMAGP-correlated genes using DAVID database. Cell viability, colony formation ability, apoptosis, and invasion were evaluated by MTT, colony formation assay, flow cytometry analysis, and Transwell invasion assay, respectively. Western blot was applied to detect the expression of SMAGP, matrix metalloproteinase (MMP)-2, and MMP-9 and analyze the changes of phosphoinositide 3-kinase (PI3K)/protein kinase B (Akt) signaling. Results showed that SMAGP was upregulated and correlated with poor prognosis in GBM. Functional annotation analysis revealed that SMAGP and 948 SMAGP-correlated genes were primarily associated with cell adhesion and PI3K/Akt pathway. SMAGP interference inhibited cell viability and colony formation ability and promoted apoptosis in GBM cells. Moreover, SMAGP interference inhibited GBM cell invasion and suppressed MMP-2 and MMP-9 expression. Additionally, SMAGP silencing inhibited the PI3K/Akt pathway in GBM cells. Overexpression of Akt abolished the effects of SMAGP knockdown on the malignant phenotypes of GBM cells. In conclusion, SMAGP silencing inhibited the malignant phenotypes of GBM cells by inactivating the PI3K/Akt pathway.

miR-454-3p exerts tumor-suppressive functions by down-regulation of NFATc2 in glioblastoma.

Glioblastoma (GBM) is the most prevalent and malignant primary brain tumor. Numerous studies have demonstrated that aberrant microRNAs (miRNAs) expression is involved in various pathogenesis events in GBM. miR-454-3p has been found to be downregulated in GBM, however, the role and underlying mechanism has not been fully investigated. The expression levels of miR-454-3p in GBM clinical tissues and cultured cell lines were measured by qRT-PCR. To evaluate the role of miR-454-3p in GBM, GBM cells were transfected with miR-454-3p inhibitor (anti-miR-454-3p)/control inhibitor (anti-miR-NC) or miR-454-3p mimic/control mimic (miR-NC). Online prediction algorithm Targetscan was used to predict the target genes of miR-454-3p. The dual luciferase reporter gene assay was performed to confirm whether nuclear factor of activated T cells c2 (NFATc2) was a direct biological target of miR-454-3p. We found that miR-454-3p expression was significantly decreased in both GBM tissues and cultured cell lines. Overexpression of miR-454-3p by transfection with miR-454-3p mimic suppressed cell proliferation and induced apoptosis of GBM cells. Nuclear factor of activated T cells c2 (NFATc2) was predicted as a target gene of miR-454-3p. We found that NFATc2 expression was significantly increased in both GBM tissues and cultured cell lines. Besides, expression levels of miR-454-3p were negatively associated with NFATc2 expression in GBM tissues. NFATc2 knockdown in GBM cells obviously inhibited cell proliferation and elevated apoptosis. Luciferase reporter assay revealed that NFATc2 was a target gene of miR-454-3p. miR-454-3p overexpression suppressed the NFATc2 expression, while inhibition of miR-454-3p induced the NFATc2 expression. Suppression of NFATc2 blocked the effects of miR-454-3p inhibitor on cell proliferation and apoptosis in GBM cells. The results indicated that miR-454-3p executed the tumor suppressive activity via downregulating NFATc2 in GBM.

Rutin alleviates hypoxia/reoxygenation-induced injury in myocardial cells by up-regulating SIRT1 expression.

Rutin possesses multiple pharmacological activities, including the cardioprotective effect. The present study aimed to evaluate the protective effects of rutin on hypoxia/reoxygenation (H/R)-induced myocardial injury and its underlying mechanism involved. H9c2 cells were pretreated with 50 µM rutin or combined with 1 µM silent information regulator 1 (SIRT1) inhibitor (EX-527) for 1 h, and subjected to hypoxia for 6 h, followed by reoxygenation for 24 h. SIRT1 expression was detected by qRT-PCR and western blot. The effects of rutin or combined with EX-527 on cell viability, myocardial injury, apoptotic rate, and oxidative stress in H/R-stimulated H9c2 cells were assayed. The results showed that rutin elevated SIRT1 expression in H9c2 cells, as well as H/R-stimulated H9c2 cells. Rutin increased cell viability in H9c2 cells exposed to H/R. H/R stimulation induced myocardial injury, as evidenced by the increased levels of lactate dehydrogenase (LDH), creatine kinase-MB (CK-MB) and aspartate transaminase (AST), which were abolished in the presence of rutin. Rutin attenuated H/R-induced increase of apoptotic rate and caspase-3 activity in H/R-treated cells. Moreover, H/R-induced decrease in the activities of superoxide dismutase (SOD), glutathione peroxidase (GSH-Px) and catalase (CAT), and increase in malondialdehyde (MDA) content were reversed by rutin treatment. The presence of EX-527 abolished these protective effects of rutin. In conclusion, rutin protected H9c2 cells against H/R injury through increasing SIRT1 expression. Our findings suggested that rutin might be a potential therapeutic agent for the treatment of myocardial H/R injury.

MicroRNA-485-5p attenuates cell proliferation in glioma by directly targeting paired box 3.

MicroRNA-485-5p (miR-485-5p) has been reported to be involved in the development and progression of human cancers; however, its role in glioma remains unclear. In the present study, we found that miR-485-5p was significantly down-regulated in both glioma tissues and cell lines. Functional experiments indicated that enhanced expression of miR-485-5p attenuated glioma cell proliferation in vitro and in vivo, and induced glioma cells cycle arrest in G1. MiR-485-5p was found to directly bind to the 3'-UTR of paired box 3 (PAX3) and decrease its expression of protein level, which further inhibits the proliferation of glioma. The decreasing of PAX3 was found to lead to the accumulation of p-JNK. Mechanistic studies revealed that restoring the expression of PAX3 alleviated miR-485-5p-induced inhibition of proliferation of glioma cells. Taken together, these findings suggest that PAX3 modulation by miR-485-5p has an important role in regulating glioma proliferation, and miR-485-5p might be a novel therapeutic target for glioma.