Neuroglobin Facilitates Neuronal Oxygenation through Tropic Migration under Hypoxia or Anemia in Rat: How Does the Brain Breathe?

The discovery of neuroglobin (Ngb), a brain- or neuron-specific member of the hemoglobin family, has revolutionized our understanding of brain oxygen metabolism. Currently, how Ngb plays such a role remains far from clear. Here, we report a novel mechanism by which Ngb might facilitate neuronal oxygenation upon hypoxia or anemia. We found that Ngb was present in, co-localized to, and co-migrated with mitochondria in the cell body and neurites of neurons. Hypoxia induced a sudden and prominent migration of Ngb towards the cytoplasmic membrane (CM) or cell surface in living neurons, and this was accompanied by the mitochondria. In vivo, hypotonic and anemic hypoxia induced a reversible Ngb migration toward the CM in cerebral cortical neurons in rat brains but did not alter the expression level of Ngb or its cytoplasm/mitochondria ratio. Knock-down of Ngb by RNA interference significantly diminished respiratory succinate dehydrogenase (SDH) and ATPase activity in neuronal N2a cells. Over-expression of Ngb enhanced SDH activity in N2a cells upon hypoxia. Mutation of Ngb at its oxygen-binding site (His64) significantly increased SDH activity and reduced ATPase activity in N2a cells. Taken together, Ngb was physically and functionally linked to mitochondria. In response to an insufficient oxygen supply, Ngb migrated towards the source of oxygen to facilitate neuronal oxygenation. This novel mechanism of neuronal respiration provides new insights into the understanding and treatment of neurological diseases such as stroke and Alzheimer's disease and diseases that cause hypoxia in the brain such as anemia.

Rapid tumor recurrence in a novel murine GBM surgical model is associated with Akt/PD-L1/vimentin signaling.

Glioblastoma multiforme (GBM) is the most common primary malignant brain tumor without curable therapy. Surgical resection remains the first choice of patients with GBM but tumors relapse rapidly even combined with conventional chemoradiotherapy. The mechanism of GBM rapid recurrence is poorly understood, which is largely due to the lack of an appropriate animal model, thus heavily impedes the improvement of postoperative therapy. Here we established a highly reproducible mouse GBM surgical model by using the syngeneic G422TN-GBM cells, which faithfully recapitulates the features of rapid recurrence of human GBM after surgery. Implanting 2 × 103-5 × 104 of G422TN-GBM cells in mouse cerebral cortex caused death in all animal within 23 days, while surgery was an effective therapy but not curable. After complete removal of visible tumors on day 5-9 of tumor growth, the tumors recurred macroscopically within 5 days accompanied by increasing infiltrative cancer foci. Mechanistically, the rapid recurrence of resected tumors was positively correlated to early Akt activation, which subsequently upregulated PD-L1/Vimentin and promoted proliferation/migration of cancer cells. In addition, environmental astrocytic activation with strong PD-L1 signal was prominent. Taken together, we provided a novel GBM surgical recurrence model for preclinical studies and suggested complicated recurring mechanisms involving in strong oncogenic signaling as well as immune inhibitory signals from both GBM cells and their neighboring astrocytes.

PD-L1 confers glioblastoma multiforme malignancy via Ras binding and Ras/Erk/EMT activation.

Glioblastoma multiforme (GBM) is the most aggressive primary brain tumor due to the lack of effective therapeutic drugs. Cancer therapy targeting programmed cell death protein 1 (PD-1) or programmed death ligand-1 (PD-L1) is of revolutionary. However, the role of intrinsic PD-L1, which determines immune-therapy outcomes, remains largely unclear. Here we demonstrated an oncogenic role of PD-L1 via binding and activating Ras in GBM cells. RNA-sequencing transcriptome data revealed that PD-L1 significantly altered gene expression enriched in cell growth/migration/invasion pathways in human GBM cells. PD-L1 overexpression and knockout or knockdown demonstrated that PD-L1 promoted GBM cell proliferation and migration in vitro and in vivo. Mechanistically, PD-L1 prominently activated epithelial mesenchymal transition (EMT) process in a MEK/Erk- but not PI3K/Akt-dependent manner. Further, we identified intracellular interactions of PD-L1 and H-Ras, which led to Ras/Erk/EMT activation. Finally, we demonstrated that PD-L1 overexpression promoted while knockdown abolished GBM development and invasion in orthotopic GBM models of rodents. Taken together, we found that intracellular PD-L1 confers GBM cell malignancy and aggressiveness via binding Ras and activating the downstream Erk-EMT signaling. Thus, these results shed important insights in improving efficacy of immune therapy for GBM as well as other malignant tumors.

Piperlongumine suppresses bladder cancer invasion via inhibiting epithelial mesenchymal transition and F-actin reorganization.

Piperlongumine (PL), a natural alkaloid isolated from longer pepper plants, is recently found to be a potent selective anti-cancer compound. We first tested its anti-cancer effects on bladder cancer, the fifth most common and aggressive cancer worldwide, to further explore the therapeutic spectrum and molecular mechanisms of PL. PL significantly suppressed bladder cancer cell proliferation, the transition of G2/M phase to next phase, migration/invasion in vitro and bladder cancer growth/development in vivo. PL markedly elevated reactive oxygen species (ROS) and the administration of antioxidants abolished PL induced cell proliferation inhibition, G2/M phase arrest and migration suppression on bladder cancer cells. In vivo studies demonstrated that PL inhibited epithelial mesenchymal transition with profoundly decreased level of Slug, ß-catenin, ZEB1 and N-Cadherin. Further, we first reported PL effects on cytoskeleton with prominently reduced lamellipodia formation and decreased F-actin intensity in bladder cancer cells. Taken together, our results first revealed that PL suppressed bladder cancer proliferation and migration in vivo and in vitro, suggesting novel mechanism underlying PL's anti-cancer effect and providing a new anticancer drug strategy for bladder cancer therapy.

Identification of repaglinide as a therapeutic drug for glioblastoma multiforme.

Glioblastoma multiforme (GBM) is a highly aggressive brain tumor with a median survival time of only 14 months after treatment. It is urgent to find new therapeutic drugs that increase survival time of GBM patients. To achieve this goal, we screened differentially expressed genes between long-term and short-term survived GBM patients from Gene Expression Omnibus database and found gene expression signature for the long-term survived GBM patients. The signaling networks of all those differentially expressed genes converged to protein binding, extracellular matrix and tissue development as revealed in BiNGO and Cytoscape. Drug repositioning in Connectivity Map by using the gene expression signature identified repaglinide, a first-line drug for diabetes mellitus, as the most promising novel drug for GBM. In vitro experiments demonstrated that repaglinide significantly inhibited the proliferation and migration of human GBM cells. In vivo experiments demonstrated that repaglinide prominently prolonged the median survival time of mice bearing orthotopic glioma. Mechanistically, repaglinide significantly reduced Bcl-2, Beclin-1 and PD-L1 expression in glioma tissues, indicating that repaglinide may exert its anti-cancer effects via apoptotic, autophagic and immune checkpoint signaling. Taken together, repaglinide is likely to be an effective drug to prolong life span of GBM patients.

Piperlongumine selectively kills hepatocellular carcinoma cells and preferentially inhibits their invasion via ROS-ER-MAPKs-CHOP.

Hepatocellular carcinomas (HCC) are highly malignant and aggressive tumors lack of effective therapeutic drugs. Piperlongumine (PL), a natural product isolated from longer pepper plants, is recently identified as a potent cytotoxic compound highly selective to cancer cells. Here, we reported that PL specifically suppressed HCC cell migration/invasion via endoplasmic reticulum (ER)-MAPKs-CHOP signaling pathway. PL selectively killed HCC cells but not normal hepatocytes with an IC50 of 10-20 µM while PL at much lower concentrations only suppressed HCC cell migration/invasion. PL selectively elevated reactive oxygen species (ROS) in HCC cells, which activated or up-regulated downstream PERK/Ire 1α/Grp78, p38/JNK/Erk and CHOP subsequently. Administration of antioxidants completely abolished PL's effects on cell death and migration/invasion. However, pharmacological inhibition of ER stress-responses or MAPKs signaling pathways with corresponding specific inhibitors only reversed PL's effect on cell migration/invasion but not on cell death. Consistently, knocking-down of CHOP by RNA interference only reversed PL-suppressed HCC cell migration. Finally, PL significantly suppressed HCC development and activated the ER-MAPKs-CHOP signaling pathway in HCC xenografts in vivo. Taken together, PL selectively killed HCC cells and preferentially inhibited HCC cell migration/invasion via ROS-ER-MAPKs-CHOP axis, suggesting a novel therapeutic strategy for the highly malignant and aggressive HCC clinically.

Piperlongumine induces apoptotic and autophagic death of the primary myeloid leukemia cells from patients via activation of ROS-p38/JNK pathways.

AIM: To investigate the effects of piperlongumine (PL), an anticancer alkaloid from long pepper plants, on the primary myeloid leukemia cells from patients and the mechanisms of action. METHODS: Human BM samples were obtained from 9 patients with acute or chronic myeloid leukemias and 2 patients with myelodysplastic syndrome (MDS). Bone marrow mononuclear cells (BMMNCs) were isolated and cultured. Cell viability was determined using MTT assay, and apoptosis was examined with PI staining or flow cytometry. ROS levels in the cells were determined using DCFH-DA staining and flow cytometry. Expression of apoptotic and autophagic signaling proteins was analyzed using Western blotting. RESULTS: PL inhibited the viability of BMMNCs from the patients with myeloid leukemias (with IC50 less than 20 µmol/L), but not that of BMMNCs from a patient with MDS. Furthermore, PL (10 and 20 µmol/L) induced apoptosis of BMMNCs from the patients with myeloid leukemias in a dose-dependent manner. PL markedly increased ROS levels in BMMNCs from the patients with myeloid leukemias, whereas pretreatment with the antioxidant N-acetyl-L-cysteine abolished PL-induced ROS accumulation and effectively reduced PL-induced cytotoxicity. Moreover, PL markedly increased the expression of the apoptotic proteins (Bax, Bcl-2 and caspase-3) and autophagic proteins (Beclin-1 and LC3B), and phosphorylation of p38 and JNK in BMMNCs from the patients with myeloid leukemias, whereas pretreatment with the specific p38 inhibitor SB203580 or the specific JNK inhibitor SP600125 partially reversed PL-induced ROS production, apoptotic/autophagic signaling activation and cytotoxicity. CONCLUSION: Piperlongumine induces apoptotic and autophagic death of the primary myeloid leukemia cells from patients via activation of ROS-p38/JNK pathways.

Piperlongumine inhibits migration of glioblastoma cells via activation of ROS-dependent p38 and JNK signaling pathways.

Piperlongumine (PL) is recently found to kill cancer cells selectively and effectively via targeting reactive oxygen species (ROS) responses. To further explore the therapeutic effects of PL in cancers, we investigated the role and mechanisms of PL in cancer cell migration. PL effectively inhibited the migration of human glioma (LN229 or U87 MG) cells but not normal astrocytes in the scratch-wound culture model. PL did not alter EdU(+)-cells and cdc2, cdc25c, or cyclin D1 expression in our model. PL increased ROS (measured by DCFH-DA), reduced glutathione, activated p38 and JNK, increased IκBα, and suppressed NFκB in LN229 cells after scratching. All the biological effects of PL in scratched LN229 cells were completely abolished by the antioxidant N-acetyl-L-cysteine (NAC). Pharmacological administration of specific p38 (SB203580) or JNK (SP600125) inhibitors significantly reduced the inhibitory effects of PL on LN229 cell migration and NF κ B activity in scratch-wound and/or transwell models. PL prevented the deformation of migrated LN229 cells while NAC, SB203580, or SP600125 reversed PL-induced morphological changes of migrated cells. These results suggest potential therapeutic effects of PL in the treatment and prevention of highly malignant tumors such as glioblastoma multiforme (GBM) in the brain by suppressing tumor invasion and metastasis.

Neuroglobin promotes neurite outgrowth via differential binding to PTEN and Akt.

Neuroglobin, the third mammalian globin with a hexa-coordinated heme, exists predominantly in neurons of the brain. Neuroglobin plays an important role in neuronal death upon ischemia and oxidative stress. The physiological function of neuroglobin remains unclear. Here, we report a novel function of neuroglobin in neurite development. Knocking-down neuroglobin exhibited a prominent neurite-deficient phenotype in mouse neuroblastoma N2a cells. Silencing neuroglobin prevented neurite outgrowth, while ectopic expression of neuroglobin but not homologous cytoglobin promoted neurite outgrowth of N2a cells upon serum withdrawal. In primary cultured rat cerebral cortical neurons, neuroglobin was upregulated and preferentially distributed in neurites during neuronal development. Overexpression of neuroglobin but not cytoglobin in cultured cortical neurons promoted axonal outgrowth, while knocking-down of neuroglobin retarded axonal outgrowth. Neuroglobin overexpression suppressed phosphatase and tensin homolog (PTEN) but increased Akt phosphorylation during neurite induction. Bimolecular fluorescence complementation and glutathione S-transferase pull-down assays revealed that neuroglobin and various mutants (E53Q, E118Q, K119N, H64A, H64L, and Y44D) bound with Akt and PTEN differentially. Neuroglobin E53Q showed a prominent reduced PTEN binding but increased Akt binding, resulting in decreased p-PTEN, increased p-Akt, and increased neurite length. Taken together, we demonstrate a critical role of neuroglobin in neuritogenesis or development via interacting with PTEN and Akt differentially to activate phosphatidylinositol 3-kinase/Akt pathway, providing potential therapeutic applications of neuroglobin for axonopathy in neurological diseases.

Piperlongumine selectively kills glioblastoma multiforme cells via reactive oxygen species accumulation dependent JNK and p38 activation.

Piperlongumine (PL), a natural alkaloid isolated from the long pepper, may have anti-cancer properties. It selectively targets and kills cancer cells but leaves normal cells intact. Here, we reported that PL selectively killed glioblastoma multiforme (GBM) cells via accumulating reactive oxygen species (ROS) to activate JNK and p38. PL at 20µM could induce severe cell death in three GBM cell lines (LN229, U87 and 8MG) but not astrocytes in cultures. PL elevated ROS prominently and reduced glutathione levels in LN229 and U87 cells. Antioxidant N-acetyl-L-cysteine (NAC) completely reversed PL-induced ROS accumulation and prevented cell death in LN229 and U87 cells. In LN229 and U87 cells, PL-treatment activated JNK and p38 but not Erk and Akt, in a dosage-dependent manner. These activations could be blocked by NAC pre-treatment. JNK and p38 specific inhibitors, SB203580 and SP600125 respectively, significantly blocked the cytotoxic effects of PL in LN229 and U87 cells. Our data first suggests that PL may have therapeutic potential for one of the most malignant and refractory tumors GBM.

Involvement of ATP-sensitive potassium channels in proliferation and differentiation of rat preadipocytes.

This paper was aimed to investigate the effects of ATP-sensitive potassium channels on the proliferation and differentiation of rat preadipocytes. We examined the expression of sulphonylurea receptor 2 (SUR2) mRNA in preadipocytes and adipocytes obtained by inducing for 5 d and the effects of the inhibitor (glibenclamide) and opener (diazoxide) of ATP-sensitive potassium channels on the expression of SUR2 mRNA in preadipocytes by real-time PCR. Preadipocyte proliferation and cell cycle were measured by MTT spectrophotometry and flow cytometer. The content of intracellular lipid was measured by oil red O staining, cell diameter was determined by Image-Pro Plus 5.0 software and the expression of peroxisome proliferator-activated receptor-gamma (PPAR-gamma) mRNA was estimated by RT-PCR. SUR2 mRNA was expressed in both preadipocytes and adipocytes obtained by inducing for 5 d, and the expression in adipocytes was obviously higher than that in preadipocytes. Glibenclamide inhibited the expression of SUR2 mRNA in preadipocyte, promoted preadipocyte proliferation in a dose-dependent manner, increased the cell percentages in G(2)/M + S phase, increased lipid content, augmented adipocyte diameter, and promoted the expression of PPAR-gamma mRNA. But the actions of diazoxide were contrary to those of glibenclamide. These results suggest that ATP-sensitive potassium channels regulate the proliferation and differentiation of preadipocytes, and PPAR-gamma is probably involved in the effect of ATP-sensitive potassium channels.

Role of histamine in airway remodeling of asthmatic guinea pig.

To investigate the role of histamine in airway remodeling, 50 healthy guinea pigs were divided into 5 groups: control group: nebulized inhalation of distilled water for 8 weeks; asthma model group: nebulized inhalation of ovalbumin (OVA) for 8 weeks after sensitization; continued asthma model group: nebulized inhalation of OVA for 14 weeks after sensitization; histamine group: nebulized inhalation of OVA for 14 weeks after sensitization and histamine was added in the last 6 weeks; antagonist group: nebulized inhalation of OVA for 14 weeks after sensitization and histamine receptor antagonists were added in the last 6 weeks. For each group, the concentration of histamine, sodium ion (Na(+)), chlorine ion (Cl(-)), arterial partial pressure of oxygen (PaO2), arterial partial pressure of carbon dioxide (PaCO2), pH, actual bicarbonate (AB), standard bicarbonate (SB) in serum, and thickness of airway mucosa, base membrane and smooth muscle were measured and compared with each other. The results showed that: (1) the concentration of histamine in serum and the thickness of airway increased, the following order was, the control group, the asthma model group, the continued asthma model group and histamine group (P<0.01); and the concentration of histamine in serum and the thickness of airway of antagonist group was lower than that of the continued asthma model group (P<0.05, 0.01). (2) PaO2 of the asthma model group was lower than that of the normal control group (P<0.01); PaO2, pH, AB, SB decreased, the following order was, the asthma model group, the continued asthma model group and the histamine group (P<0.01); and PaO2, pH, AB, SB of the antagonist group was higher than that of the continued asthma model group (P<0.01); but for PaCO2, the order was converse (P<0.01); For the concentration of Na(+) and Cl(-) in serum, there was no difference among these groups. It is concluded that: (1) Histamine is one of the mediators in the airway remodeling of asthma. (2) Histamine receptor antagonists may play a role in preventing and treating airway remodeling. (3) There is a negative correlation between the PaO2, pH and the wall thickness of the airway (P<0.01), while a positive correlation between the PaCO2, anion gap (AG) and the wall thickness of the airway (P<0.01).