ABCC4 suppresses glioblastoma progression and recurrence by restraining cGMP-PKG signalling.

BACKGROUND: Cyclic nucleotides are critical mediators of cellular signalling in glioblastoma. However, the clinical relevance and mechanisms of regulating cyclic nucleotides in glioblastoma progression and recurrence have yet to be thoroughly explored. METHODS: In silico, mRNA, and protein level analyses identified the primary regulator of cyclic nucleotides in recurrent human glioblastoma. Lentiviral and pharmacological manipulations examined the functional impact of cyclic nucleotide signalling in human glioma cell lines and primary glioblastoma cells. An orthotopic xenograft mice model coupled with aspirin hydrogels verified the in vivo outcome of targeting cyclic nucleotide signalling. RESULTS: Elevated intracellular levels of cGMP, instead of cAMP, due to a lower substrate efflux from ATP-binding cassette sub-family C member 4 (ABCC4) is engaged in the recurrence of glioblastoma. ABCC4 gene expression is negatively associated with recurrence and overall survival outcomes in glioblastoma specimens. ABCC4 loss-of-function activates cGMP-PKG signalling, promoting malignancy in glioblastoma cells and xenografts. Hydrogels loaded with aspirin, inhibiting glioblastoma progression partly by upregulating ABCC4 expressions, augment the efficacy of standard-of-care therapies in orthotopic glioblastoma xenografts. CONCLUSION: ABCC4, repressing the cGMP-PKG signalling pathway, is a tumour suppressor in glioblastoma progression and recurrence. Aspirin hydrogels impede glioblastoma progression through ABCC4 restoration and constitute a viable translational approach.

Cellular and exosomal GPx1 are essential for controlling hydrogen peroxide balance and alleviating oxidative stress in hypoxic glioblastoma.

Tumor hypoxia promotes malignant progression and therapeutic resistance in glioblastoma partly by increasing the production of hydrogen peroxide (H2O2), a type of reactive oxygen species critical for cell metabolic responses due to its additional role as a second messenger. However, the catabolic pathways that prevent H2O2 overload and subsequent tumor cell damage in hypoxic glioblastoma remain unclear. Herein, we present a hypoxia-coordinated H2O2 regulatory mechanism whereby excess H2O2 in glioblastoma induced by hypoxia is diminished by glutathione peroxidase 1 (GPx1), an antioxidant enzyme detoxifying H2O2, via the binding of hypoxia-inducible factor-1α (HIF-1α) to GPx1 promoter. Depletion of GPx1 results in H2O2 overload and apoptosis in glioblastoma cells, as well as growth inhibition in glioblastoma xenografts. Moreover, tumor hypoxia increases exosomal GPx1 expression, which assists glioblastoma and endothelial cells in countering H2O2 or radiation-induced apoptosis in vitro and in vivo. Clinical data explorations further demonstrate that GPx1 expression was positively correlated with tumor grade and expression of HIF-1α, HIF-1α target genes, and exosomal marker genes; by contrast, it was inversely correlated with the overall survival outcome in human glioblastoma specimens. Our analyses validate that the redox balance of H2O2 within hypoxic glioblastoma is clinically relevant and could be maintained by HIF-1α-promoted or exosome-related GPx1.

Hypoxia-induced CXC chemokine ligand 14 expression drives protumorigenic effects through activation of insulin-like growth factor-1 receptor signaling in glioblastoma.

Hypoxic tumor microenvironment (HTM) promotes a more aggressive and malignant state in glioblastoma. However, little is known about the role and mechanism of CXC chemokine ligand 14 (CXCL14) in HTM-mediated glioblastoma progression. In this study, we report that CXCL14 expression correlated with poor outcomes, tumor grade, and hypoxia-inducible factor (HIF) expression in patients with glioblastoma. CXCL14 was upregulated in tumor cells within the hypoxic areas of glioblastoma. Hypoxia induced HIF-dependent expression of CXCL14, which promoted glioblastoma tumorigenicity and invasiveness in vitro and in vivo. Moreover, CXCL14 gain-of-function in glioblastoma cells activated insulin-like growth factor-1 receptor (IGF-1R) signal transduction to regulate the growth, invasiveness, and neurosphere formation of glioblastoma. Finally, systemic delivery of CXCL14 siRNA nanoparticles (NPs) with polysorbate 80 coating significantly suppressed tumor growth in vivo and extended the survival time in patient-derived glioblastoma xenografts. Together, these findings suggest that HIF-dependent CXCL14 expression contributes to HTM-promoted glioblastoma tumorigenicity and invasiveness through activation of the IGF-1R signaling pathway. CXCL14 siRNA NPs as an oligonucleotide drug can inhibit glioblastoma progression and constitute a translational path for the clinical treatment of glioblastoma patients.

Blood-Brain Barrier Disruption in Preclinical Mouse Models of Stroke Can Be an Experimental Artifact Caused by Craniectomy.

The pathophysiological features of ischemia-related blood-brain barrier (BBB) disruption are widely studied using preclinical stroke models. However, in many of these models, craniectomy is required to confirm arterial occlusion via laser Doppler flowmetry or to enable direct ligation of the cerebral artery. In the present study, mice were used to construct a distal middle cerebral artery occlusion (dMCAO) model, a preclinical stroke model that requires craniectomy to enable direct ligation of the cerebral artery, or were subjected to craniectomy alone. dMCAO but not craniectomy caused neurodegeneration and cerebral infarction, but both procedures induced an appreciable increase in BBB permeability to Evans blue dye, fluorescein, and endogenous albumin but not to 10 kDa dextran-FITC, leading to cerebral edema. Using rats, we further showed that BBB disruption induced by craniectomy with no evidence of dural tearing was comparable to that induced by craniectomy involving tearing of the dura. In conclusion, our data demonstrated that craniectomy can be a major contributor to BBB disruption and cerebral edema in preclinical stroke models. The implications of this experimental artifact for translational stroke research and preclinical data interpretation are discussed.

Haloperidol Instigates Endometrial Carcinogenesis and Cancer Progression by the NF-κB/CSF-1 Signaling Cascade.

Haloperidol is a routine drug for schizophrenia and palliative care of cancer; it also has antitumor effects in several types of cancer. However, the role of haloperidol in endometrial cancer (EC) development is still unclear. Here, we show that chronic haloperidol treatment in clinically relevant doses induced endometrial hyperplasia in normal mice and promoted tumor growth and malignancy in mice with orthotopic EC. The pharmacokinetic study indicated that haloperidol highly accumulated in the uterus of mice. In vitro studies revealed that haloperidol stimulated the cellular transformation of human endometrial epithelial cells (HECCs) and promoted the proliferation, migration, and invasion of human endometrial carcinoma cells (HECCs) by activating nuclear factor kappa B (NF-κB) and its downstream signaling target, colony-stimulating factor 1 (CSF-1). Gain of function of CSF-1 promotes the cellular transformation of HEECs and the malignant progression of HECCs. Moreover, blockade of CSF-1 inhibited haloperidol-promoted EC progression in vitro and in vivo. A population-based cohort study of EC patients further demonstrated that the use of haloperidol was associated with increased EC-specific mortality. Collectively, these findings indicate that clinical use of haloperidol could potentially be harmful to female patients with EC.

Isoflurane attenuates carbogen-induced blood-brain barrier disruption independent of body temperature in mice and rats.

Isoflurane protects the blood-brain barrier (BBB) against cerebral extravasation of Evans blue dye (EBD), a commonly used serum protein tracer, in animals subjected to BBB disruption. As such, it has been implicated as a therapeutic agent that can prevent brain edema and damage caused by a number of brain insults, including focal ischemia and subarachnoid hemorrhage. Recently, it has been shown that isoflurane inhibits the cerebral extravasation of EBD following ischemic stroke chiefly by inducing hypothermia, raising the intriguing possibility that isoflurane protected against other causes of BBB disruption also through hypothermia. To test this hypothesis, we subjected mice and rats to inhalation of 20-30% carbogen, an inducer of BBB disruption, in the presence or absence of isoflurane while measuring their rectal temperature. In mice, carbogen inhalation on its own decreased rectal temperature from 36.4 ± 0.4 to 26.2 ± 0.6°C over a period of 60 minutes, and under this condition, isoflurane had no additional effect on body temperature. Nevertheless, isoflurane protected against carbogen-induced cerebral extravasation of EBD. In addition, when the body temperature was maintained in the normothermic range using an automated heating pad, isoflurane remained protective against cerebral extravasation of EBD. In rats, isoflurane also protected against cerebral extravasation of EBD, while having no effect on plasma pH, electrolyte concentrations, or osmolarity. In conclusion, isoflurane protected against BBB disruption caused by carbogen inhalation in mice and rats, but unlike isoflurane-mediated protection against ischemic BBB disruption, the effect could not be explained by anesthesia-induced hypothermia.

Carbogen inhalation opens the blood-brain barrier in rats without causing long-term metabolic or neurological deficit.

The blood-brain barrier (BBB) prevents many drugs from entering the brain. Yet, conventional methods that open the BBB are technically demanding, poorly reversible, and can be associated with long-term adverse effects. In comparison, carbogen, which is introduced nearly a century ago as a treatment for psychiatric disorders, is easy to administer and readily available to many labs and hospitals. Here, we show that carbogen inhalation opened the BBB in rats, as indicated by the extravasation of an intravenous protein tracer. When the tracer was injected immediately or hours after carbogen inhalation, less tracer was detected in the rat brains, suggesting at least partial reversibility of this response after carbogen exhalation. Despite marked increase in BBB permeability, inhalation of carbogen for 30-90 min had no acute effect on the level of neuroinflammation or apoptosis in the brain, and had no long-term effect on body weight, food intake, locomotor activity, or learning and memory performance. Our study demonstrated that carbogen inhalation is a safe method to open the BBB.

Rapid Intramitochondrial Zn2+ Accumulation in CA1 Hippocampal Pyramidal Neurons After Transient Global Ischemia: A Possible Contributor to Mitochondrial Disruption and Cell Death.

Mitochondrial Zn2+ accumulation, particularly in CA1 neurons, occurs after ischemia and likely contributes to mitochondrial dysfunction and subsequent neurodegeneration. However, the relationship between mitochondrial Zn2+ accumulation and their disruption has not been examined at the ultrastructural level in vivo. We employed a cardiac arrest model of transient global ischemia (TGI), combined with Timm's sulfide silver labeling, which inserts electron dense metallic silver granules at sites of labile Zn2+ accumulation, and used transmission electron microscopy (TEM) to examine subcellular loci of the Zn2+ accumulation. In line with prior studies, TGI-induced damage to CA1 was far greater than to CA3 pyramidal neurons, and was substantially progressive in the hours after reperfusion (being significantly greater after 4- than 1-hour recovery). Intriguingly, TEM examination of Timm's-stained sections revealed substantial Zn2+ accumulation in many postischemic CA1 mitochondria, which was strongly correlated with their swelling and disruption. Furthermore, paralleling the evolution of neuronal injury, both the number of mitochondria containing Zn2+ and the degree of their disruption were far greater at 4- than 1-hour recovery. These data provide the first direct characterization of Zn2+ accumulation in CA1 mitochondria after in vivo TGI, and support the idea that targeting these events could yield therapeutic benefits.

Mitochondrial Zn2+ Accumulation: A Potential Trigger of Hippocampal Ischemic Injury.

Ischemic stroke is a major cause of death and disabilities worldwide, and it has been long hoped that improved understanding of relevant injury mechanisms would yield targeted neuroprotective therapies. While Ca2+ overload during ischemia-induced glutamate excitotoxicity has been identified as a major contributor, failures of glutamate targeted therapies to achieve desired clinical efficacy have dampened early hopes for the development of new treatments. However, additional studies examining possible contributions of Zn2+, a highly prevalent cation in the brain, have provided new insights that may help to rekindle the enthusiasm. In this review, we discuss both old and new findings yielding clues as to sources of the Zn2+ that accumulates in many forebrain neurons after ischemia, and mechanisms through which it mediates injury. Specifically, we highlight the growing evidence of important Zn2+ effects on mitochondria in promoting neuronal injury. A key focus has been to examine Zn2+ contributions to the degeneration of highly susceptible hippocampal pyramidal neurons. Recent studies provide evidence of differences in sources of Zn2+ and its interactions with mitochondria in CA1 versus CA3 neurons that may pertain to their differential vulnerabilities in disease. We propose that Zn2+-induced mitochondrial dysfunction is a critical and potentially targetable early event in the ischemic neuronal injury cascade, providing opportunities for the development of novel neuroprotective strategies to be delivered after transient ischemia.

Vascular delivery of intraperitoneal Evans blue dye into the blood-brain barrier-intact and disrupted rat brains.

Blood-brain barrier (BBB) integrity can be determined by tracer infusion into the circulation followed by measurements of its penetration into the brain parenchyma. Tracer injection through the intraperitoneal (i.p.) route (rather than intravascular injection) avoids confounding effects of animal anesthesia or immobilization/surgical stress. Evans blue dye (EBD) can be administered by i.p. injection, and once in circulation, it binds to plasma albumin to become an endogenous protein tracer. Here, we investigated whether a similar level of EBD is extravasated into the brain following i.p. versus intravenous (i.v.) injection in rats. In comparison with i.v. EBD injection, i.p. EBD injection resulted in much of the tracer residing in the peritoneal cavity. Accordingly, comparatively less EBD was found in the blood, liver, or brain of BBB-intact rat. In addition, following unilateral osmotic BBB disruption, i.v. but not i.p. EBD stained the ipsilateral hemisphere blue. Nevertheless, following either route of tracer administration in these rats, spectrophotometric quantification detected more EBD in the ipsilateral (BBB-disrupted) than in the contralateral hemisphere. Taken together, in contrast to a recent report, we found that i.p. EBD resulted in less tracer in circulation and in peripheral/central organs than EBD delivered i.v. We nevertheless conclude that i.p. EBD delivered sufficient tracer for the detection of regional BBB disruption.

Anesthesia-Induced Hypothermia Attenuates Early-Phase Blood-Brain Barrier Disruption but Not Infarct Volume following Cerebral Ischemia.

Blood-brain barrier (BBB) disruption is thought to facilitate the development of cerebral infarction after a stroke. In a typical stroke model (such as the one used in this study), the early phase of BBB disruption reaches a peak 6 h post-ischemia and largely recovers after 8-24 h, whereas the late phase of BBB disruption begins 48-58 h post-ischemia. Because cerebral infarct develops within 24 h after the onset of ischemia, and several therapeutic agents have been shown to reduce the infarct volume when administered at 6 h post-ischemia, we hypothesized that attenuating BBB disruption at its peak (6 h post-ischemia) can also decrease the infarct volume measured at 24 h. We used a mouse stroke model obtained by combining 120 min of distal middle cerebral arterial occlusion (dMCAo) with ipsilateral common carotid arterial occlusion (CCAo). This model produced the most reliable BBB disruption and cerebral infarction compared to other models characterized by a shorter duration of ischemia or obtained with dMCAO or CCAo alone. The BBB permeability was measured by quantifying Evans blue dye (EBD) extravasation, as this tracer has been shown to be more sensitive for the detection of early-phase BBB disruption compared to other intravascular tracers that are more appropriate for detecting late-phase BBB disruption. We showed that a 1 h-long treatment with isoflurane-anesthesia induced marked hypothermia and attenuated the peak of BBB disruption when administered 6 h after the onset of dMCAo/CCAo-induced ischemia. We also demonstrated that the inhibitory effect of isoflurane was hypothermia-dependent because the same treatment had no effect on ischemic BBB disruption when the mouse body temperature was maintained at 37°C. Importantly, inhibiting the peak of BBB disruption by hypothermia had no effect on the volume of brain infarct 24 h post-ischemia. In conclusion, inhibiting the peak of BBB disruption is not an effective neuroprotective strategy, especially in comparison to the inhibitors of the neuronal death signaling cascade; these, in fact, can attenuate the infarct volume measured at 24 h post-ischemia when administered at 6 h in our same stroke model.

Estrogen and ERα enhanced ß-catenin degradation and suppressed its downstream target genes to block the metastatic function of HA22T hepatocellular carcinoma cells via modulating GSK-3ß and ß-TrCP expression.

In our previous experiments, we found ß-catenin was highly expressed in the tumor area with high invasive ability and poor prognosis. In this study, we have examined the mechanism by which ERα regulates ß-catenin expression as well as the metastasis ability of hepatocellular cancer HA22T cells. To identify whether the anticancer effect of estrogen and ERα is mediated through suppression of ß-catenin expression, we co-transfected pCMV-ß-catenin and ERα into HA22T cells, and determined the cell motility by wound healing, invasion, and migration assays. Results showed that estrogen and/or ERα inhibited ß-catenin gene expression and repressed HA22T cell motility demonstrated that similar data was observed in cells expressing the ERα stable clone. Moreover, we examined the protein-protein interaction between ERα and ß-catenin by immunostain, co-immunoprecipitation, and Western blotting. E2 enhanced the binding of ERα with ß-catenin and then triggered ß-catenin to bind with E3 ligase (ßTrCP) to promote ß-catenin degradation. Finally by employing systematic ChIP studies, we showed ERα can interact directly with the ß-catenin promoter region following E2 treatment. All our results reveal that estrogen and ERα blocked metastatic function of HA22T cells by modulating GSK3ß and ßTrCP expression and further enhanced ß-catenin degradation and suppressed its downstream target genes. © 2016 Wiley Periodicals, Inc. Environ Toxicol 32: 519-529, 2017.

Optimization of Evans blue quantitation in limited rat tissue samples.

Evans blue dye (EBD) is an inert tracer that measures plasma volume in human subjects and vascular permeability in animal models. Quantitation of EBD can be difficult when dye concentration in the sample is limited, such as when extravasated dye is measured in the blood-brain barrier (BBB) intact brain. The procedure described here used a very small volume (30 µl) per sample replicate, which enabled high-throughput measurements of the EBD concentration based on a standard 96-well plate reader. First, ethanol ensured a consistent optic path length in each well and substantially enhanced the sensitivity of EBD fluorescence spectroscopy. Second, trichloroacetic acid (TCA) removed false-positive EBD measurements as a result of biological solutes and partially extracted EBD into the supernatant. Moreover, a 1:2 volume ratio of 50% TCA ([TCA final] = 33.3%) optimally extracted EBD from the rat plasma protein-EBD complex in vitro and in vivo, and 1:2 and 1:3 weight-volume ratios of 50% TCA optimally extracted extravasated EBD from the rat brain and liver, respectively, in vivo. This procedure is particularly useful in the detection of EBD extravasation into the BBB-intact brain, but it can also be applied to detect dye extravasation into tissues where vascular permeability is less limiting.

Neuron Regeneration and Proliferation Effects of Danshen and Tanshinone IIA.

This study evaluates the proliferative effects of danshen and its monomer extract, tanshinone IIA, on Schwann cell proliferation. A piece of silicone rubber was guided across a 15-mm gap in the sciatic nerve of a rat. This nerve gap was then filled with different concentrations of danshen (0-100 mg/mL). The results showed that danshen increased the expressions of uPA, cyclin D1, E and ERK, JNK, and P38 MAP kinases via the FGF-2 signaling pathway in a dose-dependent manner. RSC96, Schwann cells were also administered with danshen (0, 20, 40, 60, 80, and 100 µg/mL) and tanshinone IIA (0, 2, 4, 6, 8, and 10 µg/mL). In lower concentrations, danshen and tanshinone IIA exhibited an apparent effect on Schwann cells. Similar effects were also demonstrated in the FGF-2-uPA regulating cascade and cell cycle proliferative protein results. Schwann cell migration was elevated as well. We used MAPK-signaling chemical inhibitors and identified the proliferative effects of danshen and tanshinone IIA as MAPK-signaling dependent. The results from the in vitro systems indicate that danshen and tanshinone IIA can be used to induce Schwann cell proliferation, and in vivo results potentially suggest that danshen and tanshinone IIA might enhance neuron regeneration.

Dangshen (Codonopsis pilosula) activates IGF-I and FGF-2 pathways to induce proliferation and migration effects in RSC96 Schwann cells.

This study evaluates the proliferative and migrative effects of dangshen on RSC96, Schwann cells. We investigated the molecular signaling pathways, which include: (1) survival signaling, IGFs-IGFIR-Akt-Bcl2 and proliferative signaling, cell cycle factors and MAPK pathways. (2) migrate and anti-scar signaling, FGF-2-uPA-MMPs. After treatment with different concentrations (20 microg/ml, 40 microg/ml, 60 microg/ml, 80 microg/ml, and 100 microg/ml) of dangshen. We observed a dose dependent proliferative effect using PCNA Western blotting assay, MTT assay and the wound healing test. We also found that dangshen stimulates the protein expressions of IGF-I pathway regulators, cell cycle controlling proteins and excites the MAPK signaling pathway regulators ERK and P38. Dangshen even stimulates the FGF-2-uPA-MMP 9 migration pathway in RSC 96 Schwann cells. Using MAPK chemical inhibitors, U0126, SB203580, and SP600125, the proliferative effects of dangshen on RSC 96 cells were identified to be ERK- and P38- dependent. Based on these results, applying an appropriate dose of dangshen with biomedical materials would be a potential approach for enhancing neuron regeneration.

The proliferation and migration effects of huangqi on RSC96 Schwann cells.

This study evaluated the proliferation effects of huangqi on neuron regeneration. We investigated the molecular mechanisms, which include: (1) cyclin D1, A, E-cell cycle factors and MAPK signaling proliferation (2) FGF-2-UPA-MMPs migration signaling. After treatment with various Huanqi concentrations (1.25, 12.5, 125, 250 and 500 microg/ml,), we observed that Huanqi can increase Rsc 96 cell proliferation at 12.5 microg/ml (p < 0.01) concentration determined by the MTT and wound healing tests. Examination by RT-PCR and Western blotting assay showed that Huangqi is able to stimulate the mRNA and protein expressions of cyclin D1, A, E, cell cycle controlling proteins and excite ERK and P38 MAPK signaling pathways to promote cell proliferation. Huangqi stimulates the FGF-2-UPA-MMP 9 migration pathway and enhances RSC 96 Schwann cells migration. Using MAPK chemical inhibitors, U0126, SB203580 and SP600125, the proliferative effects of Huangqi on RSC 96 cells were ERK and P38 signaling-dependent. Based on these results, applying an appropriate dose of Huangqi with biomedical materials would be a potential approach to enhancing neuron regeneration.

Proliferation- and migration-enhancing effects of ginseng and ginsenoside Rg1 through IGF-I- and FGF-2-signaling pathways on RSC96 Schwann cells.

The aim of the present study is to evaluate the proliferation- and migration-enhancing effects of ginseng and its component, ginsenoside (Rg1) on RSC96 Schwann cells. We investigated the molecular signaling pathways, which include: (1) survival signaling, IGFs-IGFIR-Akt-Bcl2 and proliferative signaling, cell cycle factors and mitogen-activated protein kinase (MAPK) pathways, (2) migrating and anti-scar signaling, FGF-2-uPA-MMPs.We treated RSC96 cells with different concentrations (100, 200, 300, 400, 500 microg ml(-1)) of ginseng and its constituent, Rg1 (5, 10, 15, 20, 25 microg ml(-1)). We observed a proliferative effect in a dose-dependent manner by PCNA western blotting assay, MTT assay, and wound healing test. Furthermore, we also found in the results of western blotting assay, ginseng and Rg1 enhance protein expression of IGF-I pathway regulators, cell cycle controlling proteins, and MAPK signaling pathways to promote the cell proliferation. In addition, ginseng and Rg1 also stimulated the FGF-2-uPA-MMP 9 migrating pathway to enhance the migration of RSC96 Schwann cells. Using MAPK chemical inhibitors, U0126, SB203580, and SP600125, the proliferative effects of ginseng and Rg1 on RSC96 cells were identified to be MAPK signaling-dependent. On the basis of the results, applying appropriate doses of ginseng and Rg1 with biomedical materials would be a potential approach for enhancing neuron regeneration.