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Biofilms have been recognized as a serious threat to public health as it protects microbes from antimicrobials, immune defence mechanisms, chemical treatments and nutritional stress. Biofilms are also a source of concern in industries and water treatment because their presence compromises the integrity of equipment. To overcome these problems, it is necessary to identify novel anti-biofilm compounds. Products of microorganisms have been identified as promising broad-spectrum anti-biofilm agents. These natural products include biosurfactants, antimicrobial peptides, enzymes and bioactive compounds. Anti-biofilm products of microbial origin are chemically diverse and possess a broad spectrum of activities against biofilms. The objective of this review is to give an overview of the different types of microbial anti-biofilm products and their mechanisms of action., Competing Interests: Declaration of competing interest The authors have no conflict of interest to declare., (Copyright © 2023 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved.)

Cancer is a debilitating disease that is on the increase in both developed and developing countries. Anticancer drugs are often expensive, have narrow spectrum of activities, and are associated with toxicities and side effects such as myelosuppression, immunosuppression, gastrointestinal disturbance, alopecia, skin toxicity, and hepatotoxicity. Plants have been the major source of anticancer drugs both in orthodox and traditional medicine. Many of the plants claimed by the traditional medicine practitioners (TMPs) to be effective in the treatment of cancer are yet to be evaluated scientifically. In this work, five medicinal plants used by TMPs in Borno State, Nigeria, were tested against two brain tumor cell lines. Ethanol extracts of Securidaca longepedunculata , Andira inermis subsp. rooseveltii , Annona senegalensis , Carissa edulis , and Parinari polyandra were used. U87 and U231 brain tumor cell lines were used for proliferation assay, U251 cell line was used for the invasion assay in collagen V coated inserts, and U87 cell line was used for the western blot detection of cleaved Poly-ADP-Ribose-Polymerase (PARP). The result revealed that all tested extracts significantly ( p < 0.05) inhibited the proliferation of U87 and U231 cell lines with the respective IC 50 values ranging between 8 and 20  μ g/ml for S. longepedunculata and 100 and 90  μ g/ml for P. polyandra . The five extracts significantly ( p < 0.05) inhibited the invasion of U251 cell line at the concentration of 10  μ g/ml ( S. longepedunculata ), 20  μ g/ml ( A. inermis ), 50  μ g/ml ( A. senegalensis ), 50  μ g/ml ( C. edulis ), and 50  μ g/ml ( P. polyandra ). Securidaca longepedunculata extract induced the cleavage of PARP. It was concluded that these medicinal plants have antiproliferative and anti-invasive activities and possess good prospects as source of anticancer agents especially S. longepedunculata which induced apoptosis in U87 cell line., Competing Interests: The authors declare no conflicts of interest., (Copyright © 2020 Saidu I. Ngulde et al.)

Incidence of cancer is estimated to be on the increase and current anticancer drugs are characterized by narrow margin of safety and side effects. There is the need to explore new drugs especially from plants since plants serve as major source of drugs. Securidaca longipedunculata Fresen plant is called the mother of all medicines in northern Nigeria and is used traditionally in the treatment of cancers by most traditional medicine practitioners in the region. This study is aimed at evaluating the anticancer activity of the plant extract using U87 brain tumor cell line. Ethanol extract of its root bark was prepared and fractionated by silica gel column chromatography. In vitro activity of the extract and fractions were assessed on the viability of U87 malignant brain tumor cell line by using hemacytometer, annexin V-PE and 7AAD flow cytometry and western blot detection of Poly-ADP-Ribose-Polymerase (PARP) cleavage. The results showed that the extract significantly (p<0.01) inhibited proliferation of U87 cell line with IC 50 of 20.535 μ g/ml. Apoptosis was induced by the extract (41.53 ± 10.33%) and the polar fraction (47.3 ± 2.7%) via cleavage of PARP. It was concluded that the ethanol extract of S. longipedunculata root bark inhibited proliferation of U87 cell line and induced apoptosis by cleavage of PARP, thus supporting folkloric use of the plant in the management of cancers.

Ethnopharmacological Relevance: Preparation of Laggera aurita Linn. (Asteraceae) is widely used in traditional medicine to treat various kinds of diseases such as epilepsy, malaria, fever, pain and asthma. Its efficacy is widely acclaimed among communities in Northern Nigeria., Aim of the Study: The present study is aimed at establishing the possible anticonvulsant effects of the methanol leaf extract of Laggera aurita using acute and chronic anticonvulsant models., Materials and Method: Median lethal dose (LD50) was determined in mice and rats via oral and intraperitoneal routes. Anticonvulsant screening of the extract was performed using maximal electroshock-induced seizure test in day-old chicks; pentylenetetrazole-, strychnine- and picrotoxin- induced seizure models in mice. Similarly; its effects on pentylenetetrazole-induce kindling in rats as well as when co-administered with fluphenamic and cyproheptadine in mice, were evaluated., Results: Median lethal dose (LD50) values were found to be >5000mg/kg, p.o. and 2154mg/kg, i.p., each for both rats and mice. The extract showed dose dependent protection against tonic hind limb extension (THLE) and significantly (p<0.05) decreased the mean recovery from seizure in the maximal electroshock-induced seizure. In the pentylenetetrazole-induced seizure model, the extract offered 50% protection at 600mg/kg and also increased the mean onset of seizure at all doses with significant (p<0.05) increase at the highest dose (600mg/kg). Similarly the extract produced significant (p<0.05) increase in the onset of seizures in both strychnine- and picrotoxin- induced seizure models, at all the doses except at 150mg/kg for the picrotoxin model. Co-administration of fluphenamic acid (FFA) (5mg/kg) and the extract (600mg/kg) showed an enhanced effect with percentage protection of 70% while co-administration of FFA (5mg/kg) and phenytoin (5mg/kg) as well phenytoin (5mg/kg) and the extract (600mg/kg) produced an additive effect. Administration of the extract (600mg/kg), phenytoin (20mg/kg) and cyproheptadine (4mg/kg) offered 40%, 100% and 0% protection against THLE, each respectively, while co-administration of cyproheptadine (4mg/kg) and the extract (600mg/kg) as well as co-administration of cyproheptadine (4mg/kg) and phenytoin (20mg/kg) offered reduced protection of 20% and 50% each respectively. The extract at all doses reduced the severity of seizure episodes induced by PTZ-induced kindling., Conclusion: The results suggest that the methanol leaf extract of Laggera aurita possesses anticonvulsant and antiepileptogenic properties., (Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.)

Background: Leonotis nepetifolia Linn (Lamiaceae) is used in traditional medicine for its calming (tranquilizing) effects. The aim of this study was to determine whether there is any scientific justification for this use. To achieve this purpose, we investigated the behavioural effects of the methanol extract of Leonotis nepetifolia stem (37.5, 75 and 150 mg/kg) in mice., Methods: Acute toxicity studies were carried out on the methanol stem extract of Leonotis nepetifolia to determine the LD50. The behavioural tests employed were diazepam-induced sleep onset and duration, hole board assay for exploratory activity, mouse beam walk assay for motor coordination, and the staircase test for the detection of anxiolytic compounds. Preliminary phytochemical screening was also carried out on the extract., Results: The intraperitoneal LD50 value was found to be 3.8 g/kg. The results showed that the extract significantly prolonged the duration of diazepam-induced sleep at the highest dose (150 mg/kg). There was no observable effect on exploratory activity and motor coordination at the doses tested (37.5, 75 and 150 mg/kg). The extract, however, at 150 mg/kg elicited a significant decrease in the number of rearings in the staircase test, an effect also observed in the group of mice injected with an anxiolytic dose of diazepam. The preliminary phytochemical screening revealed the presence of alkaloids, saponins, glycosides and triterpenoids., Conclusion: The results obtained suggest that the crude methanol extract of Leonotis nepetifolia stem possesses some biologically active constituents with potential anxiolytic activity and thus may justify its traditional use as a tranquilizer.

Neoplasms of the brain are often overlooked in resource-limited countries. Our literature search via AJOL and PubMed demonstrated that brain tumor research is still a rarity in these regions. We highlight the current status, importance, challenges, and methods of improving brain tumor research in West Africa. We suggest that more attention be given to basic, clinical, and epidemiological brain tumor research by national governments, private organizations, international organizations, non-governmental organizations (NGOs), and individuals in this region.

Objectives: Securinega virosa Roxb (Ex Willd) Baill (Euphorbaiceae) root bark has been reportedly used in African traditional medicine in the management of mental illnesses. Previously, the sleep-inducing potential of the crude methanol root bark of Securinega virosa extract and its butanol fraction have been reported. The study aimed to isolate and characterize the bioactive constituent that may be responsible for the sleep inducing property of the root of the plant., Materials and Methods: The phytochemical investigation of the S. virosa root bark was carried out leading to the isolation of a compound from the butanol-soluble fraction of the methanol extract. The structure of the compound was elucidated on the basis of its spectral data, including IR, 1D and 2D NMR, mass spectrometry as well as X-ray diffraction analysis. The compound was investigated for sleep-inducing potential using diazepam-induced sleeping time test and beam walking assay in mice., Results: This is the first report on the isolation of bergenin from the root of the plant. It significantly decreased the mean onset of sleep [F (2, 15) =7.167; p< 0.01] at the dose of 10 mg/kg, without significantly affecting the total sleep duration [F (2, 15) = 0.090, p=0.914]. Conversely, it did not significantly affect the number of foot slips at the doses of 5 and 10 mg/kg tested., Conclusion: Bergenin isolated from the root bark of S. virosa possesses sleep-inducing property and could be partly responsible for the sedative potential of the root of S. virosa.

Context: Carissa edulis Vahl (Apocynaceae) is used in Nigerian folk medicine to manage a plethora of diseases including epilepsy, cancer, and inflammation; its efficacy is widely acclaimed among communities of northern Nigeria., Objective: This study establishes anticonvulsant activities of aqueous fraction of ethanol root bark extract of Carissa edulis (RAF) and sub-fractions (S1 and S2) in animal models., Materials and Methods: We evaluated the acute toxicity of the RAF, S1 and S2, and the anticonvulsant activity using pentylenetetrazole (PTZ), picrotoxin, strychnine, N-methyl-d-aspartate (NMDA), isoniazid (INH), and aminophylline-induced seizures in mice. Their effects on maximal electroshock (MES) and kindling-induced seizures were studied in chicks and in rats, respectively, and in the electrophysiological study. The doses used for RAF were 150, 300, and 600 mg/kg while S1 and S2 were 250, 500, and 1000 mg/kg. Both RAF and sub-fractions were administered once during the experiment., Results: The intraperitoneal LD50 of the RAF was estimated to be 2222.61 mg/kg and that of the S1 and S2 were above 5000 mg/kg. RAF protected the mice by 50% while sub-fractions by 16.67% against PTZ-induced seizures. RAF offered 33.33 and 16.67% protection against strychnine and NMDA models, respectively. However, RAF offered 66.67-33.33% protections against aminophylline-induced seizures at doses of 150 and 600 mg/kg, but RAF, S1, and S2 had no effect on MES-induced seizures., Discussion and Conclusion: Our results validate the use of the plant traditionally in the management of epilepsy, thus supporting the appraisal of biologically active components of this plant as antiepileptic agents.

Schizophrenia is a highly disabling chronic psychiatric illness. The existing antipsychotic agents are associated with untoward effects and drug interactions leading to the intensification of search for newer agents with better efficacy and safety profile. Securinega virosa is a commonly used medicinal plant in African traditional medicine. The decoction of the leaves of the plant in combination with other plants is used in the management of mental illness. In this study, we evaluate the antipsychotic potential of the methanol leaf extract (25, 50 and 100 mg kg(-1)) of the plant using apomorphine-induced stereotypic climbing behavior and swim-induced grooming tests, all in mice. The CNS depressant effect was also evaluated using ketamine-induced sleep test mice. The extract at the highest dose tested (100 mg kg(-1)) significantly reduced the apomorphine (1 mg kg(-1))-induced stereotypic climbing behavior after 30 min. Similarly, haloperidol (2 mg kg(-1)), the standard agent significantly (p<0.001) decreased the mean climbing behavior. In the swim-induced grooming test, the extract significantly (p<0.01) and dose-dependently decreased the total grooming time. Similarly, haloperidol (2 mg kg(-1)) significantly (p<0.001) decreased the mean grooming activity. The extract significantly increased the total ketamine-induced sleep duration at doses of 50 and 100 mg kg(-1). These findings suggest that the extract possesses antipsychotic and sedative potentials and lend credence to the ethnomedical use of the leaves of the plant in the management of mental illness.

Securinega virosa (Roxb ex. Willd) Baill. is a plant which is commonly used in African traditional medicine in management of mental illness. Previous study showed that the crude methanolic root bark extract of the plant possesses antipsychotic activity. In this study, the antipsychotic potential of the residual aqueous fraction of the plant was evaluated using two experimental models, apomorphine induced stereotypic climbing behaviour and swim induced grooming, all in mice. The effect of the fraction on haloperidol-induced catalepsy was also evaluated. The fraction significantly reduced the mean climbing score at the highest dose tested (500 mg/kg). In the swim-induced grooming test, the fraction significantly and dose-dependently (125-500 mg/kg) decreased the mean number and mean duration of swim-induced grooming activity in mice. Similarly, the standard haloperidol (1 mg/kg) significantly (p < 0.001) decreased the mean grooming episodes and duration. However, the fraction did not significantly potentiate haloperidol-induced catalepsy. These results suggest that the residual aqueous fraction of methanol root bark extract of Securinega virosa contains biological active principle with antipsychotic potential.

Progress in research on the molecular aspects of glioblastoma has yet to provide a medical therapy that significantly improves prognosis. Glioblastoma invariably progress through current treatment regimens with radiotherapy as a key component. Activation of several signaling pathways is thought to be associated with this resistance to radiotherapy. Ras activity is exceptionally high in glioblastoma and may regulate sensitivity to radiotherapy. Raf-1, a downstream effector of Ras, demonstrates a high amount of activity in glioblastoma. Therefore, Raf-1 inhibition should be considered as a mechanism to increase the effectiveness of radiotherapy in treatment regimen. In vitro analysis was performed with a novel Raf-1 kinase inhibitor (BAY 54-9085) in culture with the glioblastoma cell line U1242. The cell line was treated in serum-containing media and analyzed for the effect of the BAY 54-9085 alone and BAY 54-9085 combined with radiation on cell death. BAY 54-9085 displayed a cytocidal effect on glioblastoma cells following a 3 day incubation with the drug in serum-containing media. A dose of 2.5 μM displayed moderate cell death which significantly increased with a dose of 5.0 μM. In addition, glioblastoma cells treated with both the BAY 54-9085 and gamma radiation displayed a significant increase in cell death (85.5%) as compared to either BAY 54-9085 (73.1%) or radiation (34.4%) alone. Radiation therapy is a key component of treatment for glioblastoma. A novel Raf-1 inhibitor displayed in vitro evidence of synergistically increasing cell death of glioblastoma cells in combination with radiation., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Context: Decoctions of Randia nilotica Stapf. (Rubiaceae) have been used in the Nigerian traditional medicine for the management of epilepsy, anxiety, depression and psychosis for many years and their efficacies are widely acclaimed among the rural communities of Northern Nigeria., Objective: The aim of this study is to establish whether the saponins present in R. nilotica are responsible for its acclaimed beneficial effects in Nigerian traditional medicine., Materials and Methods: The behavioural properties of the saponin-rich fraction (SFRN) of R. nilotica stem bark were studied on hole-board, diazepam-induced sleep, rota-rod and beam-walking in mice. The anticonvulsant properties of SFRN were also examined on maximal electroshock, pentylenetetrazole- and strychnine-induced seizures in mice., Results: The intraperitoneal LD₅₀ of SFRN in mice and rats were estimated to be 11.1 and 70.7 mg/kg, respectively. SFRN significantly prolonged the duration of diazepam-induced sleep; diminished head dip counts in the hole-board test and protected mice against maximal electroshock seizures. SFRN failed to protect mice against pentylenetetrazole- and strychnine-induced seizures; and had no effect on motor coordination on the rota-rod treadmill at the doses tested. SFRN significantly decreased the number of foot slips in the beam-walking assay in mice with no effect on time to reach the goal box., Discussion and Conclusion: This study provides evidence of the psychopharmacological effects of SFRN, thus supporting further development of the psychoactive components as remedies for epilepsy.

Ethnopharmacological Relevance: Preparations of Carissa edulis (Vahl) have been used in the Nigerian traditional medicine for the management of fever, sickle cell disease, epilepsy, pain and inflammation for many years and their efficacy is widely acclaimed among the Hausa communities of northern Nigeria., Aim of the Study: The present studies aimed at evaluating the toxicological properties of the standardized ethanol extract of C. edulis root bark in rats, in order to determine its safety and to complement earlier efficacy studies on this widely used medicinal plant., Materials and Methods: High performance liquid chromatography (HPLC) and preliminary phytochemical analysis of the extract were conducted and its oral median lethal dose (LD50) determined. Signs of toxicity, body weight changes, relative organs weight, feed and water consumption were monitored following 28 days of daily oral administration of graded doses of the extract in rats. Effects of the extract on sex hormones, low- and high-density lipids, hematological and biochemical parameters were examined and pathological changes of the vital organs after treatment with the extract were also investigated., Results: The oral LD50 of the extract was estimated to be >5000 mg/kg. The body weights of treated rats increased progressively, but the changes were not significantly different from the control groups. The extract neither produces significant changes in feed and water consumption nor affected the relative organs weight. Although some variations were observed in hormonal and lipid profiles hematological and biochemical indices, these important parameters were normal and within acceptable limits. No lesions or pathological changes of the organs attributable to treatment with the extract were observed from the pathological examinations. The HPLC fingerprint of the extract shows a spectrum profile characteristic of C. edulis, while the preliminary phytochemical screening revealed the presence of saponins, flavonoids, tannins, anthraquinones and cardiac glycosides., Conclusion: Our results provided evidence that short-term administration of the standardized ethanol extract of C. edulis root bark at doses lower than 1000 mg/kg is safe in rats and may not exert severe toxic effects., (Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.)

Ethnopharmacological Relevance: Securinega virosa is a commonly used medicinal plant in African traditional medicine in the management of epilepsy and mental illness. Previous studies in our laboratory showed that the crude methanol root bark extract of the plant possesses significant behavioral effect in laboratory animals. In an attempt to isolate and characterize the biological principles responsible for the observed activity, this study is aimed at evaluating the central depressant activity of the butanol fraction of the methanol root bark extract of Securinega virosa., Materials and Methods: The medial lethal dose of the butanol fraction was estimated using the method of Lorke. Preliminary phytochemical screening was conducted on the butanol fraction using standard protocol. The behavioral effect of the butanol fraction (75, 150 and 300mg/kg) was evaluated using diazepam induced sleep test, hole-board test, beam walking assay, staircase test, open field test and elevated plus maze assay, all in mice., Results: The median lethal dose of the butanol fraction was estimated to be 1256.9mg/kg. The preliminary phytochemical screening revealed the presence of tannins, saponins, alkaloids, flavonoids, cardiac glycosides, similar to those found in the crude methanol extract. The butanol fraction significantly (P<0.001) reduced the mean onset of sleep in mice and doubled the mean duration of sleep in mice at the dose of 75mg/kg. The butanol fraction and diazepam (0.5mg/kg) significantly (P<0.01-0.001) reduced the number of head dips in the hole-board test suggesting sedative effect. The sedative effect of the butanol fraction was further corroborated by its significant (P<0.01-0.001) reduction of the number of step climbed and rearing in the staircase test. The butanol fraction did not significantly increase the time taken to complete the task and number of foot slips in the beam walking assay, suggesting that it does not induce significant motor coordination deficit. Diazepam (2mg/kg), the standard agent used significantly (P<0.01) increased the number of foot slips. In the open field test, the butanol fraction significantly reduced the number of square crossed as well as the number of rearing. However, the butanol fraction did not significantly alter the behavior of mice in the elevated plus maze assay, while diazepam (0.5mg/kg) significantly (P<0.05) increased the time spent in the open arm and reduced the number of closed arm entry., Conclusion: The findings of this study suggest that the butanol fraction of Securinega virosa root bark contains some bioactive principles that are sedative in nature., (Copyright © 2012 Elsevier Ireland Ltd. All rights reserved.)

Ethnopharmacological Relevance: Balanites aegyptiaca is a native plant from the dry tropical areas of Africa and Arabia. It has been used in traditional medicine to treat psychoses, epilepsy, rheumatism and for the management of cough, liver and spleen conditions for many years. The plant is also used as antihelmintic and molluscicide., Aim of the Study: The present studies aimed at investigating the behavioral properties of ethanol extract of the root of this medicinal plant, which is already in common applications in the Nigerian traditional medicine., Materials and Methods: The intraperitoneal and oral mean lethal dose (LD(50)) of the extract was determined using the Lorke's method. The preliminary phytochemical screening of the extract was carried out to identify the secondary metabolites in the extract. Furthermore, the behavioral properties of the extract were evaluated using diazepam-induced sleep, open field test, staircase test and beam walking assay all in mice., Results: The extract significantly (p<0.001) prolonged the duration diazepam (20mg/kg i.p)-induced sleep in mice dose dependently. However, the extract showed no significant effect on the onset of diazepam-induced sleep. In the open field test, the extract (150 and 300 mg/kg) and diazepam (0.05 mg/kg) produced a significant (p<0.05, p<0.005 and p<0.001) decrease in the number of square crossings. There was no significant effect on the number of centre square crossing following the administration of the extract. The extract (75 and 150 mg/kg) and diazepam (0.05 mg/kg) produced a significant (p<0.05) decrease in the number of rearing suggestive of sedation. In the staircase experiment there was a decrease in the number of upward step climbing as well as number of rearing suggesting anxiolytic and sedative properties of the extract. In the beam walking assay the extract did not produce any significant increase in the time taken to complete task as compared to diazepam 1mg/kg which was significant at p<0.05. Furthermore, 30 mg/kg of the extract and diazepam 1mg/kg showed significant (p<0.05) mean number of foot slips, suggesting that the central nervous system depressant activity might not necessarily due to peripheral neuromuscular blockade., Conclusion: The result indicates that the extract of Balanites aegyptiaca possess biologically active compound(s) that have anxiolytic and sedative properties, which support the ethnomedicinal use of the plant as antipsychotic and antiepileptic agents., (Copyright © 2011 Elsevier Ireland Ltd. All rights reserved.)

Background: Glioblastoma (GBM) is a highly aggressive malignant primary brain tumor, characterized by rapid growth, diffuse infiltration of cells into both adjacent and remote brain regions, and a generalized resistance to currently available treatment modalities. Recent reports in the literature suggest that Signal Transducers and Activators of Transcription (STATs) play important roles in the regulation of GBM pathophysiology., Methods: STAT6 protein expression was analyzed by Western blotting in GBM cell lines and by immunohistochemistry in a tissue microarray (TMA) of glioma patient tissues. We utilized shRNA against STAT6 to investigate the effects of prolonged STAT6 depletion on the growth and invasion of two STAT6-positive GBM cell lines. Cell proliferation was assessed by measuring (3)H-Thymidine uptake over time. Invasion was measured using an in vitro transwell assay in which cells invade through a type IV collagen matrix toward a chemoattractant (Fetal Bovine Serum). Cells were then stained and counted. Kaplan-Meyer survival curves were generated to show the correlation between STAT6 gene expression and patient survival in 343 glioma patients and in a subset of patients with only GBM. Gene expression microarray and clinical data were acquired from the Rembrandt 1 public data depository (https://caintegrator.nci.nih.gov/rembrandt/). Lastly, a genome-wide expression microarray analysis was performed to compare gene expression in wild-type GBM cells to expression in stable STAT6 knockdown clones., Results: STAT6 was expressed in 2 GBM cell lines, U-1242MG and U-87MG, and in normal astrocytes (NHA) but not in the U-251MG GBM cell line. In our TMA study, STAT6 immunostaining was visible in the majority of astrocytomas of all grades (I-IV) but not in normal brain tissue. In positive cells, STAT6 was localized exclusively in the nuclei over 95% of the time. STAT6-deficient GBM cells showed a reduction in (3)H-Thymidine uptake compared to the wild-type. There was some variation among the different shRNA- silenced clones, but all had a reduction in (3)H-Thymidine uptake ranging from 35%- 70% in U-1242MG and 40- 50% in U-87MG cells. Additionally, STAT6- depleted cells were less invasive than controls in our in vitro transmembrane invasion assay. Invasiveness was decreased by 25-40% and 30-75% in U-1242MG and U-87MG cells, respectively. The microarray analysis identified matrix metalloproteinase 1 (MMP-1) and urokinase Plasminogen activator (uPA) as potential STA6 target genes involved in the promotion of GBM cell invasion. In a Kaplan-Meier survival curve based on Rembrandt 1 gene expression microarray and clinical data, there was a significant difference in survival (P < 0.05) between glioma patients with up- and down-regulated STAT6. Decreased STAT6 expression correlated with longer survival times. In two subsets of patients with either grade IV tumors (GBM) or Grade II/III astrocytomas, there was a similar trend that however did not reach statistical significance., Conclusions: Taken together, these findings suggest a role for STAT6 in enhancing cell proliferation and invasion in GBM, which may explain why up-regulation of STAT6 correlates with shorter survival times in glioma patients. This report thus identifies STAT6 as a new and potentially promising therapeutic target.

Isolation of glioblastoma stem cells requires incubation of tumor cells in a neural stem cell media. Neurospheres containing these glioblastoma stem cells are formed after approximately a five-day period. These cells can then be analyzed for the presence of stem cell markers. Immunofluorescence staining for these markers can serve as a valuable tool for analyzing the intact neurosphere directly in stem cell media. Here we present the use of a novel fixative (1,4-benzoquinone) for immunoflourescence staining of neurospheres., (Copyright © 2011 Elsevier B.V. All rights reserved.)

Aim: Protein kinase-C (PKC) and NF-kappaB are involved in cell survival, proliferation, migration and radioresistance in glioblastoma multiforme (GBM). We sought to determine the interaction between PKC and NF-kappaB pathways., Material and Methods: The activation of NF-kappaB by PKC alpha and PKC delta was assessed by Western blotting after the stimulation with Phorbol 12- Myristate 13-Acetate (PMA). Gene silencing of PKC alpha , PKC delta and NFkappaB/ p65 with siRNA interference was utilized to evaluate their roles in NFkB activation and cell proliferation., Results: PMA induced the phosphorylation of NF-kappaB/p65 by PKC alpha. Gene silencing with siRNA against NF-kappaB/p65 inhibited [3H]-thymidine incorporation in U1242 GBM cells. PKC delta decelerated the nuclear translocation of activated NF-kappaB/p65 up to 4 hours after the stimulation. PMA induced death was not observed in PKC delta silenced cells where activated NF-kappaB/p65 was located immediately in the nucleus., Conclusion: NF-kappaB/p65 is pro-survival and proliferative factor in U1242 GBM cells. PKC alpha is needed to phosphorylate NF-kappaB/p65. PKC delta delays the translocation of active NF-kappaB/p65 into the nucleus. PMA-induced cell death occurred if the phospho-NF-kappaB/p65 was prohibited from entering the nucleus in PKC delta positive cells. Translocation of phosphorylated form of NF-kappaB into the nucleus is critical in GBM cell proliferation.

One of the major pathophysiological features of malignant astrocytomas is their ability to infiltrate surrounding brain tissue. The epidermal growth factor receptor (EGFR) and proteases are known to be overexpressed in glioblastomas (GBMs), but the interaction between the activation of the EGFR and urokinase plasminogen activator (uPA) in promoting astrocytic tumor invasion has not been fully elucidated. Here, we characterized the signal transduction pathway(s) by which EGF regulates uPA expression and promotes astrocytoma invasion. We show that EGFR activation and constitutively active EGFR vIII in GBM cell lines upregulate uPA expression. Small-molecule inhibitors of mitogen-activated protein kinase, tyrosine kinase, and small interfering RNA targeting c-Src blocked uPA upregulation. Similarly, mutations in the activator protein 1 binding site of the uPA promoter reduced EGF-induced increases in uPA promoter activity. Treatment of GBM cells with EGF increased in vitro cell invasion, and the invasive phenotype was attenuated by gene silencing of uPA using small interfering RNA and short hairpin RNA. In addition, uPA knockdown clones formed smaller well-circumscribed tumors than nontarget U1242 control cells in a xenograft GBM mouse model in vivo. In summary, these results suggest that c-Src, mitogen-activated protein kinase, and a composite activator protein 1 on the uPA promoter are responsible for EGF-induced uPA expression and GBM invasion.

The lack of an intracranial human glioma model that recapitulates the extensive invasive and hypervascular features of glioblastoma (GBM) is a major hurdle for testing novel therapeutic approaches against GBM and studying the mechanism of GBM invasive growth. We characterized a high matrix metalloproteinase-9 (MMP-9) expressing U1242 MG intracranial xenograft mouse model that exhibited extensive individual cells and cell clusters in a perivascular and subpial cellular infiltrative pattern, geographic necrosis and infiltrating tumor-induced vascular proliferation closely resembling the human GBM phenotype. MMP-9 silencing cells with short hairpin RNA dramatically blocked the cellular infiltrative pattern, hypervascularity, and cell proliferation in vivo, and decreased cell invasion, colony formation, and cell motility in vitro, indicating that a high level of MMP-9 plays an essential role in extensive infiltration and hypervascularity in the xenograft model. Moreover, epidermal growth factor (EGF) failed to stimulate MMP-9 expression, cell invasion, and colony formation in MMP-9-silenced clones. An EGF receptor (EGFR) kinase inhibitor, a RasN17 dominant-negative construct, MEK and PI3K inhibitors significantly blocked EGF/EGFR-stimulated MMP-9, cell invasion, and colony formation in U1242 MG cells, suggesting that MMP-9 is involved in EGFR/Ras/MEK and PI3K/AKT signaling pathway-mediated cell invasion and anchorage-independent growth in U1242 MG cells. Our data indicate that the U1242 MG xenograft model is valuable for studying GBM extensive invasion and angiogenesis as well as testing anti-invasive and anti-angiogenic therapeutic approaches.

Cushing's disease is caused by an ACTH-producing pituitary tumor, and accounts for 10-15% of pituitary tumors. The majority of corticotroph tumors are microadenomas (<10 mm), and accurate histologic identification of these tumors can be challenging because of their small size and the presence of nests of normal corticotroph cells in the anterior pituitary. Retinoic acid has been shown to inhibit ACTH production and induce apoptosis in corticotroph tumor cells. The expression of the orphan nuclear receptor COUP-TFI antagonizes retinoic acid signaling and has been shown to be expressed in normal corticotroph cells, but absent in corticotroph tumor cell lines. We analyzed 34 corticotroph tumor specimens by immunohistochemistry using a goat polyclonal IgG antibody with epitope mapping to the N-terminus of human COUP-TFI. Segments of normal pituitary in each of the 34 specimens demonstrate COUP-TFI immunoreactivity in normal corticotroph cells. Twenty-nine of 34 ACTH producing tumors were immunonegative for COUP-TFI. All of the tumors measuring less than 5 mm by preoperative MRI were COUP-TFI immunonegative. Two tumors, measuring 9 and 11 mm, showed consistent (>90%) expression of COUP-TFI, and three adenomas (5, 11, and 18 mm) showed heterogenous (20-80%) expression of COUP-TFI. Immunohistochemistry of COUP-TFI may be a useful adjuvant diagnostic tool in distinguishing corticotroph microadenomas from nests of normal corticotroph cells in the anterior pituitary. Furthermore, this study identifies two unique corticotroph tumor populations which differ in their expression of COUP-TFI, the presence of which occurs more frequently in macroadenomas.

MMPs (matrix metalloproteinases) and the related "a disintegrin and metalloproteinases" (ADAMs) promote tumorigenesis by cleaving extracellular matrix and protein substrates, including N-cadherin. Although N-cadherin is thought to regulate cell adhesion, migration, and invasion, its role has not been characterized in glioblastomas (GBMs). In this study, we investigated the expression and function of posttranslational N-cadherin cleavage in GBM cells as well as its regulation by protein kinase C (PKC). N-Cadherin cleavage occurred at a higher level in glioblastoma cells than in non-neoplastic astrocytes. Treatment with the PKC activator phorbol 12-myristate 13-acetate (PMA) increased N-cadherin cleavage, which was reduced by pharmacological inhibitors and short interfering RNA (siRNA) specific for ADAM-10 or PKC-alpha. Furthermore, treatment of GBM cells with PMA induced the translocation of ADAM-10 to the cell membrane, the site at which N-cadherin was cleaved, and this translocation was significantly reduced by the PKC-alpha inhibitor Gö6976 [12-(2-cyanoethyl)-6,7,12,13-tetrahydro-13-methyl-5-oxo-5H-indolo[2,3-a]pyrrolo[3,4-c]carbazole] or PKC-alpha short hairpin RNA. In functional studies, N-cadherin cleavage was required for GBM cell migration, as depletion of N-cadherin cleavage by N-cadherin siRNA, ADAM-10 siRNA, or a cleavage-site mutant N-cadherin, decreased GBM cell migration. Together, these results suggest that N-cadherin cleavage is regulated by a PKC-alpha-ADAM-10 cascade in GBM cells and may be involved in mediating GBM cell migration.

Aim of the Study: To investigate the anticonvulsant activity of root bark extract of Carissa edulis., Materials and Methods: The median lethal dose (LD(50)) of Carissa edulis extract was determined using Lork's method (1983). The anticonvulsant activity of the extract was assessed in pentylenetetrazole (PTZ)-induced convulsion in mice and maximal electroshock test (MEST) in chicks, with benzodiazepine and phenytoin as standard drugs, respectively. While mechanistic studies were conducted using both flumazenil, a GABA(A)-benzodiazepine receptor complex site antagonist and naloxone a non-specific opioid receptor antagonist., Results: The median lethal dose (LD(50)) of Carissa edulis was 282.8mg/kg and over 5000mg/kg following intraperitoneal and oral administration, respectively. Carissa edulis produced 40% and 20% protection against convulsion at 5 and 20mg/kg, respectively, compared with 100% protection with benzodiazepine. The mean onset and percentage protection against convulsion in Carissa edulis extract-treated mice were reduced by flumazenil and naloxone. Carissa edulis exhibited dose-dependent inhibition of the convulsion induced by MEST with 20mg/kg providing 90% protection while phenytoin (20mg/kg) produced 100% protection., Conclusion: These results suggest that Carissa edulis possesses biologically active constituent(s) that have anticonvulsant activity which supports the ethnomedicinal claims of the use of the plant in the management of epilepsy.

Previous study reported that the activation of Ras pathway cooperated with E6/E7-mediated inactivation of p53/pRb to transform immortalized normal human astrocytes (NHA/hTERT) into intracranial tumors strongly resembling human astrocytomas. The mechanism of how H-Ras contributes to astrocytoma formation is unclear. Using genetically modified NHA cells (E6/E7/hTERT and E6/E7/hTERT/Ras cells) as models, we investigated the mechanism of Ras-induced tumorigenesis. The overexpression of constitutively active H-RasV12 in E6/E7/hTERT cells robustly increased the levels of urokinase plasminogen activator (uPA) mRNA, protein, activity and invasive capacity of the E6/E7/hTERT/Ras cells. However, the expressions of MMP-9 and MMP-2 did not significantly change in the E6/E7/hTERT and E6/E7/hTERT/Ras cells. Furthermore, E6/E7/hTERT/Ras cells also displayed higher level of uPA activity and were more invasive than E6/E7/hTERT cells in 3D culture, and formed an intracranial tumor mass in a NOD-SCID mouse model. uPA specific inhibitor (B428) and uPA neutralizing antibody decreased uPA activity and invasion in E6/E7/hTERT/Ras cells. uPA-deficient U-1242 glioblastoma cells were less invasive in vitro and exhibited reduced tumor growth and infiltration into normal brain in xenograft mouse model. Inhibitors of Ras (FTA), Raf (Bay 54-9085) and MEK (UO126), but not of phosphatidylinositol 3-kinase (PI3K) (LY294002) and of protein kinase C (BIM) pathways, inhibited uPA activity and cell invasion. Our results suggest that H-Ras increased uPA expression and activity via the Ras/Raf/MEK signaling pathway leading to enhanced cell invasion and this may contribute to increased invasive growth properties of astrocytomas., (Copyright (c) 2008 Wiley-Liss, Inc.)

Previous reports showed that urokinase plasminogen activator (uPA) converts plasminogen to plasmin which then activates matrix metalloproteinases (MMPs). Here, we report that uPA directly cleaved pro-MMP-9 in a time-dependent manner at both C- and N-terminus and generated two gelatinolytic bands. uPA-activated-MMP-9 efficiently degraded fibronectin and blocked by uPA inhibitor B428 and recombinant tissue inhibitor of metalloproteinase-1 (TIMP-1). B428 inhibited basal and PMA-induced active MMP-9 in glioblastomas (GBM) U1242 cell media as well as cell invasion in vitro. A combination of MMP-9 and uPA antibodies more significantly inhibited U1242 cell invasion than uPA or MMP-9 antibody alone. Both uPA and MMP-9 were highly expressed in U1242 cell and GBM patient specimens. Furthermore, two active MMP-9 fragments with identical molecular weights to the uPA-activated MMP-9 products were detected in GBM patient specimens. These results suggest that uPA-mediated direct activation of MMP-9 may promote GBM cell invasion.

Object: We analyzed MMP-9 expression using mRNA and protein level determinations and explored the possibility that matrix metalloproteinase-9 (MMP-9) is a potential biological marker of pituitary adenoma invasiveness and whether MMP-9 could be used to discriminate the extent of invasiveness among different hormonal subtypes, tumor sizes, growth characteristics, and primary versus recurrent tumors., Materials and Methods: 73 pituitary tumor specimens were snap frozen in liquid nitrogen immediately after surgical resection. RNA and protein were extracted. MMP-9 mRNA transcripts were analyzed by quantitative RT-PCR. MMP-9 protein activity was analyzed by gelatin zymography and validated by western blot analysis. Immunohistochemistry was performed to identify the presence and localization of MMP-9 in pituitary adenomas. Statistical differences between results were determined using Student's t-test or one way ANOVA., Results: Comparing different hormonal subtypes of noninvasive and invasive pituitary tumors, MMP-9 mRNA expression was significantly increased in the majority of invasive adenomas. Considering the protein levels, our data also showed a significant increase in MMP-9 activity in the majority of invasive adenomas and these differences were confirmed by western blot analysis and immunohistochemistry. In addition, consistent differences in MMP-9 expression levels were found according to tumor subtype, tumor size, tumor extension and primary versus redo-surgery., Conclusions: MMP-9 expression can consistently distinguish invasive pituitary tumors from noninvasive pituitary tumors and would reflect the extent of invasiveness in pituitary tumors according to tumor subtype, size, tumor extension, primary and redo surgery, even at early stages of invasiveness. MMP-9 may be considered a potential biomarker to determine and predict the invasive nature of pituitary tumors.

Aggressive and infiltrative invasion is one of the hallmarks of glioblastoma. Low-density lipoprotein receptor-related protein (LRP) is expressed by glioblastoma, but the role of this receptor in astrocytic tumor invasion remains poorly understood. We show that activation of protein kinase C-alpha (PKC-alpha) phosphorylated and down-regulated LRP expression. Pretreatment of tumor cells with PKC inhibitors, phosphoinositide 3-kinase (PI3K) inhibitor, PKC-alpha small interfering RNA (siRNA), and short hairpin RNA abrogated phorbol 12-myristate 13-acetate-induced down-regulation of LRP and inhibited astrocytic tumor invasion in vitro. In xenograft glioblastoma mouse model and in vitro transmembrane invasion assay, LRP-deficient cells, which secreted high levels of urokinase-type plasminogen activator (uPA), invaded extensively the surrounding normal brain tissue, whereas the LRP-overexpressing and uPA-deficient cells did not invade into the surrounding normal brain. siRNA, targeted against uPA in LRP-deficient clones, attenuated their invasive potential. Taken together, our results strongly suggest the involvement of PKC-alpha/PI3K signaling pathways in the regulation of LRP-mediated astrocytoma invasion. Thus, a strategy of combining small molecule inhibitors of PKC-alpha and PI3K could provide a new treatment paradigm for glioblastomas.

A survey was conducted among Hausa and Fulani, two major tribes of Northern Nigeria to identify plants and methods used traditionally in the treatment of cancers and inflammatory diseases. The ecological zones that were considered include Zaria, Kaduna and Kano in the Northern part of Nigeria. The survey involves traditional healers, hunters, farmers and Fulani nomads. This survey has identified plants useful in the treatment of cancers. The plants were identified via taxonomic means and classified according to their habitats, families, genera. Evidently the plants span families and genera, the knowledge and values of the plants was evaluated with the aim of understanding the scientific basis for the use of the plants. The inventory provides the unique opportunity of capturing plants of common uses across the communities.

Glioblastoma multiforme (GBM) is the highest grade of astrocytoma. GBM pathogenesis has been linked to receptor tyrosine kinases and kinases further down signal-transduction pathways - in particular, members of the protein kinase C (PKC) family. The expression and activity of various PKC isoforms are increased in malignant astrocytomas, but not in non-neoplastic astrocytes. This suggests that PKC activity contributes to tumor progression. The level of PKC-eta expressed correlates with the degree of phorbol-12-myristate-13-acetate (PMA)-induced proliferation of two glioblastoma cell lines, U-1242 MG and U-251 MG. Normally, U-1242 cells do not express PKC-eta, and PMA inhibits their proliferation. Conversely, PMA increases proliferation of U-1242 cells that are stably transfected with PKC-eta (U-1242-PKC-eta). PMA treatment also stimulates proliferation of U-251 cells, which express PKC-eta. Here, we determined that extracellular signal-regulated kinase (ERK) and Elk-1 are downstream targets of PKC-eta. Elk-1-mediated transcriptional activity correlates with the PKC-eta-mediated mitogenic response. Pretreatment of U-1242-PKC-eta cells with inhibitors of PKC or MAPK/ERK kinase (MEK) (bisindolyl maleimide (BIM) or U0126, respectively) blocked both PMA-induced Elk-1 transcriptional activity and PMA-stimulated proliferation. An overexpressed dominant-negative PKC-eta reduced the mitogenic response in U-251 cells, as did reduction of Elk-1 by small interfering RNA. Taken together, these results strongly suggest that PKC-eta-mediated glioblastoma proliferation involves MEK/mitogen-activated protein (MAP) kinase phosphorylation, activation of ERK and subsequently of Elk-1. Elk-1 target genes involved in GBM proliferative responses have yet to be identified.

Previously, we have shown that PKC-eta (protein kinase C-eta) positively regulates glioblastoma proliferation and confers resistance to irradiation-induced apoptosis. In this study, we investigated the efficacy of rapamycin in inhibiting cell proliferation in two glioblastoma cell lines U-251MG (PKC-eta expressing) and U-1242MG (PKC-eta deficient) following PKC-eta activation. In U-251MG cells, rapamycin (10 nM) treatment was less effective as an antiproliferative agent when cells were concurrently stimulated with 10% serum and phorbol 12-myristate 13-acetate (PMA, 100 nM), a potent activator of PKC isozymes. Rapamycin-insensitive growth was owing to PKC-eta, as U-1242MG and U-251MG cells infected with a kinase-dead form of PKC-eta (U-251kr) were susceptible to rapamycin-induced inhibition of cell proliferation. Furthermore, U-251MG cells transfected with PKC-eta antisense oligonucleotides were sensitive to rapamycin. PKC-eta-expressing cells stimulated with PMA maintained p70S6K phosphorylation on Thr389 and phosphorylation of rpS6 (ser235/36), suggesting p70S6K kinase activity was still intact. Inhibition of p70S6K expression with small interfering RNA oligonucleotides inhibited cell proliferation greater than 50% in the presence of a combination of PMA and serum. Additionally, p70S6K co-precipitated with PKC-eta, suggesting a physical interaction between PKC-eta and p70S6K regulates the observed phosphorylation. Taken together, these data demonstrate that rapamycin-insensitive glioblastoma proliferation involves PKC-eta signaling.

The complete resection of pituitary adenomas (PAs) is unlikely when there is an extensive local dural invasion and given that the molecular mechanisms remain primarily unknown. DNA microarray analysis was performed to identify differentially expressed genes between nonfunctioning invasive and noninvasive PAs. Gene clustering revealed a robust eightfold increase in matrix metalloproteinase (MMP)-9 expression in surgically resected human invasive PAs and in the (nonfunctioning) HP75 human pituitary tumor-derived cell line treated with phorbol-12-myristate-13-acetate; these results were confirmed by real-time polymerase chain reaction, gelatin zymography, reverse transcriptase-polymerase chain reaction, Western blot, immunohistochemistry, and Northern blot analyses. The activation of protein kinase C (PKC) increased both MMP-9 activity and expression, which were blocked by some PKC inhibitors (Gö6976, bisindolylmaleimide, and Rottlerin), PKC-alpha, and PKC-delta small interfering (si)RNAs but not by hispidin (PKC-beta inhibitor). In a transmembrane invasion assay, phorbol-12-myristate-13-acetate (100 nmol/L) increased the number of invaded HP75 cells, a process that was attenuated by PKC inhibitors, MMP-9 antibody, PKC-alpha siRNA, or PKC-delta siRNA. These results demonstrate that MMP-9 and PKC-alpha or PKC-delta may provide putative therapeutic targets for the control of PA dural invasion.

Renewal of nongermative epithelia is poorly understood. The novel mitogen "lacritin" is apically secreted by several nongermative epithelia. We tested 17 different cell types and discovered that lacritin is preferentially mitogenic or prosecretory for those types that normally contact lacritin during its glandular outward flow. Mitogenesis is dependent on lacritin's C-terminal domain, which can form an alpha-helix with a hydrophobic face, as per VEGF's and PTHLP's respective dimerization or receptor-binding domain. Lacritin targets downstream NFATC1 and mTOR. The use of inhibitors or siRNA suggests that lacritin mitogenic signaling involves Galpha(i) or Galpha(o)-PKCalpha-PLC-Ca2+-calcineurin-NFATC1 and Galpha(i) or Galpha(o)-PKCalpha-PLC-phospholipase D (PLD)-mTOR in a bell-shaped, dose-dependent manner requiring the Ca2+ sensor STIM1, but not TRPC1. This pathway suggests the placement of transiently dephosphorylated and perinuclear Golgi-translocated PKCalpha upstream of both Ca2+ mobilization and PLD activation in a complex with PLCgamma2. Outward flow of lacritin from secretory cells through ducts may generate a proliferative/secretory field as a different unit of cellular renewal in nongermative epithelia where luminal structures predominate.

HOX genes are a large family of regulatory genes implicated in the control of developmental processes. HOX genes are involved in malignant transformation and progression of different types of tumour. Despite intensive efforts to delineate the expression profiles of HOX genes in other cell types, nothing is known regarding the global expression profile of these genes in normal human astrocytes and astrocytomas. The present study has analysed the expression profile of the 39 class I HOX genes in normal human astrocytes (NHA and E6/E7), two well-established glioblastoma cell lines (U-87 MG and U-1242-MG), as well as neoplastic (WHO grades II/III and IV) and non-neoplastic temporal lobe specimens with hippocampal sclerosis and medically intractable epilepsy. RT-PCR, quantitative real-time PCR, immunocytochemistry, and western blot analyses revealed differential expression of nine HOX genes (A6, A7, A9, A13, B13, D4, D9, D10, and D13) in normal human astrocytic cell lines and non-neoplastic temporal lobe specimens. The data show that HOX genes are differentially expressed in neoplastic and non-neoplastic astrocytes and that multiple HOX genes are overexpressed in glioblastoma cell lines, astrocytomas (II/III), and glioblastoma multiforme. The differential expression of HOX genes in normal and neoplastic astrocytes suggests a role for these genes in brain tumourigenesis., (Copyright 2006 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.)

Primary glioblastomas (GBMs) commonly overexpress the oncogene epidermal growth factor receptor (EGFR), which leads to increased Ras activity. FTA, a novel Ras inhibitor, produced both time- and dose-dependent caspase-mediated apoptosis in GBM cell lines. EGFR-mediated increase in 3H-thymidine uptake was inhibited by FTA. FACS analysis was performed to determine the percent of apoptotic cells. The sub-Go population of GBM cells was increased from 4.5 to 13.8% (control) to over 45-53.6% in FTA-treated cells within 24 h. Furthermore, FTA also increased the activities of both caspase-3 and -9, and PARP cleavage. Treatment of GBMs with FTA before or after EGF addition to the cultures blocked phosphorylation of Akt and mitogen-activated protein kinases (MAPK). FTA also significantly reduced the amount of EGF-induced Ras-GTP as reflected by a decrease in the level of Ras bound to Raf-RBD-GST. This study demonstrates that inhibition of Ras methylation may provide a therapeutic target for the treatment of GBMs overexpressing EGFR.

Gliomas are the most common major subgroup of primary CNS tumours. Approximately 17,000 new cases are reported each year and, of these, 11,500 patients die. Glioblastoma multiforme (GBM) is highly proliferative and typically invades distal portions of the brain, thereby making complete surgical resection of these tumours nearly impossible. Moreover, GBMs are often resistant to current chemotherapy and radiation regimens. Therefore, there is a need for better therapeutic interventions. One class of proteins that is involved in the formation of malignant brain tumours is protein kinase C (PKC) and these kinases have not been thoroughly explored for their chemotherapeutic value in GBMs. The PKC isozyme, PKCeta (PKC-eta) increases cell proliferation and resistance to radiation of GBM cell lines. These properties make PKCeta an attractive target for chemotherapeutic intervention in the management of GBMs.

Both the epidermal growth factor receptor (EGFR) and protein kinase C (PKC) play important roles in glioblastoma invasive growth; however, the interaction between the EGFR and PKC is not well characterized in glioblastomas. Treatment with EGF stimulated global phosphorylation of the EGFR at Tyr(845), Tyr(992), Tyr(1068), and Tyr(1045) in glioblastoma cell lines (U-1242 MG and U-87 MG). Interestingly, phorbol 12-myristate 13-acetate (PMA) stimulated phosphorylation of the EGFR only at Tyr(1068) in the two glioblastoma cell lines. Phosphorylation of the EGFR at Tyr(1068) was not detected in normal human astrocytes treated with the phorbol ester. PMA-induced phosphorylation of the EGFR at Tyr(1068) was blocked by bisindolylmaleimide (BIM), a PKC inhibitor, and rottlerin, a PKCdelta-specific inhibitor. In contrast, Go 6976, an inhibitor of classical PKC isozymes, had no effect on PMA-induced EGFR phosphorylation. Furthermore, gene silencing with PKCdelta small interfering RNA (siRNA), siRNA against c-Src, and mutant c-Src(S12C/S48A) and treatment with a c-Src inhibitor (4-amino-5-(4-chlorophenyl)-7-(t-butyl) pyrazolo[3,4-d]pyrimidine) abrogated PMA-induced EGFR phosphorylation at Tyr(1068). PMA induced serine/threonine phosphorylation of Src, which was blocked by both BIM and rottlerin. Inhibition of the EGFR with AG 1478 did not significantly alter PMA-induced EGFR Tyr(1068) phosphorylation, but completely blocked EGF-induced phosphorylation of the EGFR. The effects of PMA on MAPK phosphorylation and glioblastoma cell proliferation were reduced by BIM, rottlerin, the MEK inhibitor U0126, and PKCdelta and c-Src siRNAs. Taken together, our data demonstrate that PMA transactivates the EGFR and increases cell proliferation by activating the PKCdelta/c-Src pathway in glioblastomas.

We previously demonstrated that protein kinase C-eta (PKC-eta) mediates a phorbol 12-myristate-13-acetate (PMA)-induced proliferative response in human glioblastoma (GBM) cells. In this report, we show that PMA-stimulated activation of PKC-eta in U-251 GBM cells resulted in activation of both Akt and the mammalian target of rapamycin (mTOR) signaling pathways and an increase in cell proliferation. Expression of a kinase dead PKC-eta (PKC-etaKR) construct reduced the basal and PMA-evoked proliferation of PKC-eta-expressing U-251 GBM cells, as well as abrogated the PMA-induced activation of Akt, mTOR, and the mTOR targets 4E-BP1 and STAT-3. Treatment of cells with the PI-3 kinase inhibitor LY294002 (10 muM) or the mTOR inhibitor rapamycin (10 nM) also reduced PMA-induced proliferation and cell-cycle progression. Expression of a constitutively active PKC-eta (PKC-etaDeltaNPS) construct in a GBM cell line with no endogenous PKC-eta (U-1242) also provided evidence that PKC-eta targets the Akt and mTOR signaling pathways. Moreover, activation of 4E-BP1 and STAT-3 in both PMA-treated U-251 and PKC-etaDeltaNPS-expressing U-1242 GBM cells was inhibited by rapamycin. However, activation of Akt, but not mTOR was inhibited by the PI-3 kinase inhibitor LY294002. This study identifies Akt and mTOR as downstream targets of PKC-eta that are involved in GBM cell proliferation.

Although Jun upregulation and activation have been established as critical to oncogenesis, the relevant downstream pathways remain incompletely characterized. In this study, we found that c-Jun blocks erythroid differentiation in primary human hematopoietic progenitors and, correspondingly, that Jun factors block transcriptional activation by GATA-1, the central regulator of erythroid differentiation. Mutagenesis of c-Jun suggested that its repression of GATA-1 occurs through a transcriptional mechanism involving activation of downstream genes. We identified the hairy-enhancer-of-split-related factor HERP2 as a novel gene upregulated by c-Jun. HERP2 showed physical interaction with GATA-1 and repressed GATA-1 transcriptional activation. Furthermore, transduction of HERP2 into primary human hematopoietic progenitors inhibited erythroid differentiation. These results thus define a novel regulatory pathway linking the transcription factors c-Jun, HERP2, and GATA-1. Furthermore, these results establish a connection between the Notch signaling pathway, of which the HERP factors are a critical component, and the GATA family, which participates in programming of cellular differentiation., (Copyright 2004 American Society for Microbiology)

The effects of the ethanol extract of Pavetta crassipes on the central nervous system (CNS) and on actions of some selected centrally acting drugs were studied in mice and rats. These studies were carried out using the spontaneous motor activity (SMA), amphetamine-induced hyperactivity and stereotyped behaviour, pentobarbital-induced hypnosis and exploratory activity, apomorphine-induced climbing and haloperidol-induced catalepsy in rats. The results demonstrated that the extract of P. crassipes dose-dependently decreased SMA in mice and attenuated amphetamine-induced hyperactivity and the different episodes of stereotypic behavioural patterns induced by amphetamine. In addition, the extract decreased the number of head dips in the exploratory activity test and potentiated pentobarbital-induced sleeping time in rats. Furthermore, the extract inhibited apomorphine-induced climbing in mice and potentiated haloperidol-induced catalepsy in rats. Our results suggest that the extract of P. crassipes contains biologically active substance(s) that might be acting centrally through the inhibition of dopaminergic pathway or a system linked to this pathway to mediate the observed pharmacological effects.

Pavetta crassipes leaf is routinely used locally in Nigeria for the management of respiratory disorders and hypertension. The hypotensive and other cardiovascular effects of Pavetta crassipes were investigated in cats and rats. The effects of the extract on rat and cat blood pressures, isolated rat atria, rat portal vein, isolated rat aorta and rat vas deferens were studied. Specific receptor antagonists (atropine, mepyramine, phentolamine, propranolol) were used to elucidate the underlying mechanism(s) involved in the cardiovascular changes induced by P. crassipes. The results revealed that the ethanolic extract of Pavetta crassipes lowered the blood pressures of cats and rats in a dose dependent manner. The extract also caused a concentration-dependent decrease in the force of contraction of the isolated rat atria and rat portal vein. The decreases in blood pressure values were attenuated in the presence of a beta-adrenoceptor antagonist, propranolol. The extract also attenuated isoprenaline-induced contraction of the rat atria. However, the extract did not affect contractions evoked by KCl, norepinephrine and 5-HT on the rat aorta. Pavetta crassipes contains biologically active substances that may be useful in the management of hypertension.