Samson Amos

Pyruvate kinase M2 is a target of the tumor-suppressive microRNA-326 and regulates the survival of glioma cells

Emerging studies have identified microRNAs (miRNAs) as possible therapeutic tools for the treatment of glioma, the most aggressive brain tumor. Their important targets in this tumor are not well understood. We recently found that the Notch pathway is a target of miRNA-326. Ectopic expression of miRNA-326 in glioma and glioma stem cells induced their apoptosis and reduced their metabolic activity. Computational target gene prediction revealed pyruvate kinase type M2 (PKM2) as another target of miRNA-326. PKM2 has recently been shown to play a key role in cancer cell metabolism. To investigate whether it might be a functionally important target of miR-326, we used RNA interference to knockdown PKM2 expression in glioma cells. Transfection of the established glioma and glioma stem cells with PKM2 siRNA reduced their growth, cellular invasion, metabolic activity, ATP and glutathione levels, and activated AMP-activated protein kinase. The cytotoxic effects exhibited by PKM2 knockdown in glioma and glioma stem cells were not observed in transformed human astrocytes. Western blot analysis of human glioblastoma specimens showed high levels of PKM2 protein, but none was observed in normal brain samples. Strikingly, cells with high levels of PKM2 expressed lower levels of miR-326, suggestive of endogenous regulation of PKM2 by miR-326. Our data suggest PKM2 inhibition as a therapy for glioblastoma, with the potential for minimal toxicity to the brain.

Anti-inflammatory and Anti-nociceptive Effects of Sphaeranthus senegalensis

Sphaeranthus senegalensis Vaill (Asteraceae) is used in traditional medicine as a remedy for rheumatic pains and other ailments. The anti-inflammatory and anti-nociceptive activity of the aqueous extract of the whole shoot of the plant was evaluated in mice and rats. Activity of the extract against egg-albumin induced hind paw oedema was measured to evaluate the anti-inflammatory activity while the anti-nociceptive potency was evaluated using three painful stimuli viz acetic acid induced abdominal constriction and hot plate test in mice, and the formalin test in rats. Results indicated that the extract possess remarkable dose dependent anti-inflammatory activities in rats. The extract also showed anti-nociceptive activities against acetic acid induced writhing, formalin and the hot plate pain models. The effects were significant (P < 0.05) when compared with the saline control group. The results suggest the presence of a potent anti-inflammatory and anti-nociceptive principle in the extract, which support the folkloric use of the plant in relieving rheumatic pains.

The Protein Kinase C-η Isoform Induces Proliferation in Glioblastoma Cell Lines Through an ERK/Elk-1 Pathway

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-η 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-η, and PMA inhibits their proliferation. Conversely, PMA increases proliferation of U-1242 cells that are stably transfected with PKC-η (U-1242-PKC-η). PMA treatment also stimulates proliferation of U-251 cells, which express PKC-η. Here, we determined that extracellular signal-regulated kinase (ERK) and Elk-1 are downstream targets of PKC-η. Elk-1-mediated transcriptional activity correlates with the PKC-η-mediated mitogenic response. Pretreatment of U-1242-PKC-η 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-η 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-η-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.

Effect of Zizyphus spina-christi Willd aqueous extract on the central nervous system in mice

The effects of the aqueous extract of Zizypus spina-christi Willd root bark against exploratory behaviour, spontaneous motor activity (SMA), motor coordination (Rota-rod performance) and pentobarbital-induced hypnosis were investigated in mice. The extract induced a significant (P<0.05) dose-dependent reduction in exploratory behaviour and SMA when administered orally. It also prolonged pentobarbital sleeping time but failed to inhibit motor coordination (rota-rod performance) in the experimental mice. These results suggest that the extract contained some constituents that depress the central nervous system, which may not be due to neuromuscular blockade.

Matrix Metalloproteinase-9, A Potential Biological Marker in Invasive Pituitary Adenomas

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.

Protein Kinase C-α–Mediated Regulation of Low-Density Lipoprotein Receptor–Related Protein and Urokinase Increases Astrocytoma Invasion

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.

Anticonvulsant properties of saponins from Ficus platyphylla stem bark

Preparations of Ficus platyphylla have been used in Nigerian traditional medicine for the management of epilepsy for many years and their efficacy is widely acclaimed among the Hausa communities of northern Nigeria. The anticonvulsant properties of the saponin rich fraction (SFG) obtained from the methanol extract of F. platyphylla stem bark were studied on pentylenetetrazole-, strychnine- and maximal electroshock seizures in mice. Effects of SFG were also examined in murine models for neurological disease and on relevant in vitro targets for anticonvulsant drugs. SFG protected mice against pentylenetetrazole- and strychnine-induced seizures; and significantly delayed the onset of myoclonic jerks and tonic seizures. SFG failed to protect mice against maximal electroshock seizures at doses tested. SFG neither abolished the spontaneous discharges induced by 4-aminopyridine in a neonatal rat brain slice model of tonic-clonic epilepsy nor could it modulate chloride currents through GABA(A) receptor channel complex in cultured cortical cells. However, it was able to non-selectively suppress excitatory and inhibitory synaptic traffic, blocked sustained repetitive firing (SRF) and spontaneous action potential firing in these cultured cells. Our results provide scientific evidence that F. platyphylla stem bark may contain psychoactive principles with potential anticonvulsant properties. SFG impaired membrane excitability; a property shared by most anticonvulsants particularly the voltage-gated sodium channel (VGSC) blocking drugs, thus supporting the isolation and development of the saponin components of this plant as anticonvulsant agents.

Pharmacological Evidence Favouring the Folkloric Use of Diospyros mespiliformis Hochst in the Relief of Pain and Fever

The methanol extract of Diospyros mespiliformis was evaluated for its claimed folkloric usage in the relief of pain and fever. Antipyretic, analgesic and anti-inflammatory effects of the extract were evaluated in rats and mice. Studies were carried out on yeast-induced pyrexia in rats, acetic acid-induced writhing in mice, formalin test and egg albumin-induced anti-inflammatory activity in rats. The extract (50 and 100 mg/kg i.p.) gave a potent antipyretic effect for 100 mg/kg and significant activity (P<0.05) against all the analgesic and anti-inflammatory models used. The LD(50) of the extract was estimated to be 513.80+/-33.92 mg/kg i.p. in mice. These results provide support for the use of the plant in relieving pain and fever.

Pharmacological evidence favouring the use of Nauclea latifolia in malaria ethnopharmacy: Effects against nociception, inflammation, and pyrexia in rats and mice

AIM OF THE STUDY Nauclea latifolia Smith is used traditionally in the treatment of uncomplicated malaria and painful conditions among its several other applications. The objective of this study is to investigate the pharmacological activities of the plant relevant to the symptomatic treatment of malaria fever and other painful conditions as an initial step towards developing an effective therapy for the symptomatic management of malaria fever and relief of other painful conditions. MATERIALS AND METHODS Various concentrations of the aqueous extract of the root bark of this plant were evaluated for its anti-nociceptive, anti-inflammatory and anti-pyretic activities in mice and rats. Investigation of the anti-nociceptive activities was performed using the acetic acid-induced abdominal constriction and hot-plate tests in mice and formalin-induced pain test in rats, as models of nociception. The extract was also investigated for its effect against inflammation induced by egg-albumin and pyrexia induced by yeast in rats. RESULTS Our data showed that the aqueous extract of Nauclea latifolia root bark (50-200mg/kg p.o.) significantly (P<0.05) attenuated writhing episodes induced by acetic acid and increased the threshold for pain perception in the hot-plate test in mice, dose-dependently. The product also remarkably decreased both the acute and delayed phases of formalin-induced pain in rats and also caused a significant reduction in both yeast-induced pyrexia and egg-albumin-induced oedema in rats. These effects were produced in a dose-dependent manner. CONCLUSION The results suggest the presence of biologically active principles in the extract with anti-nociceptive, anti-inflammatory and anti-pyretic activities that justifies its use in malaria ethnopharmacy and subsequent development for clinical application.

Farnesylthiosalicylic acid induces caspase activation and apoptosis in glioblastoma cells

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.