Activities of Some Medicinal Plants on the Proliferation and Invasion of Brain Tumor Cell Lines.

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 IC50 values ranging between 8 and 20 µg/ml for S. longepedunculata and 100 and 90 µg/ml for P. polyandra. The five extracts significantly (p < 0.05) inhibited the invasion of U251 cell line at the concentration of 10 µg/ml (S. longepedunculata), 20 µg/ml (A. inermis), 50 µg/ml (A. senegalensis), 50 µg/ml (C. edulis), and 50 µg/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.

Ethanol Extract of Securidaca longipedunculata Induces Apoptosis in Brain Tumor (U87) Cells.

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 IC50 of 20.535 µg/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.

PRELIMINARY STUDIES ON THE BEHAVIOURAL EFFECTS OF THE METHANOL EXTRACT OF LEONOTIS NEPETIFOLIA LINN STEM IN MICE.

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.

Improving Brain Tumor Research in Resource-Limited Countries: A Review of the Literature Focusing on West Africa.

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.

Sorafenib tosylate as a radiosensitizer in malignant astrocytoma.

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.

STAT6 expression in glioblastoma promotes invasive growth.

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.

A novel fixative for immunofluorescence staining of CD133-positive glioblastoma stem cells.

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.

PKC alpha phosphorylates cytosolic NF-kappaB/p65 and PKC delta delays nuclear translocation of NF-kappaB/p65 in U1242 glioblastoma cells.

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.

Epidermal growth factor receptor-mediated regulation of urokinase plasminogen activator expression and glioblastoma invasion via C-SRC/MAPK/AP-1 signaling pathways.

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.

An extensive invasive intracranial human glioblastoma xenograft model: role of high level matrix metalloproteinase 9.

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.

Immunohistochemistry of COUP-TFI: an adjuvant diagnostic tool for the identification of corticotroph microadenomas.

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.

ADAM-10-mediated N-cadherin cleavage is protein kinase C-alpha dependent and promotes glioblastoma cell migration.

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.

H-Ras increases urokinase expression and cell invasion in genetically modified human astrocytes through Ras/Raf/MEK signaling pathway.

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.

Urokinase directly activates matrix metalloproteinases-9: a potential role in glioblastoma invasion.

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.

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-alpha-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.

Matrix metalloproteinase-9 is differentially expressed in nonfunctioning invasive and noninvasive pituitary adenomas and increases invasion in human pituitary adenoma cell line.

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.

Restricted epithelial proliferation by lacritin via PKCalpha-dependent NFAT and mTOR pathways.

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.

PKC eta as a therapeutic target in glioblastoma multiforme.

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.