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Acetyl-11-keto-beta-boswellic acid (AKBA) is a naturally occurring pentacyclic triterpene isolated from the gum resin exudate of the tree Boswellia serrata (frankincense). Because pentacyclic triterpenes have antiproliferative and cytotoxic effects against different tumor types, we investigated whether AKBA would act in a similar fashion on primary human meningioma cell cultures. Primary cell cultures were established from surgically removed meningioma specimens. The number of viable cells in the absence/presence of AKBA was determined by the non-radioactive cell proliferation assay. The activation status of the proliferative cell marker, extracellular signal-regulated kinase-1 and -2 (Erk-1 and Erk-2) was determined by immunoblotting with the antibody that recognizes the activated form of these proteins. Treatment of meningioma cells by AKBA revealed a potent cytotoxic activity with half-maximal inhibitory concentrations in the range of 2 - 8 microM. At low micromolar concentrations, AKBA rapidly and potently inhibited the phosphorylation of Erk-1/2 and impaired the motility of meningioma cells stimulated with platelet-derived growth factor BB. The cytotoxic action of AKBA on meningioma cells may be mediated, at least in part, by the inhibition of the Erk signal transduction pathway. Because of the central role the Erk pathway plays in signal transduction and tumorigenesis, further investigation into the potential clinical use for AKBA and related boswellic acids is warranted.

Acetyl-11-keto-beta-boswellic acid (AKBA) is a naturally occurring pentacyclic triterpene isolated from the gum resin exudate from the stem of the tree Boswellia serrata (frankincense). AKBA has been recently identified as a novel, orally active, non-redox and non-competitive 5-lipoxygenase inhibitor that also inhibits topisomerase I and II in vitro. Because natural pentacyclic triterpenes have an antiproliferative effect against different tumor types, we investigated the effects of AKBA on the proliferation of 11 primary cell cultures established from human surgical specimens of meningiomas, common central nervous system tumors. Treatment of meningioma cells by AKBA revealed a potent cytotoxic activity with half-maximal inhibitory concentrations in the range of 2-8 microM. At similar, physiologically achievable concentrations, AKBA rapidly (within minutes) and potently inhibited the phosphorylation of extracellular signal-regulated kinase 1 and 2 (Erk-1 and Erk-2) in meningioma cells stimulated with platelet-derived growth factor BB. High expression level of 5-LO was detected in primary meningioma cells and surgical specimens by immunoblotting analysis, suggesting the possible role of 5-LO in meningioma tumorigenesis. Considering the critical importance of the Erk-1/2 signal transduction pathway not only in meningiomas but in other human neoplasms, the interruption of signaling through this evolutionarily conserved pathway might be one of the mechanisms by which AKBA induces suppression of proliferation and apoptosis of different tumor types.

Object: The neurofibromatosis Type 2 (NF2) gene is the only tumor suppressor gene that has been clearly implicated in the development of benign meningiomas. Interestingly, previous data obtained by the authors indicate that reduced NF2 protein expression seldom occurs in meningothelial meningiomas, the most common histological type of meningioma. The goal of the current study was to explore further the hypothesis of NF2 gene-independent tumorigenesis of meningothelial meningiomas., Methods: The authors performed a mutational analysis of all 17 exons of the NF2 gene by using single-stranded conformational polymorphism (SSCP). In addition, expression levels of the NF2 protein and mu-calpain, a protease suggested to inactivate the NF2 protein, were determined by immunoblotting analysis of 27 meningiomas (20 meningothelial and seven nonmeningothelial). Mutations of the NF2 gene were found in only one (5%) of 20 meningothelial meningiomas and three (43%) of seven nonmeningothelial tumors (Fisher's exact test, p = 0.042). The levels of NF2 protein were severely reduced in six (28.5%) of 21 meningothelial meningiomas, in contrast to six (86%) of seven nonmeningothelial meningiomas (Fisher's exact test, p = 0.023). Activation of IL-calpain did not correlate with the status of NF2 protein expression in the meningiomas analyzed, demonstrating that mu-calpain activation does not account for the loss of NF2 protein in meningiomas with apparently normal NF2 genes., Conclusions: These results clearly demonstrate that NF2 gene mutations and decreased NF2 protein expression rarely occur in meningothelial meningiomas compared with other histological types of meningiomas. The clinical behavior of meningothelial meningiomas, however, is similar to that of other benign meningiomas. It is likely, therefore, that the tumorigenesis of meningothelial meningiomas is the result of deleterious alterations of genes that have final phenotypical effects similar to inactivation of the NF2 gene.

Objective: Previous studies demonstrated that activation of receptor tyrosine kinases in human meningiomas by an autocrine or paracrine growth-stimulatory loop plays an important role in meningioma proliferation. Although it is well established that the proliferative signal from protein tyrosine kinase receptors is transduced through Ras proteins, the relevance of the Ras pathway in meningioma proliferation, to our knowledge, has not been studied. The purpose of this study was, therefore, to determine whether Ras proteins are functionally important in meningioma proliferation., Methods: Meningioma cells of nine primary cell cultures were infected with the recombinant adenovirus Ad-rasN17 encoding the dominant negative Ras protein or control adenovirus Ad-pAC. Ras-N17 is a Ras mutant protein with substitution of asparagine for serine at position 17 in the cellular Ha-Ras protein that inhibits function of all endogenous cellular Ras proteins. Proliferation of meningioma cells was measured using [3H]thymidine or 5-bromo-2'-deoxyuridine labeling and detection assays., Results: Infection of meningioma cells with Ad-rasN17 dramatically increased the expression levels of the Ras-N17 mutant protein and inhibited phosphorylation of the mitogen-activated protein kinases, compared with uninfected cells or cells infected with the control adenovirus. Suppression of Ras proteins inhibited proliferation of all exponentially growing and growth-arrested meningioma cells stimulated with serum., Conclusion: The obtained results suggest that proliferation of primary meningioma cells is dependent on the presence of functional Ras proteins. Therefore, inhibition of the Ras pathway may be important in preventing growth factor-stimulated meningioma proliferation.

Neurofibromin and p120 GTPase activating protein (p120 GAP) down-regulate the activity of cellular Ras proteins. How the activity of these two proteins is controlled is not yet clear. In this study, we analyzed the effects of eight nutritionally relevant fatty acids on GTPase stimulatory activity of full-length neurofibromin and p120 GAP. The fatty acids tested were: saturated stearic acid, monounsaturated oleic acid, three omega-6 and three omega-3 polyunsaturated fatty acids. The analysis was performed by Ras immunoprecipitation GTPase assay. The full-length p120 GAP expressed in insect Sf9 cells and the immunoaffinity purified full-length neurofibromin were used. Neurofibromin was readily inhibited by stearic and oleic acid, but p120 GAP was not inhibited even at high concentrations (> 80 microM). Neurofibromin was also inhibited by low concentrations of all the polyunsaturated fatty acids tested (IC50 of 6 to 16 microM). p120 GAP was 2-3 fold less sensitive to inhibition by these fatty acids. The GTPase stimulatory activity of neurofibromin was also inhibited by arachidonic and oleic acid in the presence of a lipid mixture representing the major lipid components of the cell membrane. Chimeric proteins of neurofibromin and p120 GAP were used to determine that differential sensitivity to fatty acid inhibition maps to the catalytic domain of the proteins. These results indicated that fatty acids can modulate the GTPase function of the c-Ha-Ras protein by inhibiting the GTPase stimulatory activity of two Ras regulators, full-length neurofibromin and p120 GAP, at physiologically relevant concentrations in vitro.

Hypothesis: Absent or reduced expression of schwannomin/merlin is associated with tumorigenesis of sporadic schwannomas., Background: The neurofibromatosis type 2 (NF2) gene frequently is mutated in sporadic vestibular schwannomas. The protein product of the NF2 gene is called schwannomin or merlin. Little is known about the mutated forms of schwannomin/merlin present in schwannomas., Methods: To investigate further the role of schwannomin/merlin in schwannoma tumorigenesis, immunoblotting experiments were performed. Antischwannomin/merlin-specific antibody that recognizes amino terminus of the protein was used to determine the expression levels of schwannomin/merlin in 16 sporadic vestibular schwannomas, 1 NF2-related vestibular schwannoma, and 5 spinal schwannomas., Results: The antibody detects a protein of approximately 66 kDa in the Triton X-100-insoluble fraction of tumors. The expression of schwannomin/merlin was severely reduced, <35% of control, in 11 (50%) of 22 sporadic schwannomas and in 1 NF2-related vestibular schwannoma. The intensity of 66-kDa schwannomin/merlin band was moderately reduced, from 35-60%, in 7 (32%) of 22 schwannomas compared to the expression levels found in the human brain. Truncated forms of schwannomin/merlin were identified in three tumors with moderately reduced schwannomin/merlin., Conclusions: These results provide new evidence that inactivation of schwannomin/merlin is an important factor in tumorigenesis of sporadic schwannomas.

Arachidonic acid, phosphatidic acid, and other lipids inhibit the catalytic fragment of neurofibromin more potently than that of p120 guanosine triphosphatase-activating protein (GAP). The effects of fatty acids other than arachidonic acid on full-length neurofibromin and p120 GAP, to our knowledge, have not been studied. In this study, we analyzed the effects of eight nutritionally relevant fatty acids on guanosine triphosphatase (GTPase) stimulatory activity of full-length neurofibromin and p120 GAP. The fatty acids tested were saturated stearic acid, monounsaturated oleic acid, and three n-6 and three n-3 polyunsaturated fatty acids. Analysis was performed by Ras immunoprecipitation GTPase assay. The full-length p120 GAP expressed in insect Sf9 cells and immunoaffinity-purified full-length neurofibromin were used. In contrast to neurofibromin, which was readily inhibited by stearic and oleic acid, p120 GAP was only weakly inhibited even at high concentrations (> 80 microM). Neurofibromin was also two- to threefold more sensitive to inhibition by other fatty acids tested. A chimeric protein in which the neurofibromin catalytic domain was fused to the NH2-terminal sequences of p120 GAP was used to determine that differential sensitivity to fatty acid inhibition maps to the catalytic domain of the proteins. These results indicate that nutritionally relevant fatty acids can modulate the GTPase function of c-Ha-Ras protein by inhibiting GTPase stimulatory activity of two Ras regulators, full-length neurofibromin and p120 GAP, at physiologically relevant concentrations in vitro.

We studied the effects of membrane modulation on the interaction of Plasmodium falciparum rhoptry proteins of 140/130/110 kDa (Rhop-H) with human and mouse erythrocytes. Cells treated with 2-(2-methoxyethoxy)ethyl-8-(cis-2-n-octylcyclopropyl)octanoate, myristoleyl alcohol, and proteins extracted with sublytic concentrations of membrane solubilizing detergents were used in erythrocyte binding assays. Protein binding was evaluated by immunoblotting using Rhop-H- and SERA-specific antisera, 1B9, K15, and 5E3, respectively. Protein binding to liposomes prepared with dipalmitoyl-L-alpha-phosphatidylcholine (DPPC) or dilauroyl-L-alpha-phosphatidylcholine (DLPC) was also examined. Our results show that erythrocyte membrane modulation markedly enhanced direct Rhop-H binding to intact human erythrocytes. Binding of SERA to intact human erythrocytes appeared unaffected. Both DPPC and DLPC liposomes had similar Rhop-H and SERA protein binding activities. However, binding to DLPC liposomes was reduced. Rhop-H and SERA extracted with the detergents octanoyl-N-methylglucamide, decanoyl-N-methylglucamide, sodium deoxycholate, and 3-[(3-cholamidopropyl)dimethylammonio]-1-propane sulfonate bound directly to intact human erythrocytes, probably by partitioning hydrophobically into the membranes. Sodium carbonate treatment demonstrated a nonintegral association of Rhop-H with the erythrocyte membrane during invasion. Membrane modulation may expose cryptic phospholipid binding sites in the bilayer.