Inflammatory protein sPLA(2)-IIA abrogates TNFalpha-induced apoptosis in human astroglioma cells: Crucial role of ERK.

Brain injury induces the expression of well-known cytokines, such as tumor necrosis factor-alpha (TNFalpha), and other, which functions are less understood, as secreted phospholipase A(2) group IIA (sPLA(2)-IIA). Since in pathological processes, cytokines function coordinately in networks, to further explore the actions of sPLA(2)-IIA in tumorigenesis, we investigated the effect of sPLA(2)-IIA in the presence of TNFalpha in human 1321N1 astrocytoma cells. In these cells, TNFalpha activates the apoptotic programme that is accompanied of cytoskeleton changes; however, simultaneous treatment with sPLA(2)-IIA prevents TNFalpha-mediated apoptosis and reverses the modification of the markers associated to this response. In fact, the mitogenic activity elicited by the phospholipase alone is preserved. This inhibitory effect is not found in other TNFalpha-mediated responses, even a functional cooperation is observed on COX-2 protein induction. The cross-talk between TNFalpha and sPLA(2)-IIA is associated with ERK activity since its pharmacological inhibition attenuates both synergistic and inhibitory responses. We have also observed that upon sPLA(2)-IIA stimulation, endogenous ERK has the capacity to bind and phosphorylate sequences present within the cytoplasmic domain of TNFR1/CD120a. These findings thus indicate that sPLA(2)-IIA and TNFalpha transduction pathways interact to modulate inflammatory responses and provide additional insights about the capacity of sPLA(2)-IIA to promote apoptosis resistance in astrocytoma cells.

Secreted phospholipase A2-IIA modulates key regulators of proliferation on astrocytoma cells.

Human group IIA secreted phospholipase A(2) (sPLA(2)-IIA) has been characterized in numerous inflammatory and neoplastic conditions. sPLA(2)-IIA can either promote or inhibit cell growth depending on the cellular type and the specific injury. We have previously demonstrated that exogenous sPLA(2)-IIA, by engagement to a membrane structure, induces proliferation and activation of mitogen-activated protein kinases cascade in human astrocytoma cells. In this study, we used human astrocytoma 1321N1 cells to investigate the key molecules mediating sPLA(2)-IIA-induced cell proliferation. We found that sPLA(2)-IIA promoted reactive oxygen species (ROS) accumulation, which was abrogated in the presence of allopurinol and DPI, but not by rotenone, discarding mitochondria as a ROS source. In addition, sPLA(2)-IIA triggered Ras and Raf-1 activation, with kinetics that paralleled ERK phosphorylation, and co-immunoprecipitation assays indicated an association between Ras, Raf-1 and ERK. Additionally, Akt, p70 ribosomal protein S6 kinase, and S6 ribosomal protein were also phosphorylated upon sPLA(2)-IIA treatment, effect that was abrogated by N-acetylcysteine or LY294002 treatment indicating that ROS and phosphatidylinositol 3 kinase are upstream signaling regulators. As the inhibitors N-acetylcysteine, PD98059, LY294002 or rapamycin blocked sPLA(2)-IIA-induced proliferation without activation of the apoptotic program, we suggest that inhibition of these intracellular signal transduction elements may represent a mechanism of growth arrest. Our results reveal new potential targets for therapeutic intervention in neuroinflammatory disorders and brain cancer in particular.

Natural triterpenic diols promote apoptosis in astrocytoma cells through ROS-mediated mitochondrial depolarization and JNK activation.

BACKGROUND: Triterpene alcohols and acids are multifunctional compounds widely distributed throughout the plant kingdom that exhibit a variety of beneficial health properties, being synthetic analogs of oleanolic acid under clinical evaluation as anti-tumoral therapeutic agents. However, the antineoplastic activity of two natural occurring triterpenoid alcohols extracted from olive oil, erythrodiol (an intermediate from oleanolic acid), and its isomer, uvaol, has barely been reported, particularly on brain cancer cells. Astrocytomas are among the most common and aggressive type of primary malignant tumors in the neurological system lacking effective treatments, and in this study, we addressed the effect of these two triterpenic diols on the human 1321N1 astrocytoma cell line. PRINCIPAL FINDINGS: Erythrodiol and uvaol effectively affected cell proliferation, as well as cell cycle phases and induced 1321N1 cell death. Both triterpenes successfully modulated the apoptotic response, promoting nuclear condensation and fragmentation. They caused retraction and rounding of cultured cells, which lost adherence from their supports, while F-actin and vimentin filaments disappeared as an organized cytoplasmic network. At molecular level, changes in the expression of surface proteins associated with adhesion or death processes were also observed. Moreover, triterpene exposure resulted in the production of reactive oxygen species (ROS) with loss of mitochondrial transmembrane potential, and correlated with the activation of c-Jun N-terminal kinases (JNK). The presence of catalase reversed the triterpenic diols-induced mitochondrial depolarization, JNK activation, and apoptotic death, indicating the critical role of ROS in the action of these compounds. CONCLUSIONS: Overall, we provide a significant insight into the anticarcinogenic action of erythrodiol and uvaol that may have a potential in prevention and treatment of brain tumors and other cancers.

Secreted phospholipase A2 type IIA as a mediator connecting innate and adaptive immunity: new role in atherosclerosis.

AIMS: Human atherosclerotic plaques express markers of macrophage/dendritic cells as well as high levels of inflammatory proteins such as secreted phospholipase A(2) type IIA (sPLA(2)-IIA). To understand the cellular changes associated with the progress of atherosclerosis, we evaluated the role of sPLA(2)-IIA in mediating monocyte recruitment and differentiation into antigen-presenting cells. METHODS AND RESULTS: The effect of sPLA(2)-IIA on monocyte differentiation was evaluated in human THP-1 cells, a cellular line widely used as a model for monocyte-macrophage differentiation. Changes in functional processes, morphology and expression of antigens, characteristic of differentiated cells, were monitored over a 1-3 day period. sPLA(2)-IIA inhibited CD14 expression in a time- and concentration-dependent manner and upregulated dendritic cell-specific ICAM-3 grabbing non-integrin levels at the cell surface, findings that were the same for human monocytes. In addition, sPLA(2)-IIA-differentiated cells showed a dendritic cell phenotype characterized by the generation of fine dendritic protrusions and an increase in surface markers such as CD40, CD83, CD54, CD61, and CD62L. Furthermore, cell adhesion, migration, endocytic activity, and allogeneic T cell proliferation capacity were markedly increased after sPLA(2)-IIA treatment. CONCLUSION: sPLA(2)-IIA induces the differentiation of mononuclear cells and increases their adhesive and migratory capabilities, which suggests a novel function for sPLA(2)-IIA as a mediator connecting innate and adaptive immunity. These findings may provide insight into the immuno-inflammatory processes occurring in atherosclerosis, helping us to understand the cellular changes associated with the development of atherosclerosis.

Acidic triterpenes compromise growth and survival of astrocytoma cell lines by regulating reactive oxygen species accumulation.

Several studies have shown how pentacyclic triterpenes can inhibit proliferation and induce apoptosis of some tumor cell lines; however, its effect on astrocytic tumors, one of the most malignant forms of cancer, has rarely been reported. The aim of this study was to examine how the pentacyclic triterpenes, oleanolic acid and maslinic acid, isolated from olive juice, affected astrocytoma cell morphology and survival. Cell proliferation was inhibited in 1321N1 astrocytoma cells by using 1 to 50 micromol/L of either oleanolic acid or maslinic acid, with an average IC(50) of 25 micromol/L. Growth inhibition led to morphologic and cytoskeletal alterations associated with the loss of stellate morphology and characterized by a retraction of the cytoplasm and collapse of actin stress fibers. Using 4',6-diamidino-2-phenylindole and Annexin V, we showed that astrocytoma cell death induced by oleanolic acid or maslinic acid were mainly due to apoptotic events. Furthermore, we showed that caspase-3 is activated as a consequence of triterpene treatment. Finally, we found that exposure of the cells to oleanolic acid or maslinic acid resulted in a significant increase of intracellular reactive oxygen species, followed by loss of mitochondrial membrane integrity. Importantly, enzymatic scavengers, such as catalase, or phenolic antioxidants, such as butylated hydroxytoluene, rescued cells from the triterpene-mediated apoptosis, suggesting that the potential therapeutic effect of these acidic triterpenes is dependent on oxidative stress. Our data show that acidic triterpenes play a major role in 1321N1 astrocytoma morphology and viability and support the conclusion that oleanolic acid and maslinic acid may thus be promising new agents in the management of astrocytomas.