The enzyme phospholipase A2 (PLA2) specifically hydrolyzes the 2-acyl ester bond of 1,2-diacyl-3-sn-phosphoglycerides releasing fatty acids and lysophospholipids in the presence of Ca2+. Both products represent precursors for signaling molecules that can exert a multitude of biological functions including phospholipid metabolism, exocytosis and inflammation. Consequently, PLA2 not only plays a role in regulating physiological processes, but also exhibits pharmacological effects in inflammatory diseases. Nevertheless, the signaling pathway leading to cell death still remains elusive. In the present study, the cytotoxicity of Naja naja atra PLA2 toward human neuroblastoma SK-N-SH cells and leukemia K562 cells were respectively evaluated to explore the signaling pathway of PLA2-induced cell death. Upon exposure to PLA2, p38 mitogen-activated protein kinase (p38 MAPK) or c-Jun N-terminal kinase (JNK) activation, extracellularsignal-regulated protein kinase (ERK) inactivation, reactive oxygen species (ROS) generation, increase in intracellular Ca2+ concentration, the loss of mitochondrial membrane potential (£G£Zm), cytochrome c release and upregulation of Fas/FasL were found in SK-N-SH or K562 cells. N-Acetylcysteine (ROS scavenger), BAPTA-AM (Ca2+ chelator), SB202190 (p38 MAPK inhibitor) or SP600125 (JNK inhibitor) abrogated p38 MAPK or JNK activation and rescued cell viability, £G£Zm, cytochrome c release and suppressed Fas/FasL upregulation of PLA2-treated cells, but restored phosphorylation of ERK. Activated ERK was found to attenuate p38 MAPK-mediated upregulation of Fas/FasL. Besides, sustained JNK activation was also observed in SB202190/PLA2-treated K562 cells after exterminating p38 MAPK activation, but also retained the cytotoxicity of PLA2. Knockdown of p38 MAPK or JNK1 by siRNA proved that PLA2 induced Fas/FasL upregulation through p38 MAPK/ATF-2 or JNK1/c-Jun pathways in K562 cells. Furthermore, deprivation of catalytic activity could not diminish PLA2-induced cell death and Fas/FasL upregulation. The cytotoxicity of arachidonic acid (AA) and lysophosphatidylcholine (LPC) was not related to the expression of Fas/FasL. The results showed that the cytotoxicity of AA is mediated through mitochondria-dependent death pathway, eliciting by AA-induced ROS generation and Ca2+-evoked activation of p38 MAPK and JNK. Besides, ERK activation abrogated by U0126 improved the ability of AA-mediated Fas/FasL upregulation in K562 cells. Taken together, our results indicate that PLA2-induced cell death is through Ca2+- and ROS evoked p38 MAPK or JNK activation. Upregulation of Fas/FasL partially involves in cytotoxicity of PLA2.

The survival for high-grade glioma patients is poor and the treatment may cause severe side effects. A common obstacle in the treatment is chemoresistance. To improve the quality of life and prolong survival for these patients prognostic biomarkers and new approaches for chemotherapy are needed. To this end, a strategy to evade chemoresistance was evaluated by combining chemotherapeutic drugs with agents inhibiting resistance mechanisms identified by a bioinformatic analysis (paper I). The prognostic value of 13 different proteins was analyzed in this thesis (papers II-IV). Two of them, p38 mitogen-activated protein kinase (MAPK) and protein tyrosine phosphatase non-receptor type 6 (PTPN6, also known as SHP1) were analyzed for their potential as targets in combination chemotherapy (in paper III and IV, respectively).   We found that: PTPN6 expression and methylation status may be important for survival of anaplastic glioma patients, p38 MAPK phosphorylation may be a potential negative prognostic biomarker for high-grade glioma patients and FGF2 expression may be a potential negative prognostic biomarker for proneural glioma patients. PTPN6 may be a useful target for combination chemotherapy with cisplatin, melphalan or bortezomib in high-grade gliomas. The following drug combinations; camptothecin combined with an EGFR or RAC1 inhibitor, imatinib combined with a Notch or RAC1 inhibitor, temozolomide combined with an EGFR or FAK inhibitor and vandetanib combined with a p38 MAPK inhibitor may be useful combination chemotherapy for high-grade gliomas.

Thesis (Ph.D.) -- University of Texas Southwestern Medical Center at Dallas, 2006. / Embargoed. Vita. Bibliography: 180-194.

IL-12 family cytokines IL-23 and IL-27 play an important role linking innate and adaptive immunity, and regulating T-cell responses. The production of IL-12, a structurally similar cytokine, is decreased in chronic HIV infection; therefore IL-23 and IL-27 may also be influenced by HIV infection. I hypothesized that HIV inhibits LPS-induced IL-23 and IL-27 production in human MDMs by suppressing the activation of signalling pathways regulating their expression. In vitro HIV-infection of MDMs did not have any effect on basal secretion of IL-23 or IL-27; however, HIV inhibited LPS-induced production of IL-12/23 p40 and IL-23 p19, and IL-27 EBI3 and IL-27 p28 mRNA expression, and IL-23, IL-12/23 p40 and IL-27 secretion. In order to evaluate the molecular mechanisms by which HIV inhibits IL-23 and IL-27 in LPS-stimulated MDMs, the signalling pathways regulating their expression were evaluated. The PI3K, p38 MAPK, and JNK MAPK pathways were found to positively regulate LPS-induced IL-27 secretion. Interestingly, in vitro HIV infection inhibited LPS-induced p38 and JNK MAPK activation in MDMs. In summary, I have shown that HIV inhibits IL-23 and IL-27 production in LPS-stimulated MDMs and that HIV may inhibit LPS-induced IL-27 production through the inhibition of p38 and JNK MAPK activation. It is currently unknown whether PKCs regulate LPS-induced IL-23 or IL-27 in human monocytes/macrophages. I demonstrated that classical PKCs differentially regulate LPS-induced IL-23 and IL-27 secretion within THP-1 cells, primary monocytes, and MDMs. Classical PKCs were found to positively regulate LPS-induced IL-12/23 p40 and IL-27 p28 mRNA expression and IL-12/23 p40, IL-23, and IL-27 secretion in primary human monocytes. Similarly, the classical PKCs were found to positively regulate IL-27 p28 mRNA expression and IL-27 secretion in THP-1 cells. However, classical PKCs did not regulate LPS-induced IL-27 production in MDMs, or LPS-induced IL-23 production in THP-1 cells. Overall, this demonstrates that classical PKCs differentially regulate LPS-induced IL-23 and IL-27 production in different myeloid cells.

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Cardiovascular diseases (CVD) are the leading cause of morbidity and mortality in the United States. It is also well recognized that the incidence of CVD is substantially increased in postmenopausal women due to the loss of estrogen. Experimental and clinical data support vascular protective effects of estrogen by various mechanisms. However, administration of estrogen is also associated with an increased incidence of heart disease which limits its therapeutic potential. Given the demonstrated risks of conventional estrogen therapy, a search for novel, cost-effective, alternative vasoactive agents for prevention of CVD is of major importance in the effort to decrease the burden of CVD morbidity. Genistein, a major soy isoflavone, may be one of those alternative agents because of its selective affinity to estrogen receptor-beta and various beneficial effects on CVD. However, the mechanism of the cardioprotective effects of genistein is still unclear. The objectives of this study were (1) to investigate the effect of genistein on the expression of endothelial nitric oxide synthase (eNOS) both in vitro and in vivo; (2) to define the mechanism by which genistein regulates eNOS expression; and, (3) to examine whether genistein protects against tumor necrosis factor-alpha (TNF-α)-induced apoptosis in human aortic endothelial cells (HAECs). The results demonstrated that genistein, at physiologically achievable concentrations (1-10 μM) in individuals consuming soy products, enhanced the expression of eNOS protein and subsequently elevated nitric oxie (NO) synthesis in both HAECs and human umbilical vein endothelial cells, concomitant with the increased eNOS mRNA expression (2.6-fold of control) and eNOS promoter activity, suggesting that genistein activates eNOS transcription. Furthermore, dietary supplementation of genistein to spontaneously hypertensive rats restored aortic eNOS levels, improved aortic wall thickness, and alleviated hypertension, confirming the biological relevance of the in vitro findings. However, the effects of genistein on eNOS and NO were not mediated by activation of estrogen signaling, mitogen-activated protein kinase, phosphatidylinositol 3-kinase/Akt kinase, protein kinase C or inhibition of typrosine kinases, but possibly through activating the cAMP/protein kinase A/cAMP responsive elemant binding protein pathway. These data suggest that genistein has direct genomic effects on the vascular wall that are unrelated to its known actions, leading to increase in eNOS expression and NO synthesis, thereby improving vascular homeostasis. We also found that genistein (5-10 μM) significantly inhibited TNF-α-induced apoptosis in HAECs as determined by caspase-3 activation, apoptotic cell detection and DNA laddering. The anti-apoptotic effect of genistein was associated with an enhanced expression of anti-apoptotic Bcl-2 protein and its promoter activity that was ablated by TNF-α. Moreover, this anti-apoptotic effect of genistein was not mediated by extracellular signal-regulated kinase 1/2, protein kinase A, or estrogen receptor. However, inhibition of p38 mitogen-activated protein kinase (p38) by SB203580 completely abolished the cytoprotective effect of genistein, suggesting that genistein acted through the p38-dependent pathway. Accordingly, stimulation of HAECs with genistein resulted in rapid and dose-dependent activation of p38. Unlike TNF-α which specifically activated p38α, genistein selectively induced phosphorylation of p38β, suggesting that p38β, but not p38α, is essential for the cytoprotective effect of genistein. These findings provide the evidence that genistein acts as a survival factor for vascular ECs to protect cells against apoptosis via activation of p38β. Taken together, the resuls of the present study suggest that genistein can act directly on vascular ECs, improves endothelium homeostasis by promoting eNOS expression and endothelial-derived NO synthesis through activating the cAMP/PKA/CREB cascade, and protects against TNF-α-induced apoptosis via activation of p38 β. These data potentially provide a basic mechanism underlying the physiological effects of genistein in the vasculature. / Ph. D.