Pro-apoptotic effect of curcumin on human polymorphonuclear leukocytes (PMNS): Decreased nuclear NF-KB activity

ASSOCIATION FOR ACADEMIC SURGERY—ABSTRACTS 13. Pro-Apoptotic Effect of Curcumin on Human Polymorphonuclear Leukocytes (PMNS): Decreased Nuclear NF-KB Activity. M. Hu, MD, PhD, Q. Du, MD, I. Vancurova, PhD, X. Lin, MD, E. Miller, PhD, P. Wang, MD, H. Simms, MD. North Shore University Hospital and Long Island Jewish Medical Center. Phytochemical curcumin (Cur), a promising dietary supplement for cancer prevention and liver protection, has been shown to induce tumor cell apoptosis (Ao). However, the effect of Cur on PMN Ao and the underlying mechanisms remain unknown. Methods. PMNs (10 6 cells/ml) freshly isolated from normal volunteers were incubated in vitro in the presence or absence of Cur (10 –50 ␮M). Ao was examined by morphology and TUNEL analyses. The effect of Cur on LPS (100 ng/ml)-induced Ao inhibition was also studied. Myeloidperoxidase (MPO) activity was analyzed to determine the effect of Cur on PMN degranulation. To elucidate the potential mechanism, nuclear NF-kB activity was examined by EMSA. Statistical analysis was performed by ANOVA and Tukey’s test. Results. The data demonstrated that Cur increased PMN Ao from 7.17 ⫾ 0.29 at 0 ␮M to 28.0 ⫾ 2.0% at 40 ␮M after 6 h incubation (P ⬍ 0.05). LPS-induced Ao suppression was also reversed by Cur (Fig. 1). PMN degranulation was inhibited by Cur as measured by a significant decrease in MPO activities (Table). The inhibition of nuclear NF-kB activity (58% at 1 h and 54% at 2 h) induced by LPS appears to contribute to the pro-apoptotic effect of Cur (Fig. 2). Conclusions. Since the delayed PMN Ao exacerbates acute respiratory distress syndrome and multiple organ failure, the characteristics of curcumin, including pro-apoptotic effects and anti-degranulation potential, make it a potential candidate for prevention and treatment of lung inflammation and injury.

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14. Secretory Phospholipase A2 Attacks Erythrocytes with Externalized Phosphatidylserine. N.A. Neidlinger, MD, S.K. Larkin, MS, F.A. Kuypers, PhD. Children’s Hospital Oakland Research Institute. Introduction. Membrane phospholipids are distributed asymmetrically in normal cells, with phosphatidylserine (PS) confined to the inner monolayer. Externalization of PS is a marker for apoptosis and follows ischemia-reperfusion injury. Circulating levels of secretory phospholipase A2 (sPLA2) are elevated in sepsis, the acute respiratory distress syndrome, and following trauma, pointing at an active role of the enzyme in disease-associated tissue destruction. PLA2 hydrolyzes membrane phospholipids and is an early and rate-limiting step in the production of powerful inflammatory mediators. We hypothesized that (1) normal cells are not susceptible to sPLA2, but that (2) erythrocytes with externalized PS are hemolyzed by sPLA2 at clinically relevant concentrations, (3) binding the externalized PS would protect the cells from sPLA2-induced lysis, and also that (4) inhibition of PLA2 would protect cells with externalized PS from hemolysis. Methods. Erythrocytes were experimentally induced to expose PS. Normal erythrocytes and PS-exposing erythrocytes were exposed to sPLA2, and vulnerability was measured by flow cytometry and hemolysis. Annexin V, and its dimmer, DiAnnexin V, were tested as cytoprotective agents given their affinity for binding externalized PS. Finally, PX-18 was tested for its ability to inhibit enzymatic activity of sPLA2 and protect the PSexposing cells. Results. Normal erythrocytes are not susceptible to sPLA2, however, sPLA2 hemolyzes PS-exposing erythrocytes at clinically relevant concentrations in a dose- and time-dependent fashion. Increase in hemolysis was inversely related to removal of PS-exposing cells, indicating that PLA2 targets externalized PS as a marker for cytotoxicity. Annexin V and Di-Annexin V effectively prevented PLA2induced hemolysis at 7 nmol per 5 ⫻ 10 7 cells, confirming that PLA2 requires externalization and recognition of PS for cytotoxicity. At 10 ␮M, PX-18 reduced hemolysis of PS-exposing cells to baseline levels, indicating that sPLA2 inhibition also protects PS-exposing cells. Conclusion. Secretory PLA2 effectively hemolyzes PS-exposing erythrocytes. Cellular protection can be achieved both by binding PS and by inhibiting sPLA2 activity. These inhibitors may have therapeutic efficacy in inflammatory diseases.

15. Dexamethasone Increases Protein Degradation in Cultured Myotubes Through a Calcium-Calmodulin Kinase II (CaMK-II) Dependent Mechanism. A.R. Evenson, MD, M.J. Menconi, PhD, J.C. Mitchell, MD, and P.J. Hasselgren, MD, PhD. Beth Israel Deaconess Medical Center.

TABLE—ABSTRACT 13 Group

MPO activity (absorbance ⫾ SD)

Medium LPS LPS⫹Cur (10␮M) LPS⫹Cur (30␮M) LPS⫹Cur (50␮M)

0.012 ⫾ 0.002 0.101 ⫾ 0.021* 0.103 ⫾ 0.006* 0.018 ⫾ 0.003# 0.014 ⫾ 0.002#

(6 h incubation, n ⫽ 6, * P ⬍ 0.05 versus medium. # P ⬍ 0.05 versus LPS alone)

Introduction. Previous reports suggest that glucocorticoids are important mediators of muscle protein breakdown in various musclewasting conditions, including sepsis. The mechanisms by which glucocorticoids induce muscle proteolysis are poorly understood. Previous studies provided evidence that glucocorticoids may influence cellular calcium homeostasis, and, in other experiments, calcium increased muscle protein breakdown. We tested the hypothesis that glucocorticoid-induced protein degradation in muscle cells is at least in part mediated by calcium and the calcium-activated enzyme CaMK-II. Methods. Cultured L6 myotubes, a rat skeletal muscle cell line, were treated with 1 ␮M dexamethasone for 24 h. Protein degradation was assessed by measuring the release of TCA-soluble 3 H-tyrosine from proteins that had been pre-labeled with the amino acid. The role of calcium in dexamethasone-induced protein degradation was tested by treating the cells with the intracellular calcium chelator BAPTA-AM (20 ␮M). The involvement of CaMK-II was tested by using the CaMK-II inhibitor KN-93 (20 ␮M). Results. Treatment of L6 myotubes with dexamethasone resulted in a 12% increase in protein degradation, confirming previous reports from this laboratory. This effect of dexamethasone was blocked by