Platelet activation is normally induced by primary agonists such as adenosine diphosphate (ADP), thrombin, and collagen, whereas other agonists, such as epinephrine, can play important accessory roles. It is now reported that the macrophage-derived chemokine (MDC), thymus activation-regulated chemokine (TARC), and stromal cell-derived factor one (SDF-1) are highly effective activators of platelet function under a variety of conditions, stimulating platelet shape change, aggregation, and adhesion to collagen or fibrinogen. Chemokine-mediated platelet activation was rapid and maximal (less than 5 seconds) under arterial flow conditions and depended strongly on the presence of low levels of primary agonists such as ADP or thrombin. Concentrations of ADP (0.05-0.25 microM) or thrombin (0.005-0.02 U/mL) that induced minimal aggregation caused major aggregation acting in combination with the chemokines. The ability of apyrase to block chemokine-dependent aggregation or adhesion was consistent with an important role for ADP. Chemokine-stimulated aggregation was also insensitive to indomethacin, suggesting that the activation of cyclo-oxygenase is not involved. TARC, MDC, and SDF-1 increased intracellular calcium concentrations [Ca(2+)](i) when combined with low levels of ADP. The MDC and TARC receptor CCR4 was expressed on platelets, and an anti-CCR4 antibody blocked aggregation induced by TARC or MDC. Treatment of platelets with SDF-1 and MDC rapidly exposed P-selectin (CD62P) on the cell surface but did not induce the secretion of serotonin. These findings suggest that the chemokines MDC, TARC, and SDF-1, which may be produced during inflammatory responses, coupled with low levels of ADP or thrombin, can serve as strong stimuli for activating platelet function.

Pentavalent antimony complexes, such as sodium stibogluconate and sodium antimony gluconate (SAG), are still the first choice for chemotherapy against various forms of leishmaniasis, including visceral leishmaniasis, or kala-azar. Although the requirement of a somewhat functional immune system for the antileishmanial action of antimony was reported previously, the cellular and molecular mechanism of action of SAG was not clear. Herein, we show that SAG induces extracellular signal-regulated kinase 1 (ERK-1) and ERK-2 phosphorylation through phosphoinositide 3-kinase (PI3K), protein kinase C, and Ras activation and p38 mitogen-activated protein kinase (MAPK) phosphorylation through PI3K and Akt activation. ERK-1 and ERK-2 activation results in an increase in the production of reactive oxygen species (ROS) 3 to 6 h after SAG treatment, while p38 MAPK activation and subsequent tumor necrosis factor alpha release result in the production of nitric oxide (NO) 24 h after SAG treatment. Thus, this study has provided the first evidence that SAG treatment induces activation of some important components of the intracellular signaling pathway, which results in an early wave of ROS-dependent parasite killing and a stronger late wave of NO-dependent parasite killing. This opens up the possibility of this metalloid chelate being used in the treatment of various diseases either alone or in combination with other drugs and vaccines.Visceral leishmaniasis, caused by Leishmania donovani, is fatal if left untreated. The pentavalent antimony (Sb V ) compound urea stibamine first emerged as an effective chemotherapeutic agent against Indian kala-azar (6). Although different forms of pentavalent antimony complexes (chelates, i.e., Sb V chelated to an organic backbone), namely, sodium stibogluconate (Pentostam) and meglumine antimoniate (Glucantime), are still the first choice for treatment of leishmaniases (21, 42), their mechanism of action is still largely unknown. Previous studies indicated that sodium antimony gluconate (SAG) failed to act in immunocompromised hosts, such as patients who are suffering from AIDS or receiving immunosuppressive agents (17, 38) and nude (36) and severe combined immunodeficient (SCID) mice (15). Several studies have shown that endogenous interleukin-2 (IL-2) (34), IL-4 (1, 43), and IL-12 (41) influence the effectiveness of chemotherapy with pentavalent antimony. These findings are inclined to indicate the requirement of a somewhat functional T-cell compartment for SAG action. Moreover, SAG has been found to inhibit selective protein tyrosine phosphatases (Src homology 2 domain-containing tyrosine phosphatase 1 [SHP1] and SHP2) in vitro and augment cytokine signaling and responses in hematopoietic cell lines (46), suggesting the role of phosphatases and possibly other signal transduction pathways in SAG-induced control of Leishmania infection. In addition, the dose of SAG that kills the axenic amastigotes in vitro is 50 times higher than the concentration of the drug required for killing the parasite w...

Chinese hamster lung fibroblasts V79 cells were treated with heat stress for 4 weeks with short duration (15 min) heat shock every alternate day in culture. It was observed that Hsp 70 and the antioxidant enzyme MnSOD became overexpressed during the chronic heat stress period.

BackgroundResveratrol is known to downregulate the high endogenous level of Heat shock protein 70 (Hsp70) in Chronic Myelogenous Leukemia (CML) K562 cells and induce apoptosis. Since Heat Shock Factor 1 (HSF1) controls transcription of Hsp70, we wanted to probe the signaling pathways responsible for transcriptional activation of HSF1.Methodology/Principal FindingsCells exposed to 40µM Resveratrol rapidly abolished serine473 phosphorylation of Akt and significantly reduced its kinase activity. Inactivation of Akt pathway by Resveratrol subsequently blocked serine9 phosphorylation of Gsk3β. Active non-phosphorylated Gsk3β rendered HSF1 transcriptionally inactive and reduced Hsp70 production. Blocking PI3K/Akt activity also demonstrated similar effects on Hsp70 comparable to Resveratrol. Inactivation of Gsk3β activity by inhibitors SB261763 or LiCl upregulated Hsp70. Resveratrol significantly modulated ERK1/2 activity as evident from hyper phosphorylation at T302/Y304 residues and simultaneous upregulation in kinase activity. Blocking ERK1/2 activation resulted in induction of Hsp70. Therefore, increase in ERK1/2 activity by Resveratrol provided another negative influence on Hsp70 levels through negative regulation of HSF1 activity. 17-allylamino-17-demethoxygeldanamycin (17AAG), a drug that inhibits Hsp90 chaperone and degrades its client protein Akt concomitantly elevated Hsp70 levels by promoting nuclear translocation of HSF1 from the cytosol. This effect is predominantly due to inhibition of both Akt and ERK1/2 activation by 17AAG. Simultaneously treating K562 with Resveratrol and 17AAG maintained phosho-ERK1/2 levels close to untreated controls demonstrating their opposite effects on ERK1/2 pathway. Resveratrol was found not to interfere with Bcr-Abl activation in K562 cells.Conclusion/SignificanceThus our study comprehensively illustrates that Resveratrol acts downstream of Bcr-Abl and inhibits Akt activity but stimulates ERK1/2 activity. This brings down the transcriptional activity of HSF1 and Hsp70 production in K562 cells. Additionally, Resveratrol can be used in combination with chemotherapeutic agents such as 17AAG, an Hsp90 inhibitor reported to induce Hsp70 and hence compromise its chemotherapeutic potential.

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