Yashin Sreenivasan

Mechanism of cytosine arabinoside-mediated apoptosis: role of Rel A (p65) dephosphorylation

Nuclear transcription factor kappa B (NF-κB) has been shown both to block apoptosis and to promote cell proliferation, and hence has been considered an important target for anticancer drug development. The pyrimidine analogue cytosine arabinoside (araC) is among the most effective agents used in the treatment of acute leukemia, and we demonstrate in this study that its chemotherapeutic activity may be mediated by its inhibition of NF-κB. We found that in Jurkat cells, although tumor necrosis factor (TNF), araC, or ceramide induced NF-κB, the induction was only transient in the case of araC. In both HuT-78 and serum-activated LPS-stimulated Jurkat (SA-LPS/Jkt) cells that expressed NF-κB, TNF or ceramide treatments did not affect the NF-κB expression whereas araC downregulated it. AraC, but not TNF or ceramide was able to induce apoptosis in these cells as detected by assays for lipid peroxidation, reactive oxygen intermediates generation, caspase activation, cytotoxicity, Bcl-2 degradation, and DNA fragmentation. AraC also potentiated apoptosis mediated by cis-platin, etoposide, or taxol in these cells. AraC was able to induce protein phosphatases (PP) 2A and 2B-A, and phosphorylation of p65 subunit of NF-κB in the HuT-78 and SA-LPS/Jkt cells was downregulated by araC treatment. Furthermore, calyculin A, a specific phospho-serine/phospho-threonine phosphatase inhibitor, protected HuT-78 and SA-LPS/Jkt cells from araC-mediated NF-κB downregulation and apoptosis. These observations collectively suggest that araC induces apoptosis in NF-κB-expressing cells by dephosphorylating the p65 subunit of NF-κB.

α-melanocyte-stimulating hormone down-regulates CXC receptors through activation of neutrophil elastase

Considering the role of interleukin‐8 (IL‐8) in a large number of acute and chronic inflammatory diseases, the regulation of IL‐8‐mediated biological responses is important. Alpha‐melanocyte‐stimulating hormone (α‐MSH), a tridecapeptide, inhibits most forms of inflammation by an unknown mechanism. In the present study, we have found that α‐MSH interacts predominantly with melanocortin‐1 receptors and inhibits several IL‐8‐induced biological responses in macrophages and neutrophils. It down‐regulated receptors for IL‐8 but not for TNF, IL‐4, IL‐13 or TNF‐related apoptosis‐inducing ligand (TRAIL) in neutrophils. It down‐regulated CXCR type 1 and 2 but not mRNA levels. α‐MSH did not inhibit IL‐8 binding in purified cell membrane or affinity‐purified CXCR. IL‐8 or anti‐CXCR Ab protected against α‐MSH‐mediated inhibition of IL‐8 binding. The level of neutrophil elastase, a specific serine protease, but not cathepsin G or proteinase 3 increased in α‐MSH‐treated cells, and restoration of CXCR by specific neutrophil elastase or serine protease inhibitors indicates the involvement of elastase in α‐MSH‐induced down‐regulation of CXCR. These studies suggest that α‐MSH inhibits IL‐8‐mediated biological responses by down‐regulating CXCR through induction of serine protease and that α‐MSH acts as a potent immunomodulator in neutrophil‐driven inflammatory distress.

Cardiac glycoside induces cell death via FasL by activating calcineurin and NF-AT, but apoptosis initially proceeds through activation of caspases.

Decrease in caspase activity is a common phenomenon in drug resistance. For effective therapeutic intervention, detection of such agents, which affects other pathway independent of caspases to promote cell death, might be important. Oleandrin, a polyphenolic glycoside induced cell death through activation of caspases in a variety of human tumour cells. In this report we provide evidence that besides caspases activation, oleandrin interacts with plasma membrane, changes fluidity of the membrane, disrupts Na+/K+-ATPase pump, enhances intracellular free Ca2+ and thereby activates calcineurin. Calcineurin, in turns, activates nuclear transcription factor NF-AT and its dependent genes such as FasL, which induces cell death as a late response of oleandrin. Cell death at early stages is mediated by caspases where inhibitors partially protected oleandrin-mediated cell death in vector-transfected cells, but almost completely in Bcl-xL-overexpressed cells. Overall, our data suggest that oleandrin might be important therapeutic molecule in case of tumors where cell death pathway occurs due to deregulation of caspase-mediated pathway

Cardiac glycoside inhibits IL-8-induced biological responses by downregulating IL-8 receptors through altering membrane fluidity†

Considering the potential role of interleukin‐8 (IL‐8) in inflammation, angiogenesis, tumorogenesis, and metastasis, and the involvement of different cell types especially neutrophils and macrophages in those processes, the regulation of IL‐8‐mediated biological responses is important. In this report we provide evidences that oleandrin, a cardiac glycoside potentially inhibited IL‐8‐, formyl peptide (FMLP)‐, EGF‐, or nerve growth factor (NGF)‐, but not IL‐1‐ or TNF‐induced NF‐κB activation in macrophages. Oleandrin inhibited IL‐8‐, but not TNF‐induced NF‐κB‐dependent genes expression. Oleandrin inhibited the binding of IL‐8, EGF, or NGF, but not IL‐1 or TNF. It decreased almost 79% IL‐8 binding without altering affinity towards IL‐8 receptors and this inhibition of IL‐8 binding was observed in isolated membrane. The IL‐8, anti‐IL‐8Rs antibodies, or protease inhibitors were unable to protect oleandrin‐mediated inhibition of IL‐8 binding. Phospholipids significantly protected oleandrin‐mediated inhibition of IL‐8 binding thereby restoring IL‐8‐induced NF‐κB activation. Oleandrin altered the membrane fluidity as detected by microviscosity parameter and a decrease in diphenylhexatriene, a lipid binding fluorophore binding in a dose‐dependent manner. Overall, our results suggest that oleandrin inhibits IL‐8‐mediated biological responses in diverse cell types by modulating IL‐8Rs through altering membrane fluidity and microviscosity. The study might help to regulate IL‐8‐mediated biological responses involved in inflammation, metastasis, and neovascularization. J. Cell. Physiol. 207: 195–207, 2006.

α-Melanocyte-stimulating hormone inhibits lipopolysaccharide-induced biological responses by downregulating CD14 from macrophages

Monocytes/macrophages are the first cells involved in inflammation. α‐Melanocyte‐stimulating hormone (α‐MSH) is known to possess an anti‐inflammatory role induced by a variety of stimuli; however, the molecular mechanisms underlying these effects are not clearly defined. In this report we provide evidence that α‐MSH inhibited serum‐activated lipopolysaccharide (SA‐LPS)‐induced proteolytic enzyme release, oxidative burst response, reactive oxygen intermediate generation, nitric oxide production, and adhesion molecule expression in monocyte‐derived macrophages. α‐MSH also inhibited SA‐LPS‐induced nuclear transcription factor κB activation not only in macrophages, but also in a T‐cell line and human neutrophils isolated from fresh blood. α‐MSH downregulated CD14, but not interleukin‐1 receptor, tumor necrosis factor receptor 1 or 2 from the surface of macrophages. Anti‐CD14 antibody was unable to protect α‐MSH‐mediated downregulation of CD14. Overall, our results suggest that α‐MSH exerts its anti‐inflammatory effect by a novel mechanism in macrophages through downregulating of the endotoxin receptor CD14.

Retraction Note: Oleandrin-Mediated Expression of Fas Potentiates Apoptosis in Tumor Cells

Chemotherapeutic agent is characterized by its concentration in tumor cells with minimum side effects. Oleandrin, a polyphenolic cardiac glycoside is known to induce apoptosis in tumor cells. However, no report is available on its efficacy in primary cells. In this report we are providing the evidence that oleandrin induces apoptosis, not necrosis in tumor cells but not in primary cells like peripheral blood mononuclear cells (PBMC) and neutrophils. Oleandrin inhibited NF-kappaB activation in tumor cells but not in primary cells. It induced cell death in NF-kappaB-overexpressed tumor cells. Oleandrin induced Fas expression thereby inducing apoptosis in tumor cells but not in primary cells. Dominant negative FADD inhibited oleandrin-induced cell death in tumor cells. Overall, these results suggest that oleandrin mediates apoptosis in tumor cells by inducing Fas but not in primary cells indicating its potential anti-cancer property with no or slight side effect.

α‐Melanocyte‐stimulating hormone induces cell death in mast cells: involvement of NF‐κB

Mast cells play a major role in the initiation of inflammation and allergic reactions. As cell numbers are tightly controlled by the interplay of factors affecting cell proliferation, development, and death the regulation of mast cell number may be important. Melanocyte‐stimulating hormone inhibits most forms of inflammation by an unknown mechanism. In the present study, we have found that the α‐melanocyte‐stimulating hormone (α‐MSH) inhibited endotoxin‐mediated nuclear transcription factor κB (NF‐κB) activation in different cells correlated with the expression of α‐MSH receptors. We have also found for the first time that it induces cell death alone or in endotoxin‐stimulated mast cells. α‐MSH‐mediated apoptosis was not observed in NF‐κB overexpressed cells. The inhibitory effect of α‐MSH was mediated through generation of cAMP, as inhibitors of adenylate cyclase and of protein kinase A reversed its inhibitory effect. Overall, our results suggest that NF‐κB is the key molecule involved in α‐MSH‐mediated cell death and this may help to regulate mast cell‐mediated inflammation.