Krishna B. Sainis

Potent anti-inflammatory activity of ursolic acid, a triterpenoid antioxidant, is mediated through suppression of NF-κB, AP-1 and NF-AT.

Background Ursolic acid (UA), a pentacyclic triterpenoid carboxylic acid, is the major component of many plants including apples, basil, cranberries, peppermint, rosemary, oregano and prunes and has been reported to possess antioxidant and anti-tumor properties. These properties of UA have been attributed to its ability to suppress NF-κB (nuclear factor kappa B) activation. Since NF-κB, in co-ordination with NF-AT (nuclear factor of activated T cells) and AP-1(activator protein-1), is known to regulate inflammatory genes, we hypothesized that UA might exhibit potent anti-inflammatory effects. Methodology/Principal Findings The anti-inflammatory effects of UA were assessed in activated T cells, B cells and macrophages. Effects of UA on ERK, JNK, NF-κB, AP-1 and NF-AT were studied to elucidate its mechanism of action. In vivo efficacy of UA was studied using mouse model of graft-versus-host disease. UA inhibited activation, proliferation and cytokine secretion in T cells, B cells and macrophages. UA inhibited mitogen-induced up-regulation of activation markers and co-stimulatory molecules in T and B cells. It inhibited mitogen-induced phosphorylation of ERK and JNK and suppressed the activation of immunoregulatory transcription factors NF-κB, NF-AT and AP-1 in lymphocytes. Treatment of cells with UA prior to allogenic transplantation significantly delayed induction of acute graft-versus-host disease in mice and also significantly reduced the serum levels of pro-inflammatory cytokines IL-6 and IFN-γ. UA treatment inhibited T cell activation even when added post-mitogenic stimulation demonstrating its therapeutic utility as an anti-inflammatory agent. Conclusions/Significance The present study describes the detailed mechanism of anti-inflammatory activity of UA. Further, UA may find application in the treatment of inflammatory disorders.

An immunologically active arabinogalactan from Tinospora cordifolia

An arabinogalactan of mean M(r) 2.2 x 10(6) has been isolated from the dried stems of Tinospora cordifolia and examined by methylation analysis, partial hydrolysis and carboxyl reduction. Purified polysaccharide showed polyclonal mitogenic activity against B-cells, their proliferation did not require macrophages.

Plumbagin Inhibits Proliferative and Inflammatory Responses of T Cells Independent of ROS Generation But by Modulating Intracellular Thiols

Plumbagin inhibited activation, proliferation, cytokine production, and graft‐versus‐host disease in lymphocytes and inhibited growth of tumor cells by suppressing nuclear factor‐κB (NF‐κB). Plumbagin was also shown to induce reactive oxygen species (ROS) generation in tumor cells via an unknown mechanism. Present report describes a novel role of cellular redox in modulation of immune responses in normal lymphocytes by plumbagin. Plumbagin depleted glutathione (GSH) levels that led to increase in ROS generation. The decrease in GSH levels was due to direct reaction of plumbagin with GSH as evinced by mass spectrometric and HPLC analysis. Further, addition of plumbagin to cells resulted in decrease in free thiol groups on proteins and increase in glutathionylation of proteins. The suppression of mitogen‐induced T‐cell proliferation and cytokine (IL‐2/IL‐4/IL‐6/IFN‐γ) production by plumbagin was abrogated by thiol antioxidants but not by non‐thiol antioxidants confirming that thiols but not ROS play an important role in biological activity of plumbagin. Plumbagin also abrogated mitogen‐induced phosphorylation of ERK, IKK, and degradation of IκB‐α. However, it did not affect phosphorylation of P38, JNK, and AKT. Our results for the first time show that antiproliferative effects of plumbagin are mediated by modulation of cellular redox. These results provide a rationale for application of thiol‐depleting agents as anti‐inflammatory drugs. J. Cell. Biochem. 110: 1082–1093, 2010. Published 2010 Wiley‐Liss, Inc.

Free radical scavenging activity of vanillin and o-vanillin using 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical

Abstract Vanillin, a plant derived natural product, used as food flavoring agent and its positional isomer o-vanillin, have been tested for their ability to scavenge 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical using high performance liquid chromatography (HPLC). Trolox, a water-soluble analogue of vitamin E and a well-known antioxidant was used as a reference compound. The DPPH radical was monitored at 517 nm and its retention time was 8.6 min. From the decrease in optical density of DPPH radical in the presence of the test compounds, it was observed that o-vanillin was a more effective scavenger than vanillin. At equimolar concentrations (1 mM), vanillin and o-vanillin exhibited 22.9% and 66.4% DPPH radical scavenging activity, respectively. The kinetics of the reaction of vanillin and o-vanillin with DPPH spectrophotometry and their rate constants were estimated to be 1.7 ± 0.1 M-1s-1 and 10.1 ± 0.8 M-1s-1, respectively. In comparison, the rate constant for the reaction of trolox with DPPH was estimated to be 360.2 ± 10.1 M-1s-1. These scavenging reactions involve electron/H-atom transfer from antioxidant to DPPH. To confirm this, one electron reduction potentials of these compounds were estimated using cyclic voltammetry which showed that o-vanillin was more easily oxidized than vanillin. The reduction potential for o-vanillin was about 1.5 times that of trolox. These results demonstrate that o-vanillin is a more potent antioxidant than vanillin.

Low dose radiation induced immunomodulation: Effect on macrophages and CD8+ T cells

Purpose: The aim of the present investigation was to study the effect of fractionated whole body low dose ionizing radiation (LDR) on the functional responses of T lymphocytes, their subpopulations and macrophages. Materials and methods: C57BL/6 mice were exposed to 4 cGy from a 60Co source, at 0.31 cGy/min, at 24 h intervals for 5 days (total dose 20 cGy). Phagocytic activity was measured by flow cytometry using Bioparticles® and nitric oxide generation was estimated by spectrophotometry. Proliferation of lymphocytes in response to concanavalin A (con A) and alloantigens was measured by 3H thymidine incorporation. Expression of cell surface markers was assessed by flow cytometric analysis of antibody labeled cells. Target cell killing by cytotoxic T cells (CTL) generated against allogenic cells was assessed by flow cytometry using PKH26 labeled target cells. Cytokines were estimated by enzyme linked immunosorbent assay. Results: Exposure to LDR enhanced nitric oxide secretion and phagocytosis. The expression of early activation antigen, CD69, was enhanced in CD8+ T lymphocytes concomitant with enhanced proliferation in response to con A. In addition, mixed lymphocyte reaction (MLR) and CTL response were augmented and secretion of interferon gamma (IFN-γ) was suppressed following LDR exposure. Conclusions: LDR exposure enhanced the function of macrophages and responses of CD8+ T cells in C57BL/6 mice.

Pro-oxidants ameliorate radiation-induced apoptosis through activation of the calcium–ERK1/2–Nrf2 pathway

There are no reports describing the ability of pro-oxidants to protect against radiation-induced apoptosis. Activation of the redox-sensitive transcription factor Nrf2 by low levels of ROS is known to protect against oxidative stress-induced cell death. In this study, hydrogen peroxide, diethylmaleate, and 1,4-naphthoquinone (NQ) exhibited complete protection against radiation-induced cell death in lymphocytes as estimated by propidium iodide staining. Radioprotection by NQ was demonstrated by inhibition of caspase activation, decrease in cell size, DNA fragmentation, nuclear blebbing, and clonogenic assay. Interestingly, NQ offered protection to lymphocytes even when added to cells postirradiation. NQ increased intracellular ROS levels and decreased GSH levels. NQ activated Nrf2 and increased the expression of the cytoprotective gene heme oxygenase-1 in lymphocytes. NQ increased ERK phosphorylation, which is upstream of Nrf2, and this ERK activation was through increased intracellular calcium levels. Administration of NQ to mice offered protection against whole-body irradiation (WBI)-induced apoptosis in splenic lymphocytes and loss of viability of spleen and bone marrow cells. It restored WBI-mediated changes in hematological parameters and functional responses of lymphocytes. Importantly, NQ rescued mice against WBI-induced mortality. These results demonstrated that a pro-oxidant such as NQ can protect against radiation-induced apoptosis by activation of multiple prosurvival mechanisms including activation of the calcium-ERK1/2-Nrf2 pathway.

Modification of immune response by low dose ionizing radiation: role of apoptosis

Acute as well as fractionated whole body exposures to low doses (< 50 cGy) of ionizing radiation (LDR) have been reported to alter several immunological parameters in experimental animals. It is, however, not clear whether the augmentation of immune response by LDR will be observed for all responses and across genetic barriers. Since several proteins including p53 are synthesized following radiation exposure, the role of p53 and consequently that of activation induced apoptosis in the immunomodulation by LDR also remained to be evaluated. Experiments were, therefore, carried out in two different strains of inbred mice viz. C57BL/6 and BALB/c, exposed to fractionated LDR (4 cGy/day, 5 days/week, total dose 20 cGy) and subsequently stimulated with the polyclonal mitogen Con A or immunized with Mycobacterium vaccae or dinitrofluorobenzene (DNFB) for delayed type hypersensitivity (DTH) response. The proliferation of spleen cells in response to con A as measured by [3H]thymidine incorporation was significantly higher in 20 cGy-irradiated C57BL/6 mice as compared with that in the Con A-stimulated cells from sham-irradiated controls. The same response was suppressed by LDR in BALB/c mice. On the other hand, DTH to M. vaccae as well as DNFB was suppressed in C57BL/6 mice while DTH to M. vaccae was augmented in BALB/c mice and that to DNFB was not significantly affected following same dose. The augmentation of response to con A in C57BL/6 mice was prominent in CD4- (CD8+) T cells and was marked by the decrease in the proportion of cells expressing p53 as estimated by flow cytometry. Reduction in expression of p53 was accompanied by reduced apoptosis, as measured by TUNEL assay, in the Con A-stimulated spleen cells of irradiated C57BL/6 mice when compared with that in the sham-treated controls. The spleen cells of BALB/c mice showed exactly opposite profiles in this respect. Thus alteration in the immune response following LDR depends on antigen, type of response as well as the strain of mice used. Furthermore, the alterations in the expression of pro-apoptosis gene p53 and activation induced apoptosis in the effector or regulatory cells seem to contribute to the end result.

Molecular events in the activation of B cells and macrophages by a non-microbial TLR4 agonist, G1-4A from Tinospora cordifolia

G1-4A, a polysaccharide from an Indian medicinal plant Tinospora cordifolia, was recently shown to protect mice against septic shock by modulating the proinflammatory cytokines. G1-4A also activated B cells polyclonally. The present report describes in detail the molecular events associated with G1-4A-induced immunomodulation in vitro and in vivo. G1-4A treatment led to an increase in the CD69 expression in lymphocytes. G1-4A-induced proliferation of B cells was completely inhibited by PI3K inhibitor Ly294002, mTOR inhibitor rapamycin and NF-kappaB inhibitor plumbagin. Akt, ERK and JNK were activated by G1-4A which finally resulted in the activation of IKK, degradation of IkappaB-alpha and translocation of NF-kappaB to the nucleus. Administration of G1-4A to mice led to splenomegaly and an increase in the numbers of T cells, B cells and macrophages. This increase in spleen cellularity was due to in vivo proliferation of lymphocytes and upregulation of anti-apoptotic genes. Anti-TLR4-MD2 complex antibody inhibited G1-4A-induced B cell proliferation and degradation of IkappaB-alpha suggesting that TLR-4 was a receptor for G1-4A on B cells. Activation of RAW 264.7 macrophages by G1-4A was found to be dependent on ERK and NF-kappaB-mediated signals. The phagocytosis index in peritoneal exudate cells (PEC) isolated from G1-4A treated mice was significantly higher as compared to that in PEC from control mice. G1-4A administration also increased the number of CD11b(+) cells in the PEC without an increase in the total number of PEC. Thus the present understanding of the molecular mechanism of action of G1-4A, a novel non-microbial TLR4 agonist, will pave the way for its application as an immunomodulator and adjuvant.

Delayed activation of PKCδ and NFκB and higher radioprotection in splenic lymphocytes by copper (II)–Curcumin (1:1) complex as compared to curcumin

A mononuclear 1:1 copper complex of curcumin had been found to be superior to curcumin in its anti‐oxidant properties. This paper describes the radio‐protective effects of the complex in splenic lymphocytes from swiss mice. The complex was found to be very effective in protecting the cells against radiation‐induced suppression of glutathione peroxidase, catalase and superoxide dismutase (SOD) activities. Both curcumin and the complex protected radiation‐induced protein carbonylation and lipid peroxidation in lymphocytes with the complex showing better protection than curcumin. It also showed better overall protection by decreasing the radiation‐induced apoptosis. The kinetics of activation of PKCδ and NFκB after irradiation in presence or absence of these compounds was looked at to identify the molecular mechanism involved. The modulation of irradiation‐induced activation of PKCδ and NFκB by curcumin and the complex was found different at later time periods although the initial response was similar. The early responses could be mere stress responses and the activation of crucial signaling factors at later time periods may be the determinants of the fate of the cell. In this study this delayed effect was observed in case of complex but not in case of curcumin. The delayed effect of the complex along with the fact that it is a better free radical scavenger must be the reason for its better efficacy. The complex was also found to be less cytotoxic then curcumin at similar concentration. J. Cell. Biochem. 102: 1214–1224, 2007.

TGF-β1–ROS–ATM–CREB signaling axis in macrophage mediated migration of human breast cancer MCF7 cells

Macrophages in the tumor microenvironment play an important role in tumor cell survival. They influence the tumor cell to proliferate, invade into surrounding normal tissues and metastasize to local and distant sites. In this study, we evaluated the effect of conditioned medium from monocytes and macrophages on growth and migration of breast cancer cells. Macrophage conditioned medium (MϕCM) containing elevated levels of cytokines TNF-α, IL-1β and IL-6 had a differential effect on non-invasive (MCF7) and highly invasive (MDA-MB-231) breast cancer cell lines. MϕCM induced the secretion of TGF-β1 in MCF7 cells. This was associated with apoptosis in a fraction of cells and generation of reactive oxygen and nitrogen species (ROS and RNS) and DNA damage in the remaining cells. This, in turn, increased expression of cAMP response element binding protein (CREB) and vimentin resulting in migration of cells. These effects were inhibited by neutralization of TNF-α, IL-1β and IL-6, inhibition of ROS and RNS, DNA damage and siRNA mediated knockdown of ATM. In contrast, MDA-MB-231 cells which had higher basal levels of pCREB were not affected by MϕCM. In summary, we have found that pro-inflammatory cytokines secreted by macrophages induce TGF-β1 in tumor cells, which activate pCREB signaling, epithelial-mesenchymal-transition (EMT) responses and enhanced migration.