Ruchi Pandey

Quantitative cellular uptake, localization and cytotoxicity of curcumin in normal and tumor cells.

Using absorption and fluorescence spectroscopic methods, quantitative cellular uptake of curcumin, an antioxidant and anti-tumor agent from Curcuma longa, was calculated in two types of normal cells: spleen lymphocytes, and NIH3T3 and two tumor cell lines: EL4 and MCF7. Both the uptake and fluorescence intensity of curcumin were significantly higher in tumor cells compared to the normal cells. A linear dependency on the uptake was observed with treatment concentration of curcumin. Using laser confocal microscopy, intracellular localization of curcumin was monitored and the results indicated that curcumin is located both in the cell membrane and the nucleus. Sub-cellular fractionation of curcumin-loaded MCF7 cells supported the differential distribution of curcumin in membrane, cytoplasm and nuclear compartments of cell with maximum localization in the membrane. Cytotoxicity studies in different cell lines indicated that the toxicity of curcumin increased with increasing uptake.

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.

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.

Radiation-induced bystander effects and adaptive response in murine lymphocytes.

Purpose: To study the bystander effects of γ-radiation in murine lymphocytes using irradiated conditioned medium (ICM) generated from irradiated lymphocytes. Methods: Proliferation response of unirradiated lymphocytes to mitogen concanavalin A (con A) in presence of ICM, collected from γ-irradiated lymphocytes (60Co source; 0.35 Gy/min; 0.1 – 1 Gy), was studied by 3H-thymidine incorporation and also by dye dilution using carboxyfluorescein succinimidyl ester (CFSE). Expression of proliferation markers, interleukin 2 receptor α chain (CD25) and cyclin D in ICM treated lymphocytes was analyzed by labeling with specific antibodies. Intracellular reactive oxygen species (ROS) and apoptosis were estimated by flow cytometry using dichlorodihydrofluorescein diacetate (H2DCFDA) and propidium iodide, respectively. Nitric oxide (NO) was measured using Griess reagent. Results: Proliferation response to con A in unirradiated lymphocytes was enhanced in the presence of ICM with maximum enhancement observed in the presence of 0.5 Gy ICM. Augmentation of proliferation in the presence of ICM was accompanied by an increase in CD25 and cyclin D expression, enhanced ROS and NO generation. ICM pretreated lymphocytes showed adaptive response to radiation which was not abrogated by wortmannin, a phosphatidyl inositol 3-kinase (PI3K) inhibitor. Conclusion: Soluble factors released from irradiated lymphocytes initiate a signaling cascade in unirradiated lymphocytes resulting in increased response to mitogen and radioresistance which may have an important role in radiation-induced immunomodulation.

Purification and characterization of a lectin from wild sunflower (Helianthus tuberosus L.) tubers

A lectin (HTTL) was isolated from Helianthus tuberosus L. (wild sunflower) tubers using ion-exchange chromatography, gel filtration, and affinity chromatography. The lectin agglutinated both untreated and trypsin-treated rabbit erythrocytes and did not agglutinate human blood cells of groups A, B, and O. The gel filtration showed the native molecular mass of 72 kDa and subunit molecular masses of 17 and 18.5 kDa on 12% SDS-PAGE. The lectin activity was inhibited by D-mannose. The tetrameric protein revealed a unique characteristic by forming a broad zone of protein in native PAGE at pH 8.3, which dissociated into seven subunits of varying e/m ratios on acid gel at pH 4.3. These seven bands revealed two polypeptide species of molecular masses 17 and 18.5 kDa on 12% SDS-PAGE, as in the case of the native protein. The result indicated that of the seven subunits, three were homotetramers of 17 kDa, one was a homotetramer of 18.5 kDa, and three were heterotetramers of 17 and 18.5 kDa. The lectin was thermostable with broad pH optima (pH 4-8) and had no requirement for divalent metal cations for its activity. The amino acid composition showed that the lectin contained higher amounts of glycine, alanine, and lysine, but no methionine. The sugar content was estimated to be 5.3% mannose equivalent. The HTTL was mitogenic to mouse spleen (total) cells at 25 microg/ml concentration. The lectin showed characteristics different from those of the earlier reported H. tuberosus tuber lectins and hence opens up a new avenue to investigate the structure-function relationship of lectin in Helianthus species.

Role of glutathione in augmenting the anticancer activity of pyrroloquinoline quinone (PQQ)

Abstract Pyrroloquinoline quinone (PQQ), a bacterial redox co-factor and antioxidant, is highly reactive with nucleophilic compounds present in biological fluids. PQQ induced apoptosis in human promonocytic leukemia U937 cells and this was accompanied by depletion of the major cellular antioxidant glutathione and increase in intracellular reactive oxygen species (ROS). Treatment with glutathione (GSH) or N-acetyl-L-cysteine (NAC) did not spare PQQ toxicity but resulted in a 2–5-fold increase in PQQ-induced apoptosis in U937 cells. Cellular GSH levels increased following treatment by NAC alone but were severely depleted by co-treatment with NAC and PQQ. This was accompanied by an increase in intracellular ROS. Alternatively, depletion of glutathione also resulted in increased PQQ cytotoxicity. However, the cells underwent necrosis as evidenced by dual labeling with annexin V and propidium iodide. PQQ-induced cytotoxicity is thus critically regulated by the cellular redox status. An increase in GSH can augment apoptosis and its depletion can switch the mode of cell death to necrosis in the presence of PQQ. Our data suggest that modulation of intracellular GSH can be used as an effective strategy to potentiate cytotoxicity of quinones like PQQ.

Tribulus terrestris. Fruit Extract Protects Against Oxidative Stress-Induced Apoptosis

Abstract Historically, the fruit of Tribulus terrestris. Linn. (Zygophyllaceae) has been used in India and China as a constituent of rejuvenation tonics. It is also used in traditional Indian medicine in the therapy of a variety of health conditions affecting liver, kidney, and cardiovascular and immune systems. Oxidative stress has been implicated in some of these disease conditions. Therefore, we have investigated the antioxidant activity of the aqueous extract of Tribulus terrestris. fruit (TTE) in spleen cells. Generation of intracellular reactive oxygen species (ROS) in response to γ-radiation and 2,2′-azobis.(2-propionimidinedihydrochloride) (AAPH) was measured by flow cytometry using dichlorodihydrofluorescein diacetate (H2DCFDA). TTE scavenged ROS induced by γ-radiation as well as AAPH in a concentration-dependent manner. TTE also showed protection against oxidative stress–induced apoptosis. In addition, TTE exhibited mitogenic activity in the spleen cells. We conclude that TTE attenuates oxidative stress and protects cells from lethal oxidant damage, and thus its therapeutic potential in treating many ailments may relate to its antioxidant properties.

A novel N-alkylated prodigiosin analogue induced death in tumour cell through apoptosis or necrosis depending upon the cell type

PurposeTo investigate the mechanism of cell death induced by the N-alkylated prodigiosin analogue, 2,2′-[3-methoxy-1′amyl-5′-methyl-4-(1′′-pyrryl)] dipyrryl-methene (MAMPDM) in S-180 and EL-4 tumour cell lines.MethodsEffect of MAMPDM on cell viability was assessed by MTT dye conversion. Induction of apoptosis was assessed by monitoring caspase 3 activity using a fluorogenic substrate, fragmentation of DNA by gel electrophoresis and sub-diploid DNA containing cells by flowcytometry. Necrosis was estimated by flowcytometric analysis of the uptake of propidium iodide.ResultsMAMPDM inhibited the proliferation of murine fibrosarcoma, S-180 cells and induced cell death. Investigations into the mechanism of cell death by MAMPDM in S-180 cells showed absence of hallmarks of apoptotic cell death such as activation of caspase 3, DNA fragmentation and presence of cells with sub-diploid DNA content. However, there was a rapid loss of membrane integrity as assessed by uptake of propidium iodide, which is characteristic of necrosis. In contrast to induction of necrosis in S-180 cells, MAMPDM induced apoptotic cell death in EL-4 cells as evident by activation of caspase 3, fragmentation of DNA and sub-diploid DNA containing cells.ConclusionsMAMPDM could induce cell death by either apoptosis or necrosis depending upon the cell type. This would be of advantage in elimination of tumor cells defective in apoptotic pathway and therefore, refractory to the conventional therapies.