Malini Krishna

Curcumin mediates time and concentration dependent regulation of redox homeostasis leading to cytotoxicity in macrophage cells.

The present study was designed to test a hypothesis that curcumin may be modulating oxidative stress parameters including reactive oxygen species, non-protein thiols and expression of antioxidant genes in a concentration and time dependent manner in exhibiting cytotoxic effects in macrophage cell line RAW 264.7. The results have shown that curcumin elevated the reactive oxygen species levels accompanied by a decrease in levels of intracellular non-protein thiols at 2 h after its addition to cells. However, the levels of reactive oxygen species decreased and non-protein thiols content increased at 18 h after its addition. Whereas the expression of glutathione peroxidase (GPx), catalase, Cu,Zn-superoxide dismutase (Cu,Zn-SOD) and heme oxygenase-1 (HO-1) increased with curcumin concentration and also with increase in time of incubation, the expression of Mn- superoxide dismutase (Mn-SOD) showed concentration dependant repression upon treatment with curcumin. The cell viability was significantly reduced at high concentration (25 microM) of curcumin treatment but not at low concentration (5 microM). Curcumin at 5 microM scavenged gamma-radiation induced reactive oxygen species and inhibited cell death. On the contrary, at 25 microM, curcumin increased radiation induced reactive oxygen species production and augmented cell death. Interestingly pretreatment with reducing agents glutathione (GSH) or N-acetyl-cysteine (NAC), modified the curcumin mediated redox changes and cell death differentially, due to the inhibition of cellular uptake of curcumin by GSH but not by NAC. The important finding of the study is that the concentration and time dependent dual effect of curcumin may be attributed to changes in oxidative stress and antioxidant gene expression levels leading to inhibition or promotion of cell death.

Modulation of radiation-induced protein kinase C activity by phenolics.

Natural phenolic compounds were tested in vitro for their effect on the activity of protein kinase C (PKC) isolated from the liver cytosol and the particulate fraction of unirradiated mice and mice irradiated at 5 Gy. Following irradiation, the PKC activity was found to be increased in both cytosolic and particulate fractions. Curcumin, ellagic acid and quercetin were effective in inhibiting radiation-induced PKC activity. Curcumin and ellagic acid were found to be more inhibitory towards radiation-induced PKC activity, while quercetin was the least effective. Curcumin was found to inhibit the activated cytosolic and particulate PKC at very low concentrations. Activation of PKC is one of the means of conferring radioresistance on a tumour cell. Suppression of PKC activity by phenolics may be one of the means of preventing the development of radioresistance following radiotherapy.

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.

Role of iNOS in bystander signaling between macrophages and lymphoma cells.

PURPOSE The present report describes the bystander effects of radiation between similar and dissimilar cells and the role of iNOS in such communication. MATERIALS AND METHODS EL-4 and RAW 264.7 cells were exposed to 5 Gy gamma-irradiation. The medium from irradiated cells was transferred to unirradiated cells. RESULTS Irradiated EL-4 cells as well as those cultured in the presence of medium from gamma-irradiated EL-4 cells showed an upregulation of NF-kappaB, iNOS, p53, and p21/waf1 genes. The directly irradiated and the bystander EL-4 cells showed an increase in DNA damage, apoptosis, and NO production. Bystander signaling was also found to exist between RAW 264.7 (macrophage) and EL-4 (lymphoma) cells. Unstimulated or irradiated RAW 264.7 cells did not induce bystander effect in unirradiated EL-4 cells, but LPS stimulated and irradiated RAW 264.7 cells induced an upregulation of NF-kappaB and iNOS genes and increased the DNA damage in bystander EL-4 cells. Treatment of EL-4 or RAW 264.7 cells with L-NAME significantly reduced the induction of gene expression and DNA damage in the bystander EL-4 cells, whereas treatment with cPTIO only partially reduced the induction of gene expression and DNA damage in the bystander EL-4 cells. CONCLUSIONS It was concluded that active iNOS in the irradiated cells was essential for bystander response.

Radiation‐induced bystander effect: Activation of signaling molecules in K562 erythroleukemia cells

Gap junction independent signaling mechanism was investigated using K562 human erythroleukemia cells. They were exposed to 2, 5, or 10 Gy of 60Co γ irradiation, the medium isolated 20 min post‐irradiation and added to fresh cells. Evidence of radiation‐induced bystander effect was observed wherein there was activation of p21, nuclear factor‐κB (NF‐κB), Bax, Bcl‐2 and cleavage of poly(ADP‐ribose) polymerase in bystander cells. The study implicates the involvement of signaling molecules released into the medium and factors like stable free radicals that are generated in the surrounding medium. The response elicited appears to be primarily via NF‐κB and p21 activation. J. Cell. Biochem. 100: 991–997, 2007.

DNA damage response signaling in lung adenocarcinoma A549 cells following gamma and carbon beam irradiation.

Carbon beams (5.16MeV/u, LET=290keV/μm) are high linear energy transfer (LET) radiation characterized by higher relative biological effectiveness than low LET radiation. The aim of the current study was to determine the signaling differences between γ-rays and carbon ion-irradiation. A549 cells were irradiated with 1Gy carbon or γ-rays. Carbon beam was found to be three times more cytotoxic than γ-rays despite the fact that the numbers of γ-H2AX foci were same. Percentage of cells showing ATM/ATR foci were more with γ-rays however number of foci per cell were more in case of carbon irradiation. Large BRCA1 foci were found in all carbon irradiated cells unlike γ-rays irradiated cells and prosurvival ERK pathway was activated after γ-rays irradiation but not carbon. The noteworthy finding of this study is the early phase apoptosis induction by carbon ions. In the present study in A549 lung adenocarcinoma, authors conclude that despite activation of same repair molecules such as ATM and BRCA1, differences in low and high LET damage responses might be due to their distinct macromolecular complexes rather than their individual activation and the activation of cytoplasmic pathways such as ERK, whether it applies to all the cell lines need to be further explored.

Effect of Nitric Oxide Donor and Gamma Irradiation on MAPK Signaling in Murine Peritoneal Macrophages

Irradiation (IR) of cells is known to activate enzymes of mitogen activated protein kinase (MAPK) family. These are known to be involved in cellular response to stress and are determinants of cell death or survival. When radiotherapy is delivered to malignant cells, macrophages, being radioresistant, survive, get activated, and produce large amounts of nitric oxide. As a result of activation they recognize and phagocytose tumor and normal cell apoptotic bodies leading to tumor regression. In this study, the MAPK signaling in peritoneal macrophages was investigated which plays an important role in its various functions, in an environment which is predominantly nitric oxide, as is after IR. The behavior of macrophages in such an environment was also looked at. The three MAPK (ERK1/2, p38, and JNK) respond differently to Sodium nitroprusside (SNP) alone or IR alone. All the three were activated following IR but only JNK was activated following SNP treatment. Surprisingly, when both the stresses were given simultaneously or one after the other, this differential response was lost and there was a complete inhibition of phosphorylation of all the three MAPKs, irrespective of the order of the two insults (IR and SNP). The noteworthy observation was that despite the complete inhibition of MAPK signaling there was no effect on either the viability or the phagocytic efficiency of peritoneal macrophages. J. Cell. Biochem. 103: 576–587, 2008.

Low Energy Proton Beam Induces Efficient Cell Killing in A549 Lung Adenocarcinoma Cells

ABSTRACT The aim of the current study was to determine the signaling differences between γ- and proton beam-irradiations. A549 lung adenocarcinoma cells were irradiated with 2 Gy proton beam or γ-radiation. Proton beam was found to be more cytotoxic than γ-radiation. Proton beam-irradiated cells showed phosphorylation of H2AX, ATM, Chk2, and p53. The mechanism of excessive cell killing in proton beam-irradiated cells was found to be upregulation of Bax and downregulation of Bcl-2. The noteworthy finding of this study is the biphasic activation of the sensor proteins, ATM, and DNA-PK and no activation of ATR by proton irradiation.

Inhibition of radiation induced nitration by curcumin and nicotinamide in mouse macrophages.

Nitric oxide plays an important role in inflammation and carcinogenesis and has now been implicated as an important signaling molecule under normal physiological conditions also. Increased nitric oxide (NO) results in increased nitration of proteins at tyrosine, which can cause protein dysfunction or alterations in signal transduction pathways. Irradiation of Lipopolysaccharide (LPS) activated mouse peritoneal macrophages was found to increase NO production, inducible nitric oxide synthase (iNOS) expression and nitration of proteins. The increase in iNOS expression was very less when compared to increase in NO production, indicating the possibility of post-translational activation of iNOS by LPS and ionising radiation. The addition of curcumin, nicotinamide and Jun N-terminal kinase (JNK) inhibitor, SP600125, reduced the levels of NO, iNOS expression and nitration of proteins in macrophages. Closer scrutiny of the inhibition pattern of these modulators revealed that although the JNK inhibitor did not result in significant decrease in iNOS expression it led to a significant decrease in NO production, implying the possible involvement of JNK in the regulation of iNOS activity. Curcumin and JNK inhibitor directly inhibited the nitration of proteins and JNK inhibitor and curcumin, when added together, did not show synergistic effect. (Mol Cell Biochem 276: 7–13, 2005)

Alteration in the expression of signaling parameters following carbon ion irradiation

Ionizing radiation induces DNA damage, which generates a complex array of genotoxic responses. These responses depend on the type of DNA damage, which in turn can lead to unique cellular responses. High LET radiation results in clustered damages. This evokes specific signaling responses, which can be cytotoxic or cytoprotective in nature. In the present study the effect of carbon ion irradiation on p44/42 MAPK and NF-κB, which are essentially survival factors, have been studied. Moreover, the effect of inhibition of DNA-PK, which is an important component of DNA repair mechanism, with wortmanin on these signaling factors has been studied. The expression of p44/42 MAPK was different at 0.1 Gy and 1 Gy and wortmanin was found to inhibit its expression. NF-κB expression was higher at 1 Gy than at 0.1 Gy and its expression is unaffected by inhibition of DNA-PK. The notable findings of this study are that the responses to high and low dose of high LET radiation are essentially different and the 6 h time point post irradiation is crucial in deciding the response and needs further investigation. (Mol Cell Biochem 276: 169–173, 2005)