Himanshi Narang

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.

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.

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)

Activation of DNA damage response signaling in lung adenocarcinoma A549 cells following oxygen beam irradiation.

Oxygen beams 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- and oxygen ion-irradiation. Activation of various signaling molecules was looked in A549 lung adenocarcinoma cells irradiated with 2Gy oxygen, 2Gy or 6Gy γ-radiation. Oxygen beam was found to be three times more cytotoxic than γ-radiation. By 4h there was efficient repair of DNA in A549 cells exposed to 2Gy or 6Gy gamma radiation but not in cells exposed to 2Gy oxygen beam as determined by γ-H2AX counting. Number of ATM foci was found to be significantly higher in cells exposed to 2Gy oxygen beam. Percentage of cells showing ATR foci were more with gamma however number of foci per cell were more in case of oxygen beam. Oxygen beam irradiated cells showed phosphorylation of Chk1, Chk2 and p53. Many apoptotic nuclei were seen by DAPI staining in cells exposed to oxygen beam. The noteworthy finding of this study is the activation of the sensor proteins, ATM and ATR by oxygen irradiation and the significant activation of Chk1, Chk2 and p53 only in the oxygen beam irradiated cells.

Effect of nitric oxide donor and gamma irradiation on modifications of ERK and JNK in murine peritoneal macrophages.

Mitogen activated protein kinases (MAPKs) play an important role in activation, differentiation and proliferation of macrophages. Macrophages, upon activation, produce large amounts of nitric oxide that inhibit the growth of variety of microorganisms and tumor cells. This nitric oxide which is known to interfere with tyrosine phosphorylation may result in changes in the pattern of activation of MAPKs. In a previous study we have found that tyrosine phosphorylation of MAPKs was completely abolished in the presence of nitric oxide donor and radiation but this did not affect the function of macrophages. In this study the other post translational modifications namely nitration and ubiquitination of JNK and ERK have been looked at. Both ERK and JNK were found to be nitrated. However, there was no increase in ubiquitination of ERK and JNK, indicating that ubiquitination, in this case was not a natural consequence of nitration and may serve in signaling. Additionally, when the nitration was extensive, phosphorylation was also inhibited. The activation of substrates of ERK and JNK were looked at to determine the consequences of such modifications. Inhibition of phosphorylation and extensive nitration of JNK did not prevent activation of its substrate, c-jun. This study indicates that ERK and JNK may be under regulation by different type of modifications in macrophages.

Ionizing radiation induced signaling of DNA damage response molecules in RAW 264.7 and CD4+ T cells

Ionizing radiation (IR) treatment results in activation of several DNA damage response molecules, such as ataxia telangiectasia, mutated (ATM), and DNA-dependent protein kinase (DNAPK) in mammals that are increasingly recognized for their potential roles in the sensing of DNA damage and initiating the subsequent protein kinase cascade. In vitro evidence indicates that both ATM and DNA-PK are responsible for efficient repair of DNA double strand breaks in response to IR exposure. To unravel the role of ATM and DNA-PK, we studied the mRNA and protein levels of ATM, DNA-PK and their downstream substrates in two different cell types after irradiation viz. macrophage like RAW264.7 cells and CD4+ T cells isolated from mice spleen. Our results show that despite significant increase in phosphorylation of ATM, its mRNA levels continue to remain low after IR exposure in both the cell types. Conversely, the mRNA expression of DNAPK shows a considerable increase immediately after IR exposure. Moreover, no increase in ATM mRNA levels is seen in DNAPK deficit RAW264.7 cells treated with DNAPK siRNA, indicating that ATM does not undergo any change at its transcriptional levels in response to IR treatment. However, in a similar study in CD4+ T cells, inhibition of DNAPK by siRNA, shows a considerable increase in ATM after IR exposure. Collectively, these results suggest a discrepancy in the role of the ATM and DNA-PK pathways in the cellular response to IR at the mRNA and protein levels in two different cell types.

Differential response of three cell types to dual stress of nitric oxide and radiation.

The perception of toxicity to nitric oxide (NO) and irradiation (IR) by three different cell types has been studied. The three cell types are the macrophage like RAW264.7 cells, EL4 lymphoma cells, and splenocytes, which represent the different components of a tumor. These three cell types respond differently to NO donors (SNP and SNAP) and radiation treatment. The macrophages were found to be most radio-resistant and insensitive to NO donors. The innate resistance of the macrophages was not due to its antioxidant defense system since there was no significant activation of the enzymes (superoxide dismutases, catalase, and glutathione peroxidase) in RAW264.7 cells after NO donor and irradiation. But the cell cycle arrest of the three cell types was different from each other. The EL4 cells were found to arrest in the G2/M phase while the macrophages were found arrested in the G1 phase of the cell cycle. Such specific killing of the tumor cell in response to NO donor while sparing the macrophages can be of immense importance to radiotherapy.

Differential activation of signalling factors following low and high dose of gamma radiation in vivo

The activation of signalling molecules that contribute to the development of radioresistance following radiotherapy was investigated following irradiation with very low doses (0.1 and 0.5 Gy) and high dose (1 and 3 Gy) of γ radiation. Expression of protein kinase C (PKC) was isoform specific. There was a marked difference in the response of ERK 44/42, which is known to confer radioresistance on the tumour cell. The expression at low dose was persistent till 24 hrs with a reappearance at 7 days, while the response at high dose (3 Gy) was transient, an activation at 4 hrs with a return to normal values at the subsequent time periods. These studies indicate that data obtained at high doses should not be extrapolated to low doses. The response of each signalling factor is different at different doses. Since radioadaptation and the development of radioresistance is a low dose phenomenon, ERK 44/42 seems to be the most relevant among the signalling factors.