Amit Kunwar

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.

Differential response of DU145 and PC3 prostate cancer cells to ionizing radiation: Role of reactive oxygen species, GSH and Nrf2 in radiosensitivity

BACKGROUND Radioresistance is the major impediment in radiotherapy of many cancers including prostate cancer, necessitating the need to understand the factors contributing to radioresistance in tumor cells. In the present study, the role of cellular redox and redox sensitive transcription factor, Nrf2 in the radiosensitivity of prostate cancer cell lines PC3 and DU145, has been investigated. MATERIALS AND METHODS Differential radiosensitivity of PC3 and DU145 cells was assessed using clonogenic assay, flow cytometry, and comet assay. Their redox status was measured using DCFDA and DHR probes. Expression of Nrf2 and its dependent genes was measured by EMSA and real time PCR. Knockdown studies were done using shRNA transfection. RESULTS PC3 and DU145 cells differed significantly in their radiosensitivity as observed by clonogenic survival, apoptosis and neutral comet assays. Both basal and inducible levels of ROS were higher in PC3 cells than that of DU145 cells. DU145 cells showed higher level of basal GSH content and GSH/GSSG ratio than that of PC3 cells. Further, significant increase in both basal and induced levels of Nrf2 and its dependent genes was observed in DU145 cells. Knock-down experiments and pharmacological intervention studies revealed the involvement of Nrf2 in differential radio-resistance of these cells. CONCLUSION Cellular redox status and Nrf2 levels play a causal role in radio-resistance of prostate cancer cells. GENERAL SIGNIFICANCE The pivotal role Nrf2 has been shown in the radioresistance of tumor cells and this study will further help in exploiting this factor in radiosensitization of other tumor cell types.

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.

Melanin, a promising radioprotector: Mechanisms of actions in a mice model

The radioprotective effect of extracellular melanin, a naturally occurring pigment, isolated from the fungus Gliocephalotrichum simplex was examined in BALB/C mice, and the probable mechanism of action was established. At an effective dose of 50mg/kg body weight, melanin exhibited both prophylactic and mitigative activities, increasing the 30-day survival of mice by 100% and 60%, respectively, after exposure to radiation (7Gy, whole body irradiation (WBI)). The protective activity of melanin was primarily due to inhibition of radiation-induced hematopoietic damages as evidenced by improvement in spleen parameters such as index, total cellularity, endogenous colony forming units, and maintenance of circulatory white blood cells and platelet counts. Melanin also reversed the radiation-induced decrease in ERK phosphorylation in splenic tissue, which may be the key feature in its radioprotective action. Additionally, our results indicated that the sustained activation of AKT, JNK and P38 proteins in splenic tissue of melanin pre-treated group may also play a secondary role. This was also supported by the fact that melanin could prevent apoptosis in splenic tissue by decreasing BAX/Bcl-XL ratio, and increasing the expressions of the proliferation markers (PCNA and Cyclin D1), compared to the radiation control group. Melanin also reduced the oxidative stress in hepatic tissue and abrogated immune imbalance by reducing the production of pro-inflammatory cytokines (IL6 and TNFα). In conclusion, our results confirmed that fungal melanin is a very effective radioprotector against WBI and the probable mechanisms of radioprotection are due to modulation in pro-survival (ERK) signaling, prevention of oxidative stress and immunomodulation.

Differential antioxidant/pro-oxidant activity of dimethoxycurcumin, a synthetic analogue of curcumin

Abstract Dimethoxycurcumin (Dimc), a metabolically stable analogue of curcumin, is under investigation as an anti-tumour agent. Recently a number of studies have been performed on Dimc in this laboratory and also by others. In the present article, all these results have been summarized and wherever possible compared with those of curcumin. Rate constant for reactions of Dimc with superoxide radicals was comparable with that of curcumin, while its reaction with peroxyl radicals was much slower. These results were further supported by the observations on the scavenging of basal ROS levels in lymphocytes and evaluation of antioxidant activities. In line with the earlier reports on curcumin, Dimc was a pro-oxidant and generated ROS in tumour cells. Both curcumin and Dimc were non-toxic to lymphocytes, while exhibiting comparable cytotoxicity to tumour cells. Additionally, these compounds showed higher uptake in tumour cells than in normal lymphocytes. Fluorescence studies on both the compounds revealed their binding to genomic DNA, similar sub-cellular distribution and nuclear localization. All these studies suggested that methylation of the phenolic-OH group in curcumin, although decreasing the antioxidant activity marginally, showed comparable pro-oxidant activity, making it a promising anti-tumour agent.

Interaction of a Curcumin Analogue Dimethoxycurcumin with DNA

Dimethoxycurcumin (Dimc), a synthetic analogue of curcumin, that has been reported to exhibit better in vivo stability and anti‐tumour activity, was investigated for its interaction with DNA, employing spectroscopic methods based on absorption, fluorescence, circular dichroism (CD), ethidium bromide (EtBr) competitive binding assay, 4′‐6‐diamidino‐2‐phenylindole (DAPI) displacement assay and fluorescence resonance energy transfer (FRET) assay. The mean binding constant for its interaction with calf thymus DNA (ct‐DNA) was estimated to be 4.4 ± 0.8 × 104 m−1. The studies using CD revealed that Dimc did not cause unwinding of the ct‐DNA helix or induce major conformational changes. The EtBr and DAPI assays indicated that Dimc is not an intercalator but a minor groove binder. FRET assay also confirmed that Dimc interacts with DNA strands. Furthermore, viscosity measurements of ct‐DNA solutions in the presence of Dimc supported these spectroscopic observations. Addition of Dimc to MCF‐7 cells showed nuclear localization as visualized by confocal microscopy. In conclusion, the present studies addressed the mode of interaction of Dimc with biomolecules, which may have implications in developing Dimc as a DNA‐targeted drug.

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.

Anti-unlcer and antimicrobial activities of sodium selenite against Helicobacter pylori: in vitro and in vivo evaluation.

Abstract We examined sodium selenite, an inorganic selenium supplement, for its ulcer healing properties and antimicrobial activity against gastric pathogen Helicobacter pylori. Minimum inhibitory concentrations (MIC) were determined using disk diffusion and flow cytometry. The studies were performed over a concentration range of 1 μg/ml to 500 μg/ml sodium selenite. Mild activity was seen at 10 μg/ml and 50 μg/ml, a moderate response at 100 μg/ml and strong response at 500 μg/ml with a MIC value of 10 μg/ml. The compound was found to be active at low pH without any resistance after 10 passages. Flow cytometry data showed a characteristic shift of the viability peak in comparison with the control, thereby confirming the bactericidal effects of sodium selenite. Sodium selenite administered in Wistar rats, pre-ulcerated with naproxen and infected with H. pylori, showed ulcer healing and anti-H. pylori activity at a concentration range of 10–50 μg/rat; however concentrations of 100 μg/rat and 500 μg/rat were found to be toxic in the in vivo studies. In conclusion, sodium selenite shows both ulcer healing and anti-H. pylori activity at a low concentration (10 μg/rat) without toxicity.

Antibacterial and ulcer healing effects of organoselenium compounds in naproxen induced and Helicobacter pylori infected wistar rat model.

Aim of the present study was to evaluate in vitro toxicity and in vivo antibacterial, anti-inflammatory, antiulcer, and antioxidant activities of two organoselenium compounds, selenocystine (SeCys) and ebselen (Ebs). The study was conducted in experimentally induced ulcers in rodent model infected with Helicobacter pylori (H. pylori). In vitro toxicological studies on normal splenic lymphocytes revealed that SeCys and Ebs were non-toxic to the cells even at 100 μM concentration. Antibacterial activity was observed at 500 μg/mL concentration of either of the compounds against H. pylori. In vivo studies after treatment with SeCys and Ebs (500 μg/kg/day) resulted in significant reduction in ROS production and inhibition of lipid peroxidation in gastric tissue. The antioxidant and anti-inflammatory activities of both the compounds were also confirmed by their ability to lower GSH reduction, to induce the expression of antioxidant genes such as GPx-4, and MnSOD and to suppress inflammatory genes namely COX-2, TNF-α and TGF-β. In addition, the immunomodulatory activity of both the compounds was evident by enhance of the CD4 levels and maintenance of the IgG, IL-6 and IL-10 levels. Persistent treatment (500 μg/kg, for 28 days) with both the compounds showed considerable (p<0.05) ulcer healing property supporting its role in gastro protection. In conclusion, the results of our study suggest that both SeCys and Ebs possess broad spectrum of activities without any potential toxicity.

Correlating the GPx Activity of Selenocystine Derivatives with One-Electron Redox Reactions

With an aim to develop water soluble, less toxic glutathione peroxidase (GPx) mimics, three selenocystine (SeCys) derivatives, viz., selenocystamine (SeA), diselenodipropionic acid (SeP), and methyl ester of diselenodipropionic acid (MeSeP) have been synthesized and examined for GPx activity along with SeCys. The GPx activity of the compounds was found to be in the order SeCys ≅ SeA > MeSeP > SeP. The relative affinity of these GPx mimics towards the substrates thiol and hydroperoxide were determined by Lineweaver-Burk (L-B) plots. Since the enzyme activity involves several steps of reduction and oxidation reactions, attempts have been made to understand the role of such processes in deciding the efficacy of diselenides as GPx mimics. For this, one-electron redox chemistry of these compounds was studied in aqueous solutions at pH 7 using nanosecond pulse radiolysis technique. From these studies, it was concluded that SeCys and SeA, which can undergo easy one-electron reduction, exhibit high GPx activity.