Badri N. Pandey

Induction heating studies of Fe3O4 magnetic nanoparticles capped with oleic acid and polyethylene glycol for hyperthermia

Fe3O4 magnetic nanoparticles (Fe3O4-MN) capped with either oleic acid (Fe3O4-OA-MN) or polyethylene glycol (Fe3O4-PEG-MN) were prepared by a co-precipitation method. From X-ray diffraction studies, the average crystallite sizes of Fe3O4-MN, Fe3O4-OA-MN and Fe3O4-PEG-MN were found to be 12, 6 and 8 nm, respectively. A reduction in the agglomeration of particles was observed when the magnetic nanoparticles (MN) were capped with oleic acid (OA) and polyethylene glycol (PEG), as confirmed by a transmission electron microscopy study. Magnetization of these MN was almost zero at room temperature in the absence of an applied magnetic field, indicating their superparamagnetic behavior. Magnetization was lower and the superparamagnetic fraction was higher for Fe3O4-OA-MN and Fe3O4-PEG-MN compared to Fe3O4-MN studied using a Mossbauer spectrometer. Compared to the control, an increased killing (35%) was observed in human breast cancer cells (MCF7) after Fe3O4-OA-MN treatment, which was further enhanced (65%) under induction heating conditions. However, MCF7 cells treated with Fe3O4-MN or Fe3O4-PEG-MN showed 5–10% killing after induction heating. These results showed the characterization of MN with different lipophilicity and suggests their suitability for hyperthermia applications in cancer therapy.

Synthesis of oleic acid functionalized Fe3O4 magnetic nanoparticles and studying their interaction with tumor cells for potential hyperthermia applications.

In the present study, oleic acid (OA) functionalized Fe3O4 magnetic nanoparticles (MN) were synthesized following modified wet method of MN synthesis. The optimum amount of OA required for capping of MN and the amount of bound and unbound/free OA was determined by thermogravimetric analysis (TGA). Further, we have studied the effect of water molecules, associated with MN, on the variation in their induction heating ability under alternating current (AC) magnetic field conditions. We have employed a new approach to achieve dispersion of OA functionalized MN (MN-OA) in aqueous medium using sodium carbonate, which improves their biological applicability. Interactions amongst MN, OA and sodium carbonate were studied by Fourier transform infrared spectroscopy (FT-IR). Intracellular localization of MN-OA was studied in mouse fibrosarcoma cells (WEHI-164) by prussian blue staining and confocal laser scanning microscopy (CLSM) using nile blue A as a fluorescent probe. Results showed MN-OA to be interacting mainly with the cell membrane. Their hyperthermic killing ability was evaluated in WEHI-164 cells by trypan blue method. Cells treated with MN-OA in combination with induction heating showed decreased viability as compared to respective induction heating controls. These results were supported by altered cellular morphology after treatment of MN-OA in combination with induction heating. Further, the magnitude of apoptosis was found to be ~5 folds higher in cells treated with MN-OA in combination with induction heating as compared to untreated control. These results suggest the efficacy of MN-OA in killing of tumor cells by cellular hyperthermia.

Cytokine profile of conditioned medium from human tumor cell lines after acute and fractionated doses of gamma radiation and its effect on survival of bystander tumor cells.

Cytokines are known to play pivotal roles in cancer initiation, progression and pathogenesis. Accumulating evidences suggest differences in basal and stress-induced cytokine profiles of cancers with diverse origin. However, a comprehensive investigation characterising the cytokine profile of various tumor types after acute and fractionated doses of gamma-irradiation, and its effect on survival of bystander cells is not well known in literature. In the present study, we have evaluated the cytokine secretion profile of human tumor cell lines (HT1080, U373MG, HT29, A549 and MCF-7) either before (basal) or after acute (2, 6 Gy) and fractionated doses (3×2 Gy) of gamma-irradiation in culture medium obtained from these cells by multiplex bead array/ELISA. Moreover, clonogenic assays were performed to evaluate the effect of conditioned medium (CM) on the survival and growth of respective cells. Based on the screening of 28 analytes, our results showed that the basal profiles of these cell lines varied considerably in terms of the number and magnitude of secreted factors, which was minimum in MCF-7. Interestingly, TNF-α, IL-1β, PDGF-AA, TGF-β1, fractalkine, IL-8, VEGF and GCSF were found in CM of all the cell lines. However, secretion of certain cytokines was cell line-specific. Moreover, CM caused increase in clonogenic survival of respective tumor cells (in the order HT1080>U373MG>HT29>A549>MCF-7), which was correlated with the levels of IL-1β, IL-6, IL-8, GMCSF and VEGF in their CM. After irradiation, the levels of most of the cytokines increased markedly in a dose dependent manner. The fold change in cytokine levels was lower in irradiated conditioned medium (ICM) of tumor cells collected after fractionated than respective acute dose, except in MCF-7. Interestingly, amongst these cell lines, the radiation-induced fold increase in cytokine levels was maximum in ICM of A549 cells. Moreover, bystander A549 cells treated with respective ICM showed dose dependent decrease in clonogenic survival. In conclusion, present study revealed the similarities and subtle differences in basal and radiation-induced cytokine profile of different tumor cell lines, and its influence on growth and survival of respective bystander cells. These findings may add a new dimension to our current understanding about role of cytokines in cancer biology.

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.

Autocrine IL-8 and VEGF mediate epithelial-mesenchymal transition and invasiveness via p38/JNK-ATF-2 signalling in A549 lung cancer cells.

Soluble factors in tumour microenvironment play a major role in modulating the metastatic potential of cancer cells. Herein, we investigated the effect of autocrine cytokines and growth factors in the form of self-conditioned medium (CM) on A549 lung carcinoma cells. We demonstrated that CM induced morphological and molecular changes associated with epithelial-mesenchymal transition viz change in shape from cuboidal to spindle, actin cytoskeleton remodelling, upregulation of vimentin and downregulation of E-cadherin etc. These changes were accompanied with enhanced motility, invasion, anchorage-independent growth and anoikis-resistance. Amongst the different factors of CM, IL-8 and VEGF were found to play a major role in the CM-induced motility and invasion. In the intracellular signalling cascade, CM triggered phosphorylation of JNK and p38 which was associated with the CM-enhanced invasiveness. In CM-treated cells, activated p38 and JNK further activated ATF-2 (Activating Transcription Factor-2) and knock-down of ATF-2 abrogated the CM-induced invasiveness, suggesting the signal transduction along the p38/JNK-ATF-2 axis. Furthermore, neutralising IL-8 and VEGF in CM, significantly abrogated CM-induced phosphorylation of ATF-2. Conversely, exogenous addition of these individual cytokines in plain medium, increased the activation of ATF-2 and invasiveness marginally. However, when added in combination these cytokines (IL-8 and VEGF) resulted in drastic increase in ATF-2 phosphorylation and subsequent invasiveness suggesting their synergetic interplay in the observed phenomenon. Taken together, our results identify IL-8/VEGF induced JNK/p38-ATF-2 as a novel pro-invasive pathway, which may be explored as potential therapeutic target to circumvent the invasiveness of lung malignancies.

Protection against radiation-induced oxidative damage by an ethanolic extract of Nigella sativa L.

Purpose: An ethanolic extract of Nigella sativa L. (EE-NS) was investigated for its antioxidant properties and radioprotective effects against γ-radiation-induced oxidative damage. Materials and methods: The radical scavenging activity of the extract was measured by 2,2-diphenyl-1-picrylhydrazyl (DPPH), deoxyribose degradation and plasmid relaxation assays in a cell-free system. DNA damage studies were performed using a single cell gel electrophoresis (SCGE) assay and micronuclei (MN) formation. Moreover, the alterations in lipid peroxidation and antioxidant enzymes were measured by biochemical methods. Results: EE-NS showed significant free radical scavenging and protection against DNA damage in cell free systems. Ex vivo treatment of mouse splenic lymphocytes with an ethanolic extract of N. sativa 1 h prior to irradiation (2 Gy) showed significant prevention of the formation of lipid-peroxides and intracellular reactive oxygen species (ROS), which correlated with radiation-induced apoptosis. Moreover, radiation-induced DNA damage was significantly prevented in splenocytes pre-treated with EE-NS. Swiss albino mice fed orally with the different doses of EE-NS (0–100 mg/kg bw) for five consecutive days followed by 2 Gy whole body irradiation (WBI) showed significant protection against oxidative injury to spleen and liver as measured by lipid peroxidation and the activity of antioxidant enzymes. These results were correlated with the prevention of DNA damage as measured by bone marrow micronuclei assay. Our results suggest that oral feeding of extract resulted in increased survival in mice exposed to WBI (7.5 Gy). Conclusion: The results obtained from the different experimental systems suggest the radioprotective ability of EE-NS involving prevention of radiation-induced oxidative damage.

Radiobiological basis in management of accidental radiation exposure

Purpose: With increasing utilisation of nuclear technologies in power production, medical and industrial applications, and in a scenario of nuclear terrorism/war, there is an enhanced likelihood of accidental radiation exposure to occupational workers, patients and public. The consequent health effects of the radiation exposure are resultant of interaction of radiation with biological systems and subsequent radiation injury. The present review discusses the knowledge gained in radiation biology that can be exploited for better treatment and management of radiation accident victims. Results: In comparison with planned radiation exposure during diagnosis/therapy, the management of accidental radiation exposure is quite complicated due to uncertainties in dose, duration, organs involved and radionuclides internalised, and hence, require multi-faceted approaches. However, the options available for dosimetry, decorporation of radionuclides and therapeutic protocols of patients are limited, which provides substantial scope in these areas of research. Moreover, there is a need to fill the gaps in knowledge of radiation action in different dose ranges and post-irradiation windows, which would help in improving therapeutic approaches. Cytogenetic approaches are ‘gold standard’ for biodosimetry but with limited applications in mass casualty scenario. State-of-the-art technological advancement and high throughput in metabolomics, proteomics and genomics could be employed successfully in developing better biodosimetry for triage in accidental radiation exposure. Furthermore, identification of targets at organs/organelles level of internalised radionuclides would be helpful to develop effective decorporation strategies. Despite substantial research investigating several agents, which could modify radiation effects, only a few could reach up to practical application due to poor bioavailability or toxicity. Conclusions: Deeper insight into the mechanisms of radiation injury under accidental radiation conditions would be helpful in achieving better biodosimetry, decorporation strategies and improvement in prevention/post-irradiation management of radiation accident patients.

Radiosensitization in human breast carcinoma cells by thymoquinone: role of cell cycle and apoptosis

TQ (thymoquinone), the bioactive constituent of black seed (Nigella sativa), has been shown to inhibit the growth of various human cancers both in vitro and in vivo. This study reports the radiosensitizing effect of TQ on human breast carcinoma cells (MCF7 and T47D). TQ in combination with single dose of ionizing radiation (2.5 Gy) was found to exert supra‐additive cytotoxic effects on both the carcinomas as measured by cell proliferation and colony‐formation assays. Annexin V binding and FACS analysis revealed the role of enhanced apoptosis and cell cycle modulation in the mechanism of TQ‐mediated radiosensitization, thus supporting TQ as an adjuvant for preclinical testing in cancer chemo‐radiotherapy.

Radiosensitization by diospyrin diethylether in MCF-7 breast carcinoma cell line

The development of radio-resistant tumor cells might be overcome by the use of tumor selective cytotoxic agents in combination with radiation treatment of cancer. Thus, we are exploring the radiomodifying potential of D7, a tumor-inhibitory compound derived from a plant product, diospyrin, in breast carcinoma cells, MCF-7. The present study indicated that D7 could enhance the radiation-induced cytotoxicity and apoptosis through down-regulation of the anti-apoptotic Bcl-2 and COX-2 gene expression, and up-regulation of pro-apoptotic genes, like p53 and p21. The higher expression of PUMA, a pro-apoptotic protein was also observed in the combination treatment. Effect of D7 on up-regulation of p21 expression in irradiated MCF-7 cells was concomitant with the cell cycle arrest in the G1 phase. Thus, it was concluded that D7 could sensitize the effect of radiation in breast carcinoma by regulating the gene expression involved in cell cycle and apoptosis.

Diospyrin derivative, an anticancer quinonoid, regulates apoptosis at endoplasmic reticulum as well as mitochondria by modulating cytosolic calcium in human breast carcinoma cells

Diospyrin diethylether (D7), a bisnaphthoquinonoid derivative, exhibited an oxidative stress-dependent apoptosis in several human cancer cells and tumor models. The present study was aimed at evaluation of the increase in cytosolic calcium [Ca(2+)](c) leading to the apoptotic cell death triggered by D7 in MCF7 human breast carcinoma cells. A phosphotidylcholine-specific phospholipase C (PC-PLC) inhibitor, viz. U73122, and an antioxidant, viz. N-acetylcysteine, could significantly prevent the D7-induced rise in [Ca(2+)](c) and PC-PLC activity. Using an endoplasmic reticulum (ER)-Ca(2+) mobilizer (thapsigargin) and an ER-IP3R antagonist (heparin), results revealed ER as a major source of [Ca(2+)](c) which led to the activation of calpain and caspase12, and cleavage of fodrin. These effects including apoptosis were significantly inhibited by the pretreatment of Bapta-AM (a cell permeable Ca(2+)-specific chelator), or calpeptin (a calpain inhibitor). Furthermore, D7-induced [Ca(2+)](c) was found to alter mitochondrial membrane potential and induce cytochrome c release, which was inhibited by either Bapta-AM or ruthenium red (an inhibitor of mitochondrial Ca(2+) uniporter). Thus, these results provided a deeper insight into the D7-induced redox signaling which eventually integrated the calcium-dependent calpain/caspase12 activation and mitochondrial alterations to accentuate the induction of apoptotic cell death.