Kaushala Prasad Mishra

Potential of radiosensitizing agents in cancer chemo-radiotherapy.

Potential of herbs and other plant-based formulations have been increasingly recognized in prevention and treatment of human diseases including cancer. There exist enormous prospect for screening and evaluation of herbal/plant products for developing effective radiosensitization and radioprotection relevant to nuclear research program. Investigations in our laboratory have focused on the mechanism of activity of variety of anticancer and antioxidant agents, namely, Eugenol, (EU), Ellagic acid (EA), Triphala (TPL), Tocopherol Succinate (TOS) and Arachidonic acid on normal and cancer cells with view to design effective protocols in practical radioprotection and cancer radiotherapy. This paper is mainly focused on studies on cytotoxic effects on cancer cell lines. Results have shown that these agents produced radiosensitizing action involving oxidative damage, membrane alteration and damage to nucleic acid in various human cell lines. Studies were performed employing fluorescence probes and electron spin resonance methods and gel electrophoresis protocols. It has been found that cytotoxic effect was induced by initiating membrane oxidative damage and by triggering intracellular generation of reactive oxygen species (ROS) by gamma radiation in combination with phytochemicals like TPL, EA and TOS in tumor cell line Ehrlich Ascites (EAC), Human cervical (HeLa) and breast (MCF-7) cells. Membrane damage and ROS generation was measured by DPH and DCF-FDA fluorescent probes respectively after exposure to low to moderate doses of gamma radiation. This talk will present the cytotoxic effects of phytochemicals in combination with ionizing radiation. It is emphasized that modulation of membrane peroxidative damage and intra cellular ROS may help achieve efficient killing of cancer cells which may provide a new approach to developing effective treatment of cancer.

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.

Silibinin-Induced Apoptosis in MCF7 and T47D Human Breast Carcinoma Cells Involves Caspase-8 Activation and Mitochondrial Pathway

Silibinin, a natural flavonoid, under phase I/II clinical trial in prostate cancer patients was aimed to evaluate its chemotherapeutic potential in human breast cancer cell MCF7 and T47D. Results showed that T47D cells were found to be more sensitive to silibinin than MCF7 as observed by proliferation, clonogenic, and apoptotic assays, which was abrogated by pan-caspase inhibitor but remained unaffected by p53 inhibitor. Apoptotic events in both cell types differ temporally and also by magnitude that involved mitochondrial and caspase-8 activation pathway. These results have relevance in understanding silibinin treatment to breast tumor.

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.

Prevention of isoproterenol-induced cardiac hypertrophy by eugenol, an antioxidant.

Recent reports on the involvement of calcineurin in cardiac hypertrophy and its susceptibility to free radicals, prompted us to examine possible beneficial effects of dietary antioxidants in this regard. In continuation of initialin vitro studies revealing eugenol to be a potent calcineurin inhibitor, we investigated its ability to reverse isoproterenol-induced cardiac hypertrophy in rats. Intraperitoneal administration of isoproterenol (1 mg/kg body wt/day for 10 days) induced cardiac hypertrophy with increased heart weight and enhanced apoptosis of myocytes concomitant with accumulation of reactive oxygen species, decreased glutathione contents, increased activities of calcineurin and protein kinase C in ventricular tissue. Administering eugenol for 3 days (1 mg/kg body wt/twice a day), followed by combined administration of isoproterenol and eugenol resulted in significant reversal of cardiac hypertrophy and restoration of above changes. These results suggest that eugenol, a natural antioxidant of dietary origin, may offer potential benefits in the management of cardiac hypertrophy.

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.

Biologic Evaluation of a Novel 188Re-Labeled Porphyrin in Mice Tumor Model

The aim of this study was to develop a (188)Re-labeled porphyrin-based tumor-specific agent and to evaluate its biologic behavior, including tumor-regressing effectiveness, in mouse tumor models for possible use in achieving targeted cancer radiotherapy. (188)Re was obtained from an alumina-column-based (188)W-(188)Re generator constructed in-house. The compound, 5,10,15,20-tetrakis[3,4-bis(carboxymethyleneoxy)phenyl]porphyrin, was synthesized and labeled with (188)ReO(4)(-). (188)Re-labeled porphyrin complex was produced with a radiochemical purity of approximately 98% with reasonably good in vitro stability (>24 hours at 4 degrees C). Swiss mice bearing thymic lymphoma and fibrosarcoma were used as tumor models. The biodistribution studies revealed satisfactory tumor retention (2.07% +/- 0.80% injected activity per g) with insignificant activities in blood (0.53%), liver (0.26%) and kidney (0.04%) at 24 hours. The radiolabeled conjugate treatment increased the average tumor-doubling time and decreased the average specific growth rate substantially in thymic lymphoma, compared to fibrosarcoma tumor. (188)Re-labeled 5,10,15,20-tetrakis[3,4 bis(carboxymethyleneoxy)phenyl] porphyrin has specific affinity toward the fibrosarcoma and thymic lymphoma tumors in mice. Thymic lymphoma was found to be more sensitive to the radionuclide complex, compared to fibrosarcoma. The (188)Re-labeled porphyrin complex showed promising results and warrants further investigations.

Thorium-induced oxidative stress mediated toxicity in mice and its abrogation by Diethylenetriamine pentaacetate

Purpose: Thorium (232Th, IV) preferentially accumulates in the liver, femur and spleen, which necessitates evaluation of its toxic effect in these organs. The present study was aimed at evaluation of liver function, oxidative stress and histological alterations in these organs. Materials and methods: Swiss albino mice were administered either with Thorium nitrate (10 mg/kg body weight/day equivalent to 1090 pCi/kg body weight/day) for 30 days (1/40th dose of LD50/30; the dose of thorium required to kill 50% of the test cohort within 30 days) intraperitoneally or with calcium salt of diethylenetriamine pentaacetate (Ca-DTPA, 100 μmole/kg body/weight) intravenously or both. Liver function tests and oxidative damage was assessed. The concentration of Th in the tissues was determined by Inductively Coupled Plasma-Atomic Emission Spectroscopy (ICP-AES) method. Results: Administration of Th prevented the increase in the body and liver weight and altered liver functions. Th treatment to mice showed a decrease in the activities and gene expression of antioxidant enzymes, and increased lipid peroxidation and protein carbonylation. The extent of observed oxidative damage was correlated with accumulation of Th in examined organs and further associated with histological alterations. Furthermore it was found that these effects were significantly lower when the chelating agent, Ca-DTPA, was given 1 h after Th injection. Conclusion: Administration of subtoxic concentration of Th to mice markedly altered the liver functions and induced oxidative stress in the liver, femur and spleen of mice. The results further demonstrated that Ca-DTPA significantly protected mice against the toxic effects of Th.