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.

Studies on efficacy of a novel 177Lu-labeled porphyrin derivative in regression of tumors in mouse model.

OBJECTIVE: The aim of the present study was to develop a (177)Lu-labeled porphyrin derivative having favorable characteristics for use in targeted radiotherapy of cancer and to evaluate its biological behavior in mouse tumor models with respect to its effectiveness in tumor regression. Owing to the inherent affinity of porphyrins to accumulate in the tumors, suitably modified porphyrin derivative was chosen as the vehicle for the targeted delivery of the radionuclide. (177)Lu was preferred as the radionuclide of choice due to its suitable nuclear decay characteristics [E(ß(max)) = 497 keV, Eγ = 208 keV (11%), 113 keV (6.4%)], comparatively longer half-life (6.73 d) and ease of production in adequate quantity and sufficiently high specific activity using medium flux research reactors. METHODS: A novel porphyrin analogue, 5,10,15,20-tetrakis[4-carboxymethyleneoxyphenyl]porphyrin was synthesized inhouse and coupled with a macrocyclic bi-functional chelating agent, namely p-amino-benzyl-1,4,7,10- tetraazacyclododecane-1,4,7,10-tetraacetic acid. The porphyrin-BFCA conjugate was labeled with (177)Lu and the biological behavior of the radiolabeled conjugate was studied by biodistribution and imaging in Swiss mice bearing either fibrosarcoma or thymic lymphoma tumors. Effectiveness of the agent in controlling the growth of tumor volumes was also studied by administering various doses of the radiolabeled preparation in the mouse tumor models. RESULTS: (177)Lu-labeled porphyrin-BFCA conjugate was prepared with high radiochemical purity ( > 99%) and adequate invitro stability. Biodistribution and imaging studies revealed good uptake and retention of the agent in the tumors with encouraging tumor to blood and tumor to muscle ratios at various post-administration time points. Tumor regression studies showed that the administration of the agent increased the average tumor doubling time and decreased the average specific growth rate substantially in both the types of tumors. However, thymic lymphoma was found to be more sensitive to the radiolabeled conjugate compared to fibrosarcoma. CONCLUSION: Preliminary biological evaluation and tumor regression studies carried out in two different tumor models in Swiss mice exhibited the promising nature of (177)Lu-labeled porphyrin-BFCA conjugate as an agent for targeted tumor therapy. However, further detailed investigations are warranted to evaluate the true potential of the developed agent.

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.

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.

Role of membrane sialic acid and glycophorin protein in thorium induced aggregation and hemolysis of human erythrocytes.

Thorium-232 ((232)Th), a natural radionuclide from the actinide family, is abundantly present in monazite and other ores. It is used as one of the prime fuel materials in nuclear industry and may pose an exposure risk to nuclear workers and members of the public. Human erythrocytes, as a classical cellular membrane model, were coincubated with (232)Th in order to elucidate whether this naturally occurring important radionuclide produced perturbations to cell membrane. Present study revealed that erythrocytes underwent aggregation or lysis depending on the ratio of (232)Th to cell. Scanning electron micrographs showed that erythrocytes transformed into equinocytes and/or spherocytes after (232)Th treatment. Further examination of erythrocyte by atomic force microscopy suggested significant increase in surface roughness after (232)Th treatment. Experiments on neuraminidase treated and/or anti-GpA antibody blocked erythrocytes suggested significant role of membrane sialic acid and glycophorin A (GpA) protein in aggregation or hemolytic effects of (232)Th. Further results showed that (232)Th caused hemolysis by colloid osmotic mechanism, as evidenced by potassium efflux, osmotic protection and osmotic fragility studies. Osmoprotection experiments indicated that hemolysis get elicited through the formation of membrane pores of approximately 2.0 nm in size. Hemolysis studies in presence of inhibitors (TEA, bumetanide, DIDS and amiloride) revealed the role of K(+) channel, Na(+)/K(+)/2Cl(-) channel, Cl(-)/HCO(3)(-) anion exchanger and Na(+)/H(+) antiporter in (232)Th induced erythrolysis. Presence of non-diffusible cation (N-methyl d-glucasamine) or anion (gluconate) in erythrocyte suspending medium further confirm the role of Na(+) and Cl(-) influx in hemolytic effect of (232)Th. These findings provide significant insight in structural, biochemical and osmotic toxic effects of (232)Th on human erythrocytes.

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.

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.

Impairment of mitochondrial respiratory chain enzyme activities in tetralogy of Fallot.

BACKGROUND: During the last decade, disorders of the respiratory chain, so-called mitochondrial disorders, have emerged as a major clinical entity. Tetralogy of fallot (TOF) children>2 month of age are at risk for postoperative myocardial contractile failure. Myocardial ischemia is associated with a reduction in mitochondrial enzyme activity and have impaired metabolism resulting in decreased postoperative myocardial adenosine triphosphate (ATP) concentrations and increased lactate levels. With this in view, we measured the mitochondrial energy system (respiration and OXPHOS) and to study morphological changes from the right ventricular outflow tract (RVOT) muscle of patients with TOF. METHODS: 30 infants with TOF were studied with age-matched control group consisted of 12 normal patients who died due to extracardiac causes. Mitochondrial respiratory chain complexes, OXPHOS, cytochrome content and ATPase activity were measured by documented standard procedure. Morphological changes examined with a transmission electron microscope. RESULTS: In the presence of glutamate and succinate as substrates, the rate of mitochondrial oxygen consumption was significantly lower in RVOT muscles (p<0.001) by using with and without addition of ADP. The ADP/O ratio indices for glutamate and succinate were not significantly affected. The activities of rotenone-sensitive NADH cytochrome c reductase (complexes I+III), cytochrome c oxidase (complex IV) and the ratio of I and III to II and III complexes (complex I) were significantly lower in TOF (p<0.001). A significant reduction of total cytochrome content and ATPase activity (p<0.001) was noted in study group. Morphological changes were also seen in study group as compared with control. CONCLUSIONS: OXPHOS, mitochondrial respiratory chain complex I, I+III and IV, cytochrome content and ATPase activity are more impaired in RVOT muscles in patients with TOF.

Modulation of DNA methyltransferase profile by methyl donor starvation followed by gamma irradiation.

DNA methylation is an important epigenetic mechanism of transcriptional control, which plays an essential role in maintaining cellular function. Role of one-carbon transfer agents/methyl donors namely folate, choline and methionine in DNA methylation has been the subject of extensive investigation. The methylation pattern of DNA is established during embryogenesis by DNA methyltransferase 3 (dnmt3) and is subsequently maintained by maintenance methylation activity of the enzyme DNA methyltransferase 1 (dnmt1). Ionizing radiation is known to extensively damage the DNA. Sufficient dietary availability of methyl donors is known to contribute towards one-carbon transfer mediated repair of damaged DNA where folate is involved in nucleotide base synthesis. In the present study, modification in activities of dnmt1 and dnmt3 by methyl donor starvation followed by gamma-irradiation was observed. Assays were based on the catalytic transfer of (3)H-methyl groups from S-adenosyl-L: -methionine to a DNA substrate. Experiments showed a dose and methyl donors starvation dependent attenuation in dnmt1 activity. Attenuation of dnmt1 activity was most significant for diet deprived of all the three-methyl donors. No significant change in nuclear or cytoplasmic dnmt3 activity was observed when either or all the three possible source of dietary methyl group supply were removed. Ionizing radiation and methyl donor deficiency were observed to act synergistically towards inhibiting dnmt1 activity. Present results suggested possibility of interaction among folate, methionine and choline deficiency to potentiate symptoms of ionizing radiation stress. These enzymatic modifications might contribute to altered DNA methylation after chronic feeding of methyl donor free diets followed by gamma irradiation. These results suggested that dietary availability of methyl donors and gamma-radiation stress might significantly alter the dnmt1 profile.

Modification of p53 protein profile by gamma irradiation followed by methyl donor starvation.

The possible beneficial radio-protective effects of one-carbon transfer agents namely folate, choline and methionine have been the subject of extensive investigation. Ionizing radiation is known to extensively damage the DNA. One-carbon transfer agents have been proposed to have important role in context of DNA repair via their role in purine and thymidylate synthesis and in DNA methylation. Sufficient dietary availability of one-carbon transfer agents therefore, might have ability to modify radiation effects. In present study modifications in level of tumor suppressor protein p53 by gamma irradiation followed by methyl donor starvation was observed. Experiments showed an increase in nuclear and cytoplasmic p53 protein concentration in liver, spleen and thymus. The overall rise in the level of p53 protein in liver was found to be less than that in spleen and thymus. Moreover significant heterogeneity in the basal level of expression of the p53 protein in liver, spleen and thymus was observed as the level of p53 protein in spleen and thymus was found to be 7-8 fold more than that in liver. Results indicated that radiation stress followed by methyl donor starvation could significantly induce p53 protein in spleen and thymus where there was a dramatic accumulation of p53 following irradiation, while in other tissues, particularly the liver, no such dramatic response was seen. Folate contribution of intestinal bacteria was found to influence p53 protein levels. Our observations indicated a prominent role played by the methyl donors in protecting the cell against harmful effects of ionizing radiation.

Electroporation enhances radiation and doxorubicin-induced toxicity in solid tumor in vivo.

Treatment of cancer patients is subject to limitations in radiotherapy and chemotherapy. This necessitates development of new protocols, and the present work reports on the effects of a combination of local electroporation with ionizing radiation and/or anticancer drug doxorubicin hydrochloride (DOX) on subcutaneous solid tumor murine fibrosarcoma. Localized treatment of fibrosarcoma tumor, grown in right hind leg of Swiss mice, has been carried out using DOX (0.6 mg/kg body weight), radiation (Co 60 gamma-rays, dose rate 0.37 Gy/min) and electroporation (1 kV/cm, 200 micros, 8 pulses per burst, 10 bursts) individually or in combinations. Measurements of the tumor growth kinetics after treatment with combinations have revealed significant growth delay. The treatment groups, (i) radiation and electroporation, (ii) DOX and electroporation, and (iii) radiation, DOX and electroporation, have yielded tumor growth delays (TGDs) of 1.22, 1.5, and 1.73 days, respectively, compared to control with the tumor volumes being 53%, 57%, and 49% that of control on the final day of observation. These results suggest that the antitumor effects of a moderate dose of gamma radiation and low concentration of DOX can be significantly enhanced by combination with electroporation.

Apoptotic index as predictive marker for radiosensitivity of cervical carcinoma: evaluation of membrane fluidity, biochemical parameters and apoptosis after the first dose of fractionated radiotherapy to patients.

BACKGROUND: This study was aimed to develop possible predictive response of cervical carcinoma in stage IIIA and B patients by evaluating the changes in physical parameter, such as, membrane fluidity, biochemical parameters, such as, intracellular calcium, antioxidant enzymes [superoxide dismutase (SOD), catalase, glutathione peroxidase (GPx)] and apoptotic cell death in cervical cancer cells from patients after treating with the first fractionated dose of 2 Gy in radiation therapy protocol. METHODS: Biopsies of cervical carcinoma patients were collected before and 24h after first fractionated radiation dose of 2 Gy. Cell suspensions and tissue of cervix cancer biopsies were used to measure various physical and biochemical parameters. RESULTS AND CONCLUSIONS: A negative correlation was found to exist between observed fluidity of membrane/SOD level with the degree of apoptosis in cervical cells. On the other hand, a positive correlation was observed between intracellular calcium level and percent cellular apoptosis. These results suggest that changes in membrane fluidity, SOD and calcium level were involved in the mechanism of radiation induced cervical apoptosis as measured by TUNEL assay. Moreover, apoptotic sensitivity of these cells after the first dose of radiation treatment showed a direct correlation with the radiation treatment outcome in patients after completion of radiotherapy protocol (70 Gy) in the clinic suggesting that apoptotic index may form a basis for prognosis in radiotherapy in stage III cervix cancer patients.

Enhancement of radiation cytotoxicity in murine cancer cells by electroporation: in vitro and in vivo studies.

Increasing evidence has accumulated in recent years to suggest that the cell membrane forms the vital common target for action by ionizing radiation and electroporation. The present work describes the use of electric pulses for enhancement of radiation-induced cytotoxicity of cancer cells both in vitro and in vivo. In vitro: low dose rate (0.37 Gy/min) Co60 gamma-rays (2 Gy) in combination with electric pulses of square wave (2 kV/cm, 200 micros duration, 8 pulses/burst, 10 times) significantly enhanced the cytotoxicity in Ehrlich ascites carcinoma cells (EAC), probably through enhanced production of intracellular reactive oxygen species (ROS). The intracellular generation of ROS and changes in oxidative damage-mediated membrane fluidity were determined by fluorescence using DCH-FDA and DPH, respectively, as probes. Both radiation and electroporation, separately, have been observed to produce ROS in a dose-dependent fashion. We show that the combined treatment of cells with radiation and electroporation significantly increased intracellular ROS and changed membrane fluidity of EAC cells as compared to the effects by each individual treatment. In vivo studies have been carried out with murine fibrosarcoma as a model system. The localized treatment of a fibrosarcoma tumor, grown in the right hind leg of Swiss mice, had been carried out using radiation (Co60 gamma-rays, 2 Gy, dose rate: 0.37 Gy/min) and electric pulses (1 kV/cm, 200 micros, 8 pulses/burst, 10 times). Studies on tumor growth kinetics have shown a significant growth delay (by 50% to that of control) 7 days after treatment of tumor with radiation and electroporation. The results suggest that radiocytotoxicity of tumor cells in vitro as well as in vivo were enhanced significantly by electric pulses, which may offer a potentially improved treatment of cancer.

Modification of thymocytes membrane radiooxidative damage and apoptosis by eugenol.

Radiation-induced membrane oxidative damage, generation of reactive oxygen species (ROS), and cellular apoptosis, as well as their modification by a natural antioxidant from plant source, eugenol, have been investigated in mouse thymocytes. Thymocyte suspension was irradiated by gamma-rays, and the malondialdehyde (MDA) formation was measured with the thiobarbituric acid reactive species (TBARS) method. The results showed an increase in MDA in irradiated (2 Gy) thymocytes, which was inhibited in samples treated with increasing concentrations of eugenol (10-200 microM) prior to irradiation. The concentration of eugenol required to inhibit half of the MDA formation (IC(50)) in irradiated thymocytes was 100 microM. A dose-dependent increase in the generation of ROS was observed in irradiated thymocytes (0.5-200 cGy) as measured by 2,7-dichlorodihydro fluorescein diacetate (DCH-FDA), which was, however, inhibited by eugenol administered before irradiation. Furthermore, experiments showed a significant decrease in thymocytes apoptosis by antioxidant as measured by annexin-V labeling protocol. The inhibition of apoptosis by membrane-localized antioxidants such as eugenol, isoeugenol, and alpha-tocopherol acetate was more effective than a cytosolic antioxidant such as ascorbic acid. The results suggest an effective prevention of membrane and cellular damage in irradiated thymocytes by eugenol. It is inferred that damage to membrane played a significant role in radiation-induced cellular apoptotic death, which was markedly modified by membrane-specific antioxidants.

Cell membrane oxidative damage induced by gamma-radiation and apoptotic sensitivity.

Ionizing radiation-generated reactive oxygen species (ROS) resulting in oxidative damage to the cell membrane and its consequent role in the mechanism of apoptotic cell death have been receiving growing attention in cellular radiobiology. In recent years, evidence has accumulated to suggest that it is the damage to the cell membrane that contributes to the radiation cell killing. It has been demonstrated that degradation of membrane-bound sphingomyelinase (SMase) after irradiation of bovine endothelial cell produces ceramide, which initiates an apoptotic cascade, suggesting membrane-triggered events in the mechanism of cellular apoptosis. Fluorescence and electron spin resonance (ESR) studies from gamma-irradiation ofliposomal vesicles have shown that radiation-mediated lipid damage was modified by the inclusion of structure-modulating agents (e.g., cholesterol) and antioxidants (e.g., tocopherol, eugenol). The magnitude of the modification of the damage was found to be dependent on the concentration of these modifiers. Moreover, experiments on dipalmitoyl phosphatidyl choline (DPPC) unilamellar liposomes demonstrated a biphasic behavior of radiation damage, which was remarkably modified by ascorbic acid and alpha-tocopherol in a concentration-dependent fashion. The comparison of their protective effects showed that ascorbic acid was less effective than tocopherol against radiation damage to liposomes. Studies on irradiated mouse thymocytes employing FDA fluorescence probe have suggested post-irradiation time- and dose-dependent changes in membrane permeability. The determination of induction of apoptosis in irradiated thymocytes showed a time-dependent DNA fragmentation, suggesting that radiation-induced permeability changes and occurrence of apoptotic death in thymocytes were closely correlated. These results are discussed, with an emphasis on membrane-damage-mediated apoptotic death with relevance to improvement of cancer radiotherapy.