Nucleoside analogs as a radiosensitizer modulating DNA repair, cell cycle checkpoints, and apoptosis.

The combination of low dose of radiation and an anticancer drug is a potent strategy for cancer therapy. Nucleoside analogs are known to have a radiosensitizing effects via the inhibition of DNA damage repair after irradiation. Certain types of nucleoside analogs have the inhibitory effects on RNA synthesis, but not DNA synthesis, with multiple functions in cell cycle modulation and apoptosis. In this review, the most up-to-date findings regarding radiosensitizing nucleoside analogs will be discussed, focusing especially on the mechanisms of action.

The prospective application of a hypoxic radiosensitizer, doranidazole to rat intracranial glioblastoma with blood brain barrier disruption.

BACKGROUND: Glioblastoma is one of the intractable cancers and is highly resistant to ionizing radiation. This radioresistance is partly due to the presence of a hypoxic region which is widely found in advanced malignant gliomas. In the present study, we evaluated the effectiveness of the hypoxic cell sensitizer doranidazole (PR-350) using the C6 rat glioblastoma model, focusing on the status of blood brain barrier (BBB). METHODS: Reproductive cell death in the rat C6 glioma cell line was determined by means of clonogenic assay. An intracranial C6 glioma model was established for the in vivo experiments. To investigate the status of the BBB in C6 glioma bearing brain, we performed the Evans blue extravasation test. Autoradiography with [(14)C]-doranidazole was performed to examine the distribution of doranidazole in the glioma tumor. T2-weighted MRI was employed to examine the effects of X-irradiation and/or doranidazole on tumor growth. RESULTS: Doranidazole significantly enhanced radiation-induced reproductive cell death in vitro under hypoxia, but not under normoxia. The BBB in C6-bearing brain was completely disrupted and [(14)C]-doranidazole specifically penetrated the tumor regions. Combined treatment with X-irradiation and doranidazole significantly inhibited the growth of C6 gliomas. CONCLUSIONS: Our results revealed that BBB disruption in glioma enables BBB-impermeable radiosensitizers to penetrate and distribute in the target region. This study is the first to propose that in malignant glioma the administration of hydrophilic hypoxic radiosensitizers could be a potent strategy for improving the clinical outcome of radiotherapy without side effects.

Tolerance of anhydrobiotic eggs of the Tardigrade Ramazzottius varieornatus to extreme environments.

Tardigrades are tiny (less than 1 mm in length) invertebrate animals that have the potential to survive travel to other planets because of their tolerance to extreme environmental conditions by means of a dry ametabolic state called anhydrobiosis. While the tolerance of adult tardigrades to extreme environments has been reported, there are few reports on the tolerance of their eggs. We examined the ability of hydrated and anhydrobiotic eggs of the tardigrade Ramazzottius varieornatus to hatch after exposure to ionizing irradiation (helium ions), extremely low and high temperatures, and high vacuum. We previously reported that there was a similar pattern of tolerance against ionizing radiation between hydrated and anhydrobiotic adults. In contrast, anhydrobiotic eggs (50% lethal dose; 1690 Gy) were substantially more radioresistant than hydrated ones (50% lethal dose; 509 Gy). Anhydrobiotic eggs also have a broader temperature resistance compared with hydrated ones. Over 70% of the anhydrobiotic eggs treated at either -196°C or +50°C hatched successfully, but all the hydrated eggs failed to hatch. After exposure to high-vacuum conditions (5.3×10(-4) Pa to 6.2×10(-5) Pa), the hatchability of the anhydrobiotic eggs was comparable to that of untreated control eggs.

The influence of gender- and age-related differences in the radiosensitivity of hematopoietic progenitor cells detected in steady-state human peripheral blood.

To investigate the importance of gender and aging on the individual radiosensitivity of lineage-committed myeloid hematopoietic stem/progenitor cells (HSPCs) detected in mononuclear cells (MNCs) of steady-state human peripheral blood (PB), the clonogenic survival of HPCs, including colony-forming unit-granulocyte macrophage; burst-forming unit-erythroid; colony-forming unit-granulocyte-erythroid-macrophage-megakaryocyte cells derived from MNCs exposed to 0.5 Gy and 2 Gy X-irradiation were estimated. MNCs were prepared from the buffy-coats of 59 healthy individual blood donors. The results showed that large individual differences exist in the number of HSPCs, as well as in the surviving fraction of cells. Furthermore, the number of progenitor cells strongly correlated with their surviving fraction, suggesting that the radiosensitivity of hematopoietic progenitor cells decreases with the number of cells in the 10(5) cells population. A statistically significant negative correlation was observed between the surviving fraction observed at a dose of 0.5 Gy and the age of an individual, however, none of these correlations were observed after 2 Gy irradiation. No statistically significant difference was observed in individual radiosensitivity between males and females at either radiation dose. The present results indicated a correlation between the individual responsiveness of HSPCs to ionizing irradiation, especially to low dose irradiation, and aging.

Induction of neurite outgrowth by alpha-phenyl-N-tert-butylnitrone through nitric oxide release and Ras-ERK pathway in PC12 cells.

It has previously been suggested that the spin trap agent alpha-phenyl-N-tert-butylnitrone (PBN) induces neurite outgrowth through activation of the Ras-ERK pathway in PC12 cells. However, the chemical properties of PBN contributing to its biological function and the detailed mechanism for the activation of Ras by PBN remain unknown. This study demonstrates that the hydrophobic structure of PBN is related to the activation of Ras, by comparing with hydrophilic analogues of PBN. [(14)C]-labelled PBN was found to localize in the lipid fraction and activate Ras indirectly. On the other hand, neurite outgrowth by PBN was inhibited by a nitric oxide (NO) scavenger. Moreover, the neurite outgrowth induced by PBN and the NO donor NOR4 was inhibited by the dominant negative Ras or MAPK/ERK inhibitor. Taken together, these results suggest that PBN is incorporated into the plasma membrane and induces neurite outgrowth in PC12 cells by activating the Ras-ERK pathway through NO release.

A novel copper(II) coordination at His186 in full-length murine prion protein.

To explore Cu(II) ion coordination by His(186) in the C-terminal domain of full-length prion protein (moPrP), we utilized the magnetic dipolar interaction between a paramagnetic metal, Cu(II) ion, and a spin probe introduced in the neighborhood of the postulated binding site by the spin labeling technique (SDSL technique). Six moPrP mutants, moPrP(D143C), moPrP(Y148C), moPrP(E151C), moPrP(Y156C), moPrP(T189C), and moPrP(Y156C,H186A), were reacted with a methane thiosulfonate spin probe and a nitroxide residue (R1) was created in the binding site of each one. Line broadening of the ESR spectra was induced in the presence of Cu(II) ions in moPrP(Y148R1), moPrP(Y151R1), moPrP(Y156R1), and moPrP(T189R1) but not moPrP(D143R1). This line broadening indicated the presence of electron-electron dipolar interaction between Cu(II) and the nitroxide spin probe, suggesting that each interspin distance was within 20 A. The interspin distance ranges between Cu(II) and the spin probes of moPrP(Y148R1), moPrP(Y151R1), moPrP(Y156R1), and moPrP(T189R1) were estimated to be 12.1 A, 18.1 A, 10.7 A, and 8.4 A, respectively. In moPrP(Y156R1,H186A), line broadening between Cu(II) and the spin probe was not observed. These results suggest that a novel Cu(II) binding site is involved in His186 in the Helix2 region of the C-terminal domain of moPrP(C).

Super paramagnetic iron oxide MRI shows defective Kupffer cell uptake function in non-alcoholic fatty liver disease.

BACKGROUND: The pathogenesis of non-alcoholic fatty liver disease (NAFLD) is incompletely understood. Kupffer cells (KCs), phagocytic liver-resident macrophages, provide a protective barrier against egress of endotoxin from the portal to the systemic circulation. It is not known if KC phagocytic function is impaired in NAFLD. Super-paramagnetic iron oxide (SPIO) magnetic resonance imaging is a comparative technology dependent on KC phagocytic function. OBJECTIVE: To evaluate KC uptake function, in patients and experimental animals with NAFLD, using SPIO. METHODS: Abdominal CT and histological examination of liver biopsy specimens were used to estimate the degree of steatosis in patients with NAFLD and controls with chronic hepatitis C. SPIO-MRI was then performed in all patients. Normal rats fed a methionine-choline-deficient diet to induce non-alcoholic steatohepatitis (NASH), the more severe stage of NAFLD, and obese, insulin resistant, Zucker fa/fa rats with steatohepatitis, were also studied with SPIO-MRI and analysed for hepatic uptake of fluorescent microbeads. Immunohistochemical analysis evaluated the numbers of KCs in patients and rat livers. RESULTS: Relative signal enhancement (RSE), inversely proportional to KC function, was higher in patients with NAFLD than in controls and with the degree of steatosis on CT. RSE also positively correlated with the degree of steatosis on histology and was similarly higher in rats with induced severe NAFLD (NASH). On immunohistochemistry, defective phagocytic function was the result of reduced phagocytic uptake and not due to reduced KC numbers in rats or patients with NAFLD. CONCLUSIONS: KC uptake function is significantly impaired in patients with NAFLD and experimental animals with NASH, worsens with the degree of steatosis and is not due to a reduction of KC numbers.

Redox regulation in radiation-induced cytochrome c release from mitochondria of human lung carcinoma A549 cells.

Mitochondria in mammalian cells are well-known to play an important role in the intrinsic pathway of genotoxic-agent-induced apoptosis by releasing cytochrome c into cytosol and to be a major source of reactive oxygen species (ROS). The aim of this study was to examine whether mitochondrial ROS are involved in radiation-induced apoptotic signaling in A549 cells. Post-irradiation treatment with N-acetyl-L-cysteine (NAC) inhibited cytochrome c release from mitochondria but did not affect expression levels of Bcl-2, Bcl-X(L) and Bax, suggesting that late production of ROS triggered cytochrome c release. Experiments using DCFDA (a classical ROS fluorescence probe) and MitoAR (a novel mitochondrial ROS probe) demonstrated that intracellular and mitochondrial ROS were enhanced 6h after X irradiation. Furthermore, the O(2)(-*) production ability of mitochondria isolated from A549 cells was evaluated by ESR spectroscopy combined with a spin-trapping reagent (CYPMPO). When isolated mitochondria were incubated with NADH, succinate and CYPMPO, an ESR spectrum due to CYPMPO-OOH was detected. This NADH/succinate-dependent O(2)(-*) production from mitochondria of irradiated cells was significantly increased in comparison with that of unirradiated cells. These results indicate that ionizing radiation enhances O(2)(-*) production from mitochondria to trigger cytochrome c release in A549 cells.

A BOLD-fMRI study of cerebral activation induced by injection of algesic chemical substances into the anesthetized rat forepaw.

This study was performed to examine whether the brain activities induced by noxious algesic chemical substances in anesthetized animals could be detected by blood oxygen-level-dependent functional magnetic resonance imaging (BOLD-fMRI). Multislice gradient echo images of the primary somatosensory cortex were obtained using a 7.05 T superconducting system and a one-turned surface coil centered over the primary somatosensory cortex of the 1.0%-isoflurane-anesthetized rat. The Z-score t-map of BOLD signals and its time-course analysis revealed that subcutaneous injection of formalin into the left forepaw immediately induced an early response in the contralateral primary sensory cortex lasting for a few minutes, followed by a late response until 20 min after stimulation. In contrast, injection of capsaicin into the left forepaw evoked only the early response. Furthermore, pretreatment with morphine completely abolished these responses induced by the chemical algesic substances. Thus BOLD-fMRI is a useful method to analyze the brain activities of painful stimulation in anesthetized animals.

Regulation of cell survival and death signals induced by oxidative stress.

Oxidative stress stimulates two opposite signaling pathways leading to cell death and cell survival. Preferential selection of survival signals leads to the protection of cells against damage induced by reactive oxygen species, whereas preferential acceleration of death signals can be used to advantage in tumor therapy with oxidizing agents such as ionizing radiation and anticancer drugs. In vitro and in vivo experiments using cultured mammalian cells and experimental animals showed that ERK was included in survival signals and SAPK and p38 MAPK in death signals in oxidative stress. The activation of SAPK/JNK and subsequent expression of death receptor Fas on the cell surface caused the induction of cell death. The results mean that the acceleration of the activation of SAPK/JNK might lead to the enhancement of cell death by oxidizing agents like ionizing radiation and anticancer drugs. In fact, when cultured mammalian cells were exposed to ionizing radiation with 2-nitroimidazole derivatives having electrophilicity, the lethal effect of ionizing radiation was found to be enhanced together with the activation of SAPK/JNK and the enhancement of Fas expression. The activation of both survival and death signals was suppressed by the antioxidants N-acetylcystein and Trolox, suggesting that both signaling pathways are redox-regulated.

The antiproliferative effect of bovine lactoferrin on canine mammary gland tumor cells.

Lactoferrin has several biological activities, including antitumor activities in some human and animal tumor cells. Clinical trials have been carried out in human medicine based on these effects. However, the antitumor effects of lactoferrin in veterinary medicine remain unknown. In this in vitro study, we demonstrated that co-incubation of canine mammary gland tumor cells (CIPp and CHMp) and bovine lactoferrin induced growth arrest of tumor cells. This growth arrest was associated with induction of G1 arrest. Furthermore, this effect was stronger in tumor cells than in normal cells. These findings demonstrate that bovine lactoferrin has anti-tumor activity in canine mammary tumors and has the potential for use in tumor-bearing dogs.

Purvalanol A induces apoptosis and downregulation of antiapoptotic proteins through abrogation of phosphorylation of JAK2/STAT3 and RNA polymerase II.

To clarify the mechanisms of purvalanol A in the induction of apoptosis, we investigated whether purvalanol A influenced the RNA synthesis and expression of RNA polymerase II and signal transducer and activator of transcription 3 (STAT3). When MKN45 cells were treated with 30 micromol/l purvalanol A, mitochondrial dysfunction occurred before the induction of the apoptosis and the expression of antiapoptotic proteins survivin, Bcl-XL, and Bcl-2 was reduced. The treatment with parvalanol A was also shown to reduce not only mRNA for these proteins but also global RNA synthesis. The phosphorylation of the carboxy-terminal domain of RNA polymerase II, which was involved in transcriptional regulation, was strongly inhibited by purvalanol A, followed by the partial inhibition of the expression of RNA polymerase II. Furthermore, the phosphorylation at Tyr705 of STAT3, which is known to be a phosphorylation site for Janus kinase 2 (JAK2), was completely inhibited by purvalanol A early (3 h) after drug treatment, although the phosphorylation of STAT3 at Ser727, which is a phosphorylation site for Ras/Raf/MEK and extracellular signal-regulated protein kinase 1/2, was still detectable until late (12 h) after treatment. In addition, the tyrosine phosphorylation of JAK2 was efficiently inhibited by purvalanol A. These results suggest that the inhibition of JAK2/STAT3 and RNA polymerase II is crucial in the downregulation of antiapoptotic proteins leading to the apoptotic cell death induced by parvalanol A.

X irradiation combined with TNF alpha-related apoptosis-inducing ligand (TRAIL) reduces hypoxic regions of human gastric adenocarcinoma xenografts in SCID mice.

Our previous study showed that X irradiation induced the expression of death receptor DR5 on the cell surface in tumor cell lines under not only normoxia but also hypoxia. X irradiation combined with TNF alpha-related apoptosis-inducing ligand (TRAIL), which is the ligand of DR5, induced apoptosis in vitro (Takahashi et al., (2007) Journal of Radiation Research, 48: 461-468). In this report, we examined the in vivo antitumor efficacy of X irradiation combined with TRAIL treatment in tumor xenograft models derived from human gastric adenocarcinoma MKN45 and MKN28 cells in SCID mice. X irradiation combined with TRAIL synergistically suppressed the tumor growth rates in the xenograft models derived from MKN45 and MKN28 cells, which have wild type Tp53 and mutated Tp53, respectively, indicating that the antitumor effects occurred in a Tp53-independent manner. Histological analysis showed that the combination of X irradiation and TRAIL induced caspase-3-dependent apoptotic cell death. Moreover, the immunohistochemical detection of hypoxic regions using the hypoxic marker pimonidazole revealed that caspase-3-dependent apoptosis occurred in the hypoxic regions in the tumors. These results indicated that X irradiation combined with TRAIL may be a useful treatment to reduce tumor growth in not only normoxic but also hypoxic regions.

Individual differences in the radiosensitivity of hematopoietic progenitor cells detected in steady-state human peripheral blood.

The aim of this study is to evaluate the individual differences in radiosensitivity of lineage-committed myeloid hematopoietic progenitors, colony-forming cells (CFC), detected in steady-state human peripheral blood (PB). Mononuclear cells were prepared from the buffy-coat of 30 individuals PB, and were assayed for CFC by semi-solid culture supplemented with cytokines. X irradiation was performed in the range of 0.5-4 Gy at a dose rate of about 80 cGy/min. The mean number of hematopoietic progenitor cells is 5866 alpha 3408 in 1 ml of buffy-coat, suggesting that the erythroid progenitor cells are the major population. The total CFC radiosensitivity parameter D(0) and n value are 1.18 alpha 0.24 and 1.89 alpha 0.98, respectively. Using a linear regression analysis, a statistically significant correlation is observed between the D(0) value and the surviving fraction at 4 Gy (r = 0.611 p < 0.001). Furthermore, we evaluate the relationship between individual radiosensitivity and the level of antioxidants, plasma uric acid, plasma bilirubin, and intracellular glutathione. No statistically significant correlations are observed, however, between the D(0) parameter and the level of antioxidants, plasma uric acid, plasma bilirubin, and intracellular glutathione. The present study demonstrates that there are large individual differences in the radiosensitivity of hematopoietic progenitor cells as detected in steady-state human PB. These differences demonstrate almost no correlation with plasma or intracellular antioxidants. The prediction of individual differences in radiosensitivity of CFC can only be measured by 4 Gy irradiation.

Instability of familial spongiform encephalopathy-related prion mutants.

We examined the influence of D177N (D178N in humans) mutation on the conformational stability of the S2 region of moPrP(C) with varying pHs by using the SDSL-ESR technique. The ESR spectrum of D177N at pH 7.5 was narrower than that of Y161R1, referred to as WT( *). The ESR spectrum of D177N did not change when pH in the solution decreased to pH 4.0. Our results suggested that the disappearance of a salt bridge (D177-R163) induced the increase in the instability of S2 region. Moreover, the line shape of the ESR spectrum obtained from H176S neighboring the salt bridge linked to the S2 region was similar to D177N. These results indicate that the protonation of H176 is strongly associated with the stability of S2 region. These findings are important for understanding the mechanism by which the disruption of the salt bridge in the S2 region forms the pathogenic PrP(Sc) structure in hereditary prion disease.

Radiation-induced apoptosis of tumor cells is facilitated by inhibition of the interaction between Survivin and Smac/DIABLO.

To investigate the mechanism of radioresistance of solid tumor cells, we created two expression vectors encoding Survivin mutants, T34A and D53A. When T34A and D53A were overexpressed in NIH3T3, A549 and HeLa cells, radiation-induced apoptosis was significantly enhanced. Furthermore, we examined the binding capability of Survivin with Smac/DIABLO in the cells that overexpressed these mutants. Coimmunoprecipitation analysis revealed that mutant form of Survivin, D53A and T34A could bind to Smac/DIABLO, but with much less affinity compared to the authentic form. These results suggest that radiation-induced apoptosis of tumor cells is increased by inhibition of the interaction between Survivin and Smac/DIABLO through overexpression of T34A and D53A.

Costimulatory effects of complement receptor type 3 and Fc receptor for IgG (FcgammaR) on superoxide production and signal transduction in bovine neutrophils.

The present study evaluated the costimulatory effects of complement receptor type 3 (CR3) and Fc receptor for IgG (FcgammaR) on superoxide production and intracellular signal transduction in bovine neutrophils. Stimulation with opsonized zymosan (OPZ) and heat-aggregated bovine IgG (Agg-IgG) resulted in much greater superoxide production and chemiluminescent (CL) responses in normal neutrophils compared with those stimulated with OPZ or Agg-IgG only. Superoxide production and CL response were closely associated with the stimulant-induced rise of the intracellular calcium ([Ca2+]i) concentration, amount of tyrosine phosphorylated 100 kDa protein, and activation of p38 mitogen-activated protein kinase (p38 MAPK). No costimulatory effect was found for these receptors on superoxide production in CR3-deficient neutrophils. Costimulation of CR3 and FcgammaR on bovine neutrophils leads to enhancement of superoxide production and their signaling pathways and appears to be associated with enhancement of neutrophil functions.

A new amphiphilic derivative, N-{[4-(lactobionamido)methyl]benzylidene}-1,1-dimethyl-2-(octylsulfanyl)ethylamine N-oxide, has a protective effect against copper-induced fulminant hepatitis in Long-Evans Cinnamon rats at an extremely low concentration compared with its original form alpha-phenyl-N-(tert-butyl) nitrone.

An amphiphilic alpha-phenyl-N-(tert-butyl) nitrone (PBN) derivative, N-{[4-(lactobionamido)methyl]benzylidene}-1,1-dimethyl-2-(octylsulfanyl)ethylamine N-oxide (LPBNSH), newly synthesized from its original form PBN in hopes of clinical use, was intraperitoneally administered to Long-Evans Cinnamon (LEC) rats every 2 days at the concentrations of 0.1, 0.5, 1.0, and 2.0 mg/kg. We found that LPBNSH protected against copper-induced hepatitis with jaundice in LEC rats at concentrations of 0.1 and 0.5 mg/kg, which were extremely low compared with that of PBN. It also effectively prevented the loss of body weight, reduced the death rate, and suppressed the increase in serum aspartate aminotransferase and alanine aminotransferase values arising from fulminant hepatitis with jaundice at the same concentrations. Similar results were observed when PBN was administered at the concentration of 150 mg/kg. Immunohistochemical analysis of 8-hydroxy-2'-deoxyguanosine and measurement of thiobarbituric acid-reactive substances in the liver showed that LPBNSH largely suppressed the formation of these oxidative products at same concentrations. No difference in the abnormal accumulation of copper in the liver between the LPBNSH administered and control groups was observed. From these results, it was concluded that LPBNSH exhibited liver-protective effects against fulminant hepatitis with jaundice at ca. 1/1000, 500 the molar concentration of PBN and, therefore, was clinically promising.

Enhancement of cell death by TNF alpha-related apoptosis-inducing ligand (TRAIL) in human lung carcinoma A549 cells exposed to x rays under hypoxia.

Our previous study showed that ionizing radiation induced the expression of death receptor DR5 on the cell surface in tumor cell lines and that the death receptor of the TNF alpha-related apoptosis-inducing ligand TRAIL enhanced the apoptotic pathway (Hamasu et al., (2005) Journal of Radiation Research, 46:103-110). The present experiments were performed to examine whether treatment with TRAIL enhanced the cell killing in tumor cells exposed to ionizing radiation under hypoxia, since the presence of radioresistant cells in hypoxic regions of solid tumors is a serious problem in radiation therapy for tumors. When human lung carcinoma A549 cells were irradiated under normoxia and hypoxia, respectively, radiation-induced enhancement of expression of DR5 was observed under both conditions. Incubation in the presence of TRAIL enhanced the caspase-dependent and chymotrypsin-like-protease-dependent apoptotic cell death in A549 cells exposed to X rays. Furthermore, it was shown that treatment with TRAIL enhanced apoptotic cell death and loss of clonogenic ability in A549 cells exposed to X rays not only under normoxia but also under hypoxia, suggesting that combination treatment with TRAIL and X irradiation is effective for hypoxic tumor cells.

Oral administration of Antioxidant Biofactor (AOBtrade mark) ameliorates ischemia/reperfusion- induced neuronal death in the gerbil.

Oxidative damage due to ischemia/reperfusion has been implicated as one of the leading causes for delayed neuronal cell death in a number of neurodegenerative diseases, including stroke. The purpose of this research was to investigate whether oral administration of a fermented grain food mixture (AOB(R)) might offer protective effects against ischemia/reperfusion-induced neuronal damage in Mongolian gerbils, a model known for delayed neuronal death in the hippocampal CA1 region. Histological analysis revealed that AOB administration ad libitum for 3 weeks (preoperative administration) and 1 week (postoperative administration) dose-dependently suppressed the induction of transient ischemia/reperfusion-induced neuronal cell death. TUNEL assay also revealed that AOB suppressed it by inhibiting the induction of apoptosis. A significant increase of superoxide dismutase-like (SOD-like) activity was observed in the hippocampal CA1 region of the AOB-treated gerbil. Furthermore, immunoblot analysis showed that AOB administration down-regulated the expression of heat shock proteins HSP27 and HSP70 in the same region. These results indicated that oral administration of AOB protected against ischemia/reperfusion-induced brain injury by minimizing oxidative damage via its SOD-like activity and inhibiting apoptosis.