Masaki Nagane

ER stress suppresses DNA double-strand break repair and sensitizes tumor cells to ionizing radiation by stimulating proteasomal degradation of Rad51

In this study, we provide evidence that endoplasmic reticulum (ER) stress suppresses DNA double‐strand break (DSB) repair and increases radiosensitivity of tumor cells by altering Rad51 levels. We show that the ER stress inducer tunicamycin stimulates selective degradation of Rad51 via the 26S proteasome, impairing DSB repair and enhancing radiosensitivity in human lung cancer A549 cells. We also found that glucose deprivation, which is a physiological inducer of ER stress, triggered similar events. These findings suggest that ER stress caused by the intratumoral environment influences tumor radiosensitivity, and that it has potential as a novel target to improve cancer radiotherapy.

Radiosensitization of tumor cells through endoplasmic reticulum stress induced by PEGylated nanogel containing gold nanoparticles

High atomic number molecules, such as gold and platinum, are known to enhance the biological effect of X-irradiation. This study was aimed to determine the radiosensitizing potential of PEGylated nanogel containing gold nanoparticles (GNG) and the cellular mechanism involved. GNG pretreatment increased the levels of reproductive cell death and apoptosis induced by X-irradiation. GNG accumulated in cytoplasm and increased the expression of endoplasmic reticulum (ER) stress-related protein. GNG suppressed the repair capacity of DNA after X-irradiation by down-regulating DNA repair-related proteins. Our results suggest that GNG radiosensitized cells by enhancing apoptosis and impairing DNA repair capacity via ER stress induction.

ErbB2 overexpression upregulates antioxidant enzymes, reduces basal levels of reactive oxygen species, and protects against doxorubicin cardiotoxicity

Levels of the HER2/ErbB2 protein in the heart are upregulated in some women during breast cancer therapy, and these women are at high risk for developing heart dysfunction after sequential treatment with anti-ErbB2/trastuzumab or doxorubicin. Doxorubicin is known to increase oxidative stress in the heart, and thus we considered the possibility that ErbB2 protein influences the status of cardiac antioxidant defenses in cardiomyocytes. In this study, we measured reactive oxygen species (ROS) in cardiac mitochondria and whole hearts from mice with cardiac-specific overexpression of ErbB2 (ErbB2(tg)) and found that, compared with control mice, high levels of ErbB2 in myocardium result in lower levels of ROS in mitochondria (P = 0.0075) and whole hearts (P = 0.0381). Neonatal cardiomyocytes isolated from ErbB2(tg) hearts have lower ROS levels and less cellular death (P < 0.0001) following doxorubicin treatment. Analyzing antioxidant enzyme levels and activities, we found that ErbB2(tg) hearts have increased levels of glutathione peroxidase 1 (GPx1) protein (P < 0.0001) and GPx activity (P = 0.0031) in addition to increased levels of two known GPx activators, c-Abl (P = 0.0284) and Arg (P < 0.0001). Interestingly, although mitochondrial ROS emission is reduced in the ErbB2(tg) hearts, oxygen consumption rates and complex I activity are similar to control littermates. Compared with these in vivo studies, H9c2 cells transfected with ErbB2 showed less cellular toxicity and produced less ROS (P < 0.0001) after doxorubicin treatment but upregulated GR activity (P = 0.0237) instead of GPx. Our study shows that ErbB2-dependent signaling contributes to antioxidant defenses and suggests a novel mechanism by which anticancer therapies involving ErbB2 antagonists can harm myocardial structure and function.

Radiation-induced nitric oxide mitigates tumor hypoxia and radioresistance in a murine SCCVII tumor model

Tumor hypoxia, which occurs mainly as a result of inadequate tissue perfusion in solid tumors, is a well-known challenge for successful radiotherapy. Recent evidence suggests that ionizing radiation (IR) upregulates nitric oxide (NO) production and that IR-induced NO has the potential to increase intratumoral circulation. However, the kinetics of NO production and the responsible isoforms for NO synthase in tumors exposed to IR remain unclear. In this study, we aimed to elucidate the mechanism by which IR stimulates NO production in tumors and the effect of IR-induced NO on tumor radiosensitivity. Hoechst33342 perfusion assay and electron spin resonance oxymetry showed that IR increased tissue perfusion and pO2 in tumor tissue. Immunohistochemical analysis using two different hypoxic probes showed that IR decreased hypoxic regions in tumors; treatment with a nitric oxide synthase (NOS) inhibitor, L-NAME, abrogated the effects of IR. Moreover, IR increased endothelial NOS (eNOS) activity without affecting its mRNA or protein expression levels in SCCVII-transplanted tumors. Tumor growth delay assay showed that L-NAME decreased the anti-tumor effect of fractionated radiation (10Gy×2). These results suggested that IR increased eNOS activity and subsequent tissue perfusion in tumors. Increases in intratumoral circulation simultaneously decreased tumor hypoxia. As a result, IR-induced NO increased tumor radiosensitivity. Our study provides a new insight into the NO-dependent mechanism for efficient fractionated radiotherapy.

Inhibition of the mitochondrial fission protein dynamin-related protein 1 (Drp1) impairs mitochondrial fission and mitotic catastrophe after x-irradiation

The role of mitochondrial dynamics in cellular responses to ionizing radiation (IR) is still largely unknown. This study demonstrates that IR triggers Drp1-dependent mitochondrial fission and that Drp1 inhibition attenuates radiation-induced mitotic catastrophe, suggesting that Drp1 is involved in determining the fate of cells after irradiation.

In vivo tumour extracellular pH monitoring using electron paramagnetic resonance: the effect of X-ray irradiation

The in vivo quantification of extracellular pH (pHe) in tumours may provide a useful biomarker for tumour cell metabolism. In this study, we assessed the viability of continuous‐wave electron paramagnetic resonance (CW‐EPR) spectroscopy with a pH‐sensitive nitroxide for the measurement of extracellular tumour pH in a mouse model. CW‐EPR spectroscopy (750 MHz) of C3H HeJ mice hind leg squamous cell tumour was performed after intravenous tail vein injection of pH‐sensitive nitroxide (R‐SG, 2‐(4‐((2‐(4‐amino‐4‐carboxybutanamido)‐3‐(carboxymethylamino)‐3‐oxoproylthio)methyl)phenyl)‐4‐pyrrolidino‐2,5,5‐triethyl‐2,5‐dihydro‐1Н‐imidazol‐1‐oxyl) during stages of normal tumour growth and in response to a single 10‐Gy dose of X‐ray irradiation. An inverse relationship was observed between tumour volume and pHe value, whereby, during normal tumour growth, a constant reduction in pHe was observed. This relationship was disrupted by X‐ray irradiation and, from 2–3 days post‐exposure, a transitory increase in pHe was observed. In this study, we demonstrated the viability of CW‐EPR spectroscopy using R‐SG nitroxide to obtain high‐sensitivity pH measurements in a mouse tumour model with an accuracy of <0.1 pH units. In addition, the measured changes in pHe in response to X‐ray irradiation suggest that this may offer a useful method for the assessment of the physiological change in response to existing and novel cancer therapies. Copyright

Evaluation of the relative biological effectiveness of spot-scanning proton irradiation in vitro

Variations in relative biological effectiveness (RBE) from a fixed value of 1.1 are critical in proton beam therapy. To date, studies estimating RBE at multiple positions relative to the spread-out Bragg peak (SOBP) have been predominantly performed using passive scattering methods, and limited data are available for spot-scanning beams. Thus, to investigate the RBE of spot-scanning beams, Chinese hamster fibroblast V79 cells were irradiated using the beam line at the Hokkaido University Hospital Proton Therapy Center. Cells were placed at six different depths, including the entrance of the proton beam and the proximal and distal part of the SOBP. Surviving cell fractions were analyzed using colony formation assay, and cell survival curves were obtained by the curve fitted using a linear–quadratic model. RBE10 and RBE37 were 1.15 and 1.21 at the center of the SOBP, respectively. In contrast, the distal region showed higher RBE values (1.50 for RBE10 and 1.85 for RBE37). These results are in line with those of previous studies conducted using passive scattering proton beams. Taken together, these data strongly suggest that variations in RBE should be considered during treatment planning for spot-scanning beams as well as for passive scattering proton beams.

Activation of eNOS in endothelial cells exposed to ionizing radiation involves components of the DNA damage response pathway

In this study, the involvement of ataxia telangiectasia mutated (ATM) kinase and heat shock protein 90 (HSP90) in endothelial nitric oxide synthase (eNOS) activation was investigated in X-irradiated bovine aortic endothelial cells. The activity of nitric oxide synthase (NOS) and the phosphorylation of serine 1179 of eNOS (eNOS-Ser1179) were significantly increased in irradiated cells. The radiation-induced increases in NOS activity and eNOS-Ser1179 phosphorylation levels were significantly reduced by treatment with either an ATM inhibitor (Ku-60019) or an HSP90 inhibitor (geldanamycin). Geldanamycin was furthermore found to suppress the radiation-induced phosphorylation of ATM-Ser1181. Our results indicate that the radiation-induced eNOS activation in bovine aortic endothelial cells is regulated by ATM and HSP90.

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

BackgroundGlioblastoma 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).MethodsReproductive 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 [14C]-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.ResultsDoranidazole 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 [14C]-doranidazole specifically penetrated the tumor regions. Combined treatment with X-irradiation and doranidazole significantly inhibited the growth of C6 gliomas.ConclusionsOur 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.

3-Methyl pyruvate enhances radiosensitivity through increasing mitochondria-derived reactive oxygen species in tumor cell lines

Considerable interest has recently been focused on the special characteristics of cancer metabolism, and several drugs designed to modulate cancer metabolism have been tested as potential anticancer agents. To date, however, very few studies have been conducted to investigate the combined effects of anticancer drugs and radiotherapy. In this study, to evaluate the role of mitochondria-derived reactive oxygen species (ROS) in the radiation-induced cell death of tumor cells, we have examined the effect of 3-methyl pyruvate (MP). MP is a membrane-permeable pyruvate derivative that is capable of activating mitochondrial energy metabolism in human lung carcinoma A549 cells and murine squamous carcinoma SCCVII cells. Pretreatment with MP significantly enhanced radiation-induced cell death in both cell lines, and also led to increases in the mitochondrial membrane potential, intracellular adenosine triphosphate content, and mitochondria-derived ROS production following the exposure of the cells to X-rays. In A549 cells, MP-induced radiosensitization was completely abolished by vitamin C. In contrast, it was partially abolished in SCCVII cells. These results therefore suggest that the treatment of the cells with MP induced radiosensitization via the production of excess mitochondria-derived ROS in tumor cells.