Tetsuo Nonaka

Heavy‐ion‐induced bystander killing of human lung cancer cells: Role of gap junctional intercellular communication

The aim of the present study was to clarify the mechanisms of cell death induced by heavy‐ion irradiation focusing on the bystander effect in human lung cancer A549 cells. In microbeam irradiation, each of 1, 5, and 25 cells under confluent cell conditions was irradiated with 1, 5, or 10 particles of carbon ions (220 MeV), and then the surviving fraction of the population was measured by a clonogenic assay in order to investigate the bystander effect of heavy‐ions. In this experiment, the limited number of cells (0.0001–0.002%, 5–25 cells) under confluent cell conditions irradiated with 5 or 10 carbon ions resulted in an exaggerated 8–14% increase in cell death by clonogenic assay. However, these overshooting responses were not observed under exponentially growing cell conditions. Furthermore, these responses were inhibited in cells treated with an inhibitor of gap junctional intercellular communication (GJIC), whereas they were markedly enhanced by the addition of a stimulator of GJIC. The present results suggest that bystander cell killing by heavy‐ions was induced mainly by direct cell‐to‐cell communication, such as GJIC, which might play important roles in bystander responses. (Cancer Sci 2009; 100: 684–688)

Heat shock protein 90 (Hsp90) chaperone complex inhibitor, radicicol, potentiated radiation-induced cell killing in a hormone-sensitive prostate cancer cell line through degradation of the androgen receptor.

Until now, there has not been enough information on how androgens or androgen deprivation may influence the response of cancer cells to radiation. In this study, the effect of dihydrotestosterone (DHT) on cellular proliferative activity and radiosensitivity was examined in a hormone-sensitive human prostate cancer cell line, LNCaP. In addition, the study also examined how a heat shock protein 90 (Hsp90) chaperone complex inhibitor modified the effect of DHT on the radiosensitivity of the cells, because binding of the androgen receptor (AR) to Hsp90 is required to maintain the stability and functioning of AR. The hormone-sensitive human prostate cancer cell line, LNCaP, was used. Radicicol was used as one of the known Hsp90 chaperone complex inhibitors, and the cells were incubated in the presence of this compound at a concentration of 500 nM. Cellular radiosensitivity was determined by the clonogenic assay; the changes in the protein expression were examined by Western blotting or immunofluorescence. DHT at a concentration of 1 nM caused enhancement of the proliferative activity and reduction of the radiosensitivity of the cells. Radicicol at a concentration of 500 nM abolished the DHT-induced decrease in cellular radiosensitivity and potentiated the radiation-induced cell killing synergistically. Consistent with the changes in the cellular radiosensitivity, radicicol degraded AR, Raf-1 and HER2/neu via reduced binding of AR to Hsp90, although selective degradation of HER2/neu caused by Herceptin, a monoclonal antibody against HER2, did not affect the cellular radiosensitivity. The results suggest that the Hsp90 chaperone complex may be a potential molecular target for potentiation of radiation-induced cell killing in a hormone-sensitive prostate cancer cell line.

Rapid rise in FDG uptake in an irradiated human tumour xenograft

In order to investigate early changes in the glucose metabolism of irradiated tumours, tumour uptake of 2-[18F]fluoro-2-deoxy-d-glucose (18FDG) was studied in human tumour xenografts. Three human tumour lines [ependymoblastoma (NNE), small cell lung cancer (GLS), and glioblastoma (KYG)] showing different radiosensitivities and incidences of radiation-induced apoptosis were subcutaneously transplanted into nude mice, and were irradiated at a single dose of 10 Gy. Then 0.5 mCi of18FDG was intravenously administered 1 h before sacrifice. The animals were sacrificed at 2, 4 and 6 h following irradiation, and18FDG accumulation in the tumours was examined. Before irradiation, GLS and KYG tumours showed significantly higher rates of18FDG accumulation compared with NNE tumours (P <0.004 andP <0.001, respectively). NNE (the most radiosensitive tumour with the highest incidence of radiation-induced apoptosis), however, displayed a 2.3-fold higher rate of18FDG accumulation at 2 h following irradiation compared with a non-irradiated group (P <0.01), and thereafter showed a plateau up to 6 h. The accumulation did not increase significantly in the other tumours with lower radiosensitivity and much less radiation-induced apoptosis. The rapidity of the increase in18FDG accumulation in the most radiosensitive tumour line, occurring as early as 2 h following irradiation, suggests that the increase was independent of recovery phenomena following radiation damage.

Radiation therapy alone for stage I (UICC T1N0M0) squamous cell carcinoma of the esophagus: Indications for surgery or combined chemoradiotherapy

Background and Aim:  The aim of this study was to clarify the efficacy and limitations of radiation therapy (RT) for superficial esophageal carcinoma, and to explore the indications for more aggressive therapy, such as combined chemo‐radiotherapy.

Dose-Volume Histogram Parameters and Clinical Factors Associated With Pleural Effusion After Chemoradiotherapy in Esophageal Cancer Patients

PURPOSE To investigate the dose-volume histogram parameters and clinical factors as predictors of pleural effusion in esophageal cancer patients treated with concurrent chemoradiotherapy (CRT). METHODS AND MATERIALS Forty-three esophageal cancer patients treated with definitive CRT from January 2001 to March 2007 were reviewed retrospectively on the basis of the following criteria: pathologically confirmed esophageal cancer, available computed tomography scan for treatment planning, 6-month follow-up after CRT, and radiation dose ≥ 50 Gy. Exclusion criteria were lung metastasis, malignant pleural effusion, and surgery. Mean heart dose, mean total lung dose, and percentages of heart or total lung volume receiving ≥ 10-60 Gy (Heart-V(10) to V(60) and Lung-V(10) to V(60), respectively) were analyzed in relation to pleural effusion. RESULTS The median follow-up time was 26.9 months (range, 6.7-70.2) after CRT. Of the 43 patients, 15 (35%) developed pleural effusion. By univariate analysis, mean heart dose, Heart-V(10) to V(60), and Lung-V(50) to V(60) were significantly associated with pleural effusion. Poor performance status, primary tumor of the distal esophagus, and age ≥ 65 years were significantly related with pleural effusion. Multivariate analysis identified Heart-V(50) as the strongest predictive factor for pleural effusion (p = 0.01). Patients with Heart-V(50) <20%, 20%≤ Heart-V(50) <40%, and Heart-V(50) ≥ 40% had 6%, 44%, and 64% of pleural effusion, respectively (p < 0.01). CONCLUSION Heart-V(50) is a useful parameter for assessing the risk of pleural effusion and should be reduced to avoid pleural effusion.

The synchronization of chemotherapy to circadian rhythms and irradiation in pre-operative chemoradiation therapy with hyperthermia for local advanced rectal cancer.

Purpose: The therapeutic and adverse effects of pre-operative chrono-chemoradiation with local hyperthermia for patients with rectal adenocarcinoma were evaluated. Materials and methods: Pre-operative radiation therapy of a total dose of 40 Gy (n = 10) or 50 Gy (n = 19) on the whole pelvis and hyperthermia once a week during the radiation therapy for 1 h were performed for patients with T2–T4 rectal adenocarcinoma. Chemotherapy consisted of 5-FU (250 mg m−2 per day) and LV (25 mg m−2 per day) administered by continuous infusion in the night for 5 days a week in the second and fourth weeks of radiation. Results: Grade 3+ toxicities were seen only in two patients (6.9%). A significant down staging was seen in 41.4% of all cases and 52.6% of cases with a radiation dose of 50 Gy. Of the patients who had received surgical resection of a tumour, three (11.1%) had no residue pathologically in the specimen and eight (29.6%) had microscopic lesions. Conclusions: These results yielded a high response rate with minimal toxicities for advanced low-rectal adenocarcinoma. The administration of 5-FU during the sleeping time before irradiation might have an advantage not only as a chronotherapy but also as a radiation sensitizer.

Usefulness of Intraluminal Brachytherapy Combined With External Beam Radiation Therapy for Submucosal Esophageal Cancer: Long-Term Follow-Up Results

PURPOSE To assess the efficacy of radiation therapy (RT) by using intraluminal brachytherapy (IBT) combined with external beam RT (EBRT) for submucosal esophageal cancer. METHODS AND MATERIALS Between 1991 and 2005, 59 consecutive patients received definitive RT without chemotherapy. IBT was performed after patients completed EBRT as a booster therapy for 17 patients, using low-dose-rate Cs-137 sources until 1997, and for 19 patients, using high-dose-rate Ir-192 sources thereafter. The long-term outcomes were investigated with a median follow-up time of 61 months. RESULTS Logoregional recurrences and distant metastases were observed in 14 patients and in 2 patients in the lung, respectively, and 5 patients were rescued by salvage treatments. The 5-year logoregional control and cause-specific survival rates were 75% and 76%, respectively. The 5-year cause-specific survival rate in the EBRT group was 62%, whereas the corresponding rate in the IBT group was 86% (p = 0.04). Multivariate analysis revealed that IBT was the most powerful predictor of survival but did not reach a significant level (p = 0.07). There were five esophageal ulcers in the IBT group, but no ulcers developed with small fractions of 3 Gy. Grade 2 or higher cardiorespiratory complications developed in 2 patients (5.6%) in the IBT group and in 3 patients (13.0%) in the EBRT group. CONCLUSIONS Combining IBT with EBRT is suggested to be one of the preferable treatment modalities for medically inoperable submucosal esophageal cancer because of its preferable local control and survival probabilities, with appreciably less morbidity.

Effect of histologic type on recurrence pattern in radiation therapy for medically inoperable patients with stage I non-small-cell lung cancer.

Japanese randomized trials showed that there was a significant impact on survival from stage I adenocarcinoma (AD) of the lung by adjuvant chemotherapy with uracil-tegaful after complete resection but there was no effect for patients with squamous cell carcinoma (SQ). The purpose of this study was to examine the correlation of tumor histology and clinical outcome of radiation therapy (RT) for stage I non-small-cell lung cancer (NSCLC) and to consider the necessity of adjuvant chemotherapy after RT for these patients. The subjects were 83 patients, 54 with SQ and 29 with AD; they had received definitive RT with the total dose ranging from 60 to 80 Gy with conventional fractionation at a daily dose of 2 Gy. The differences between SQ and AD with respect to survival and recurrence pattern were investigated. The 5-year overall survival and cause-specific survival rates were 26.5% and 49.1%, respectively. No difference in survival was observed between SQ and AD patients, and the recurrence rates were almost identical (44% for SQ and 45% for AD). However, the 5-year primary control rate of SQ was significantly poorer than that of AD (SQ: 61.5%; AD: 87.6%; p = 0.03). Conversely, the 5-year metastasis-free survival rate of SQ was significantly better than that of AD (SQ: 88.2%; AD: 53.0%; p = 0.005). The different failure pattern, according to tumor histology, indicates that taking into consideration the difference in their clinical behaviors would also be important for planning RT and surgery for early lung cancer.

Attenuation of chronic thermotolerance by KNK437, a benzylidene lactam compound, enhances thermal radiosensitization in mild temperature hyperthermia combined with low dose-rate irradiation.

Purpose: We investigated whether the attenuation of chronic thermotolerance by KNK437, a heat shock protein inhibitor, can modify the effect of thermal radiosensitization in mild temperature hyperthermia (MTH) combined with low dose-rate irradiation (LDRI). Materials and methods: The human lung adenocarcinoma cell line A549 was simultaneously exposed to LDRI with MTH at 41°C and KNK437 at a dose of 100 μM. Cell survival was estimated by a clonogenic assay. Cell cycle change during treatment was analyzed by flow cytometry. Expression levels of the heat shock proteins hsp72, hsp27 and heat shock factor 1 (HSF-1) were measured by Western blotting. Results: KNK437 inhibited the expression of inducible hsp72 and hsp27, but produced no change in the mobility shift of HSF-1. The cytotoxicity of LDRI was enhanced by MTH. The survival curve for LDRI + MTH revealed no development of chronic thermotolerance up to 48 h. Simultaneous LDRI and KNK437 treatment also resulted in enhanced cell killing. The radiosensitizing effect of KNK437 was enhanced by simultaneous exposure of the cells to MTH. Flow cytometry analysis of cell cycle progression demonstrated marked G2 arrest and mild G1 arrest with LDRI alone, but mild G1 arrest with MTH alone, and mild G2-M, S-phase accumulation with KNK437 alone. The marked G2 arrest caused by LDRI was partially suppressed by the addition of MTH, and was also suppressed by KNK437 treatment. Conclusions: Exposure of A549 cells to KNK437 caused inhibition of hsp72 and hsp27 expression. The addition of KNK437 increased not only thermosensitivity to MTH, but also radiosensitivity to LDRI. KNK437 also enhanced the MTH-induced radiosensitization under these experimental conditions.

Radicicol potentiates heat-induced cell killing in a human oesophageal cancer cell line: the Hsp90 chaperone complex as a new molecular target for enhancement of thermosensitivity

Purpose: To examine the ability of a heat shock protein 90 (Hsp90) chaperone complex inhibitor, radicicol, to modify thermal response and heat‐induced cell killing, and to clarify the underlining mechanisms. Materials and methods: A human oesophageal cancer cell line (TE‐1), with a mutant p53 gene, was used. To examine the effect of radicicol on heat‐induced cell killing, radicicol at a concentration of 100 nM was incubated with the cells for 7 h during heat treatment. Changes in the expression of proteins were examined by Western blot and immunofluorescence analysis. Results: Radicicol in combination with heat synergistically potentiated heat‐induced cellular killing despite an increase in the expression of Hsp72 and Hsp27 caused by radicicol. Heat alone activated Raf‐1 and p42/p44 extracellular signal‐regulated kinase (Erk), and heat in combination with radicicol inhibited the activation of Raf‐1 and p42/p44 Erk through reduced binding of Raf‐1 to Hsp90. Phosphorylation of Akt was also decreased by radicicol. Conclusions: The Hsp90 chaperone complex inhibitor, radicicol, potentiated heat‐induced cellular killing, and inhibition of p42/p44 Erk and Akt activation rather than modification of Hsp expression might be involved in enhancing cellular thermosensitivity. Results suggest that the Hsp90 chaperone complex could be a new molecular target for the modification of the cellular response to heat.