Nobuteru Kubo

C646, a selective small molecule inhibitor of histone acetyltransferase p300, radiosensitizes lung cancer cells by enhancing mitotic catastrophe

BACKGROUND AND PURPOSE Chromatin remodeling through histone modifications, including acetylation, plays an important role in the appropriate response to DNA damage induced by ionizing radiation (IR). Here we investigated the radiosensitizing effect of C646, a selective small molecule inhibitor of p300 histone acetyltransferase, and explored the underlying mechanisms. MATERIALS AND METHODS A549, H157 and H460 human non-small cell lung carcinoma (NSCLC) cells, and HFL-III human lung fibroblasts were assessed by clonogenic survival assay. Apoptosis and necrosis were assessed by annexin V staining. Senescence was assessed by Senescence-associated β-galactosidase staining. Mitotic catastrophe was assessed by evaluating nuclear morphology with DAPI staining. Cell cycle profiles were analyzed by flow cytometry. Protein expression was analyzed by immunoblotting. RESULTS C646 sensitized A549, H460 and H157 cells to IR with a dose enhancement ratio at 10% surviving fraction of 1.4, 1.2 and 1.2, respectively. C646 did not radiosensitize HFL-III cells. In A549 cells, but not in HFL-III cells, C646 (i) enhanced mitotic catastrophe but not apoptosis, necrosis, or senescence after IR; (ii) increased the hyperploid cell population after IR; and (iii) suppressed the phosphorylation of CHK1 after IR. CONCLUSIONS C646 radiosensitizes NSCLC cells by enhancing mitotic catastrophe through the abrogation of G2 checkpoint maintenance.

Radiosensitizing effect of carboplatin and paclitaxel to carbon-ion beam irradiation in the non-small-cell lung cancer cell line H460

Abstract The present study investigated the ability of carboplatin and paclitaxel to sensitize human non-small-cell lung cancer (NSCLC) cells to carbon-ion beam irradiation. NSCLC H460 cells treated with carboplatin or paclitaxel were irradiated with X-rays or carbon-ion beams, and radiosensitivity was evaluated by clonogenic survival assay. Cell proliferation was determined by counting the number of viable cells using Trypan blue. Apoptosis and senescence were evaluated by terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) staining and senescence-associated β-galactosidase (SA-β-gal) staining, respectively. The expression of cleaved caspase-3, Bax, p53 and p21 was analyzed by western blotting. Clonogenic survival assays demonstrated a synergistic radiosensitizing effect of carboplatin and paclitaxel with carbon-ion beams; the sensitizer enhancement ratios (SERs) at the dose giving a 10% survival fraction (D10) were 1.21 and 1.22, respectively. Similarly, carboplatin and paclitaxel showed a radiosensitizing effect with X-rays; the SERs were 1.41 and 1.29, respectively. Cell proliferation assays validated the radiosensitizing effect of carboplatin and paclitaxel with both carbon-ion beam and X-ray irradiation. Carboplatin and paclitaxel treatment combined with carbon-ion beams increased TUNEL-positive cells and the expression of cleaved caspase-3 and Bax, indicating the enhancement of apoptosis. The combined treatment also increased SA-β-gal-positive cells and the expression of p53 and p21, indicating the enhancement of senescence. In summary, carboplatin and paclitaxel radiosensitized H460 cells to carbon-ion beam irradiation by enhancing irradiation-induced apoptosis and senescence.

Evaluation of therapeutic gain for fractionated carbon-ion radiotherapy using the tumor growth delay and crypt survival assays

BACKGROUND AND PURPOSE The aim of the study was to evaluate the therapeutic gain of carbon ion (C-ion) radiotherapy using a mouse model. MATERIALS AND METHODS Transplanted fibrosarcoma (NFSa) growing in C3H/He mice and murine small intestine were irradiated with 290 MeV/nucleon C-ion beams (C-ions) in 1-12 fractions separated by 4h. The cell killing efficiencies of C-ions were measured using jejunum crypt survival and tumor growth delay (TGD) assays. RESULTS The equieffect dose for crypt survival and TGD increased with increasing number of fractions after X-rays and 20 keV/μm C-ions, whereas TGD after 77 keV/μm C-ions rather decreased. Crypts showed stronger LET-dependent increase in α terms than the tumor while β terms less depended on LET irrespective of tissues. Therapeutic gain factor, i.e., a ratio of tumor RBE over crypt RBE, of 77 keV/μm C-ions was more than unity at any doses while that of 20 keV/μm C-ions increased with an increase in dose per fraction. CONCLUSIONS These specific data imply that use of large dose per fraction would be suitable for C-ion radiotherapy irrespective of LET from the point of view of therapeutic gain, though small dose per fraction by high-LET radiation decreases total dose for tumor.

Dosimetric comparison of carbon ion and X-ray radiotherapy for Stage IIIA non–small cell lung cancer

The present study compared the dose–volume histograms of patients with Stage IIIA non–small cell lung cancer (NSCLC) treated with carbon ion radiotherapy with those of patients treated with X-ray radiotherapy. Patients with Stage IIIA NSCLC (n = 10 patients for each approach) were enrolled. Both radiotherapy plans were calculated with the same targets and organs at risk on the same CT. The treatment plan for the prophylactic lymph node and primary tumor (PTV1) delivered 40 Gy for X-ray radiotherapy and 40 Gy (relative biological effectiveness; RBE) for carbon ion radiotherapy. The total doses for the primary tumor and clinically positive lymph nodes (PTV2) were 60 Gy for X-ray radiotherapy and 60 Gy (RBE) for carbon ion radiotherapy. The homogeneity indexes for PTV1 and PTV2 were superior for carbon ion radiotherapy in comparison with X-ray radiotherapy (PTV1, 0.57 vs 0.65, P = 0.009; PTV2, 0.07 vs 0.16, P = 0.005). The normal lung mean dose, V5, V10 and V20 for carbon ion radiotherapy were 7.7 Gy (RBE), 21.4%, 19.7% and 17.0%, respectively, whereas the corresponding doses for X-ray radiotherapy were 11.9 Gy, 34.9%, 26.6% and 20.8%, respectively. Maximum spinal cord dose, esophageal maximum dose and V50, and bone V10, V30 and V50 were lower with carbon ion radiotherapy than with X-ray radiotherapy. The present study indicates that carbon ion radiotherapy provides a more homogeneous target dose and a lower dose to organs at risk than X-ray radiotherapy for Stage IIIA non–small cell lung cancer.

Dose–volume histogram parameters of high-dose-rate brachytherapy for Stage I–II cervical cancer (≤4cm) arising from a small-sized uterus treated with a point A dose-reduced plan

We investigated the rectal dose-sparing effect and tumor control of a point A dose-reduced plan in patients with Stage I–II cervical cancer (≤4 cm) arising from a small-sized uterus. Between October 2008 and August 2011, 19 patients with Stage I–II cervical cancer (≤4 cm) were treated with external beam radiotherapy (EBRT) for the pelvis and CT-guided brachytherapy. Seven patients were treated with brachytherapy with standard loading of source-dwell positions and a fraction dose of 6 Gy at point A (conventional brachy-plan). The other 12 patients with a small uterus close to the rectum or small intestine were treated with brachytherapy with a point A dose-reduction to match D2cc of the rectum and <6 Gy as the dose constraint (‘point A dose-reduced plan’) instead of the 6-Gy plan at point A (‘tentative 6-Gy plan’). The total doses from EBRT and brachytherapy were added up and normalized to a biological equivalent dose of 2 Gy per fraction (EQD2). The median doses to the high-risk clinical target volume (HR-CTV) D90 in the conventional brachy-plan, tentative 6-Gy plan and point A dose-reduced plan were 62 GyEQD2, 80 GyEQD2 and 64 GyEQD2, respectively. The median doses of rectal D2cc in the corresponding three plans were 42 GyEQD2, 62 GyEQD2 and 51 GyEQD2, respectively. With a median follow-up period of 35 months, three patients developed Grade-1 late rectal complications and no patients developed local recurrence. Our preliminary results suggested that CT-guided brachytherapy using an individualized point A dose-reduced plan might be useful for reducing late rectal complications while maintaining primary tumor control.

Changes in Rectal Dose Due to Alterations in Beam Angles for Setup Uncertainty and Range Uncertainty in Carbon-Ion Radiotherapy for Prostate Cancer

Background and Purpose Carbon-ion radiotherapy of prostate cancer is challenging in patients with metal implants in one or both hips. Problems can be circumvented by using fields at oblique angles. To evaluate the influence of setup and range uncertainties accompanying oblique field angles, we calculated rectal dose changes with oblique orthogonal field angles, using a device with fixed fields at 0° and 90° and a rotating patient couch. Material and Methods Dose distributions were calculated at the standard angles of 0° and 90°, and then at 30° and 60°. Setup uncertainty was simulated with changes from −2 mm to +2 mm for fields in the anterior-posterior, left-right, and cranial-caudal directions, and dose changes from range uncertainty were calculated with a 1 mm water-equivalent path length added to the target isocenter in each angle. The dose distributions regarding the passive irradiation method were calculated using the K2 dose algorithm. Results The rectal volumes with 0°, 30°, 60°, and 90° field angles at 95% of the prescription dose were 3.4±0.9 cm3, 2.8±1.1 cm3, 2.2±0.8 cm3, and 3.8±1.1 cm3, respectively. As compared with 90° fields, 30° and 60° fields had significant advantages regarding setup uncertainty and significant disadvantages regarding range uncertainty, but were not significantly different from the 90° field setup and range uncertainties. Conclusions The setup and range uncertainties calculated at 30° and 60° field angles were not associated with a significant change in rectal dose relative to those at 90°.

Solitary cardiac metastasis of uterine cervical cancer with antemortem diagnosis: A case report and literature review

Cardiac metastasis of uterine cervical cancer with antemortem diagnosis is extremely rare. Therefore, its landscape epidemiology has not been well elucidated to date. In the present study, a case of solitary cardiac metastasis of uterine cervical cancer diagnosed antemortem is reported, and a review of the currently available literature (which includes 18 cases of cardiac metastasis of uterine cervical cancer) is conducted. In January 2013, a 78-year-old woman with squamous cell carcinoma (SCC) of the uterine cervix (International Federation of Gynecology and Obstetrics stage IIIb) underwent definitive radiotherapy at Gunma University Hospital (Gunma, Japan). Follow-up examination at 5 months after completion of the treatment indicated no evidence of recurrence or metastasis. In April 2014, the patient reported epigastric discomfort and general malaise. Electrocardiogram suggested myocardial dysfunction. Transthoracic echocardiography revealed the presence of a mass occupying the right ventricle and pericardial effusion. Cine magnetic resonance imaging demonstrated a filling defect in the right ventricle, and transcatheter biopsy confirmed SCC. The patient was diagnosed with a solitary cardiac metastasis of uterine cervical cancer. Despite aggressive medical therapy, the patient succumbed to disease 31 days after admission to hospital. A review of the current literature revealed that 84% of cases of cardiac metastasis develop within 2 years of completion of the initial treatment, and that electrocardiogram and echocardiography reveal findings of myocardial dysfunction and the presence of a mass in the right ventricle, respectively. A treatment strategy for cardiac metastasis of uterine cervical cancer has not been standardized thus far, and the prognosis is very poor, as the majority of patients succumbed to disease within 1 year. In summary, the current case and the literature review conducted in the present study suggest that: i) Cardiac metastasis should be included in the differential diagnosis in cases with nonspecific complaints such as epigastric discomfort and general malaise when patients have a history of uterine cervical cancer, particularly within the previous 2 years; and ii) electrocardiogram and echocardiography are convenient and effective modalities for the diagnosis of cardiac metastasis of uterine cervical cancer.

Interfractional change of high-risk CTV D90 during image-guided brachytherapy for uterine cervical cancer

The purpose of this study was to evaluate interfractional changes of the minimum dose delivered to 90% of the high-risk clinical target volume (HR-CTV D90) and D2cc of the bladder and rectum during brachytherapy for uterine cervical cancer patients. A total of 52 patients received external beam radiotherapy and high-dose-rate intracavitary brachytherapy (ICBT). For each of four ICBT applications, a pelvic CT scan was performed and the HR-CTV was delineated. Retrospectively, these patients were divided into two groups: (i) the standard dose group with 6 Gy to point A in each ICBT, and (ii) the adaptive dose group with a modified dose to point A to cover the HR-CTV with the 6-Gy isodose line as much as possible. The HR-CTV D90 was assessed in every session, and analyzed as interfractional changes. In the standard dose group, the interfractional changes of the HR-CTV D90 showed a linear increase from the first to the third of the four ICBT (average 6.1, 6.6, 7.0 and 7.1 Gy, respectively). In contrast, those of the adaptive dose group remained almost constant (average 7.2, 7.2, 7.3 and 7.4 Gy, respectively). Especially, in the case of a large HR-CTV volume (≥35 cm3) at first ICBT, the total HR-CTV D90 of the adaptive dose group with brachytherapy was significantly higher than that of the standard dose group. There were no significant differences in total D2cc in bladder and rectum between the two groups. Image-guided adaptive brachytherapy based on interfractional tumor volume change improves the dose to the HR-CTV while keeping rectal and bladder doses within acceptable levels.

Mutational analysis of uterine cervical cancer that survived multiple rounds of radiotherapy

Radiotherapy is an essential component of cancer therapy. Despite advances in cancer genomics, the mutation signatures of radioresistant tumors have not yet been fully elucidated. To address this issue, we analyzed a unique set of clinical specimens from a uterine cervical cancer that repeatedly locally recurred after multiple rounds of radiotherapy. Exon sequencing of 409 cancer-related genes in the treatment-naïve tumor and the tumors that recurred after initial and secondary radiotherapy identified (i) activating mutations in PIK3CA and KRAS, and putative inactivating mutations in SMAD4, as trunk mutation signatures that persisted over the clinical course; and (ii) mutations in KMT2A, TET1, and NLRP1 as acquired mutation signatures observed only in recurrent tumors after radiotherapy. Comprehensive mining of published in vitro genomics data pertaining to radiosensitivity revealed that simultaneous mutations in KRAS and SMAD4, which have not been described previously in uterine cervical cancer, are associated with cancer cell radioresistance. The association between this mutation signature and radioresistance was validated by isogenic cell-based experiments. These results provide proof-of-principle for the analytical pipeline employed in this study, which explores clinically relevant mutation signatures for radioresistance, and demonstrate that this approach is worth pursuing with larger cohorts in the future.