Kaori Imadome

Expression profiles are different in carbon ion-irradiated normal human fibroblasts and their bystander cells

Evidence has accumulated that ionizing radiation induces biological effects in non-irradiated bystander cells having received signals from directly irradiated cells; however, energetic heavy ion-induced bystander response is incompletely characterized. Here we performed microarray analysis of irradiated and bystander fibroblasts in confluent cultures. To see the effects in bystander cells, each of 1, 5 and 25 sites was targeted with 10 particles of carbon ions (18.3 MeV/u, 103 keV/microm) using microbeams, where particles traversed 0.00026, 0.0013 and 0.0066% of cells, respectively. diated cells, cultures were exposed to 10% survival dose (D), 0.1D and 0.01D of corresponding broadbeams (108 keV/microm). Irrespective of the target numbers (1, 5 or 25 sites) and the time (2 or 6h postirradiation), similar expression changes were observed in bystander cells. Among 874 probes that showed more than 1.5-fold changes in bystander cells, 25% were upregulated and the remainder downregulated. These included genes related to cell communication (PIK3C2A, GNA13, FN1, ANXA1 and IL1RAP), stress response (RAD23B, ATF4 and EIF2AK4) and cell cycle (MYCN, RBBP4 and NEUROG1). Pathway analysis revealed serial bystander activation of G protein/PI-3 kinase pathways. Instead, genes related to cell cycle or death (CDKN1A, GADD45A, NOTCH1 and BCL2L1), and cell communication (IL1B, TCF7 and ID1) were upregulated in irradiated cells, but not in bystander cells. Our results indicate different expression profiles in irradiated and bystander cells, and imply that intercellular signaling between irradiated and bystander cells activate intracellular signaling, leading to the transcriptional stress response in bystander cells.

Upregulation of stress-response genes with cell cycle arrest induced by carbon ion irradiation in multiple murine tumors models

Objective: To elucidate the in vivo biological effects induced by carbon-ion irradiation using comprehensive expression analysis. Materials and Methods: We examined gene expression changes after carbon-ion (C-ion) irradiation (290 MeV/m, SOBP 6 cm middle, 50 kev/μm) with a single dose of 30 Gy in four mouse tumors (NR-S1, SCCVII, NFSa, and #8520) transplanted into the hind legs of C3H/HeNrs mice, using 44K single-color oligo-microarrays at 6 hours (h), 1 day, and 3 days after irradiation. Gamma rays of 30 Gy and 50 Gy were used as a reference beam. Identification of C-ion-responsive genes was based on a false discovery rate of < 5% using the Wilcoxon test (P < 0.001) and the Benjamini-Hochberg correction. Results: In all tumors, the level of expression of several tens of genes, including Ccl3, Ccng1, Cd80, Cdkn1a, Cxcl2, IL7r, Lrdd, Mgmt, Mmp8, and Polk, was significantly altered 6 h and day 1 following C-ion irradiation. At day 3, several hundred genes, many of which are also classified as stress-response or cell-communication genes, including Tnfrsf5, Ikbke, and Icam1, were upregulated following C-ion irradiation. The expression level of the majority of these genes was similar following γ-ray treatment, although the change was not as extensive and intertumor variance was apparent. Several genes, including Ikbke, Serpina3n, and Saa3, responded differentially following C-ion irradiation than after γ-ray irradiation. Pathological investigation and immunohistochemical analysis of Cdkn1a revealed cell cycle arrest with mitotic catastrophe in tumors irradiated by C-ions. Conclusions: This study revealed significant C-ion induced upregulation of stress-responsive and cell-communication genes common to different tumor types. These findings provide evidence for the efficacy of this modality for the treatment of local tumors.

Gene expression analysis in human malignant melanoma cell lines exposed to carbon beams.

Purpose: To elucidate the molecular changes in response to carbon beams (C-ions) in melanoma. Materials and methods: We examined expression profiles of 6 melanoma cell lines exposed to C-ions or X-rays with 2 Gy using single-color microarrays. Results: Twenty-two genes, including nuclear factor of kappa light polypeptide gene enhancer in B-cells inhibitor, alpha (NFKBIA), responded to C-ions in all six cell lines, based on analysis of variance (ANOVA) filtering (p < 0.001). We found 173 genes that responded in common to C-ions in four cell lines. We identified many down-regulated genes including the cell cycle – related genes that were more responsive to C-ions than X-rays. In contrast, most of the up-regulated genes including the tumor protein p53 (p53) target genes responded to both C-ions and X-rays. C-ions induced G2/M arrest significantly more than X-rays at 30 h (p < 0.05). Conclusion: Our findings suggest that down-regulation of gene expression plays a key role in the response to C-ions. Regulation of cell cycle – related genes and induction of prolonged G2/M arrest may be responsible for the extra sensitivity to C-ions, whereas p53-related genes may have similar roles in the sensitivities to both C-ions and X-rays.

Carbon-ion radiation enhances migration ability and invasiveness of the pancreatic cancer cell, PANC-1, in vitro.

Pancreatic cancer is an aggressive disease that responds poorly to conventional photon radiotherapy. Carbon‐ion (C‐ion) radiation has advantages compared with conventional radiotherapy, because it enables more accurate dose distribution and more efficient tumor cell killing. To elucidate the effects of local radiotherapy on the characteristics of metastatic tumors, it is necessary to understand the nature of motility in irradiated tumor cells; this will, in turn, facilitate the development of effective strategies to counter tumor cell motility, which can be used in combination with radiotherapy. The aim of the present study was to examine the invasiveness of pancreatic cancer cells exposed to C‐ion irradiation. We found that C‐ion irradiation suppressed the migration of MIAPaCa‐2, BxPC‐3 and AsPC‐1; diminished the invasiveness of MIAPaCa‐2; and tended to reduce the invasion of BxPC‐3 and AsPC‐1. However, C‐ion irradiation increased the invasiveness of PANC‐1 through the activation of plasmin and urokinase‐type plasiminogen activator. Administration of serine protease inhibitor (SerPI) alone failed to reduce C‐ion‐induced PANC‐1 invasiveness, whereas the combination of SerPI and Rho‐associated coiled‐coil forming protein kinase (ROCK) inhibitor suppressed it. Furthermore, PANC‐1 showed mesenchymal–amoeboid transition when we treated with SerPI alone. In conclusion, C‐ion irradiation is effective in suppressing the invasive potential of several pancreatic tumor cell lines, but not PANC‐1; this is the first study showing that C‐ion irradiation induces the invasive potential of a tumor cell line. Further in vivo studies are required to examine the therapeutic effectiveness of radiotherapy combined with inhibitors of both mesenchymal and amoeboid modes of tumor cell motility. (Cancer Sci 2012; 103: 677–683)

Application of Carbon-Ion Beams or Gamma-Rays on Primary Tumors Does Not Change the Expression Profiles of Metastatic Tumors in an In Vivo Murine Model

PURPOSE To clarify how carbon-ion radiotherapy (C-ion) on primary tumors affects the characteristics of subsequently arising metastatic tumor cells. METHODS AND MATERIALS Mouse squamous cell carcinomas, NR-S1, in synergic C3H/HeMsNrs mice were irradiated with a single dose of 5-50 Gy of C-ion (290 MeV per nucleon, 6-cm spread-out Bragg peak) or gamma-rays ((137)Cs source) as a reference beam. The volume of the primary tumors and the number of metastatic nodules in lung were studied, and histologic analysis and microarray analysis of laser-microdissected tumor cells were also performed. RESULTS Including 5 Gy of C-ion and 8 Gy of gamma-rays, which did not inhibit the primary tumor growth, all doses used in this study inhibited lung metastasis significantly. Pathologic findings showed no difference among the metastatic tumor nodules in the nonirradiated, C-ion-irradiated, and gamma-ray-irradiated groups. Clustering analysis of expression profiles among metastatic tumors and primary tumors revealed a single cluster consisting of metastatic tumors different from their original primary tumors, indicating that the expression profiles of the metastatic tumor cells were not affected by the local application of C-ion or gamma-ray radiotherapy. CONCLUSION We found no difference in the incidence and histology, and only small differences in expression profile, of distant metastasis between local C-ion and gamma-ray radiotherapy. The application of local radiotherapy per se or the type of radiotherapy applied did not influence the transcriptional changes caused by metastasis in tumor cells.

The Proangiogenic Factor Ephrin-A1 Is Up-Regulated in Radioresistant Murine Tumor by Irradiation

While the pre-treatment status of cancer is generally correlated with outcome, little is known about microenvironmental change caused by anti-cancer treatment and how it may affect outcome. For example, treatment may lead to induction of gene expression that promotes resistance to therapy. In the present study, we attempted to find a gene that was both induced by irradiation and associated with radioresistance in tumors. Using single-color oligo-microarrays, we analyzed the gene expression profiles of two murine squamous cell carcinomas, NR-S1, which is highly radioresistant, and SCCVII, which is radiosensitive, after irradiation with 137-Cs gamma rays or carbon ions. Candidate genes were those differentially regulated between NR-S1 and SCCVII after any kind of irradiation. Four genes, Efna1 (Ephrin-A1), Sprr1a (small proline-rich protein 1A), Srgap3 (SLIT-ROBO Rho GTPase activating protein 3) and Xrra1 [RIKEN 2 days neonate thymus thymic cells (NOD) cDNA clone E430023D08 3′], were selected as candidate genes associated with radiotherapy-induced radioresistance. We focused on Efna1, which encodes a ligand for the Eph receptor tyrosine kinase known to be involved in the vascular endothelial growth factor (VEGF) pathway. We used immunohistochemical methods to detect expression of Ephrin-A1, VEGF, and the microvascular marker CD31 in radioresistant NR-S1 tumor cells. Ephrin-A1 was detected in the cytoplasm of NR-S1 tumor cells after irradiation, but not in SCCVII tumor cells. Irradiation of NR-S1 tumor cells also led to significant increases in microvascular density, and up-regulation of VEGF expression. Our results suggest that radiotherapy-induced changes in gene expression related with angiogenesis might also modulate microenvironment and influence responsiveness of tumors.

The Radiation-Induced Cell-Death Signaling Pathway is Activated by Concurrent Use of Cisplatin in Sequential Biopsy Specimens from Patients with Cervical Cancer

Objective: To identify changes in gene expression related to the concurrent use of platinum compounds with radiotherapy, in the treatment of cervical cancer. Patients and Methods: Biopsy specimens were obtained from 39 patients with squamous cell carcinoma of the uterine cervix, before and during fractionated radiotherapy. Twenty patients were treated with radiotherapy (RT) alone, while 19 received the same radiotherapy plus concomitant chemotherapy with cisplatin (CRT). Changes in gene expression induced by treatment were investigated using single-color oligo-microarrays consisting of 44K human sequences. Paraffin-embedded samples were used to examine apoptosis and the expression of protein by treatment-responsive genes. Changes in mRNA expression were assessed for these genes by real-time reverse transcriptase-polymerase chain reaction. Aberrant genomic change (detected using microarray-based comparative genomic hybridization), human papillomavirus infection, and p53 status were also evaluated. Results: The expression of CDKN1A, BAX, TNFSF8, and RRM2B was consistently upregulated by CRT (9 Gy with a single administration of cisplatin). Similar expression changes were induced by RT (9 Gy) alone, although the variability between tumors was greater. Apoptotic cells were significantly increased in both groups. CRT significantly increased the numbers of cases with diffusely distributed CDKN1A-positive cells. Genetic losses at 2q33-ter and gains of 3q26-ter were detected in the samples with high frequency; 60% were positive for human papillomavirus DNA; and 3 tumors had deletions/mutations of the p53 gene. There was no difference in the incidence of these genomic changes between the groups, and no association was found with the changes in expression of CDKN1A, BAX, TNFSF8, or RRM2B. Conclusions: Using biopsy samples from pretreatment and midtreatment cervical tumors, we identified therapy-induced genes related to the cell death signaling pathway. CRT produced a homogenous pattern of changes in expression of known radiation-responsive genes.

Nitric oxide increases the invasion of pancreatic cancer cells via activation of the PI3K–AKT and RhoA pathways after carbon ion irradiation

Previous studies have shown that serine proteases and Rho‐associated kinase contribute to carbon ion radiation‐enhanced invasion of the human pancreatic cancer cell line PANC‐1. The results presented here show that nitric oxide synthase (NOS) also plays a critical role in this process. Irradiation of PANC‐1 cells promoted invasion and production of nitric oxide (NO), which activated the PI3K–AKT signaling pathway, while independently activating RhoA. Inhibition of PI3K, Rho‐associated kinase, and serine protease alone or in conjunction with NOS suppressed the radiation‐enhanced invasion of PANC‐1 cells, suggesting that they could serve as possible targets for the management of tumor metastasis.

Carbon-Ion Irradiation Suppresses Migration and Invasiveness of Human Pancreatic Carcinoma Cells MIAPaCa-2 via Rac1 and RhoA Degradation

PURPOSE To investigate the mechanisms underlying the inhibition of cancer cell migration and invasion by carbon (C)-ion irradiation. METHODS AND MATERIALS Human pancreatic cancer cells MIAPaCa-2, AsPC-1, and BxPC-3 were treated by x-ray (4 Gy) or C-ion (0.5, 1, 2, or 4 Gy) irradiation, and their migration and invasion were assessed 2 days later. The levels of guanosine triphosphate (GTP)-bound Rac1 and RhoA were determined by the active GTPase pull-down assay with or without a proteasome inhibitor, and the binding of E3 ubiquitin ligase to GTP-bound Rac1 was examined by immunoprecipitation. RESULTS Carbon-ion irradiation reduced the levels of GTP-bound Rac1 and RhoA, 2 major regulators of cell motility, in MIAPaCa-2 cells and GTP-bound Rac1 in AsPC-1 and BxPC-3 cells. Proteasome inhibition reversed the effect, indicating that C-ion irradiation induced Rac1 and RhoA degradation via the ubiquitin (Ub)-proteasome pathway. E3 Ub ligase X-linked inhibitor of apoptosis protein (XIAP), which directly targets Rac1, was selectively induced in C-ion--irradiated MIAPaCa-2 cells and coprecipitated with GTP-bound Rac1 in C-ion--irradiated cells, which was associated with Rac1 ubiquitination. Cell migration and invasion reduced by C-ion radiation were restored by short interfering RNA--mediated XIAP knockdown, indicating that XIAP is involved in C-ion--induced inhibition of cell motility. CONCLUSION In contrast to x-ray irradiation, C-ion treatment inhibited the activity of Rac1 and RhoA in MIAPaCa-2 cells and Rac1 in AsPC-1 and BxPC-3 cells via Ub-mediated proteasomal degradation, thereby blocking the motility of these pancreatic cancer cells.

Villin1, a novel diagnostic marker for cervical adenocarcinoma

The number of new cervical adenocarcinoma (AD) cases has risen slowly, however, its histological similarity to other tumor types and the difficulty of identifying the site of the original tumor makes the diagnosis of cervical AD particularly challenging. We investigated a novel molecular biomarker for cervical AD through the integration of multiple methods of genomic analysis. Tumor samples in discovery set were obtained from 87 patients who underwent radiotherapy, including 31 cervical AD. Microarray analysis and quantitative polymerase chain reaction analysis were performed to screen a candidate diagnostic molecule for cervical AD, and its clinical significance was investigated by immunohistochemical analysis (IHC). We found a difference between biopsy samples of AD and squamous cell carcinoma (SCC) in the expression and genomic copy number of Villin1 (VIL1), which maps to 2q35. IHC revealed 14 VIL1-positive tumors; 13 cervical AD and 1 small cell carcinoma of cervix, while none of SCC or endometrial AD was VIL1-positive. Kaplan-Meier survival curves revealed worse disease-free survival in VIL1-positive tumors. The marker was validated by newly enrolled 65 patients, and VIL1 positive staining showed 52% of sensitivity and 100% of selectivity for cervical AD. In conclusion, we have identified VIL1 as a novel biomarker of cervical AD. VIL1, a major structural component of the brush border cytoskeleton, which was recently found to be an epithelial cell-specific anti-apoptotic protein. Our study suggests the existence of a subtype of cervical tumors which are VIL1 positive with poor radioresponse.