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)

Tumor cells can migrate and invade tissue by two modes of motility: mesenchymal and amoeboid. X-ray or c-ray irradiation increases the invasiveness of tumor cells with mesenchymal motility through the induction of matrix metalloproteinases (MMP), and this increase is suppressed by MMP inhibitors (MMPI). However, the effects of X-ray or c-ray irradiation on the invasiveness of tumor cells with amoeboid motility remain unclear. We investigated the effect of irradiation on amoeboid motility by using cells of the human pancreatic cancer line, MIAPaCa-2, which exhibits both modes of motility. The X-ray-induced invasiveness of MIAPaCa-2 cells was associated with the upregulation of MMP2 at both the RNA and protein levels and was inhibited by MMPI treatment. Amoeboid-mesenchymal transition was slightly induced after irradiation. The MMPI treatment caused mesenchymal-amoeboid transition without significant increase in invasiveness, while the ROCK inhibitor (ROCKI) stimulated amoeboid-mesenchymal transition and enhanced invasiveness under both non-irradiated and irradiated conditions. This ROCKI-induced transition was accompanied by the upregulation of MMP2 mRNA and protein.Exposure to both irradiation and ROCKI further enhanced MMP2 expression and had an additive effect on the invasiveness of MIAPaCa-2 cells. Additionally, exposure to MMPI led to significant suppression of both radiation-induced and the basal invasiveness of MIAPaCa-2 cells. This suggests that ROCKI treatment, especially with concomitant X-ray irradiation, can induce invasion of cancer cells and should be used only for certain types of cancer cells. Simultaneous use of inhibitors, ROCKI and MMPI may be effective in suppressing invasiveness under both X-ray-irradiated and nonirradiated conditions. (Cancer Sci 2011; 102: 792-798) P ancreatic cancer is one of the most aggressive diseases with an extremely low 5-year survival rate. (1,2) Most patients with pancreatic cancer have advanced disease at the time of diagnosis, and this disease is associated with a high metastatic potential, which is a major clinical problem. (1,3) Chemotherapy and radiotherapy are used as adjuvants to surgery and for palliative purposes in cases where surgical resection is not feasible. However, it has been reported that there is no clear evidence to indicate that intraoperative radiotherapy is more effective than other therapies in treating pancreatic cancer in locally advanced and metastatic stages. (4) Further characterization of pancreatic cancer cells exposed to anti-tumor drugs and irradiation is required to develop methods for improving treatment strategies.It remains to be determined whether the application of local radiotherapy affects the characteristics of subsequently appearing metastatic tumors. Several studies have shown that conventional X-ray or c-ray irradiation itself induces the metastasis of pancreatic tumor cells both in vitro and in vivo. (1,3,(5)(6)(7) These studies revealed that photon-irradiated cells produce proteases, such as matrix metalloproteinase...

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In the present work, a convenient microarray SNP typing system has been developed using a plastic base that covalently immobilizes amino-modified oligonucleotides. Reliable SNP allele discrimination was achieved by using allelic specificity-enhanced enzymatic extension of immobilized oligonucleotide primer, with a locked nucleic acid (LNA) modification at the SNP-discriminating 3′-end nucleotide. Incorporation of multiple biotin-dUTP molecules during primer extension, followed by binding of alkaline phosphatase-conjugated streptavidin, allowed optical detection of the genotyping results through precipitation of colored alkaline phosphatase substrates onto the surface of the plastic base. Notably, rapid primer extension was demonstrated without a preliminary annealing step of double-stranded template DNA, allowing overall processes to be performed within a couple of hours. Simultaneous evaluation of three SNPs in the genes TGFB1, SOD2 and APEX1, previously investigated for association with radiation sensitivity, in 25 individuals has shown perfect assignment with data obtained by another established technique (MassARRAY system).