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

PurposeTo investigate the mechanisms underlying the inhibition of cancer cell migration and invasion by carbon (C)-ion irradiation.Methods and MaterialsHuman 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.ResultsCarbon-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.ConclusionIn 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

Metabolic characterization of invaded cells of the pancreatic cancer cell line, PANC-1.

We previously reported that about 0.4% of cells in the cultured human pancreatic cancer cell line, PANC-1, can invade matrigel during the transwell invasion assay, suggesting that these invaded PANC-1 cells may have specific characteristics to keep their invasive potential. To identify the metabolic characterization specific in the invaded PANC-1 cells, metabolome analysis of the invaded PANC-1 compared with the whole cultured PANC-1 was performed using CE-TOFMS, and concentrations of 110 metabolites were measured. In contrast to the whole cultured cells, the invaded PANC-1 was characterized as a population with reduced levels of amino acids and TCA cycle intermediates, and decreased and increased intermediates in glycolysis and nucleic acid metabolism. In particular, the ratio of both adenosine and guanosine energy charge was reduced in the invaded cells, revealing that the consumption of ATP and GTP was high in the invaded cells, and thus suggesting that ATP- or GTP-generating pathways are stimulated. In addition, the GSH/GSSG ratio was low in the invaded cells, but these cells had a higher surviving fraction after exposure to hydrogen peroxide. Thus, the invaded cells were the population resistant to oxidative stress. Furthermore, reduction in intracellular GSH content inhibited PANC-1 invasiveness, indicated that GSH has an important role in PANC-1 invasiveness. Overall, we propose the invaded cells have several unique metabolic profiles. This article is protected by copyright. All rights reserved

Search of inhibitors effective in suppressing the altered invasiveness of irradiated cancer cell line.

Tumor cells invade by two modes of motility—mesenchymal and amoeboid. Tumor cells with the mesenchymal mode of motility are known to use proteolytic enzymes to create a path to move through the ECM. In contrast, cells with the amoeboid mode of motility, which are rounded, exhibit a protease-independent mechanism of invasion; this mechanism is based on actomyosin contractility. Evidence shows that some cells can shift between these two modes of motility, depending on the environmental conditions; this may limit the effectiveness of therapeutic agents, such as protease inhibitors, which are directed at inhibiting a single mode of tumor cell motility. Therefore, an understanding of cancer cell motility is critical for the effective use of inhibitors. Irradiation alters the invasiveness of several tumor cell lines, however the effects of irradiation on the modes of motility remain unknown. Here we report that X-ray irradiation enhanced human pancreatic cancer cell lines MIAPaCa-2 and PANC-1 invasion via matrix metalloproteinase-2 (MMP-2), whereas Carbon-ion (C-ion) irradiation reduced MIAPaCa-2 invasion but increased PANC-1 invasion via serine proteases (SerP). Treatment of MMP inhibitor (MMPI) or SerP inhibitor (SerPI) failed to decrease the radiation-enhanced MIAPaCa-2 or PANC-1 invasion accompanied by mesenchymal–amoeboid transition. ROCK inhibitor plus those protease inhibitor suppressed the enhanced invasiveness, suggested that both modes of motility were significant in MIAPACa-2 and PANC-1. We further found that irradiation affects the activity of small GTPase, Rac1 and RhoA, the key factors involved in two modes of motility. Rac1 was activated in X-ray-irradiated MIAPaCa-2, whereas, both Rac1 and RhoA activities were diminished in C-ion-irradiated MIAPaCa-2. In contrast, RhoA was activated in C-ion-irradiated PANC-1. These results suggested that irradiation alters the invasive potential through protease activity, and also Rac1 and RhoA activities. Overall, the study indicated that the inappropriate use of inhibitors, i.e., the use of protease_I or ROCKI alone, may lead to the induction of invasive potential in some cell types. Therefore, the use of inhibitors of both mesenchymal and amoeboid modes of motility is the effective strategy for altered invasiveness of irradiated MIAPaCa-2 and PANC-1 invasiveness.Heavy Ion in Therapy and Space Radiation Symposium 2013 (HITSRS2013

The effects of irradiation on invasive potential of human pancreatic cancer cell lines, MIAPaCa-2 and PANC-1

[Objectives] The aim of this study was to compare the effects of X-ray or carbon-ion (C-ion) irradiation on invasive potential of human pancreatic cancer cell lines, MIAPaCa-2 and PANC-1.[Methods] MIAPaCa-2 and PANC-1 was irradiated with either X-ray (0, 2, or 4 Gy) or C-ion (0, 0.5, 1, 2, and 4 Gy). We examined the migration, invasion, protease expression and invasiveness in response to protease inhibitors and/or ROCK inhibitors of irradiated cells, and the activation of Rac1 and Rho.[Results] X-ray irradiation enhanced MIAPaCa-2 and PANC-1 invasion via MMP-2, whereas, Carbon-ion irradiation reduced MIAPaCa-2 invasion but increased PANC-1 invasion via activation of serine proteases (SerP). Treatment of MMP inhibitor or SerP inhibitor failed to decrease the radiation-enhanced MIAPaCa-2 or PANC-1 invasion accompanied by mesenchymal–amoeboid transition. ROCK inhibitor plus those protease inhibitor suppressed the enhanced invasiveness, suggested that both modes of motility were significant in MIAPACa-2 and PANC-1. We further found that irradiation affects the activity of small GTPase, Rac1 and RhoA, the key factors involved in two modes of motility. Rac1 was activated in X-ray-irradiated MIAPaCa-2, whereas, both Rac1 and RhoA activities were diminished in C-ion-irradiated MIAPaCa-2. In contrast, RhoA was activated in C-ion-irradiated PANC-1.[Conclusions] X-ray and C-ion irradiation show differential effects on the invasive potential of MIAPaCa-2 and PANC-1 cells with corresponding alterations in the MMP-2 activity or SerP activity, and also the activation of Rac1 and Rho signalings.The 2nd Japan-China Symposium on Cancer Researc

Irradiation alters the invasive potential of PANC-1 cell line thorough NO and NOS-PI3K-AKT pathway activities in addition to the SerP activity

We have previously reported that Carbon ion (C-ion) irradiation suppresses the invasiveness of several pancreatic cancer cell lines, MIAPaCa-2, BxPC-3, and AsPC-1 cells, however, we also observed that C-ion irradiation enhanced invasion in PANC-1 cells, which occurred via the activation of serine proteases (SerP), plasmin, and uPA. Treatment of C-ion irradiated PANC-1 with SerP inhibitor and ROCK inhibitor resulted in reduced invasiveness, but only by 50%, suggesting that there are more factors that have a role in C-ion-irradiation-induced PANC-1 invasion. To study the molecular factors involved in irradiation-induced PANC-1 invasion, we used the Screening Committee of Anticancer Drugs (SCADS) compound library and found that nitric oxide synthase (NOS) inhibitors and phosphatidylinositol 3-kinases (PI3K) inhibitors were both effective in reducing PANC-1 invasion. Most invaded PANC-1 cells were nitric oxide (NO)-producing cells, and the NOS-PI3K-AKT pathway was activated in these cells. This effect could be abrogated by NOS inhibitor treatment, which also reduced invasion. The population of NO-producing cells was increased by C-ion irradiation, thereby enhancing C-ion-irradiation-induced PANC-1 invasion. These results suggested that irradiation alters the invasive potential through NO and NOS-PI3K-AKT pathway activities in addition to the SerP activity.Ninth AACR-Japanese Cancer Association Joint Conferenc

Effects of Irradiation on Cellular Invasiveness with Regard to Cancer Cell Heterogeneity

Tumor metastasis is the major cause of cancer-related mortality. We and other groups have reported that photon irradiation enhances the invasiveness of tumor cell lines, whereas carbon-ion (C-ion) irradiation reduces invasion. However, each of these studies used only a few particular cell lines and did not demonstrate the heterogeneity of the cell lines. Heterogeneity of mutated genes among cancer cells may cause the cells to have different responses to irradiation; thus, it is necessary to use a more diverse set of cell lines to clarify the effects of irradiation on invasiveness with regard to the heterogeneity. To date, we have used 30 tumor cell lines to examine the effects of X-ray (4 Gy) or C-ion (2 Gy) irradiation on invasiveness. In contrast to X-ray irradiation, C-ion irradiation was effective in suppressing the invasion of many cell lines. Indeed, 11 cell lines showed an over 25% reduction in invasiveness. However, C-ion irradiation also enhanced the invasiveness of two cell lines. To elucidate the molecular factors involved in the irradiation-altered invasiveness, we used two pancreatic cancer cell lines, MIAPaCa-2 and PANC-1, which exhibited diminished or enhanced invasiveness after C-ion irradiation, respectively. In MIAPaCa-2, C-ion irradiation reduced the expression of GTP-Rac1 and GTP-RhoA, two master regulators of cell motility. In contrast, no reduction in GTP-Rac1 was detected in C-ion-irradiated PANC-1 cells, and GTP-RhoA levels were increased. Moreover, the reductions in GTP-Rac1 and GTP-RhoA observed in irradiated MIAPaCa-2 were recovered by treatment with a proteasome inhibitor, indicating that these proteins underwent degradation via the ubiquitin-proteasome pathway. Among the E3 ubiquitin ligases affecting Rac1, XIAP was selectively induced and was coprecipitated with GTP-Rac1 in C-ion-irradiated MIAPaCa-2. In conclusion, C-ion irradiation was effective in reducing the invasiveness of many cell lines. However, some cells were not affected, suggesting that those effects were influenced by the genomic heterogeneity. Indeed, GTP-Rac1 and GTP-RhoA expression levels were differentially regulated in MIAPaCa-2 and PANC-1. Therefore, genes affecting GTP-Rac1 and GTP-RhoA levels may be promising candidates for modulating the irradiation-dependent invasiveness of cancer cells.15th International Congress of Radiation Research (ICRR 2015

The effect of C-ion irradiation on metastatic potential of tumor cell lines

Blocking metastatic potential of tumor cells is crucial to improve the clinical outcome of cancer treatment. Carbon-ion (C-ion) radiotherapy offers several advantages over conventional photon radiotherapy with aspects being a high local control. However, it remains to be elucidated whether the local radiotherapy affects the characteristics of subsequently appearing metastatic tumors. Several evidences have shown that the conventional photon irradiation could enhance the metastatic potential of human tumor cells both in vitro and in vivo. On the other hand, C-ion irradiation diminishes the invasive potential of cell lines, but, to date, these studies performed only for 5 cancer cell lines, and thus, further characterization of other tumor cells is required to develop methods for improving the treatment strategies. In this study, we used 20 tumor cell lines including 10 different kinds of human tumors to investigate the effect of C-ion irradiation on metastatic potential of tumor cell lines. Cells were irradiated with C-ion (0 or 2 Gy), and examined the invasive potential by using the transwell chambers containing matrigel on a 6.5-mm filter with a pore size of 8 m. Here we show that 9 cell lines resulted in over 30 % reduction in invasiveness by C-ion irradiation, whereas two cell lines, SF-126 and Caski resulted in induction of invasiveness. Further investigation is needed to clarify the mechanisms of C-ion induced invasiveness found in minority of tumor cells.34th Annual Meeting of the Molecular Biology Society of Japan(MBSJ2011