Calpain regulates thymidylate synthase-5-fluoro-dUMP complex levels associated with response to 5-fluorouracil in gastric cancer cells.

Thymidylate synthase (TS) plays a major role in the response to 5-fluorouracil (5-FU) by binding directly to the 5-FU metabolite, 5-fluoro-dUMP (FdUMP). The change in the TS expression levels after 5-FU administration was examined in parallel to 5-FU responsiveness in six human gastric adenocarcinoma cell lines to elucidate the source of variability of 5-FU sensitivity. MKN-1, SH-10-TC and MKN-74 cells were more resistant to 5-FU than MKN-28, KATO III and MKN-45 cells. Western blotting analysis revealed that the 5-FU sensitivity of these cells did not correlate with the basal TS expression levels but did correlate with rapid detection of the TS-FdUMP complex after exposure to 5-FU. In 5-FU-resistant cells, very low levels of the TS-FdUMP complex early after 5-FU exposure were elevated by pretreatment with calpain inhibitors such as benzyloxycarbonyl-leucyl-leucinal (ZLLH), benzyloxycarbonyl-leucyl-leucyl-leucinal (ZLLLH) and ALLN, but not by other protease inhibitors. In contrast, ONO-3403, which causes calpain activation, stimulated downregulation of the TS-FdUMP complex in 5-FU-sensitive cells. The expression levels of calpastatin, an endogenous calpain inhibitor, were higher in 5-FU-sensitive cells than in 5-FU-resistant cells. ZLLH increased the 5-FU sensitivity of 5-FU-resistant cells, whereas ONO-3403 decreased the sensitivity of 5-FU-sensitive cells. In addition, knockdown of m-calpain by siRNA increased the 5-FU sensitivity in 5-FU-resistant cells, while knockdown of calpastatin reduced the sensitivity in 5-FU-sensitive cells. These results suggest that calpain might reduce the chemosensitivity of human gastric cancer cells to 5-FU possibly by rapid degradation of the TS-FdUMP complex, a finding that is considered to have novel therapeutic implications.

Sustained and NK/CD4+ T cell-dependent efficient prevention of lung metastasis induced by dendritic cells harboring recombinant Sendai virus.

We recently demonstrated efficient antitumor immunity against murine tumors using dendritic cells (DCs) activated by recombinant Sendai viruses (rSeVs), and proposed a new concept, "immunostimulatory virotherapy," for cancer immunotherapy. However, there has been little information on the efficacy of this method in preventing metastatic diseases. In this study, we investigated the efficacy of vaccinating DCs activated by fusion gene-deleted nontransmissible rSeV (rSeV/dF) using a murine model of lung metastasis. Bolus and i.v. administration of DCs harboring rSeV/dF-expressing GFP without pulsation of tumor Ag (DC-rSeV/dF-GFP) 2 days before tumor inoculation showed efficient prevention against lung metastasis of c1300 neuroblastoma, but not of RM-9 prostatic cancer. We found that the timing of DC therapy was critical for the inhibition of pulmonary metastasis of RM-9, and that the optimal effect of DCs was seen 28 days before tumor inoculation. Interestingly, the antimetastatic effect was sustained for over 3 mo, even when administered DCs were already cleared from the lung and organs related to the immune system. Although NK cell activity had already declined to baseline at the time of tumor inoculation, Ab-mediated depletion studies revealed that CD4+ cells as well as the presence of, but not the activation of, NK cells were crucial to the prevention of lung metastasis. These results are the first demonstration of efficient inhibition of lung metastasis via bolus administration of virally activated DCs that was sustained and NK/CD4+ cell-dependent, and may suggest a potentially new mechanism of DC-based immunotherapy for advanced malignancies.

Urokinase-targeted fusion by oncolytic Sendai virus eradicates orthotopic glioblastomas by pronounced synergy with interferon-ß gene.

Glioblastoma multiforme (GM), the most frequent primary malignant brain tumor, is highly invasive due to the expression of proteases, including urokinase-type plasminogen activator (uPA). Here, we show the potential of our new and powerful recombinant Sendai virus (rSeV) showing uPA-specific cell-to-cell fusion activity [rSeV/dMFct14 (uPA2), named "BioKnife"] for GM treatment, an effect that was synergistically enhanced by arming BioKnife with the interferon-ß (IFN-ß) gene. BioKnife killed human GM cell lines efficiently in a uPA-dependent fashion, and this killing was prevented by PA inhibitor-1. Rat gliosarcoma 9L cells expressing both uPA and its functional receptor uPAR (9L-L/R) exhibited high uPA activity on the cellular surface and were highly susceptible to BioKnife. Although parent 9L cells (9L-P) were resistant to BioKnife and to BioKnife expressing IFN-ß (BioKnife-IFNß), cell-cell fusion of 9L-L/R strongly facilitated the expression of IFN-ß, and in turn, IFN-ß significantly accelerated the fusion activity of BioKnife. A similar synergy was seen in a rat orthotopic brain GM model with 9L-L/R in vivo; therefore, these results suggest that BioKnife-IFNß may have significant potential to improve the survival of GM patients in a clinical setting.

Cytokine-based log-scale expansion of functional murine dendritic cells.

BACKGROUND: Limitations of the clinical efficacy of dendritic cell (DC)-based immunotherapy, as well as difficulties in their industrial production, are largely related to the limited number of autologous DCs from each patient. We here established a possible breakthrough, a simple and cytokine-based culture method to realize a log-scale order of functional murine DCs (>1,000-fold), which cells were used as a model before moving to human studies. METHODOLOGY/PRINCIPAL FINDINGS: Floating cultivation of lineage-negative hematopoietic progenitors from bone marrow in an optimized cytokine cocktail (FLT3-L, IL-3, IL-6, and SCF) led to a stable log-scale proliferation of these cells, and a subsequent differentiation study using IL-4/GM-CSF revealed that 3-weeks of expansion was optimal to produce CD11b+/CD11c+ DC-like cells. The expanded DCs had typical features of conventional myeloid DCs in vitro and in vivo, including identical efficacy as tumor vaccines. CONCLUSIONS/SIGNIFICANCE: The concept of DC expansion should make a significant contribution to the progress of DC-based immunotherapy.