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.

Modelling of tumour--host coexistence In vitro in the presence of serine protease inhibitors.

The activities of cell surface serine proteases are markedly enhanced in malignant tumours. Proteolytic degradation of the extracellular matrix and basal membrane of normal cells is an important event for tumour cell growth and invasion. Two well-known broad-spectrum inhibitors of serine protease, Foy-305 and Ono-3403, were evaluated for their ability to affect the growth rate and survival of MCF7 breast cancer cells co-cultured with MRC5 lung fibroblasts as feeder cells in the absence of serum. Flow cytometry and differential staining demonstrated that in the mixed culture, the rate of tumor growth was dependent upon the presence of the feeder MRC5 lung fibroblasts and could be obviated by the additional presence of the inhibitors of serine proteases.

Antimetastatic activity of a synthetic serine protease inhibitor, FOY-305 (Foypan).

Metastasis is one of the major causes of mortality in cancer. It is well known that the activities of cell surface serine proteases are especially enhanced in malignant tumors. Proteolytic degradation of the extracellular matrix and basal membrane is a crucial event for tumor cell invasion and metastasis formation. FOY-305 (Foypan), a remedy for tumor pancreatitis, is a broad spectrum synthetic serine protease inhibitor which inhibits enzymatic activities including trypsin, thrombin, kallikrein and plasmin. Using Lewis lung carcinoma cell, we found that FOY-305 inhibited both spontaneous and experimental pulmonary metastasis. Furthermore, the combined treatment of FOY-305 and a traditional anti-cancer drug, 5-FU or bleomycin, resulted in marked enhancement of anti-pulmonary metastatic activity.

Growth inhibition of mouse autochthonous skin cancer by oral administration of new serine protease inhibitor ONO-3403.

The orally active serine protease inhibitor ONO-3403 is an analog of FOY-3403 that has more potent protease-inhibitory activity. In the present study, oral administration of ONO-3403 was used to challenge 3-methylcholanthrene-induced carcinoma. This drug was administered 3 times daily for 9 weeks via a stomach tube at a dose of 10 mg/kg in a 1-ml volume in 6 mice harboring solid tumors. This protease inhibitor significantly inhibited tumor growth (p<0.001) and prolonged survival-time (p<0.01). These results indicated that oral administration of the potent serine protease inhibitor ONO-3403 has an antitumor effect on malignant tumors.

Decrease in growth factor receptors after treatment with serine protease inhibitor ONO-3403.

FOY-305 is a synthetic serine protease inhibitor and ONO-3403 is a derivative with a higher protease-inhibitory activity. The growth-suppressive effects of ONO-3403 were more prominent in Ha-ras-transformed NIH3T3 (ras-NIH) cells than in non-transformed NIH3T3 cells. After treatment of ras-NIH cells with ONO-3403 at 100-200 microg/ml, the percentage of cells found in G(1) phase decreased and, concomitantly, that in S phase increased. Molecular events caused by ONO-3403 were investigated by Western blot analysis using anti-phosphotyrosine antibody. The results showed a marked decrease in the tyrosine phosphorylation level of a 180-kDa protein after treatment with ONO-3403. This 180-kDa phosphotyrosine-containing molecule which was tentatively designated pY-p180 might be platelet-derived growth factor (PDGF) receptor since addition of PDGF to serum-starved NIH3T3 cells induced a marked tyrosine phosphorylation of the same size within 5 min. This was further confirmed by immunoprecipitation of cell extract with anti-PDGF-receptor antibody followed by Western blot analysis using anti-phosphotyrosine antibody. Treatment of T.Tn human esophageal carcinoma cells with ONO-3403 caused also decrease in pY-p180, which appeared to be epidermal growth factor receptor. Thus, ONO-3403 may induce growth suppression by down-regulation of cell surface growth factor receptors.