Enhancement of hyperthermic killing in L5178Y cells by protease inhibitors.

We have investigated the effect of protease inhibitors on hyperthermic cell killing using cultured mammalian cells (L5178Y) and found that protease inhibitors were potent hyperthermia sensitizers. At 37 degrees C, phenylmethylsulfonyl fluoride (PMSF), a serine protease inhibitor, was not cytotoxic at the concentration of 400 micrograms/ml for up to 6 h. When cells were exposed to PMSF (200-400 micrograms/ml) during heating at 43 degrees C, significant potentiation of hyperthermic cell killing was observed. Other protease inhibitors, such as chymostatin and diisopropylfluorophosphate (both are serine protease inhibitors); (2S,3S)-trans-epoxy-succinyl-L-leucylamido-3-methylbutane ethyl ester (cysteine protease inhibitor) and pepstatin-A (aspartate protease inhibitor) showed similar effects. However, when cells were heated at 43 degrees C in the presence of cycloheximide (a protein synthesis inhibitor) together with PMSF, hyperthermic enhancement by PMSF decreased markedly. A decrease in potentiating the effect of PMSF was also noted with thermotolerant cells. These facts suggest that protease inhibitors may exert their hyperthermic cell killing by inhibiting proteases and ubiquitin, which are necessary to degrade denatured proteins induced by heat.

Possible explanation of radioresistance of glioblastoma in situ.

The response of human glioblastoma to radiation was studied in the logarithmic phase and in the plateau phase of growth, and was compared with those of HeLa S3 irradiated under identical conditions. There was no major difference in the in vitro survival curve parameters between glioblastoma and HeLa in the logarithmic growth phase. However, the surviving fractions for glioblastoma with radiation doses in the range used clinically were higher not only when irradiated in the logarithmic growth phase, but also when subcultured 6 hours after irradiation in the plateau phase, than those for HeLa treated under identical conditions. This suggests that the relatively low cure rate of glioblastoma can be partially explained by the intrinsic radiosensitivity in the logarithmic growth phase and by a high surviving fraction for cells irradiated in the plateau phase.

[Effect of etoposide on a human pancreatic cancer cell line exposed to high dose X-ray irradiation].

Effects of etoposide on the cell deaths of a human pancreatic cancer cell line by exposure to high-dose X-ray irradiation were examined. Both high dose X-ray irradiation and etoposide killed the pancreatic cancer cells by apoptosis. With 30 gray irradiation, simultaneous treatment of etoposide did not alter the percentage of cell deaths; however, 24-hr prior treatment of etoposide killed a significantly larger percentage of the cells, accompanying the accumulation to the S phase of the cell cycle. The results indicate that apoptosis of human pancreatic cancer cell line induced by high dose X-ray irradiation can be enhanced by prior treatment with etoposide.

[Protection of cultured mammalian cells by rebamipide].

Rebamipide which is used as a drug for gastritis and stomach ulcer has large capability for OH radical scavenging. It is expected that rebamipide has protective effect against ionizing radiations. The present paper deals with protective effect of rebamipide for cultured mammalian cells exposed to ionizing radiations. As rebamipide is insoluble in water, three solvents were used to dissolve. Rebamipide dissolved in dimethyl sulfoxide (DMSO), dimethyl formamide (DMFA) and 0.02 N NaOH was added to the cells in Eagle's minimum essential medium (MEM) supplemented with 10% fetal calf serum and the cells were irradiated with X-rays. After irradiation, the cells were trypsinized, plated in MEM with 10% fetal calf serum and incubated for 7 days in a CO2 incubator to form colonies. Rebamipide dissolved in 0.02 N NaOH exhibited the protective effect expected its OH radical scavenging capability. However, the protective effect of rebamipide dissolved in DMSO was about half of that expected by its radical scavenging capability and that of rebamipide dissolved in DMFA was not observed. Uptake of rebamipide labeled with 14C increased with increasing contact time with rebamipide. These rebamipide mainly distributed in nucleous rather than cytoplasm.

Promotion of heat-induced apoptosis in FM3A cells by protease inhibitors.

Although it has been shown that proteases may play a positive role in in causing apoptosis of some cells, we report here that, on the contrary, protease inhibitors can promote heat-induced apoptosis in FM3A cells. Cysteine protease inhibitor, trans-Epoxy-succinyl-L-leucylamido-(4-guanidino)butane (E-64, 100 micrograms/ml) and aspartate protease inhibitor, pepstatin-A (100 micrograms/ml) were used to test hyperthermic effect on FM3A cells and showed remarkable cytotoxicity when they were present in cell suspension during heating at 44 degrees C. The cytotoxicity was due to promotion of heat-induced apoptosis as judged by DNA agarose electrophoresis. Furthermore, using flow cytometric analysis, we observed a decrease in the G0/G1 phase cell and an increase in the S phase cell as well as increased apoptosis after heat shock. E-64 and pepstatin-A exhibited a promotive effect on the changes of cell cycle induced by heat. The data presented suggest that the enhancement of hyperthermic cell killing by protease inhibitors may be related to promotion of heat-induced apoptosis and changes of cell cycle.