Kenji Sugita

Antitumor efficacy of hypothemycin, a new ras-signaling inhibitor

We have devised a new drug screening assay to discover anti‐cancer drugs which inhibit Ras‐mediated cellular signals, by utilizing a Ras‐responsive element (RRE)‐driven reporter gene system. We found that hypothemycin, an anti‐bacterial, reduces RRE‐dependent transcription. Treatment of tumor cells with hypothemycin resulted in reduced expression of Ras‐inducible genes, including MMP (matrix metalloproteinase)‐1, MMP‐9, transforming growth factor‐β (TGF‐β), and vascular endothelial growth factor (VEGF), but not that of the constitutively expressed gene, MMP‐2. The results of zymography demonstrated that hypothemycin reduced the production of MMP‐9 and MMP‐3, another Ras‐inducible MMP, in the culture medium. Hypothemycin selectively inhibits anchorage‐independent growth of Ras‐transformed cells in comparison with anchorage‐dependent growth. These findings suggest that hypothemycin inhibits Ras‐mediated cellular signaling. Daily treatment of tumor‐bearing mice with hypothemycin resulted in significant inhibition of tumor growth. Since MMP‐1, MMP‐3 and MMP‐9 play important roles in tumor invasion and TGF‐? and VEGF are involved in tumor angiogenesis, hypothemycin is considered to be an example of a new class of antitumor drugs, whose antitumor efficacy can be at least partly attributed to inhibition of Ras‐inducible genes.

A Novel Tetracyclic Peptide, Trapoxin, Induces Phenotypic Change from Transformed to Normal in sis-Oncogene-transformed NIH3T3 Cells

A novel tetra cyclic peptide, trapoxin [cyclo(L‐phenylalanyl‐L‐phenylalanyl‐D‐pipecolinyl‐L‐2‐amino‐8‐oxo‐9,10‐epoxy‐decanoyl)], was found to induce the flat phenotype in v‐sis‐transformed NIH3T3 cells at a quite low concentration of 1 ng/ml. Actin stress fiber could be detected after trapoxin treatment. Almost complete reversion into the flat phenotype was observed at 6 h after the administration of the compound. The effect of trapoxin was reversible, when the cell culture was incubated for more than 24 h after its removal. The intracellular level of sis‐mRNA did not decrease with trapoxin treatment at a concentration (50 ng/ml), sufficient to reverse the transformed morphology. Substitution of pipecolinic acid with proline in trapoxin did not change the activity. WF3161, in which leucine was substituted for a phenylalanine of trapoxin, showed only one‐sixteenth of the activity of trapoxin. Reduction of the epoxide residue of trapoxin destroyed the activity.

A novel compound, depudecin, induces production of transformation to the flat phenotype of NIH3T3 cells transformed by ras-oncogene

A novel compound, depudecin, induced production of the flat phenotype of Ki-ras-transformed NIH3T3 cells at the low concentration of 1 microgram/ml. This effect was reversible. Actin stress fiber was detected in these cells after depudecin treatment. Almost complete reversion to the flat phenotype was observed at 6 h after depudecin addition. The synthesis of ras-mRNA did not decrease enough with depudecin treatment at the concentration of 10 micrograms/ml to reverse the transformed morphology.

Suppression of oncogenic transformation by hypothemycin associated with accelerated cyclin D1 degradation through ubiquitin-proteasome pathway.

Hypothemycin was originally isolated as an antifungal metabolite of Hypomyces trichothecoides. Here we report that treatment on v-K-ras-transformed NIH3T3 cells (DT cells) with hypothemycin caused drastic decrease in amount of cyclin D1 protein with concomitant prolongation of G1 phase in their cell cycle. Analysis using hypothemycin-resistant mutant of Schizosaccharomyces pombe (S. pombe) was carried out to show that S. pombe rhp6+ (homologue of Saccharomyces cerevisiae RAD6) and mammalian ubiquitin-conjugating enzyme 2 (ubc2) are the targets of hypothemycin or its downstream molecules in ubiquitin-conjugation process. Furthermore, in the presence of lactacystin, a specific inhibitor for proteasome, hypothemycin greatly enhanced the accumulation of multi-ubiquitinated form of cyclin D1 in DT cells. Therefore, it is indicated that hypothemycin facilitates ubiquitinating process of cyclin D1. In terms of malignant phenotype, hypothemycin inhibited anchorage-independent growth and reverted the morphology of DT cells. On the contrary, their morphology still remained transformed in the additional presence of lactacystin. Our results suggest that cyclin D1 is a key molecule working downstream in ras-signaling and that the transformation can be inhibited by the compound which can activate ubiquitin-proteasome pathway including degradation of cyclin D1.

Combination Chemotherapy with Nedaplatin and Cyclophosphamide in Human Ovarian Cancer Model

The antitumor efficacy of the combination of nedaplatin (NDP) with cyclophosphamide (CPM) was evaluated using human ovarian cancer models. Since NDP has been found to have greater anti‐tumor activity and lower nephrotoxicity than cisplatin (CDDP), we also compared the antitumor activity of NDP plus CPM with that of CDDP plus CPM. Increasing doses of NDP (16.5, 33 and 66 mg/kg as a total dose) and a fixed amount of CPM (174 or 348 mg/kg as a total dose) were injected three times at intervals of 7 days via the tail vein into mice implanted with RMUG‐S, OC9‐JCK or KF‐28 human ovarian cancer. Simultaneous administration of NDP with CPM resulted in markedly enhanced inhibition of tumor growth for all cancers tested. The growth inhibition and survival effect of the combination therapy of NDP with CPM against KF‐28 and OC9‐JCK were as potent as those of CDDP plus CPM. Neither increased hematotoxicity nor a significant difference in maximum concentration, half time or area under the curve of platinum or CPM in plasma between the single and combined treatment was found. These results suggest that the combination of NDP with CPM may be clinically effective.