[Future directions of anticancer drug development in Japan].

At the 13th Oncology Forum, future directions of anticancer drug development in Japan were discussed. Development of anticancer drugs in the 1990s was based on the concept of total cell kill, but now development of molecular targeted drugs becomes the mainstream. Unfortunately, molecular targeted drugs and antibody agents are mostly foreign products and translational research in Japan is poor as it stands now. As future directions of anticancer drug development, international collaborative development is considered essential, but there are various obstacles to the conduct of international collaborative studies. Companies, medical institutions and regulatory agencies must make collaborative efforts to overcome these obstacles. As future development of anticancer agents in individual cancer regions in Japan is considered, gastric cancer therapy is progressing considerably with the advent of S-1 and in the future, development of multi-agent combination therapy including molecular targeted agents is expected. Much progress in colon cancer therapy has been made owing to accumulation of evidence in recent years. Multi-agent chemotherapy combined with antibody agent, which is advancing overseas, is introduced to Japan. Clinical development of combination therapy with a high therapeutic index, including compounds discovered in Japan, is expected in the future. Although conventionally hormone therapy has been considered as first-line treatment of breast cancer and used in combination with chemotherapy, with the advent of antibody agents in recent years, HER2 sensitivity has greatly affected the algorithm of treatment. Future development of molecular targeted drugs and individualised diagnosis using cDNA array, etc. are likely to advance individualisation of treatment. On the other hand, large-scale clinical trials are required to prove a small difference in adjuvant therapy, etc. and accordingly international studies are becoming indispensable. For urological cancers, molecular targeted drugs have been proved effective in renal cancer and future development of molecular targeted drugs for prostate cancer and testicular tumors is desirable. At that time, elucidation of the mechanism of action of molecular targeted drug and strategic drug development designed to increase its efficacy are expected. As a future direction of anticancer drug development, there are many cancers in whose international collaborative studies Japan can participate. Studies of prostate cancer and renal cell carcinoma can be internationalised while internationalisation of studies in ovarian and pancreatic cancers is essential. Phase III should be performed as international collaborative studies and depending on the type of cancer and drug, collaborative studies in an Asian region are effective. When participating in an international collaborative study, Japan needs to recruit subjects at a speed similar to the rest of the world, but differences in medical environment including clinical trials pose a problem. To solve this problem, it is considered effective not only to pursue the Western environment but also to improve staff such as nurses and CRC. The number of Japanese patients necessary for Phase III studies is individual developmental strategy and needs to be examined by both companies and regulatory agencies.

Bioactive triterpene saponins from the roots of Phytolacca americana.

Five new triterpene saponins named phytolaccasaponins N-1 (1), N-2 (2), N-3 (3) N-4 (4), and N-5 (5) were isolated from the roots of Phytolacca americana together with seven known triterpene saponins (6-12). The structures of the five new saponins were established as shown in structures 1-5 on the basis of their spectroscopic data. The MDR-reversal activity of 1-12 was evaluated on the basis of the amount of calcein accumulated in MDR human ovarian cancer 2780 AD cells in the presence of each compound. The most effective compound was 8 (155% of control at 25 microg/mL).

Bioactive cardenolides from the stems and twigs of Nerium oleander.

Four new cardenolide monoglycosides, cardenolides N-1 (1), N-2 (2), N-3 (3), and N-4 (4), were isolated from Nerium oleander, together with two known cardenolides, 5 and 12, and seven cardenolide monoglycosides, 6-11 and 13. The structures of compounds 1-4 were established on the basis of their spectroscopic data. The in vitro anti-inflammatory activity of compounds 1-13 was examined on the basis of inhibitory activity against the induction of the intercellular adhesion molecule-1 (ICAM-1). Compounds 1, 5, 6, and 11-13 were active at an IC50 value of less than 1 microM. The cytotoxicity of compounds 1-13 was evaluated against three human cell lines, normal human fibroblast cells (WI-38), malignant tumor cells induced from WI-38 (VA-13), and human liver tumor cells (HepG2). Compounds 1, 4, 6, and 11-13 were active toward V-13 cells, and compounds 1, 11, and 12 were active toward HepG2 cells at IC50 values of less than 1 microM. Compounds 4, 5, 10, and 12 showed selective cell growth inhibitory activity toward V-13 tumor cells compared with that of parental normal WI-38 cells. The MDR-reversal activity of compounds 1-13 was evaluated on the basis of the amount of calcein accumulated in MDR human ovarian cancer 2780AD cells in the presence of each compound. Compounds 4, 9, and 10 showed significant effects on calcein accumulation, compound 4 showing stronger activity than that of verapamil.

Bioactive polyketides from Peperomia duclouxii.

Four new compounds (1-4) were isolated along with 16 known compounds from whole plants of Peperomia duclouxii. The new structures were elucidated as 4-hydroxy-2-[(3,4-methylenedioxyphenyl)nonanoyl]cyclohexane-1,3-dione (1), 4-hydroxy-2-[(3,4-methylenedioxyphenyl)undecanoyl]cyclohexane-1,3-dione (2), 4-hydroxy-2-[(3,4-methylenedioxyphenyl)tridecanoyl]cyclohexane-1,3-dione (3), and 2-[(3,4-methylenedioxyphenyl)dodecyl]-4-hydroxy-2,3,4,6,7,8-hexahydro-2H-1-benzopyran-5-one (4), by analysis of their spectroscopic data. The known polyketides, surinone A and oleiferinone, showed cell growth inhibitory activity against the WI-38, VA-13, and HepG2 cell lines with IC50 values that ranged from 4.4 to 9.6 microg/mL. The known sesquiterpenoid, sinugibberodiol, showed a more potent effect on calcein accumulation than verapamil at 2.5 and 25 microg/mL. Compounds 3 and 4, surinone A, and oleiferinone showed moderate to weak inhibitory activity on the induction of the intercellular adhesion molecule-1 (ICAM-1) in the presence of IL-1alpha or TNF-alpha.

Bioactive lignans from Peperomia duclouxii.

Six new lignans (1-6), along with 14 known compounds, were obtained from Peperomia duclouxii. The new structures were elucidated mainly by the analysis of their NMR and MS data. The absolute configurations of 1-6 were determined by comparing their optical rotations or CD spectra with those of known compounds. In cytotoxic and MDR reversal cell activity assays, compound 3 showed cancer cell growth inhibitory activity against VA-13 and HepG2 cells, with IC50 values of 5.3 and 13.2 microg/mL, and more potent effects on calcein accumulation in MDR 2780AD cells than verapamil, a positive control. Compound 6 showed anti-inflammatory activity using an ICAM-1 assay (induction of the intercellular adhesion molecule-1), stimulated by IL-1alpha and TNF-alpha.

Chelating compound, chrysoidine, is more effective in both antiprion activity and brain endothelial permeability than quinacrine.

1. As an extension of our previous study of quinacrine and its derivatives, chelating chemicals were screened to obtain more effective, better brain-permeable antiprion compounds using either prion-infected neuroblastoma cells or brain capillary endothelial cells.2. Eleven chemicals were found to have antiprion activity. Most of them shared a common structure consisting of benzene or naphthalene at either end of an azo bond. Structure-activity data suggest that chelating activity is not necessary but might contribute to the antiprion action.3. Chrysoidine, a representative compound found here, was about 27 times more effective in the antiprion activity and five times more efficiently permeable through the brain capillary endothelial cells than quinacrine was.4. These chemicals might be useful as compounds for development of therapeutics for prion diseases.

Bioactive dibenzylbutyrolactone and dibenzylbutanediol lignans from Peperomia duclouxii.

Six new dibenzylbutyrolactone (6-11) and two new dibenzylbutanediol lignans (12, 13) were obtained from Peperomia duclouxii. The structures were elucidated mainly by the analysis of NMR and MS data. The anticancer activity against a normal (WI-38) and a simian virus 40-transformed human lung fibroblast cell (VA-13) and a hepatoma G2 cell (HepG2) and the MDR reversal activity of the isolated compounds were examined. Compound 7 showed moderate inhibitory activity against VA-13 and HepG2 with IC(50) values of 23.2 and 26.4 microM, respectively. Compound 2 inhibited the growth of HepG2 cells with an IC(50) of 42.8 microM. Compounds 2 and 13 exhibited stronger MDR reversal activity than verapamil, at 25 and 2.5 microg/mL, respectively, and 4, 5, and 7 showed comparable activity with verapamil, at 25, 25, and 2.5 microg/mL, respectively.

Bioactive tetrahydrofuran lignans from Peperomia dindygulensis.

Five new tetrahydrofuran lignans (1-5), accompanied by four known compounds, were isolated from the ethyl acetate extract of Peperomia dindygulensis. Structures were elucidated mainly using 1D NMR, 2D NMR, and mass spectroscopic studies. The relative configurations of 1-5 were determined by NOE correlations. Several of the compounds showed weak growth inhibitory activity against three cell lines (WI-38, VA-13, and HepG2). Compound 5 exhibited stronger MDR (multidrug resistance) reversal activity than verapamil at 2.5 microg/mL in a cellular calcein accumulation assay. Compounds 4 and 5 showed weak inhibitory activity against induction of the intercellular adhesion molecule-1 (ICAM-1) in anti-inflammatory activity experiments.

Taxoids and abietanes from callus cultures of Taxus cuspidata.

Seventeen known taxoids and 10 abietanes were isolated from the dark brown callus culture of Taxus cuspidata cultivated on a modified Gamborg's B5 medium with 0.5 or 1.0 mg/L NAA. Seven known taxoids and four abietanes were obtained from the callus culture incubated under light irradiation on the medium with 1.0 mg/L NAA. Eight taxoids and five abietanes were also separated from the callus culture on the medium with 10 mmol/L beta-cyclodextrin and 1.0 mg/L NAA. The new compounds were identified by analyses of the spectroscopic data and were found to be abieta-6,8,11,13-tetraene-3beta,12-diol (1), 3beta,20-epoxy-12-methoxy-abieta-8,11,13-triene-3alpha,11-diol (2), 3alpha-hydroxy-9(10-->20)abeo-abieta-1,5,8,10(20),13-pentaene-7,11,12-trione (3), 2-hydroxy-9(10-->20)abeo-abieta-1,5,8,10(20),13-pentaene-3,7,11,12-tetraone (4), and 3,7-dioxo-9(10-->20)abeo-12-norabieta-1,5,8,10(20),13-pentaene-11,13-lactone (5), respectively. The yield of paclitaxel and its analogues was markedly decreased in the calluses mentioned above compared with that of standard callus. Instead, abietanes and some taxoids related to biosynthesis of paclitaxel were produced. Taxusin (6) exhibited stronger MDR-reversing activity than verapamil toward 2780 AD tumor cells.

Production of biologically active taxoids by a callus culture of Taxus cuspidata.

Ten known taxoids, paclitaxel, 7-epi-taxol, taxol C, baccatin VI, taxayuntin C, taxuyunnanine C and its analogues (2-5), and yunnanxane (6), and an abietane, taxamairin A, were produced in the callus culture of Taxus cuspidata cultivated on a modified Gamborg's B5 medium in the presence of 0.5 mg/L NAA. After stimulation with 100 microM methyl jasmonate, five more taxoids, cephalomannine, 1beta-dehydroxybaccatin VI, taxinine NN-11 (1), baccatin I, and 2alpha-acetoxytaxusin, and one more abietane, taxamairin C, were found in addition to the above-mentioned compounds. It was also observed that the content of the products increased over three times. Taxinine NN-11 (1) is a new taxane whose structure was assigned as 5alpha,13alpha-diacetoxy-9alpha-cinnamoyloxy-4(20),11-taxadien-10beta-ol by analysis of its spectral data. Taxinine NN-11 (1) exhibited significant MDR reversal activity toward 2780 AD tumor cells. The results of primary screening based on 39 human cancer cell lines suggest that 1 also belongs to a new mechanistic class. Efficient production of 1 was investigated using the callus culture of T. cuspidata.

Survival-signaling pathway as a promising target for cancer chemotherapy.

The serine/threonine kinase AKT, also known as PKB or RAC-PK, is a key molecule for protecting cells from undergoing apoptosis. Several studies have suggested that the AKT-mediated survival-signaling pathway is an attractive target for cancer chemotherapy: (1) the AKT pathway is relatively inactive in resting cells; (2) amplification of the AKT gene occurs in some tumors; (3) loss of the tumor suppressor gene PTEN (phosphatase and tensin homolog deleted on chromosome 10) is common in tumors and its loss constitutively activates AKT; (4) AKT is activated at the cancer invasion front. To clarify which drugs exhibit their cytotoxicity by inhibiting the AKT pathway, we screened anticancer drugs that could downregulate phospho-AKT levels and AKT kinase activity. We found that UCN-01 (7-hydroxystaurosporine), heat-shock protein 90 (HSP90) inhibitors, and topotecan (10-hydroxy-9-dimethylaminomethyl-(S)-camptothecin) possessed the ability to interfere with the AKT pathway. UCN-01 directly suppressed upstream AKT kinase 3-phosphoinositide-dependent protein kinase-1 (PDK1) (IC(50) <33 nM) both in vitro and in tumor xenografts. HSP90 inhibitors and topotecan suppressed AKT activity via indirectly downregulating PDK1 and phosphatidylinositide-3-OH kinase activities. Transfection of the constitutively active AKT complementary DNA into cells attenuated the cytotoxic effects of the drugs, indicating that inhibition of the AKT pathway plays an important role in exerting their cytotoxic effects. These results strongly suggest that the AKT-mediated survival-signaling pathway is a promising and attractive target for cancer chemotherapy.

Regulation of kinase activity of 3-phosphoinositide-dependent protein kinase-1 by binding to 14-3-3.

3-Phosphoinositide-dependent protein kinase-1 (PDK1) plays a central role in activating the protein kinase A, G, and C subfamily. In particular, PDK1 plays an important role in regulating the Akt survival pathway by phosphorylating Akt on Thr-308. PDK1 kinase activity was thought to be constitutively active; however, recent reports suggested that its activity is regulated by binding to other proteins, such as protein kinase C-related kinase-2 (PRK2), p90 ribosomal protein S6 kinase-2 (RSK2), and heat-shock protein 90 (Hsp90). Here we report that PDK1 binds to 14-3-3 proteins in vivo and in vitro through the sequence surrounding Ser-241, a residue that is phosphorylated by itself and is critical for its kinase activity. Mutation of PDK1 to increase its binding to 14-3-3 decreased its kinase activity in vivo. By contrast, mutation of PDK1 to decrease its interaction with 14-3-3 resulted in increased PDK1 kinase activity. Moreover, incubation of wild-type PDK1 with recombinant 14-3-3 in vitro decreased its kinase activity. These data indicate that PDK1 kinase activity is negatively regulated by binding to 14-3-3 through the PDK1 autophosphorylation site Ser-241.