15-Deoxyspergualin Inhibits Akt Kinase Activation and Phosphatidylcholine Synthesis
2002; Elsevier BV; Volume: 277; Issue: 31 Linguagem: Inglês
10.1074/jbc.m200318200
ISSN1083-351X
AutoresManabu Kawada, Tohru Masuda, Masaaki Ishizuka, TOMIO TAKEUCHI,
Tópico(s)Healthcare and Venom Research
Resumo15-Deoxyspergualin (DSG) strongly inhibited growth of mouse EL-4 lymphoma cells in vitro and in vivo. It significantly prolonged the survival days of EL-4-transplanted mice. In vitro study revealed that its antiproliferative effect appeared only after 2 days of treatment. At that time, protein synthesis was significantly inhibited rather than DNA and RNA syntheses. Furthermore, DSG induced apoptosis without arresting the cell cycle. p70 S6 kinase (p70S6K), a key molecule in protein synthesis, was inhibited by 2 days of treatment of DSG. Akt, an upstream kinase of p70S6K, was also deactivated by 2 days of treatment of DSG. Hsp90 is reported to bind to and stabilize Akt kinase and also to bind to DSG. Yet DSG did not inhibit the binding of Hsp90 to Akt kinase. PI3-kinase, an activator of Akt, was not affected by DSG treatment. However, when we looked into phospholipid synthesis, we found that DSG inhibited phosphatidylcholine (PC) synthesis strongly rather than phosphatidylinositol even by 1 day of treatment. Moreover, DSG failed to inhibit Akt kinase activation and PC synthesis in DSG-less sensitive human K562 leukemia cells. These results demonstrate that DSG inhibits tumor cell growth through the inhibition of protein synthesis and induction of apoptosis, which is caused by the down-regulation of Akt kinase and p70S6K. It is also indicated that the down-regulation of Akt kinase by DSG should not depend on PI3-kinase and Hsp90. There might be possible involvement of PC in Akt kinase activity. 15-Deoxyspergualin (DSG) strongly inhibited growth of mouse EL-4 lymphoma cells in vitro and in vivo. It significantly prolonged the survival days of EL-4-transplanted mice. In vitro study revealed that its antiproliferative effect appeared only after 2 days of treatment. At that time, protein synthesis was significantly inhibited rather than DNA and RNA syntheses. Furthermore, DSG induced apoptosis without arresting the cell cycle. p70 S6 kinase (p70S6K), a key molecule in protein synthesis, was inhibited by 2 days of treatment of DSG. Akt, an upstream kinase of p70S6K, was also deactivated by 2 days of treatment of DSG. Hsp90 is reported to bind to and stabilize Akt kinase and also to bind to DSG. Yet DSG did not inhibit the binding of Hsp90 to Akt kinase. PI3-kinase, an activator of Akt, was not affected by DSG treatment. However, when we looked into phospholipid synthesis, we found that DSG inhibited phosphatidylcholine (PC) synthesis strongly rather than phosphatidylinositol even by 1 day of treatment. Moreover, DSG failed to inhibit Akt kinase activation and PC synthesis in DSG-less sensitive human K562 leukemia cells. These results demonstrate that DSG inhibits tumor cell growth through the inhibition of protein synthesis and induction of apoptosis, which is caused by the down-regulation of Akt kinase and p70S6K. It is also indicated that the down-regulation of Akt kinase by DSG should not depend on PI3-kinase and Hsp90. There might be possible involvement of PC in Akt kinase activity. p70 S6 kinase 15-deoxyspergualin phosphatidylinositol phosphatidylinositol 3-kinase phosphatidylinositol-3,4-bisphosphate phosphatidylinositol-3,4,5-triphosphate phosphatidylcholine FKBP12-rapamycin-associated protein 3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide heat shock proteins Protein synthesis is regulated by many molecules. Among them, p70 S6 kinase (p70S6K),1 a serine/threonine kinase, is one of the key molecules. p70S6K phosphorylates the 40-S ribosomal S6 protein, resulting in the translational up-regulation of mRNAs (1Terada N. Patel H.R. Takase K. Kohno K. Nairn A.C. Gelfand E.W. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 11477-11481Crossref PubMed Scopus (318) Google Scholar, 2Jefferies H.B.J. Reinhard C. Kozma S.C. Thomas G. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 4441-4445Crossref PubMed Scopus (550) Google Scholar). p70S6K is activated by the phosphatidylinositol 3-kinase (PI3K)-Akt pathway or FRAP (a mammalian target for rapamycin) (3Heitman J. Movva N.R. Hall M.N. Science. 1991; 253: 905-909Crossref PubMed Scopus (1521) Google Scholar) upon stimulation of growth signals. Rapamycin is an inhibitor of p70S6K activation (4Price D.J. Grove J.R. Calvo V. Avruch J. Bierer B.E. Science. 1992; 257: 973-977Crossref PubMed Scopus (585) Google Scholar, 5Kuo C.J. Chung J. Fiorentino D.F. Flanagan W.M. Blenis J. Crabtree G.R. Nature. 1992; 358: 70-73Crossref PubMed Scopus (561) Google Scholar) and inhibits FRAP by forming a complex with the immunophilin FK506-binding protein (6Schreiber S.L. Science. 1991; 251: 283-287Crossref PubMed Scopus (1335) Google Scholar), which binds to and inhibits FRAP (7Brown E.J. Beal P.A. Keith C.T. Chen J. Shin T.B. Schreiber S.L. Nature. 1995; 377: 441-446Crossref PubMed Scopus (616) Google Scholar). PI3K consists of p110 catalytic and p85 regulatory subunits (8Vlahos C.J. Matter W.F. FEBS Lett. 1992; 309: 242-248Crossref PubMed Scopus (53) Google Scholar) and generates the intracellular amounts of phosphatidylinositol-3,4-bisphosphate (PIP2) and phosphatidylinositol-3,4,5-triphosphate (PIP3) (9Dekker L.V. Segal A.W. Science. 2000; 287: 982-985Crossref PubMed Scopus (97) Google Scholar). Wortmannin is an inhibitor of PI3K and inhibits various downstream events of PI3K including Akt and p70S6K activations (10Klippel A. Kavanaugh W.M. Pot D. Williams L.T. Mol. Cell. Biol. 1997; 17: 338-344Crossref PubMed Scopus (445) Google Scholar, 11Franke T.F. Yang S.I. Chan T.O. Datta K. Kazlauskas A. Morrison D.K. Kaplan D.R. Tsichlis P.N. Cell. 1995; 81: 727-736Abstract Full Text PDF PubMed Scopus (1820) Google Scholar, 12Burgering B.M.T. Coffer P.J. Nature. 1995; 376: 599-602Crossref PubMed Scopus (1871) Google Scholar, 13Franke T.F. Kaplan D.R. Cantley L.C. Toker A. Science. 1997; 275: 665-668Crossref PubMed Scopus (1293) Google Scholar). Akt (also known as protein kinase B) is also a serine/threonine kinase, which mediates PI3K-regulated biological events. By stimulation, Akt is recruited from the cytosol to the plasma membrane and is phosphorylated at two key regulatory sites, Thr-308 in the catalytic domain and Ser-473 at the C terminus (14Alessi D.R. Andjelkovic M. Caudwell B. Cron P. Morrice N. Cohen P. Hemmings B.A. EMBO J. 1996; 15: 6541-6551Crossref PubMed Scopus (2495) Google Scholar). This translocation requires PI3K activation and the pleckstrin homology domain, through which Akt directly interacts with PIP2 or PIP3 (13Franke T.F. Kaplan D.R. Cantley L.C. Toker A. Science. 1997; 275: 665-668Crossref PubMed Scopus (1293) Google Scholar, 15James S.R. Downes C.P. Gigg R. Grove S.J. Holmes A.B. Alessi D.R. Biochem. J. 1996; 315: 709-713Crossref PubMed Scopus (270) Google Scholar,16Andjelkovic M. Alessi D.R. Meier R. Fernandez A. Lamb N.J.C. Frech M. Cron P. Cohen P. Lucocq J.M. Hemmings B.A. J. Biol. Chem. 1997; 272: 31515-31524Abstract Full Text Full Text PDF PubMed Scopus (895) Google Scholar). The membrane-bound Akt is then phosphorylated by 3-phosphoinositide-dependent kinase-1 (PDK1) (17Stephens L. Anderson K. Stokoe D. Erdjument-Bromage H. Painter G.F. Holmes A.B. Gaffney P.R.J. Reese C.B. McCormick F. Tempst P. Coadwell J. Hawkins P.T. Science. 1998; 279: 710-714Crossref PubMed Scopus (910) Google Scholar). One of targets of Akt is p70S6K, and the PI3K-Akt pathway activates p70S6K (12Burgering B.M.T. Coffer P.J. Nature. 1995; 376: 599-602Crossref PubMed Scopus (1871) Google Scholar). Akt also plays an important role in a cell survival signaling pathway and inactivates BAD, a Bcl-2 family protein, rendering it incapable of blocking Bcl-2 activity (18Datta S.R. Dudek H. Tao X. Masters S., Fu, H. Gotoh Y. Greenberg M.E. Cell. 1997; 91: 231-241Abstract Full Text Full Text PDF PubMed Scopus (4914) Google Scholar, 19Peso L. Gonzalez-Garcia M. Page C. Herrera R. Nunez G. Science. 1997; 278: 687-689Crossref PubMed Scopus (1978) Google Scholar).15-Deoxyspergualin (DSG) is the most potent synthetic analogue of spergualin, which was isolated as an antitumor compound from microbial cultured broth (20Takeuchi T. Iinuma H. Kunimoto S. Masuda T. Ishizuka M. Takeuchi M. Hamada M. Naganawa H. Kondo S. Umezawa H. J. Antibiot. (Tokyo). 1981; 34: 1619-1621Crossref PubMed Scopus (194) Google Scholar, 21Umeda Y. Moriguchi M. Kuroda H. Nkamura T. Iinuma H. Takeuchi T. Umezawa H. J. Antibiot. (Tokyo). 1985; 38: 886-898Crossref PubMed Scopus (90) Google Scholar, 22Iwasawa H. Kondo S. Ikeda D. Takeuchi T. Umezawa H. J. Antibiot. (Tokyo). 1982; 35: 1665-1669Crossref PubMed Scopus (86) Google Scholar). DSG inhibits growth of various tumor cell lines in vitro and in vivo (23Plowman J. Steadman D. Harrison J. Trader M.W. Daniel P. Griswold J. Chadwick M. McComish M.F. Silveira D.M. Zaharko D. Cancer Res. 1987; 47: 685-689PubMed Google Scholar, 24Nishikawa K. Shibasaki C. Hiratsuka M. Arakawa M. Takahasi K. Takeuchi T. J. Antibiot. (Tokyo). 1991; 44: 1101-1109Crossref PubMed Scopus (14) Google Scholar). Furthermore, DSG has a potent immunosuppressive effect and has been used clinically as an immunosuppressant (25Nemoto K. Hayaxhi M. Abe F. Nkamura T. Ishizuka M. Umezawa H. J. Antibiot. (Tokyo). 1987; 40: 561-562Crossref PubMed Scopus (65) Google Scholar, 26Masuda T. Mizutani S. Iijima M. Odai H. Suda H. Ishizuka M. Takeuchi T. Umezawa H. J. Antibiot. (Tokyo). 1987; 40: 1612-1618Crossref PubMed Scopus (24) Google Scholar). It is reported that DSG binds to heat shock proteins (HSPs) such as Hsc70 and Hsp90 and inhibits their functions (27Nadeau K. Nadler S.G. Saulnier M. Tepper M.A. Walsh C.T. Biochemistry. 1994; 33: 2561-2567Crossref PubMed Scopus (114) Google Scholar, 28Nadler S.G. Tepper M.A. Schacter B. Mazzucco C.E. Science. 1992; 258: 484-486Crossref PubMed Scopus (221) Google Scholar). In respect to the antitumor effect, DSG is reported to inhibit the cell cycle progression at G1 phase (29Nishikawa K. Shibasaki C. Uchida T. Takahasi K. Takeuchi T. J. Antibiot. (Tokyo). 1991; 44: 1237-1246Crossref PubMed Scopus (9) Google Scholar, 30Hiratsuka M. Kuramochi H. Takahasi K. Takeuchi T. Oshimura M. Jpn. J. Cancer Res. 1991; 82: 1065-1068Crossref PubMed Scopus (14) Google Scholar) and tumorigenic angiogenesis (31Oikawa T. Shimamura M. Ashino-Fuse H. Iwaguchi T. Ishizuka M. Takeuchi T. J. Antibiot. (Tokyo). 1991; 44: 1033-1035Crossref PubMed Scopus (16) Google Scholar, 32Oikawa T. Hasegawa M. Morita I. Murota S.-I. Ahisno H. Shimamura M. Kiue A. Hamanaka R. Kuwano M. Ishizuka M. Takeuchi T. Anti-Cancer Drugs. 1992; 3: 293-299Crossref PubMed Scopus (16) Google Scholar). However, the precise mechanism of DSG action remains to be elucidated. We have been studying the mechanism of DSG action concerning its antitumor effect. DSG has a spermidine and a guanidine moiety in its structure. Focusing on its structural characteristic, we have recently reported that the spermidine moiety has cell binding activity and that the guanidine moiety has cytotoxic activity (33Kawada M. Someno T. Iinuma H. Masuda T. Ishizuka M. Takeuchi T. J. Antibiot. (Tokyo). 2000; 53: 705-710Crossref PubMed Scopus (5) Google Scholar). Moreover, it is suggested that its action should not be related to polyamine synthesis. In this study, we have further investigated the precise mechanism of DSG action on tumor cell growth. Finally, we have found that DSG inhibits Akt kinase activation and phosphatidylcholine synthesis.DISCUSSIONBecause DSG has a spermidine moiety, mechanistic studies of DSG had been first done to study polyamine synthesis. Then, DSG was reported to inhibit spermidine synthase, spermine synthase, and polyamine oxidase (38Hibasami H. Tsukada T. Suzuki R. Takano K. Takaji S. Takeuchi T. Shirakawa S. Murata T. Nakashima K. Anticancer Res. 1991; 11: 325-330PubMed Google Scholar). Moreover, DSG was reported to be unstable in serum because of degradation by amine oxidase and found to be a good inhibitor of amine oxidase (39Kunimoto S. Takeuchi T. J. Antibiot. (Tokyo). 1994; 47: 1130-1135Crossref PubMed Scopus (3) Google Scholar). Using other amine oxidase inhibitors, two modes of cytotoxic action of DSG were suggested, one dependent on and one independent of amine oxidase in serum (40Kunimoto S. Nosaka C., Xu, C.Z. Takeuchi T. J. Antibiot. (Tokyo). 1989; 42: 116-122Crossref PubMed Scopus (8) Google Scholar, 41Kuramochi H. Hiratsuka M. Nagamine S. Takehashi K. Nakamura T. Takeuchi T. Umezawa H. J. Antibiot. (Tokyo). 1988; 41: 234-238Crossref PubMed Scopus (16) Google Scholar). However, using EL-4 cells, our recent results have suggested other possibilities. As reported previously, modification of polyamine metabolism did not affect DSG action on EL-4 cells (33Kawada M. Someno T. Iinuma H. Masuda T. Ishizuka M. Takeuchi T. J. Antibiot. (Tokyo). 2000; 53: 705-710Crossref PubMed Scopus (5) Google Scholar). Erwin and Pegg (42Erwin B.G. Pegg A.E. Biochem. J. 1986; 238: 581-587Crossref PubMed Scopus (49) Google Scholar) reported that DSG activates spermidine/spermine acetyl transferase activity . However, DSG did not increase spermidine/spermine acetyl transferase activity in EL-4 cells even by 2 days of treatment (data not shown). Therefore, we focused on the growth inhibitory mechanism instead of the polyamine metabolism in this study. The antiproliferative effect of DSG is unique, and it inhibited the growth only after 2 days of treatment even at high doses (Fig. 2). Because the cell growth seemed to be arrested, it is first assumed that DSG could arrest the cell cycle. We then assessed flow cytometric analysis (Fig. 2 B) and evaluated the changes in various cell cycle-related molecules including cyclins, cyclin-dependent kinases, and cyclin-dependent kinase inhibitors (data not shown). Although we could not obtain any evidence of the cell cycle arrest, we found that DSG induced apoptosis in EL-4 cells (Fig. 2, Band C). It is reported that DSG arrests the cell cycle at G1 phase (29Nishikawa K. Shibasaki C. Uchida T. Takahasi K. Takeuchi T. J. Antibiot. (Tokyo). 1991; 44: 1237-1246Crossref PubMed Scopus (9) Google Scholar, 30Hiratsuka M. Kuramochi H. Takahasi K. Takeuchi T. Oshimura M. Jpn. J. Cancer Res. 1991; 82: 1065-1068Crossref PubMed Scopus (14) Google Scholar). In contrast, Odaka et al.(43Odaka C. Toyoda E. Nemoto K. Immunology. 1998; 95: 370-376Crossref PubMed Scopus (10) Google Scholar) reported that DSG induced apoptosis in T-cell hybridomas. Therefore, DSG action on the cell cycle is different and is dependent on the cell line used, suggesting that its direct effect will not act on the cell cycle.Macromolecular synthesis revealed that DSG inhibited protein synthesis strongly rather than inhibiting DNA synthesis strongly (Fig. 3). Hibasami et al. (38Hibasami H. Tsukada T. Suzuki R. Takano K. Takaji S. Takeuchi T. Shirakawa S. Murata T. Nakashima K. Anticancer Res. 1991; 11: 325-330PubMed Google Scholar) also reported that DSG inhibited protein synthesis greatly in other cell lines. Therefore, the inhibition of protein synthesis is considered to be a common effect in DSG-treated cells. To study the mechanism of DSG action on protein synthesis, we first examined the effect of DSG on p70S6K, a key molecule in protein synthesis. We then found that DSG inhibited p70S6K activation (Fig. 4). However, its inhibitory effect was also retarded and only apparent after 2 days of treatment (Fig. 4). Because DSG did not inhibit p70S6K directly, we next examined Akt kinase, an upstream molecule of p70S6K. As a result, we found that DSG inhibited Akt activation (Fig. 5). Hsp90 binds to and stabilizes Akt kinase (37Sato S. Fujita N. Tsuruo T. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 10832-10837Crossref PubMed Scopus (826) Google Scholar), and DSG associates with Hsp90 (27Nadeau K. Nadler S.G. Saulnier M. Tepper M.A. Walsh C.T. Biochemistry. 1994; 33: 2561-2567Crossref PubMed Scopus (114) Google Scholar, 28Nadler S.G. Tepper M.A. Schacter B. Mazzucco C.E. Science. 1992; 258: 484-486Crossref PubMed Scopus (221) Google Scholar). It is easily postulated that DSG should modulate Akt through interfering with their association. In EL-4 cells, Hsp90 associated with Akt, but DSG did not affect it (Fig.5), suggesting that DSG inhibits Akt kinase Hsp90-independently. Since Akt suppresses apoptosis through phosphorylation of BAD (18Datta S.R. Dudek H. Tao X. Masters S., Fu, H. Gotoh Y. Greenberg M.E. Cell. 1997; 91: 231-241Abstract Full Text Full Text PDF PubMed Scopus (4914) Google Scholar, 19Peso L. Gonzalez-Garcia M. Page C. Herrera R. Nunez G. Science. 1997; 278: 687-689Crossref PubMed Scopus (1978) Google Scholar), it is considered that DSG induces apoptosis in EL-4 cells through the down-regulation of Akt kinase.PI3K is an upstream effector of Akt, so we then examined the effect of DSG on PI3K activity (Fig. 6 A). As a result, DSG weakly inhibited PI3K activity only at a high dose (10 μg/ml). Because Akt is phosphorylated by PDK-1 (14Alessi D.R. Andjelkovic M. Caudwell B. Cron P. Morrice N. Cohen P. Hemmings B.A. EMBO J. 1996; 15: 6541-6551Crossref PubMed Scopus (2495) Google Scholar, 17Stephens L. Anderson K. Stokoe D. Erdjument-Bromage H. Painter G.F. Holmes A.B. Gaffney P.R.J. Reese C.B. McCormick F. Tempst P. Coadwell J. Hawkins P.T. Science. 1998; 279: 710-714Crossref PubMed Scopus (910) Google Scholar), we cannot exclude the possibility that DSG inhibits PDK-1 activity. When we evaluated the effect of DSG on phospholipid synthesis, we found that DSG significantly inhibited PC synthesis rather than PI synthesis (Fig. 6 B). Although partial inhibition of PI synthesis may contribute to the down-regulation of PI3K and Akt kinase, it is interesting that PC synthesis was inhibited even by 1 day of treatment of DSG (Fig.6 D). PC is a main constituent of plasma membrane and plays an important role in various enzyme activities. Because PC reduction by DSG precedes Akt down-regulation (Figs. 5 and 6 D), there might be a possibility that PC could regulate Akt kinase directly or indirectly. Furthermore, DSG failed to inhibit PC synthesis and Akt kinase in DSG-less sensitive K562 cells. Therefore, there is a correlation between strong growth inhibition and the down-regulation of PC synthesis and Akt kinase.In conclusion, the results obtained in this study demonstrate that DSG inhibits tumor cell growth through the inhibition of protein synthesis and induction of apoptosis by the downregulation of Akt kinase in a PI3K- and Hsp90-independent manner. Although EL-4 cells rapidly grew (<13h doubling time), the antiproliferative effect of DSG clearly appeared only by 2 days of treatment. Therefore, DSG will modulate some other intracellular events first, and the accumulated intracellular changes must result in the down-regulation of Akt kinase. The PC synthesis pathway is one of candidates for a real target of DSG. To evaluate the inhibition of PC synthesis by DSG on Akt pathway, we are now studying the precise mechanism of DSG action on the PC synthesis pathway. Protein synthesis is regulated by many molecules. Among them, p70 S6 kinase (p70S6K),1 a serine/threonine kinase, is one of the key molecules. p70S6K phosphorylates the 40-S ribosomal S6 protein, resulting in the translational up-regulation of mRNAs (1Terada N. Patel H.R. Takase K. Kohno K. Nairn A.C. Gelfand E.W. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 11477-11481Crossref PubMed Scopus (318) Google Scholar, 2Jefferies H.B.J. Reinhard C. Kozma S.C. Thomas G. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 4441-4445Crossref PubMed Scopus (550) Google Scholar). p70S6K is activated by the phosphatidylinositol 3-kinase (PI3K)-Akt pathway or FRAP (a mammalian target for rapamycin) (3Heitman J. Movva N.R. Hall M.N. Science. 1991; 253: 905-909Crossref PubMed Scopus (1521) Google Scholar) upon stimulation of growth signals. Rapamycin is an inhibitor of p70S6K activation (4Price D.J. Grove J.R. Calvo V. Avruch J. Bierer B.E. Science. 1992; 257: 973-977Crossref PubMed Scopus (585) Google Scholar, 5Kuo C.J. Chung J. Fiorentino D.F. Flanagan W.M. Blenis J. Crabtree G.R. Nature. 1992; 358: 70-73Crossref PubMed Scopus (561) Google Scholar) and inhibits FRAP by forming a complex with the immunophilin FK506-binding protein (6Schreiber S.L. Science. 1991; 251: 283-287Crossref PubMed Scopus (1335) Google Scholar), which binds to and inhibits FRAP (7Brown E.J. Beal P.A. Keith C.T. Chen J. Shin T.B. Schreiber S.L. Nature. 1995; 377: 441-446Crossref PubMed Scopus (616) Google Scholar). PI3K consists of p110 catalytic and p85 regulatory subunits (8Vlahos C.J. Matter W.F. FEBS Lett. 1992; 309: 242-248Crossref PubMed Scopus (53) Google Scholar) and generates the intracellular amounts of phosphatidylinositol-3,4-bisphosphate (PIP2) and phosphatidylinositol-3,4,5-triphosphate (PIP3) (9Dekker L.V. Segal A.W. Science. 2000; 287: 982-985Crossref PubMed Scopus (97) Google Scholar). Wortmannin is an inhibitor of PI3K and inhibits various downstream events of PI3K including Akt and p70S6K activations (10Klippel A. Kavanaugh W.M. Pot D. Williams L.T. Mol. Cell. Biol. 1997; 17: 338-344Crossref PubMed Scopus (445) Google Scholar, 11Franke T.F. Yang S.I. Chan T.O. Datta K. Kazlauskas A. Morrison D.K. Kaplan D.R. Tsichlis P.N. Cell. 1995; 81: 727-736Abstract Full Text PDF PubMed Scopus (1820) Google Scholar, 12Burgering B.M.T. Coffer P.J. Nature. 1995; 376: 599-602Crossref PubMed Scopus (1871) Google Scholar, 13Franke T.F. Kaplan D.R. Cantley L.C. Toker A. Science. 1997; 275: 665-668Crossref PubMed Scopus (1293) Google Scholar). Akt (also known as protein kinase B) is also a serine/threonine kinase, which mediates PI3K-regulated biological events. By stimulation, Akt is recruited from the cytosol to the plasma membrane and is phosphorylated at two key regulatory sites, Thr-308 in the catalytic domain and Ser-473 at the C terminus (14Alessi D.R. Andjelkovic M. Caudwell B. Cron P. Morrice N. Cohen P. Hemmings B.A. EMBO J. 1996; 15: 6541-6551Crossref PubMed Scopus (2495) Google Scholar). This translocation requires PI3K activation and the pleckstrin homology domain, through which Akt directly interacts with PIP2 or PIP3 (13Franke T.F. Kaplan D.R. Cantley L.C. Toker A. Science. 1997; 275: 665-668Crossref PubMed Scopus (1293) Google Scholar, 15James S.R. Downes C.P. Gigg R. Grove S.J. Holmes A.B. Alessi D.R. Biochem. J. 1996; 315: 709-713Crossref PubMed Scopus (270) Google Scholar,16Andjelkovic M. Alessi D.R. Meier R. Fernandez A. Lamb N.J.C. Frech M. Cron P. Cohen P. Lucocq J.M. Hemmings B.A. J. Biol. Chem. 1997; 272: 31515-31524Abstract Full Text Full Text PDF PubMed Scopus (895) Google Scholar). The membrane-bound Akt is then phosphorylated by 3-phosphoinositide-dependent kinase-1 (PDK1) (17Stephens L. Anderson K. Stokoe D. Erdjument-Bromage H. Painter G.F. Holmes A.B. Gaffney P.R.J. Reese C.B. McCormick F. Tempst P. Coadwell J. Hawkins P.T. Science. 1998; 279: 710-714Crossref PubMed Scopus (910) Google Scholar). One of targets of Akt is p70S6K, and the PI3K-Akt pathway activates p70S6K (12Burgering B.M.T. Coffer P.J. Nature. 1995; 376: 599-602Crossref PubMed Scopus (1871) Google Scholar). Akt also plays an important role in a cell survival signaling pathway and inactivates BAD, a Bcl-2 family protein, rendering it incapable of blocking Bcl-2 activity (18Datta S.R. Dudek H. Tao X. Masters S., Fu, H. Gotoh Y. Greenberg M.E. Cell. 1997; 91: 231-241Abstract Full Text Full Text PDF PubMed Scopus (4914) Google Scholar, 19Peso L. Gonzalez-Garcia M. Page C. Herrera R. Nunez G. Science. 1997; 278: 687-689Crossref PubMed Scopus (1978) Google Scholar). 15-Deoxyspergualin (DSG) is the most potent synthetic analogue of spergualin, which was isolated as an antitumor compound from microbial cultured broth (20Takeuchi T. Iinuma H. Kunimoto S. Masuda T. Ishizuka M. Takeuchi M. Hamada M. Naganawa H. Kondo S. Umezawa H. J. Antibiot. (Tokyo). 1981; 34: 1619-1621Crossref PubMed Scopus (194) Google Scholar, 21Umeda Y. Moriguchi M. Kuroda H. Nkamura T. Iinuma H. Takeuchi T. Umezawa H. J. Antibiot. (Tokyo). 1985; 38: 886-898Crossref PubMed Scopus (90) Google Scholar, 22Iwasawa H. Kondo S. Ikeda D. Takeuchi T. Umezawa H. J. Antibiot. (Tokyo). 1982; 35: 1665-1669Crossref PubMed Scopus (86) Google Scholar). DSG inhibits growth of various tumor cell lines in vitro and in vivo (23Plowman J. Steadman D. Harrison J. Trader M.W. Daniel P. Griswold J. Chadwick M. McComish M.F. Silveira D.M. Zaharko D. Cancer Res. 1987; 47: 685-689PubMed Google Scholar, 24Nishikawa K. Shibasaki C. Hiratsuka M. Arakawa M. Takahasi K. Takeuchi T. J. Antibiot. (Tokyo). 1991; 44: 1101-1109Crossref PubMed Scopus (14) Google Scholar). Furthermore, DSG has a potent immunosuppressive effect and has been used clinically as an immunosuppressant (25Nemoto K. Hayaxhi M. Abe F. Nkamura T. Ishizuka M. Umezawa H. J. Antibiot. (Tokyo). 1987; 40: 561-562Crossref PubMed Scopus (65) Google Scholar, 26Masuda T. Mizutani S. Iijima M. Odai H. Suda H. Ishizuka M. Takeuchi T. Umezawa H. J. Antibiot. (Tokyo). 1987; 40: 1612-1618Crossref PubMed Scopus (24) Google Scholar). It is reported that DSG binds to heat shock proteins (HSPs) such as Hsc70 and Hsp90 and inhibits their functions (27Nadeau K. Nadler S.G. Saulnier M. Tepper M.A. Walsh C.T. Biochemistry. 1994; 33: 2561-2567Crossref PubMed Scopus (114) Google Scholar, 28Nadler S.G. Tepper M.A. Schacter B. Mazzucco C.E. Science. 1992; 258: 484-486Crossref PubMed Scopus (221) Google Scholar). In respect to the antitumor effect, DSG is reported to inhibit the cell cycle progression at G1 phase (29Nishikawa K. Shibasaki C. Uchida T. Takahasi K. Takeuchi T. J. Antibiot. (Tokyo). 1991; 44: 1237-1246Crossref PubMed Scopus (9) Google Scholar, 30Hiratsuka M. Kuramochi H. Takahasi K. Takeuchi T. Oshimura M. Jpn. J. Cancer Res. 1991; 82: 1065-1068Crossref PubMed Scopus (14) Google Scholar) and tumorigenic angiogenesis (31Oikawa T. Shimamura M. Ashino-Fuse H. Iwaguchi T. Ishizuka M. Takeuchi T. J. Antibiot. (Tokyo). 1991; 44: 1033-1035Crossref PubMed Scopus (16) Google Scholar, 32Oikawa T. Hasegawa M. Morita I. Murota S.-I. Ahisno H. Shimamura M. Kiue A. Hamanaka R. Kuwano M. Ishizuka M. Takeuchi T. Anti-Cancer Drugs. 1992; 3: 293-299Crossref PubMed Scopus (16) Google Scholar). However, the precise mechanism of DSG action remains to be elucidated. We have been studying the mechanism of DSG action concerning its antitumor effect. DSG has a spermidine and a guanidine moiety in its structure. Focusing on its structural characteristic, we have recently reported that the spermidine moiety has cell binding activity and that the guanidine moiety has cytotoxic activity (33Kawada M. Someno T. Iinuma H. Masuda T. Ishizuka M. Takeuchi T. J. Antibiot. (Tokyo). 2000; 53: 705-710Crossref PubMed Scopus (5) Google Scholar). Moreover, it is suggested that its action should not be related to polyamine synthesis. In this study, we have further investigated the precise mechanism of DSG action on tumor cell growth. Finally, we have found that DSG inhibits Akt kinase activation and phosphatidylcholine synthesis. DISCUSSIONBecause DSG has a spermidine moiety, mechanistic studies of DSG had been first done to study polyamine synthesis. Then, DSG was reported to inhibit spermidine synthase, spermine synthase, and polyamine oxidase (38Hibasami H. Tsukada T. Suzuki R. Takano K. Takaji S. Takeuchi T. Shirakawa S. Murata T. Nakashima K. Anticancer Res. 1991; 11: 325-330PubMed Google Scholar). Moreover, DSG was reported to be unstable in serum because of degradation by amine oxidase and found to be a good inhibitor of amine oxidase (39Kunimoto S. Takeuchi T. J. Antibiot. (Tokyo). 1994; 47: 1130-1135Crossref PubMed Scopus (3) Google Scholar). Using other amine oxidase inhibitors, two modes of cytotoxic action of DSG were suggested, one dependent on and one independent of amine oxidase in serum (40Kunimoto S. Nosaka C., Xu, C.Z. Takeuchi T. J. Antibiot. (Tokyo). 1989; 42: 116-122Crossref PubMed Scopus (8) Google Scholar, 41Kuramochi H. Hiratsuka M. Nagamine S. Takehashi K. Nakamura T. Takeuchi T. Umezawa H. J. Antibiot. (Tokyo). 1988; 41: 234-238Crossref PubMed Scopus (16) Google Scholar). However, using EL-4 cells, our recent results have suggested other possibilities. As reported previously, modification of polyamine metabolism did not affect DSG action on EL-4 cells (33Kawada M. Someno T. Iinuma H. Masuda T. Ishizuka M. Takeuchi T. J. Antibiot. (Tokyo). 2000; 53: 705-710Crossref PubMed Scopus (5) Google Scholar). Erwin and Pegg (42Erwin B.G. Pegg A.E. Biochem. J. 1986; 238: 581-587Crossref PubMed Scopus (49) Google Scholar) reported that DSG activates spermidine/spermine acetyl transferase activity . However, DSG did not increase spermidine/spermine acetyl transferase activity in EL-4 cells even by 2 days of treatment (data not shown). Therefore, we focused on the growth inhibitory mechanism instead of the polyamine metabolism in this study. The antiproliferative effect of DSG is unique, and it inhibited the growth only after 2 days of treatment even at high doses (Fig. 2). Because the cell growth seemed to be arrested, it is first assumed that DSG could arrest the cell cycle. We then assessed flow cytometric analysis (Fig. 2 B) and evaluated the changes in various cell cycle-related molecules including cyclins, cyclin-dependent kinases, and cyclin-dependent kinase inhibitors (data not shown). Although we could not obtain any evidence of the cell cycle arrest, we found that DSG induced apoptosis in EL-4 cells (Fig. 2, Band C). It is reported that DSG arrests the cell cycle at G1 phase (29Nishikawa K. Shibasaki C. Uchida T. Takahasi K. Takeuchi T. J. Antibiot. (Tokyo). 1991; 44: 1237-1246Crossref PubMed Scopus (9) Google Scholar, 30Hiratsuka M. Kuramochi H. Takahasi K. Takeuchi T. Oshimura M. Jpn. J. Cancer Res. 1991; 82: 1065-1068Crossref PubMed Scopus (14) Google Scholar). In contrast, Odaka et al.(43Odaka C. Toyoda E. Nemoto K. Immunology. 1998; 95: 370-376Crossref PubMed Scopus (10) Google Scholar) reported that DSG induced apoptosis in T-cell hybridomas. Therefore, DSG action on the cell cycle is different and is dependent on the cell line used, suggesting that its direct effect will not act on the cell cycle.Macromolecular synthesis revealed that DSG inhibited protein synthesis strongly rather than inhibiting DNA synthesis strongly (Fig. 3). Hibasami et al. (38Hibasami H. Tsukada T. Suzuki R. Takano K. Takaji S. Takeuchi T. Shirakawa S. Murata T. Nakashima K. Anticancer Res. 1991; 11: 325-330PubMed Google Scholar) also reported that DSG inhibited protein synthesis greatly in other cell lines. Therefore, the inhibition of protein synthesis is considered to be a common effect in DSG-treated cells. To study the mechanism of DSG action on protein synthesis, we first examined the effect of DSG on p70S6K, a key molecule in protein synthesis. We then found that DSG inhibited p70S6K activation (Fig. 4). However, its inhibitory effect was also retarded and only apparent after 2 days of treatment (Fig. 4). Because DSG did not inhibit p70S6K directly, we next examined Akt kinase, an upstream molecule of p70S6K. As a result, we found that DSG inhibited Akt activation (Fig. 5). Hsp90 binds to and stabilizes Akt kinase (37Sato S. Fujita N. Tsuruo T. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 10832-10837Crossref PubMed Scopus (826) Google Scholar), and DSG associates with Hsp90 (27Nadeau K. Nadler S.G. Saulnier M. Tepper M.A. Walsh C.T. Biochemistry. 1994; 33: 2561-2567Crossref PubMed Scopus (114) Google Scholar, 28Nadler S.G. Tepper M.A. Schacter B. Mazzucco C.E. Science. 1992; 258: 484-486Crossref PubMed Scopus (221) Google Scholar). It is easily postulated that DSG should modulate Akt through interfering with their association. In EL-4 cells, Hsp90 associated with Akt, but DSG did not affect it (Fig.5), suggesting that DSG inhibits Akt kinase Hsp90-independently. Since Akt suppresses apoptosis through phosphorylation of BAD (18Datta S.R. Dudek H. Tao X. Masters S., Fu, H. Gotoh Y. Greenberg M.E. Cell. 1997; 91: 231-241Abstract Full Text Full Text PDF PubMed Scopus (4914) Google Scholar, 19Peso L. Gonzalez-Garcia M. Page C. Herrera R. Nunez G. Science. 1997; 278: 687-689Crossref PubMed Scopus (1978) Google Scholar), it is considered that DSG induces apoptosis in EL-4 cells through the down-regulation of Akt kinase.PI3K is an upstream effector of Akt, so we then examined the effect of DSG on PI3K activity (Fig. 6 A). As a result, DSG weakly inhibited PI3K activity only at a high dose (10 μg/ml). Because Akt is phosphorylated by PDK-1 (14Alessi D.R. Andjelkovic M. Caudwell B. Cron P. Morrice N. Cohen P. Hemmings B.A. EMBO J. 1996; 15: 6541-6551Crossref PubMed Scopus (2495) Google Scholar, 17Stephens L. Anderson K. Stokoe D. Erdjument-Bromage H. Painter G.F. Holmes A.B. Gaffney P.R.J. Reese C.B. McCormick F. Tempst P. Coadwell J. Hawkins P.T. Science. 1998; 279: 710-714Crossref PubMed Scopus (910) Google Scholar), we cannot exclude the possibility that DSG inhibits PDK-1 activity. When we evaluated the effect of DSG on phospholipid synthesis, we found that DSG significantly inhibited PC synthesis rather than PI synthesis (Fig. 6 B). Although partial inhibition of PI synthesis may contribute to the down-regulation of PI3K and Akt kinase, it is interesting that PC synthesis was inhibited even by 1 day of treatment of DSG (Fig.6 D). PC is a main constituent of plasma membrane and plays an important role in various enzyme activities. Because PC reduction by DSG precedes Akt down-regulation (Figs. 5 and 6 D), there might be a possibility that PC could regulate Akt kinase directly or indirectly. Furthermore, DSG failed to inhibit PC synthesis and Akt kinase in DSG-less sensitive K562 cells. Therefore, there is a correlation between strong growth inhibition and the down-regulation of PC synthesis and Akt kinase.In conclusion, the results obtained in this study demonstrate that DSG inhibits tumor cell growth through the inhibition of protein synthesis and induction of apoptosis by the downregulation of Akt kinase in a PI3K- and Hsp90-independent manner. Although EL-4 cells rapidly grew (<13h doubling time), the antiproliferative effect of DSG clearly appeared only by 2 days of treatment. Therefore, DSG will modulate some other intracellular events first, and the accumulated intracellular changes must result in the down-regulation of Akt kinase. The PC synthesis pathway is one of candidates for a real target of DSG. To evaluate the inhibition of PC synthesis by DSG on Akt pathway, we are now studying the precise mechanism of DSG action on the PC synthesis pathway. Because DSG has a spermidine moiety, mechanistic studies of DSG had been first done to study polyamine synthesis. Then, DSG was reported to inhibit spermidine synthase, spermine synthase, and polyamine oxidase (38Hibasami H. Tsukada T. Suzuki R. Takano K. Takaji S. Takeuchi T. Shirakawa S. Murata T. Nakashima K. Anticancer Res. 1991; 11: 325-330PubMed Google Scholar). Moreover, DSG was reported to be unstable in serum because of degradation by amine oxidase and found to be a good inhibitor of amine oxidase (39Kunimoto S. Takeuchi T. J. Antibiot. (Tokyo). 1994; 47: 1130-1135Crossref PubMed Scopus (3) Google Scholar). Using other amine oxidase inhibitors, two modes of cytotoxic action of DSG were suggested, one dependent on and one independent of amine oxidase in serum (40Kunimoto S. Nosaka C., Xu, C.Z. Takeuchi T. J. Antibiot. (Tokyo). 1989; 42: 116-122Crossref PubMed Scopus (8) Google Scholar, 41Kuramochi H. Hiratsuka M. Nagamine S. Takehashi K. Nakamura T. Takeuchi T. Umezawa H. J. Antibiot. (Tokyo). 1988; 41: 234-238Crossref PubMed Scopus (16) Google Scholar). However, using EL-4 cells, our recent results have suggested other possibilities. As reported previously, modification of polyamine metabolism did not affect DSG action on EL-4 cells (33Kawada M. Someno T. Iinuma H. Masuda T. Ishizuka M. Takeuchi T. J. Antibiot. (Tokyo). 2000; 53: 705-710Crossref PubMed Scopus (5) Google Scholar). Erwin and Pegg (42Erwin B.G. Pegg A.E. Biochem. J. 1986; 238: 581-587Crossref PubMed Scopus (49) Google Scholar) reported that DSG activates spermidine/spermine acetyl transferase activity . However, DSG did not increase spermidine/spermine acetyl transferase activity in EL-4 cells even by 2 days of treatment (data not shown). Therefore, we focused on the growth inhibitory mechanism instead of the polyamine metabolism in this study. The antiproliferative effect of DSG is unique, and it inhibited the growth only after 2 days of treatment even at high doses (Fig. 2). Because the cell growth seemed to be arrested, it is first assumed that DSG could arrest the cell cycle. We then assessed flow cytometric analysis (Fig. 2 B) and evaluated the changes in various cell cycle-related molecules including cyclins, cyclin-dependent kinases, and cyclin-dependent kinase inhibitors (data not shown). Although we could not obtain any evidence of the cell cycle arrest, we found that DSG induced apoptosis in EL-4 cells (Fig. 2, Band C). It is reported that DSG arrests the cell cycle at G1 phase (29Nishikawa K. Shibasaki C. Uchida T. Takahasi K. Takeuchi T. J. Antibiot. (Tokyo). 1991; 44: 1237-1246Crossref PubMed Scopus (9) Google Scholar, 30Hiratsuka M. Kuramochi H. Takahasi K. Takeuchi T. Oshimura M. Jpn. J. Cancer Res. 1991; 82: 1065-1068Crossref PubMed Scopus (14) Google Scholar). In contrast, Odaka et al.(43Odaka C. Toyoda E. Nemoto K. Immunology. 1998; 95: 370-376Crossref PubMed Scopus (10) Google Scholar) reported that DSG induced apoptosis in T-cell hybridomas. Therefore, DSG action on the cell cycle is different and is dependent on the cell line used, suggesting that its direct effect will not act on the cell cycle. Macromolecular synthesis revealed that DSG inhibited protein synthesis strongly rather than inhibiting DNA synthesis strongly (Fig. 3). Hibasami et al. (38Hibasami H. Tsukada T. Suzuki R. Takano K. Takaji S. Takeuchi T. Shirakawa S. Murata T. Nakashima K. Anticancer Res. 1991; 11: 325-330PubMed Google Scholar) also reported that DSG inhibited protein synthesis greatly in other cell lines. Therefore, the inhibition of protein synthesis is considered to be a common effect in DSG-treated cells. To study the mechanism of DSG action on protein synthesis, we first examined the effect of DSG on p70S6K, a key molecule in protein synthesis. We then found that DSG inhibited p70S6K activation (Fig. 4). However, its inhibitory effect was also retarded and only apparent after 2 days of treatment (Fig. 4). Because DSG did not inhibit p70S6K directly, we next examined Akt kinase, an upstream molecule of p70S6K. As a result, we found that DSG inhibited Akt activation (Fig. 5). Hsp90 binds to and stabilizes Akt kinase (37Sato S. Fujita N. Tsuruo T. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 10832-10837Crossref PubMed Scopus (826) Google Scholar), and DSG associates with Hsp90 (27Nadeau K. Nadler S.G. Saulnier M. Tepper M.A. Walsh C.T. Biochemistry. 1994; 33: 2561-2567Crossref PubMed Scopus (114) Google Scholar, 28Nadler S.G. Tepper M.A. Schacter B. Mazzucco C.E. Science. 1992; 258: 484-486Crossref PubMed Scopus (221) Google Scholar). It is easily postulated that DSG should modulate Akt through interfering with their association. In EL-4 cells, Hsp90 associated with Akt, but DSG did not affect it (Fig.5), suggesting that DSG inhibits Akt kinase Hsp90-independently. Since Akt suppresses apoptosis through phosphorylation of BAD (18Datta S.R. Dudek H. Tao X. Masters S., Fu, H. Gotoh Y. Greenberg M.E. Cell. 1997; 91: 231-241Abstract Full Text Full Text PDF PubMed Scopus (4914) Google Scholar, 19Peso L. Gonzalez-Garcia M. Page C. Herrera R. Nunez G. Science. 1997; 278: 687-689Crossref PubMed Scopus (1978) Google Scholar), it is considered that DSG induces apoptosis in EL-4 cells through the down-regulation of Akt kinase. PI3K is an upstream effector of Akt, so we then examined the effect of DSG on PI3K activity (Fig. 6 A). As a result, DSG weakly inhibited PI3K activity only at a high dose (10 μg/ml). Because Akt is phosphorylated by PDK-1 (14Alessi D.R. Andjelkovic M. Caudwell B. Cron P. Morrice N. Cohen P. Hemmings B.A. EMBO J. 1996; 15: 6541-6551Crossref PubMed Scopus (2495) Google Scholar, 17Stephens L. Anderson K. Stokoe D. Erdjument-Bromage H. Painter G.F. Holmes A.B. Gaffney P.R.J. Reese C.B. McCormick F. Tempst P. Coadwell J. Hawkins P.T. Science. 1998; 279: 710-714Crossref PubMed Scopus (910) Google Scholar), we cannot exclude the possibility that DSG inhibits PDK-1 activity. When we evaluated the effect of DSG on phospholipid synthesis, we found that DSG significantly inhibited PC synthesis rather than PI synthesis (Fig. 6 B). Although partial inhibition of PI synthesis may contribute to the down-regulation of PI3K and Akt kinase, it is interesting that PC synthesis was inhibited even by 1 day of treatment of DSG (Fig.6 D). PC is a main constituent of plasma membrane and plays an important role in various enzyme activities. Because PC reduction by DSG precedes Akt down-regulation (Figs. 5 and 6 D), there might be a possibility that PC could regulate Akt kinase directly or indirectly. Furthermore, DSG failed to inhibit PC synthesis and Akt kinase in DSG-less sensitive K562 cells. Therefore, there is a correlation between strong growth inhibition and the down-regulation of PC synthesis and Akt kinase. In conclusion, the results obtained in this study demonstrate that DSG inhibits tumor cell growth through the inhibition of protein synthesis and induction of apoptosis by the downregulation of Akt kinase in a PI3K- and Hsp90-independent manner. Although EL-4 cells rapidly grew (<13h doubling time), the antiproliferative effect of DSG clearly appeared only by 2 days of treatment. Therefore, DSG will modulate some other intracellular events first, and the accumulated intracellular changes must result in the down-regulation of Akt kinase. The PC synthesis pathway is one of candidates for a real target of DSG. To evaluate the inhibition of PC synthesis by DSG on Akt pathway, we are now studying the precise mechanism of DSG action on the PC synthesis pathway. We thank Dr. S. Kunimoto and Dr. H. Kumagai for helpful discussions, S. Ohba and I. Usami for technical assistance, and K. Miyaji for secretarial assistance.
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