15-Deoxyspergualin Inhibits Akt Kinase Activation and Phosphatidylcholine Synthesis

Manabu Kawada,Tohru Masuda,Masaaki Ishizuka,Tomio Takeuchi

doi:10.1074/jbc.m200318200

Manabu Kawada, Tohru Masuda + Show 2 more

Open Access

https://doi.org/10.1074/jbc.m200318200

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Abstract

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.

Highlights

Protein synthesis is regulated by many molecules
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 downregulation of Akt kinase and p70 S6 kinase (p70S6K)
DSG Inhibits Protein Synthesis and Induces Apoptosis—In vitro study of time course evaluation of growth revealed that the antiproliferative effect of DSG appeared only after 2 days of treatment

Summary

EXPERIMENTAL PROCEDURES

Reagents and Antibodies—DSG was provided from Takara Shuzou Co. Ltd. (Otsu, Japan). [3H]Thymidine, [3H]uridine, [3H]leucine, [␥-32P]ATP, [32P]phosphoric acid, and [14C]choline chloride were purchased from PerkinElmer Life Sciences. DNA Fragmentation—Cells were cultured in 10-cm dishes with or without DSG for 3 days, and 1 ϫ 106 cells were washed with phosphate-buffered saline and lysed in 100 ␮l of lysis buffer containing 0.5% Triton X-100, 10 mM Tris-HCl (pH 7.4), and 10 mM EDTA at room temperature for 10 min. The immune complexes were collected on protein A-Sepharose beads (Amersham Biosciences) or protein G-agarose beads (Santa Cruz Biotechnology) and washed four times in immunoprecipitation buffer (20 mM Hepes, pH 7.5, 150 mM NaCl, 1% Triton X-100, 1 mM EDTA, 50 mM NaF, 50 mM ␤-glycerophosphate, 0.1 mM phenylmethylsulfonyl fluoride, and 0.2 mM Na3VO4). Cells were resuspended for 30 min at 4 °C in a lysis buffer (1% Triton X-100, 20 mM Tris-HCl, pH 8.0, 100 mM NaCl, 10 mM Na4P2O7, 2 mM EDTA, 50 mM NaF, 10% glycerol, 1 mM phenylmethylsulfonyl fluoride, 1 mM Na3VO4, and 25 ␮g/ml each of antipain, leupeptin, and pepstatin).

Mean survival days

RESULTS

DISCUSSION