Daisuke Iizuka

A Novel Anticancer Ribonucleoside, 1-(3-C-Ethynyl-β-D-ribo-pentofuranosyl)Cytosine, Enhances Radiation-Induced Cell Death in Tumor Cells

Abstract Inanami, O., Iizuka, D., Iwahara, A., Yamamori, T., Kon, Y., Asanuma, T., Matsuda, A., Kashiwakura, I., Kitazato, K. and Kuwabara, M. A Novel Anticancer Ribonucleoside, 1-(3-C-Ethynyl-β-d-ribo-pentofuranosyl)Cytosine, Enhances Radiation-Induced Cell Death in Tumor Cells. Radiat. Res. 162, 635–645 (2004). 1-(3-C-Ethynyl-β-d-ribo-pentofuranosyl)cytosine (ECyd, TAS106) is a newly developed anti-tumor agent that targets RNA synthesis. We report here that a low dose of ECyd induces radiosensitization of caspase-dependent apoptosis and reproductive cell death in cells of the gastric tumor cell lines MKN45 and MKN28 and murine rectum adenocarcinoma Colon26. Flow cytometry demonstrated that TAS106 induced the abrogation of the X-ray-induced G2/M checkpoint. Western blot analysis showed that X rays increased the expression of cyclin B1, phospho-Cdc2 and Wee1, whereas co-treatment with X rays and TAS106 decreased the expression of these cell cycle proteins associated with the G2/M checkpoint. Furthermore, TAS106 was shown to decrease the radiation-induced expression of survivin but not Bcl2 and BclXL regardless of TP53 status and cell type. Overexpression of wild-type survivin in MKN45 cells inhibited the induction of apoptosis induced by co-treatment with X rays and TAS106. These results suggest that TAS106 enhances X-ray-induced cell death through down-regulation of survivin and abrogation of the cell cycle machinery.

X irradiation induces the proapoptotic state independent of the loss of clonogenic ability in chinese hamster V79 cells

Abstract Iizuka, D., Inanami, O., Matsuda, A., Kashiwakura, I., Asanuma, T. and Kuwabara, M. X Irradiation Induces the Proapoptotic State Independent of the Loss of Clonogenic Ability in Chinese Hamster V79 Cells. Radiat. Res. 164, 36–44 (2005). The clonogenic ability (reproductive cell death) of Chinese hamster V79 cells was measured after treatment with X radiation and a newly developed anti-cancer drug, 1-(3-C-ethynyl-β-d-ribo-pentofuranosyl)cytosine (ECyd, TAS106). Amplification in the loss of clonogenicity was observed compared to that obtained for cells exposed to X rays alone. Addition of benzyloxycarbonyl-val-ala-asp-fluoromethylketone (Z-VAD-FMK), a broad-spectrum caspase inhibitor, attenuated the increased lethality, but the dose–response curve obtained was found to merely revert to that obtained for cells exposed to X rays alone. Flow cytometric analysis showed that the number of cells arrested at the G2/M phase by X irradiation was decreased by co-treatment with TAS106, and instead the number of cells in the sub-G1 phase increased. Western blot analysis proved that TAS106 treatment down-regulated the expression of the G2/M arrest-related proteins cyclin B1, phospho-CDC2 and WEE1. From these results, it was concluded that (1) no apoptosis was included in the dose–response curve obtained from cells exposed to X rays alone, (2) X radiation induced a potentially apoptotic (proapoptotic) state in cells independent of the loss of their clonogenic ability, and (3) TAS106 enhanced the loss of their clonogenic ability by converting the proapoptotic cells to apoptotic cells through the abrogation of arrest at the G2/M phase.

Purvalanol A Enhances Cell Killing by Inhibiting Up-Regulation of CDC2 Kinase Activity in Tumor Cells Irradiated with High Doses of X Rays

Abstract Iizuka, D., Inanami, O., Kashiwakura, I. and Kuwabara, M. Purvalanol A Enhances Cell Killing by Inhibiting Up-Regulation of CDC2 Kinase Activity in Tumor Cells Irradiated with High Doses of X Rays. Radiat. Res. 167, 563–571 (2007). To clarify the relationship between CDC2 kinase activity and radiation-induced apoptosis, we examined whether the cyclin-dependent kinase (CDK) inhibitor purvalanol A enhanced radiation-induced apoptosis in gastric tumor cells. MKN45 cells exposed to 20 Gy of X rays increased the CDC2 kinase activity and the expression of regulatory proteins (phospho-CDC2 and cyclin B1) of the G2/M phase, followed by activation of the G2/M checkpoint, whereas the treatment of X-irradiated MKN45 cells with 20 μM purvalanol A suppressed the increase in the CDC2 kinase activity and expression of the G2/M-phase regulatory proteins and reduced the fraction of the cells in the G2/M phase in the cell cycle. Furthermore, this treatment resulted in not only a significant increase in radiation-induced apoptosis but also the loss of clonogenicity in both MKN45 (p53-wild) and MKN28 (p53-mutated) cells. The expression of anti-apoptosis proteins, inhibitor of apoptosis protein (IAP) family members (survivin and XIAP) and BCL2 family members (Bcl-XL and Bcl-2), in purvalanol A-treated cells with and without X rays was significantly lower than for cells exposed to X rays alone. These results suggest that the inhibition of radiation-induced CDC2 kinase activity by purvalanol A induces apoptosis through the enhancement of active fragments of caspase 3.

Inhibition of cell proliferation by SARS-CoV infection in Vero E6 cells.

Abstract Severe acute respiratory syndrome (SARS) is caused by SARS‐coronavirus (SARS‐CoV). Infection of Vero E6 cells with SARS‐CoV inhibits cell proliferation. Our previous study indicated that Akt, which is poorly phosphorylated in confluent cultures of Vero E6 cells, is phosphorylated and then dephosphorylated upon infection by SARS‐CoV. In the present study, we showed that a serine residue of Akt was phosphorylated in Vero E6 cells in subconfluent culture and that Akt was dephosphorylated rapidly after SARS‐CoV infection without up‐regulation of its phosphorylation. Phosphorylation of glycogen synthase kinase‐3β, which is one of the downstream targets of Akt, was prevented in SARS‐CoV‐infected cells. However, treatment with glycogen synthase kinase‐3β small interfering RNA indicated that the glycogen synthase kinase‐3β signaling pathway was not related to inhibition of cell proliferation. Treatment of Vero E6 cells with the phosphatidylinositol 3′‐kinase/Akt inhibitor, LY294002, which induces dephosphorylation of Akt, inhibited cell proliferation. As shown in our previous studies, apoptosis occurred in virus‐infected cells within 18u2003h postinfection. Cellular mRNA transcription, which was reported to be up‐regulated in SARS‐CoV‐infected Caco‐2 cells, was not up‐regulated in virus‐infected Vero E6 cells, partially as a result of apoptosis. These results suggested that inhibition of cell proliferation is regulated by both the phosphatidylinositol 3′‐kinase/Akt signaling pathway and by apoptosis in SARS‐CoV‐infected Vero E6 cells. This is the first study to analyze SARS‐CoV‐induced cell growth inhibition.

Purvalanol A induces apoptosis and downregulation of antiapoptotic proteins through abrogation of phosphorylation of JAK2/STAT3 and RNA polymerase II.

To clarify the mechanisms of purvalanol A in the induction of apoptosis, we investigated whether purvalanol A influenced the RNA synthesis and expression of RNA polymerase II and signal transducer and activator of transcription 3 (STAT3). When MKN45 cells were treated with 30u2009μmol/l purvalanol A, mitochondrial dysfunction occurred before the induction of the apoptosis and the expression of antiapoptotic proteins survivin, Bcl-XL, and Bcl-2 was reduced. The treatment with parvalanol A was also shown to reduce not only mRNA for these proteins but also global RNA synthesis. The phosphorylation of the carboxy-terminal domain of RNA polymerase II, which was involved in transcriptional regulation, was strongly inhibited by purvalanol A, followed by the partial inhibition of the expression of RNA polymerase II. Furthermore, the phosphorylation at Tyr705 of STAT3, which is known to be a phosphorylation site for Janus kinase 2 (JAK2), was completely inhibited by purvalanol A early (3u2009h) after drug treatment, although the phosphorylation of STAT3 at Ser727, which is a phosphorylation site for Ras/Raf/MEK and extracellular signal-regulated protein kinase 1/2, was still detectable until late (12u2009h) after treatment. In addition, the tyrosine phosphorylation of JAK2 was efficiently inhibited by purvalanol A. These results suggest that the inhibition of JAK2/STAT3 and RNA polymerase II is crucial in the downregulation of antiapoptotic proteins leading to the apoptotic cell death induced by parvalanol A.