Ryoko Nakanishi

Luteolin induces apoptosis via death receptor 5 upregulation in human malignant tumor cells

Luteolin, a naturally occurring flavonoid, induces apoptosis in various cancer cells. Little is known however concerning the underlying molecular mechanisms responsible for this activity. In this report, we reveal a novel mechanism by which luteolin-induced apoptosis occurs, and show for the first time that the apoptosis by luteolin is mediated through death receptor 5 (DR5) upregulation. Luteolin markedly induced the expression of DR5, along with Bcl-2-interacting domain cleavage and the activation of caspase-8, -10, -9 and -3. In addition, suppression of DR5 expression with siRNA efficiently reduced luteolin-induced caspase activation and apoptosis. Human recombinant DR5/Fc also inhibited luteolin-induced apoptosis. On the other hand, luteolin induced neither DR5 protein expression nor apoptosis in normal human peripheral blood mononuclear cells. These results suggest that DR5 induced by luteolin plays a role in luteolin-induced apoptosis, and raises the possibility that treatment with luteolin might be promising as a new therapy against cancer.

Genistein induces Gadd45 gene and G2/M cell cycle arrest in the DU145 human prostate cancer cell line.

Genistein is the most abundant isoflavone of soybeans and has been shown to cause growth arrest in various human cancer cell lines. However, the precise mechanism for this is still unclear. We report here that the growth arrest and DNA damage‐inducible gene 45 (gadd45) gene is induced by genistein via its promoter in a DU145 human prostate cancer cell line. The binding of transcription factor nuclear factor‐Y to the CCAAT site of the gadd45 promoter appears to be important for this activation by genistein.

Cyclin‐dependent kinase inhibitor SU9516 enhances sensitivity to methotrexate in human T‐cell leukemia Jurkat cells

(Cancer Sci 2010; 101: 728–734)

Polymorphisms in Promoter Sequences of the p15 INK4B and PTEN Genes of Normal Japanese Individuals

Gene promoter regions of p15INK4B, a cyclin-dependent kinase inhibitor, and phosphatase and tensin homolog (PTEN), a dual-function protein and lipid phosphatase, interact with regulatory factors for gene transcription and methylation. Normal individuals exhibit sequence polymorphisms in these regulatory genes. We isolated genomic DNA from whole blood of healthy Japanese individuals and sequenced promoter regions of the p15INK4B and PTEN genes. We also examined the influence of polymorphisms on promoter activity in several cell lines. We identified polymorphisms at positions −699, −394, and −242 and an insertion at position −320 in the p15INK4B gene and a polymorphism at position −1142 in the PTEN gene. Reporter gene analysis revealed that these polymorphisms influenced transcriptional regulation in their cell lines. Our results indicate for the first time that promoter sequences of the p15INK4B and PTEN genes differ among normal Japanese individuals and that promoter polymorphisms can influence gene transcription.

Abstract 2560: Cyclin-dependent kinase inhibitor SU9516 enhances sensitivity to methotrexate in human T-cell Jurkat cells

Proceedings: AACR 101st Annual Meeting 2010‐‐ Apr 17‐21, 2010; Washington, DC Methotrexate (MTX), a classical anti-folate drug, has been used to treat various hematological malignancies. Since MTX prevents tumor cells from proliferating by inhibiting dihydrofolate reductase (DHFR), DHFR expression is a key determinant of resistance to MTX in malignant hematological tumor cells. In fact, it is well known that elevated expression of DHFR is a direct factor in clinical therapeutic resistance to MTX. First of all, we demonstrated that the sensitivity to MTX was enhanced by the knockdown of the DHFR gene in human T-cell leukemia cell. Concretely, the anti-proliferative effect of MTX was significantly enhanced by the knockdown of DHFR expression by siRNA in Jurkat cells. Therefore, a novel strategy down-regulating DHFR expression seems promising for enhancing sensitivity to MTX. On the other hand, the expression of DHFR is controlled by the transcription factor E2F. Unphosphorylated retinoblastoma (RB) protein is known to bind and inhibit the E2F. Cyclin-dependent kinase (CDK) inhibitors activate RB function and inhibit E2F function. Then we tested whether CDK inhibitor acted as a suppressor of DHFR through the E2F site of the promoter. We found that SU9516, a cyclin-dependent kinase inhibitor, decreased phospho-RB (Ser780) and reduced the expression of both DHFR mRNA and protein, and the DHFR promoter activity was attenuated by SU9516 dependent on the E2F site. Furthermore, pretreatment with SU9516 significantly enhanced sensitivity to MTX in a colony formation assay. To expand these results in Jurkat cells, we have also examined other leukemic cell lines (human T cell leukemia CCRF-CEM cells and human erythroleukemia K562 cells) and confirmed that SU9516 also repressed the levels of DHFR protein and phopho-RB (Ser780), and enhanced the cytotoxicity of MTX in a colony formation assay in both of cell lines. To expand these results by SU9516, we have examined other CDK inhibitors (Purvalanol A and Cdk4/6 Inhibitor IV) and confirmed that these agents also decreased DHFR protein and phospho-RB (Ser780), and enhanced the cytotoxicity of MTX in a colony formation assay in Jurkat cells as similar as SU9516. Based on these findings, we conclude that a combination of cyclin-dependent kinase inhibitors and MTX may be useful for overcoming resistance to MTX. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2560.