Toshiaki Hitomi

Sesamin, a lignan of sesame, down‐regulates cyclin D1 protein expression in human tumor cells

Sesamin is a major lignan constituent of sesame and possesses multiple functions such as antihypertensive, cholesterol‐lowering, lipid‐lowering and anticancer activities. Several groups have previously reported that sesamin induces growth inhibition in human cancer cells. However, the nature of this growth inhibitory mechanism remains unknown. The authors here report that sesamin induces growth arrest at the G1 phase in cell cycle progression in the human breast cancer cell line MCF‐7. Furthermore, sesamin dephosphorylates tumor‐suppressor retinoblastoma protein (RB). It is also shown that inhibition of MCF‐7 cell proliferation by sesamin is correlated with down‐regulated cyclin D1 protein expression, a proto‐oncogene that is overexpressed in many human cancer cells. It was found that sesamin‐induced down‐regulation of cyclin D1 was inhibited by proteasome inhibitors, suggesting that sesamin suppresses cyclin D1 protein expression by promoting proteasome degradation of cyclin D1 protein. Sesamin down‐regulates cyclin D1 protein expression in various kinds of human tumor cells, including lung cancer, transformed renal cells, immortalized keratinocyte, melanoma and osteosarcoma. Furthermore, depletion of cyclin D1 protein using small interfering RNA rendered MCF‐7 cells insensitive to the growth inhibitory effects of sesamin, implicating that cyclin D1 is at least partially related to the antiproliferative effects of sesamin. Taken together, these results suggest that the ability of sesamin to down‐regulate cyclin D1 protein expression through the activation of proteasome degradation could be one of the mechanisms of the antiproliferative activity of this agent. (Cancer Sci 2007; 98: 1447–1453)

p15INK4b in HDAC inhibitor‐induced growth arrest

Histone deacetylase (HDAC) inhibitors arrest human tumor cells at the G1 phase of the cell cycle and activate the cyclin‐dependent kinase inhibitor, p21WAF1/Cip1. However, several studies have suggested the existence of a p21WAF1/Cip1‐independent molecular pathway. We report here that HDAC inhibitors, trichostatin A (TSA) and sodium butyrate, activate the p15INK4b gene, a member of the INK4 gene family, through its promoter in HaCaT cells. Furthermore, we show that up‐regulation of p15INK4b by TSA is associated with cell growth inhibition of HCT116 p21 (−/−) cells. Our findings suggest that p15INK4b is one of the important molecular targets of HDAC inhibitors.

Indole-3-carbinol activates the cyclin-dependent kinase inhibitor p15INK4b gene

Indole‐3‐carbinol (I3C) is a naturally occurring compound found in vegetables such as broccoli and cauliflower, and has been shown to arrest human tumor cells in the G1 phase of the cell cycle. However, the molecular mechanism responsible for this effect has not been sufficiently elucidated. We report here that I3C activates the cyclin‐dependent kinase (CDK) inhibitor p15INK4b gene through its promoter, accompanied by cell growth inhibition in HaCaT cells. Treatment with I3C almost did not affect the expressions of the other CDK inhibitors such as p19INK4d, p21WAF1 and p27Kip1. These results suggest that p15INK4b is an important molecular target of I3C among CDK inhibitors.

ZD1839 induces p15INK4b and causes G1 arrest by inhibiting the mitogen-activated protein kinase/extracellular signal–regulated kinase pathway

Inactivation of the retinoblastoma protein pathway is the most common abnormality in malignant tumors. We therefore tried to detect agents that induce the cyclin-dependent kinase inhibitor p15INK4b and found that ZD1839 (gefitinib, Iressa) could up-regulate p15INK4b expression. ZD1839 has been shown to inhibit cell cycle progression through inhibition of signaling pathways such as phosphatidylinositol 3′-kinase-Akt and mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase (ERK) cascades. However, the mechanism responsible for the differential sensitivity of the signaling pathways to ZD1839 remains unclear. We here showed that ZD1839 up-regulated p15INK4b, resulting in retinoblastoma hypophosphorylation and G1 arrest in human immortalized keratinocyte HaCaT cells. p15INK4b induction was caused by MAPK/ERK kinase inhibitor (PD98059), but not by Akt inhibitor (SH-6, Akt-III). Moreover, mouse embryo fibroblasts lacking p15INK4b were resistant to the growth inhibitory effects of ZD1839 compared with wild-type mouse embryo fibroblasts. Additionally, the status of ERK phosphorylation was related to the antiproliferative activity of ZD1839 in human colon cancer HT-29 and Colo320DM cell lines. Our results suggest that induction of p15INK4b by inhibition of the MAPK/ERK pathway is associated with the antiproliferative effects of ZD1839. [Mol Cancer Ther 2007;6(5):1579–1587]

Activation of protein kinase C promotes human cancer cell growth through downregulation of p18 INK4c

p18INK4c, a member of INK4 family of cyclin-dependent kinase inhibitors, negatively regulates the cyclin D-cyclin-dependent kinase 4/6 complexes which promote G1/S transition by phosphorylating the retinoblastoma tumor-suppressor gene product. Several recent studies using p18INK4c-null mice revealed that the p18INK4c plays an important role in cell proliferation and tumor development. We report here that 12-O-tetradecanoylphorbol-13-acetate (TPA), widely used as a protein kinase C (PKC) activator, suppresses the expression of p18INK4c through its promoter, accompanied by the induction of human cancer cell growth. Reduction of p18INK4c using small interfering RNA (siRNA) also enhanced cell growth, suggesting that p18INK4c is a critical target of TPA. Ro 31-8425, a potent and highly specific PKC inhibitor abrogated the suppressive effect of TPA on p18INK4c gene expression. However, the expression of dominant-negative c-Jun (TAM-67) did not inhibit the action of TPA on p18INK4c. These findings suggest that activation of PKC promotes human cancer cell growth through downregulation of p18INK4c in an AP-1 activation-independent manner. These results suggest that the accelerated cellular proliferation of some human tumors caused by enhanced PKC activity at least partially involves the suppression of p18INK4c, which is a ubiquitously expressed cyclin-dependent kinase inhibitor.

Oct‐1 is involved in the transcriptional repression of the p15INK4b gene

p15INK4b functions as a tumor suppressor and implicated in cellular senescence. Here, we show that the Oct‐1 binding site in the human p15INK4b gene promoter functions as a silencer. Oct‐1 specifically interacts with this binding site in vitro and in vivo and SMRT and HDAC1 are present in the p15INK4b proximal promoter region. Moreover, mouse embryo fibroblasts (MEFs) lacking Oct‐1 have shown significantly increased levels of p15INK4b protein compared to their normal counterparts. Treatment with a histone deacetylase (HDAC) inhibitor has activated the expression of p15INK4b in wild‐type MEFs but has no effect in MEFs lacking Oct‐1, suggesting that Oct‐1 represses p15INK4b gene expression in an HDAC‐dependent manner. Finally, we show that the expression of Oct‐1 protein significantly decreases, whereas p15INK4b protein significantly increases with the cellular aging process. Taken together, these results suggest that Oct‐1 is an important transcriptional repressor for p15INK4b gene and the transcriptional repression of the p15INK4b gene by Oct‐1 may be one of the regulatory mechanisms of cellular senescence.

Perillyl alcohol causes G1 arrest through p15INK4b and p21WAF1/Cip1 induction

The monoterpene perillyl alcohol (POH) is a naturally occurring compound derived from citrus fruits, mint and herbs. It exhibited chemotherapeutic potential against various malignant tumors in preclinical models and is currently being tested in clinical trials in patients with refractory advanced cancers. POH inhibits cellular proliferation at the G1 phase of the cell cycle in vitro. However, the molecular mechanisms responsible for this effect have not been sufficiently elucidated. Here we showed that 1.0 mM POH upregulates p15(INK4b) and p21(WAF1/Cip1), resulting in hypophosphorylation of the retinoblastoma (RB) protein and subsequent G1 arrest in human immortalized keratinocyte HaCaT cells. The induction of p15(INK4b) was mediated through its promoter, but that of p21(WAF1/Cip1) was not. The small interfering RNA (siRNA) of either p15(INK4b) or p21(WAF1/Cip1) significantly attenuated the increase in the G1 cell population caused by POH. The induction of p15(INK4b) and p21(WAF1/Cip1) and sub-sequent G1 arrest by POH was also observed in other cancer cell lines. These results suggest that the induction of p15(INK4b) as well as p21(WAF1/Cip1) is associated with the antiproliferative effect of POH.

Molecular cloning and characterization of the human p19INK4d gene promoter

p19INK4d, a member of the INK4 family of cyclin‐dependent kinase (CDK) inhibitors, negatively regulates the cyclin D–CDK4/6 complexes, which promote G1/S transition by phosphorylating the retinoblastoma tumor‐suppressor gene product. To investigate the mechanism of transcriptional regulation of the p19INK4d gene, we characterized the 5′‐flanking region of the human p19INK4d gene. The cap‐site hunting method revealed that the transcription starts at −16 nucleotide (nt) upstream of the initiation codon. The 5′‐flanking region of the human p19INK4d gene was ligated to a luciferase reporter gene and possessed functional promoter activity. Luciferase assay with a series of truncated 5′‐flanking regions indicated that the region from −81 to −2 nt could drive the transcription of the p19INK4d gene. Several Sp1 and activating protein 2 binding sites are located within the region from −81 to −2 nt. Mutation of the second Sp1 binding site from −33 to −25 nt decreased the promoter activity. Collectively, it was demonstrated that the human p19INK4d gene is under the control of TATA‐less promoter and the Sp1 binding site is involved in the transcription.