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

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1 Sesamin, a lignan of sesame, down-regulates cyclin Blackwell Publishing Asia D1 protein expression in human tumor cells Tomoya Yokota, 1,7 Youichirou Matsuzaki, 1,7 Makoto Koyama 1 Toshiaki Hitomi 1 Mayumi Kawanaka, 1 Masako Enoki-Konishi, 1 Yusuke Okuyama, 1,5 Junko Takayasu, 2 Hoyoku Nishino, 2 Akiyoshi Nishikawa, 3 Toshihiko Osawa 4 and Toshiyuki Sakai 1,6 Departments of 1 Molecular-Targeting Cancer Prevention, and 2 Biochemistry and Molecular Biology, Graduate School of Medical Science, Kyoto Prefectural University of Medicine, Kawaramachi-Hirokoji, Kamigyo-ku, Kyoto ; 3 Division of Pathology, National Institute of Health Sciences, Kamiyoga, Setagaya-ku, Tokyo ; 4 Laboratory of Food and Biodynamics, Graduate School of Bioagricultural Sciences, Nagoya University, Nagoya ; 5 Department of Gastroenterology, Kyoto First Red Cross Hospital, Honmachi, Higashiyama-ku, Kyoto , Japan (Received April 11, 2007/Revised May 24, 2007/Accepted May 29, 2007/Online publication July 12, 2007) 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 downregulate 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: ) Sesamin is a major lignan constituent of sesame and sesame oil, which have been traditional health foods in eastern countries for thousands of years. Many studies have revealed that sesamin is effective in preventing hypertension, thrombogenesis, (1) and hypercholesteremia by increasing hepatic fatty acid oxidation. (2,3) Additionally, sesamin exhibits antioxidative properties, by reducing peroxidation products in the plasma and liver of rats, (4 6) and exerts an inhibitory effect on chemically induced cancers. (7) Several groups have previously reported that treatment with sesamin induces growth inhibition and apoptosis in human lymphoid leukemia Molt 4B cells and human stomach cancer KATO III cells, respectively. (8,9) Furthermore, sesamin inhibits the incorporation of tritiated thymidine into human leukemia (HL-60) cells. (10) However, the growth inhibitory mechanism of sesamin remains to be elucidated. Cell cycle progression is regulated by cyclin-dependent kinases (CDK) that form complexes with cyclin in a phase-specific manner during the cell cycle. (11) Cyclin D1/D2/D3-CDK4/6 and cyclin E/A-CDK2 play important roles in promoting the G1-to-S phase transition of the cell cycle by phosphorylating tumor-suppressor retinoblastoma protein (RB). (11,12) Activation of cyclins/ CDK is counterbalanced by CDK inhibitors (CKI). The first family of CKI, referred to as the CIP/KIP family, consists of the related proteins known as p21 WAF1/Cip1, p27 Kip1 and p57 Kip2, and each member inhibits cyclin E/A-CDK2 complexes. (13,14) The second family of CDK inhibitors is called the INK4 family of proteins. The four members of the INK4 family, p16 INK4a, p15 INK4b, p18 INK4c and p19 INK4d, specifically and directly bind to CDK4/6 and inhibit their activities. (14 16) In the present study, it is shown that sesamin induces growth arrest at the G1 phase and dephosphorylates RB protein in the human breast cancer cell line MCF-7. It is also shown that sesamin specifically down-regulates the expression of cyclin D1 protein among the cell-cycle related molecules examined. The authors 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 suppresses cyclin D1 protein expression in various kinds of human tumor cells, including lung cancer, transformed renal cells, immortalized keratinocyte, melanoma and osteosarcoma. Depletion of cyclin D1 protein using small interfering RNA (sirna) rendered MCF-7 cells insensitive to the growth inhibitory effects of sesamin, suggesting that cyclin D1 is at least partially associated with the antiproliferative effects of sesamin. Overall, these results suggest that sesamin down-regulates cyclin D1 protein expression through the activation of proteasome degradation, and that this contributes to its growth inhibitory effects. Materials and Methods Cell culture, cell proliferation studies and treatment with sesamin. The human breast cancer cell line MCF-7, the human lung cancer cell line A549, the human transformed renal cell line 293T, the human immortalized keratinocyte cell line HaCaT and the human osteosarcoma cell line MG63 were maintained in Dulbecco s modified Eagle s medium (DMEM) with 10% fetal bovine serum (FBS). The human melanoma cell line UACC-62 was maintained in Roswell Park Memorial Institute (RPMI) 1640 medium with 10% FBS. The human breast cancer cell line T- 47D was maintained in RPMI 1640 medium with 10% FBS and 10 µg/ml insulin. All of these cells were incubated at 37 C in a humidified atmosphere of 5% CO 2. For the cell proliferation studies, MCF-7 cells were plated at cells in 12-well plates. 24 h after cell plating, various concentrations of sesamin (Takemoto Oil & Fat) were added to the culture medium. The number of viable cells was counted using the Trypan-blue dye exclusion test. 6 To whom correspondence should be addressed. tsakai@koto.kpu-m.ac.jp 7 The first two authors contributed equally to this work. doi: /j x Cancer Sci September 2007 vol. 98 no

2 Analysis of cell cycle progression. Unsynchronized MCF-7 cells were exposed to sesamin for 24 h and harvested. The cells were then fixed in 70% ethanol, treated with RNase, and their nuclei were stained with propidium iodide before their DNA content was measured using a Becton Dickinson FACScaliber. Protein isolation and western blot analysis. Cells were lysed using lysis buffer (50 mm Tris-HCl, ph 7.5, and 0.1% SDS). The protein extract was then boiled for 5 min and loaded onto a 12% polyacrylamide gel for p15 INK4b, p18 INK4c, p19 INK4d, p21 WAF1/Cip1, p27 Kip1, p57 Kip2, CDK2, CDK4, CDK6, cyclin A, cyclin D1, cyclin D3, and cyclin E detection or 7% polyacrylamide gel for RB detection, electrophoresed, and transferred to a nitrocellulose membrane. A mouse monoclonal antibody against human cyclin D1 (DCS-6, MBL) and CDK6 (DCS-83, MBL), and rabbit polyclonal antibodies against human p15 INK4b (C-20, Santa Cruz Biotechnology), p18 INK4c (N-20, Santa Cruz Biotechnology), p19 INK4d (C-20, Santa Cruz Biotechnology), p21 WAF1/Cip1 (C-19, Santa Cruz Biotechnology), p27 Kip1 (C-19, Santa Cruz Biotechnology), p57 Kip2 (P-0357, Sigma), CDK2 (M-2, Santa Cruz Biotechnology), CDK4 (H-22, Santa Cruz Biotechnology), cyclin A (C-19, Santa Cruz Biotechnology), cyclin D3 (C-16, Santa Cruz Biotechnology), cyclin E (HE-12, Santa Cruz Biotechnology) and RB (PM A, Pharmingen) were used as primary antibodies. Signals were then detected with an enhanced chemiluminescence (ECL) system (Amersham Pharmacia Biotech). RNA isolation and quantitative real-time reverse-transcription polymerase chain reaction (RT-PCR) analysis. Total RNA was isolated from MCF-7 cells treated with 100 µm sesamin for the indicated times using Sepasol-RNA I (Nakalai Tesque Inc.) according to the manufacturer s instructions. Total RNA (10 µg) was reverse transcribed to cdna in a 20 µl reaction volume, with Superscript Reverse transcriptase (Invitrogen Corporation, Carlsbad, CA, USA), using oligo (dt) primers (Toyobo Co., Ltd, Osaka, Japan). The reaction mixture was incubated at 42 C for 50 min, then at 70 C for 15 min to stop the reaction. An equivalent volume (1 µl) of cdna solution was used for the quantification of specific cdna using quantitative real-time RT- PCR. Quantitative real-time RT-PCR was carried out using an RT-PCR system GeneAmp5700 (Applied Biosystems, Foster, CA, USA). The expression level of cyclin D1 mrna was normalized to that of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mrna of the same sample. sirna transfection. Cyclin D1 sirna#1 (#16810, Ambion), cyclin D1 sirna#2 (#118854, Ambion) and negative control sirna (#4611, Ambion) were used. The targeted exon of cyclin D1 sirna#1 or #2 is exon 3 or 5, respectively. MCF-7 cells were plated at cells in 24-well plates and transfected with 0.5 µm of sirna using lipofectamine 2000 reagent (Invitrogen). Fig. 1. The effect of sesamin on the growth of MCF-7 cells. One day after the inoculation of MCF-7 cells, sesamin at 12.5, 25, 50, or 100 µm was added, and cell growth was compared with a control culture with equivalent dimethylsulfoxide (DMSO; white square). Results Sesamin induces growth arrest at the G1 phase in cell cycle progression in MCF-7 human breast cancer cells. The effect of sesamin on the proliferation of the human breast cancer cell line MCF-7 was first examined. Fig. 1 shows the growth of MCF-7 cells in the presence or absence of various concentrations of sesamin. Sesamin inhibited the proliferation of MCF-7 cells in a dosedependent manner and 100 µm sesamin had a cytostatic effect. The percentage of viable cells was 88, 70, 60 or 45% of the control level following treatment with 12.5, 25, 50 or 100 µm sesamin for 24 h, respectively (Fig. 1). To investigate the effect of sesamin on the cell cycle progression of MCF-7 cells, the DNA content of the cell nuclei was measured using flow cytometric analysis. Treatment with 100 µm sesamin for 24 h significantly (P < 0.05) increased the percentage of cells in the G1 phase from 31.0 to 66.3%, and significantly (P < 0.05) decreased those in the S phase from 44.1 to 19.1% (Fig. 2). These data show that sesamin arrests the cell cycle of MCF-7 cells at the G1 phase. Sesamin dephosphorylates the RB protein in MCF-7 cells. Cyclins CDK play important roles in promoting the G1-to-S phase transition of the cell cycle by phosphorylating RB protein. (12,14) Fig. 2. The effect of sesamin on the cell cycle progression of MCF-7 cells. Unsynchronized cells were incubated in the presence of dimethylsulfoxide (DMSO) or 100 µm sesamin for 24 h, and the DNA content of the cells was determined by flow cytometry. The data represents means of triplicate experiments and is shown as means ± SD (n = 3) doi: /j x

3 Fig. 3. The effect of sesamin on the protein expressions of retinoblastoma (RB), cyclin-dependent kinase inhibitor (CKI), cyclin-dependent kinase (CDK) and cyclins in MCF-7 cells. (a d) MCF-7 cells were exposed to dimethylsulfoxide (DMSO) alone ( ) or 100 µm sesamin (+), and were lysed at the indicated times after stimulation. The protein expressions of (a) RB, (b) CKI, (c) CDK and (d) cyclins were then analyzed. α-tubulin was chosen as a loading control for all blots. (e) MCF-7 cells were treated with various concentrations of sesamin for 3 h and the expression of cyclin D1 protein was then examined. 0, DMSO alone. Whether sesamin can alter the phosphorylation status of RB protein was examined using western blotting. Whole-cell lysates were isolated after 0, 1, 3, 6 h of culturing with or without 100 µm sesamin, and subjected to immunoblotting against RB protein. A hyperphosphorylated form of RB protein began to be converted into a hypophosphorylated form 3 h after treatment (Fig. 3a). Sesamin down-regulates the expression of cyclin D1 protein in MCF-7 cells. Among the CKI, p16 INK4a is homozygously deleted, (17) whereas other members of the CIP/KIP family and the INK4 Yokota et al. Cancer Sci September 2007 vol. 98 no

4 family are normally expressed in MCF-7 cells. The authors screened for the expression of CKI to clarify the molecular mechanisms underlying the G1 phase arrest induced by sesamin in MCF-7 cells. Whole-cell lysates were isolated after 0, 1, 3, 6 h of culturing with or without 100 µm sesamin and then subjected to immunoblotting for various CKI. Sesamin did not affect the expression of the CKI examined, including p21 WAF1/Cip1, p27 Kip1, p57 Kip2, p15 INK4b, p18 INK4c, and p19 INK4d (Fig. 3b). Next, the effect of sesamin on the expression of CDK was investigated. As shown in Fig. 3c, sesamin did not change the expression of the CDK examined, CDK2, CDK4 and CDK6. The transition of cells from G1 to S phase is also regulated by cyclins. Whether the expression of cyclins can be altered by treatment with sesamin in MCF-7 cells was next evaluated. Among the cyclins that were examined, cyclin D1 started to decrease after 1 h of culture (Fig. 3d). In contrast, sesamin did not affect the expression of cyclins D3, A and E (Fig. 3d). Cyclin D2 protein could not be detected. Western blot analysis showed that treatment with various concentrations of sesamin for 3 h reduced the expression of cyclin D1 protein in a dose-dependent manner (Fig. 3e). These data are consistent with the result that sesamin dose-dependently inhibited the growth of MCF-7 cells. Proteasome inhibitors block sesamin-induced down-regulation of cyclin D1 protein. To elucidate the molecular mechanism of sesamin-induced down-regulation of cyclin D1 protein, the effect of sesamin on cyclin D1 mrna expression was first investigated. Total RNA were isolated after 0, 1, 3, 6 h of culturing with or without 100 µm sesamin and examined using quantitative realtime RT-PCR analysis. As shown in Fig. 4, sesamin did not significantly affect the cyclin D1 mrna level. This result suggests that sesamin down-regulates cyclin D1 protein expression through post-transcriptional mechanism. Previous studies have found that cyclin D1 is regulated by proteolysis via the proteasome pathway. (18,19) To clarify whether sesamin down-regulates cyclin D1 protein expression through this pathway, the ability of the proteasome inhibitor MG132 or lactacystin to block sesamin-induced suppression of cyclin D1 protein in MCF-7 cells, respectively, was examined. Lactacystin specifically and irreversibly inhibits the proteasome by covalently modifying the proteasome-subunit. (20) MG132 is a general, potent, and reversible proteolysis inhibitor. (20) Cells were pretreated with 10 µm lactacystin for 4 h and then exposed to 50 or 100 µm sesamin for 3 h. Thereafter, whole-cell extracts were prepared and analyzed for cyclin D1 protein. As shown in Fig. 5a, down-regulated cyclin D1 protein expression in sesamin-treated cells was restored by the addition of lactacystin. MG132 also suppressed the sesamin-induced down-regulation of cyclin D1 protein in MCF-7 cells (Fig. 5b). These results suggest that sesamin down-regulates cyclin D1 protein expression through facilitating the proteasome degradation of cyclin D1 protein. Sesamin suppresses cyclin D1 protein expression in various kinds of human tumor cells. To investigate whether the down-regulation of cyclin D1 protein expression by sesamin is a general event, the effect of sesamin on cyclin D1 protein expression in other human tumor cell lines was examined. For this, the human breast cancer cell line T-47D, the human lung cancer cell line A549, the human transformed renal cell line 293T, the human immortalized keratinocyte cell line HaCaT, the human melanoma cell line UACC-62 and the human osteosarcoma cell line MG63 were used. As shown in Fig. 6, sesamin suppressed the expression of cyclin D1 protein in all of these cell lines, suggesting that the sesamin-induced down-regulation of cyclin D1 protein is a ubiquitous event in human tumor cells. Cyclin D1 is one of the important targets of sesamin for its growth inhibitory effect in MCF-7 cells. The present results suggest the possibility that down-regulation of cyclin D1 protein by sesamin contributes to its growth inhibitory effect. If cyclin D1 is one of the key targets of sesamin, cyclin D1-depleted MCF-7 cells are expected to be insensitive to the growth inhibitory action of sesamin. To test this hypothesis, cyclin D1 sirna were used to deplete cyclin D1 protein in MCF-7 cells. As shown in Fig. 7a, treatment with cyclin D1 sirna#1 or cyclin D1 sirna#2, respectively, almost depleted cyclin D1 protein. However, the cyclin D1 sirna had no effect on the expression of cyclin D3 protein (Fig. 7a). As shown in Fig. 7b,c, 50 µm sesamin inhibited growth in mock- or control sirna-treated MCF-7 cells. In contrast, cyclin D1 sirna-treated cells grew more slowly than mock- or control sirna-treated cells, and became almost insensitive to the growth inhibitory effect of 50 µm sesamin. Taken together with the result that sesamin down-regulates cyclin D1 protein expression in MCF-7 cells, this suggests that cyclin D1 is one of the important targets of sesamin for its growth inhibitory function. Fig. 4. The effect of sesamin on the expression of cyclin D1 mrna in MCF-7 cells. MCF-7 cells were treated with dimethylsulfoxide (DMSO) alone ( ) or 100 µm sesamin (+) for the indicated times, and the expression of cyclin D1 mrna was examined using real time reverse transcription-polymerase chain reaction as described in Materials and Methods. The mrna level of cyclin D1 was standardized against that of glyceraldehyde-3-phosphate dehydrogenase (GAPDH). The data represents means of triplicate experiments and are shown as means ± SD (n = 3). Fig. 5. Proteasome inhibitors block sesamin-induced down-regulation of cyclin D1 protein in MCF-7 cells, respectively. MCF-7 cells were plated at cells in 2 ml of medium in 6-well plates. After 24 h, the cells were pretreated with (a) 10 µm lactacystin or (b) 20 µm MG132 for 4 h and then exposed to 50 or 100 µm sesamin for 3 h. Thereafter, whole cell extracts were prepared and the expression of cyclin D1 protein was examined doi: /j x

5 Fig. 6. The effect of sesamin on the expression of cyclin D1 protein in various kinds of human cancer cells. The human breast cancer cell line T-47D, the human lung cancer cell line A549, the human transformed renal cell line 293T, the human immortalized keratinocyte HaCaT, the human melanoma cell line UACC-62 and the human osteosarcoma MG63 were treated with 100 µm sesamin for 3 h and the expression of cyclin D1 protein was then examined using western blotting. Discussion Sesamin, a major lignan of sesame seed, has pluripotent functions including antihypertensive, cholesterol-lowering, lipid-lowering and anticancer activities. (1 7) Several studies have reported that sesamin induces growth inhibition in human cancer cells. (8 10) However, its growth suppressive mechanism remained to be elucidated. The present results indicated that sesamin, a major lignan constituent of sesame, inhibits the growth of MCF-7 cells at the G1 phase in the cell cycle, and that it dephosphorylates tumor-suppressor RB protein. It was also shown that inhibition of MCF-7 cell growth by sesamin is correlated with downregulated cyclin D1 protein expression. Furthermore, the proteasome inhibitor lactacystin or MG132 inhibited the sesamin-induced down-regulation of cyclin D1 protein, suggesting that sesamin suppresses cyclin D1 protein expression by enhancing proteasome degradation of cyclin D1 protein. Sesamin suppressed cyclin D1 protein expression in various kinds of human tumor cells, including lung cancer, transformed renal cells, immortalized keratinocytes, melanoma and osteosarcoma. Finally, the authors indicated that depletion of cyclin D1 protein using cyclin D1 sirna rendered MCF-7 cells insensitive to the growth inhibitory effects of sesamin, implicating cyclin D1 as a key target of sesamin. Taken together, these results suggest that sesamin down-regulates cyclin D1 protein expression through the activation of proteasome degradation, and that this at least partially contributes to the growth inhibition induced by sesamin. The results presented in this study show that sesamin inhibits the growth of the human breast cancer cell line MCF-7. These results are consistent with previous reports that sesamin suppresses the proliferation of human lymphoid leukemia Molt 4B cells and human stomach cancer KATO III cells. (8,9) To investigate the effect of sesamin on the cell cycle progression of MCF- 7 cells, the DNA content of the cell nuclei was measured using flow cytometric analysis. The results indicate that sesamin arrests the cell cycle of MCF-7 cells at the G1 phase. This is the first report to indicate that sesamin induces G1 arrest. Cyclins CDK play important roles in promoting the G1-to-S phase transition of the cell cycle by phosphorylating RB protein. (12,14) Phosphorylated RB releases E2F, which leads to DNA synthesis and cell cycle progression. The present results show that RB phosphorylation is inhibited by sesamin seen using western blot analysis. To gain insight into the molecular mechanisms underlying G1 arrest induced by sesamin, the authors examined the effect of sesamin on the expression of cell-cycle related molecules such as CKI, CDK and cyclins. It was found that sesamin specifically down-regulates cyclin D1 protein expression, and that the antiproliferative effect of sesamin is correlated with the suppression of cyclin D1 protein. To clarify whether the cyclin D1-RB pathway plays an important role in growth inhibition induced by sesamin, we examined the effect of sesamin on the proliferation of the human osteosarcoma cell line Saos2 and the human prostate cancer cell line DU145, which harbor deleted or mutated RB, respectively. These cells were resistant to the growth inhibitory effects of sesamin compared to MCF-7 cells with wild-type RB expression (data not shown). These results suggest that cyclin D1-RB pathway is at least partially associated with the antiproliferative effects of sesamin. In addition, sesamin also inhibited cell growth of the normal human fibroblast WI-38 cells, although WI-38 cells were more insensitive to the growth inhibitory effect of sesamin than these malignant tumor cells (data not shown). The cyclin D1 gene is regulated by multiple mechanisms. To gain insight into the molecular mechanisms of sesamin-induced down-regulation of cyclin D1 protein, the effect of sesamin on cyclin D1 mrna expression was examined using quantitative real-time RT-PCR analysis. Time-course studies demonstrated that, unlike cyclin D1 protein expression, almost no alteration in cyclin D1 mrna was observed by the treatment with sesamin, suggesting that sesamin suppresses cyclin D1 protein expression through post-transcriptional mechanism. Previous studies have shown that cyclin D1 protein is degraded through the proteasome pathway. (18,19) To investigate whether sesamin down-regulates cyclin D1 protein expression through this pathway, lactacystin or MG132 was used as a proteasome inhibitor, and it was discovered that these proteasome inhibitors suppressed the sesamin-induced down-regulation of cyclin D1 protein. These results suggest that sesamin down-regulates cyclin D1 protein expression by enhancing the proteasome degradation. The present results are similar to those previously reported with retinoic acid or curcumin, which down-regulates cyclin D1 protein expression. Retinoic acid or curcumin-induced down-regulation of cyclin D1 protein is also inhibited by proteasome inhibitors. (21,22) However, unlike cyclin D1 protein, no apparent change in p27 Kip1 expression was observed by the treatment with sesamin. It is well known that p27 Kip1 is also regulated by proteolysis via the ubiquitin/proteasome pathway and is degraded by Skp2- Cul1-F box (SCF) ubiquitin ligase complex. (23,24) In contrast, Skp1 is involved in ubiquitin-dependent degradation of cyclin D1. (25) These findings raise a possibility that the variety of F-box protein or the difference in Skp subunit might facilitate substrate-specific response to proteasome. Cyclin D1/ D2/D3, a regulator of the cell-cycle transition from the G1 to S phase, forms a holoenzyme with CDK4/6 that phosphorylates RB. Therefore, it is possible that the growth inhibitory effects of sesamin are due to the repression of cyclin D1 protein expression. To investigate whether the down-regulation of cyclin D1 protein by sesamin contributes to cell growth inhibition in MCF-7 cells, the effect of sesamin on cyclin D1- depleted MCF-7 cells was examined. If cyclin D1 is one of the key targets of sesamin, cyclin D1-depleted MCF-7 cells are expected to be insensitive to the growth inhibitory effect of sesamin. To test this hypothesis, cyclin D1 sirna were used to specifically deplete cyclin D1 protein in MCF-7 cells. In fact, the treatment with cyclin D1 sirna almost depleted cyclin D1 protein. In contrast, the cyclin D1 sirna did not affect the expression of cyclin D3 protein. Cyclin D2 protein could not be detected in MCF-7 cells. It was shown that the cyclin D1- depleted MCF-7 cells grow more slowly than mock- or control sirna-treated MCF-7 cells, and become almost insensitive to the growth inhibitory effect of 50 µm sesamin. Furthermore, 100 µm sesamin and cyclin D1 sirna#1 co-treatment provided slightly additive but no synergistic antiproliferative effect on Yokota et al. Cancer Sci September 2007 vol. 98 no

6 Fig. 7. Depletion of cyclin D1 protein renders MCF-7 cells insensitive to the growth inhibitory effect of sesamin. (a) MCF-7 cells were plated at cells in 24-well plates and transfected with 0.5 µm of small interfering RNA (sirna) using lipofectamine 2000 reagent. Cyclin D1 sirna#1, cyclin D1 sirna#2 and negative control sirna were used. Mock: MCF-7 cells treated with lipofectamine 2000 reagent only. 24 h after transfection, cell lysates were prepared and the expression of cyclin D1 or cyclin D3 protein was examined using western blotting. (b) 24 h after transfection, sesamin at 50 µm was added, and cell growth was compared with a control culture. The data represents means of triplicate experiments and is shown as means ± SD (n = 3). *P < (c) Relative cell viability of MCF-7 cells treated with dimethylsulfoxide (DMSO; white square) or 50 µm sesamin (black square) for 24 h. MCF-7 cells (data not shown). Taken together with the result that sesamin down-regulates cyclin D1 protein in MCF-7 cells, this suggests that loss of cyclin D1 at least partially influences the cellular response to the antiproliferative effects of sesamin. Sesamin suppresses the sequential development of hypertension, cardiovascular hypertrophy and renal damage in various experimental models of hypertension in vivo, (26 28) by inhibiting enhanced vascular O production. (4) 2 However, it does not possess antioxidant properties in vitro. (29) The authors therefore examined whether the down-regulation of cyclin D1 protein induced by sesamin is associated with its antioxidative activity. The production of reactive oxygen species (ROS) after treatment with sesamin or N-acetylcysteine (NAC), which is widely used as an antioxidant, was then measured in MCF-7 cells. However, sesamin did not alter the production of ROS at concentrations that reduce cyclin D1 protein expression, although NAC 1452 doi: /j x

7 significantly reduced production (data not shown). From these results, it appears that the down-regulation of cyclin D1 by sesamin is not related to its antioxidative properties in MCF-7 cells. Genetic alterations in the regulatory components that regulate the G1-to-S phase transition in the cell cycle frequently occur in human cancers. (30) Cyclin D1 is a proto-oncogene that is overexpressed in many human malignant tumors. For example, the cyclin D1 gene is amplified in 15% and overexpressed in 30 50% of human breast cancers. (31) Furthermore, transgenic mice that overexpress cyclin D1 in mammary glands develop breast cancer, suggesting that cyclin D1 functions as an oncogene. (32) Therefore, regulated agents of the cyclin D1 expression may contribute to new strategies aimed at the prevention or therapy References 1 Noguchi T, Ikeda K, Sasaki Y et al. Effects of vitamin E and sesamin on hypertension and cerebral thrombogenesis in stroke-prone spontaneously hypertensive rats. 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