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,2
* Department of Food Science and Nutrition and
Silver Biotechnology Research Center, Hallym University, Chuncheon, Korea 200-702;
** Department of Sports Science, Seoul Sports Graduate University, Seoul, Korea; and
Korea Food Research Institute, Songnam, 463-746, Korea
2To whom correspondence should be addressed. E-mail: jyoon{at}hallym.ac.kr.
ABSTRACT
Fisetin, a natural flavonol present in edible vegetables, fruits, and wine, was reported to exert anticarcinogenic effects. The objective of the current study was to examine the effect of fisetin on the cell cycle progression of the human colon cancer cell line HT-29. HT-29 cells were cultured in serum-free medium with 0, 20, 40, or 60 µmol/L fisetin. Fisetin dose dependently inhibited both cell growth and DNA synthesis (P < 0.05), with a 79 ± 1% decrease in cell number observed 72 h after the addition of 60 µmol/L fisetin. Perturbed cell cycle progression from the G1 to S phase was observed at 8 h with 60 µmol/L fisetin treatment, whereas a G2/M phase arrest was observed after 24 h (P < 0.05). The phosphorylation state of the retinoblastoma proteins shifted from hyperphosphorylated to hypophosphorylated in cells treated with 40 µmol/L fisetin. (P < 0.05). Fisetin decreased the activities of cyclin-dependent kinases (CDK)2 and CDK4; these effects were likely attributable to decreases in the levels of cyclin E and D1 and an increase in p21CIP1/WAF1 levels (P < 0.05). However, fisetin also inhibited CDk4 activity in a cell-free system (P < 0.05), indicating that it may directly inhibit CDk4 activity. The protein levels of cell division cycles (CDC)2 and CDC25C and the activity of CDC2 were also decreased in fisetin-treated cells (P < 0.05). These results indicate that inhibition of cell cycle progression in HT-29 cells after treatment with fisetin can be explained, at least in part, by modification of CDK activities.
KEY WORDS: • G1 phase arrest • G2/M phase arrest • cell division cycle 2 • p21CIP1/WAF1 • retinoblastoma proteins
Colon cancer remains one of the leading causes of cancer death in Western countries, and modifications of diet and life style offer measures for reducing the risk of developing colon cancer. Epidemiologic studies indicate that a diet containing plentiful vegetables and fruits can reduce the risk of several cancers, including colon cancer (1). Flavonoids are a group of naturally occurring polyphenolic compounds present in fruits, vegetables, and other edible plants. Some of these flavonoids possess chemopreventive properties, and numerous studies were performed recently to assess the mechanisms whereby each flavonoid prevents cancer, including induction of cell cycle arrest, apoptosis, and antiproliferation [reviewed in (2)].
The mammalian cell cycle is divided into 4 distinct phases; the G1, S, G2, and M phases. Cyclin-dependent protein kinases (CDKs)3 are important regulators of the eukaryotic cell cycle progression, and the activities of these kinases require the binding of a positive regulatory subunit, known as a cyclin. The protein levels of cyclins (e.g., cyclins D, E, and A) vary during the G1/S cell cycle transition (3), leading to activation of CDK2 and CDK4/6. A main function of these cyclin/CDK complexes is to phosphorylate members of the retinoblastoma protein (Rb) family. This event, in turn, promotes cellular proliferation and S-phase progression through the release of a family of transcriptional regulators, collectively named the E2Fs, which are normally bound to hypophosphorylated Rb (4). Released E2F elicits the expression of various genes associated with S-phase progression (5). On the other hand, CDK inhibitors (CDKIs), such as p21WAF/CIP1 and p27KIP1, play important roles in cell cycle regulation by coordinating internal and external signals that inhibit cell cycle progression at important checkpoints (6).
The cell division cycle (CDC)2 kinase is required for the onset of mitosis, and its activity is subject to multiple levels of regulation. CDC2 is active only at the G2/M border, and turned off as the cells enter the anaphase stage of mitosis. The first step in generating active CDC2 is its association with a cyclin. In animal cells, CDC2 associates with an A- or B-type cyclin. The CDC2-cyclin B complex must enter the nucleus and be phosphorylated by cyclin-dependent kinase-activating kinase at threonine 161 to be active (7,8). In addition, it must be activated by the dephosphorylation of tyrosine 15 and threonine 14 of CDC2 at the G2/M boundary by CDC25 (9).
Fisetin, 3,3',4',7-tetrahydroxyflavone, is found in fruits and vegetables, such as strawberry, apple, persimmon, grape, onion, and cucumber at concentrations of 2–160 µg/g (10). Fisetin was reported to inhibit proliferation (11) and induce apoptosis (12,13) of various tumor cells at concentrations of 1–100 µmol/L in cell culture models. To our knowledge, the effects of fisetin on colon cancer have not been studied in detail, with the exception of a study that reported the decreased viability of HT-29 and Caco-2 colon cancer cells in the presence of fisetin (14). The initial stage of colon cancer development is often characterized by an increased proliferation of the epithelium leading to the formation of adenomas. This is primarily a result of dysregulated cell cycle control and/or decreased apoptosis, as is usually observed in colorectal cancers (15,16). In the present study, we investigated the mechanisms of fisetin inhibition of HT-29 cell growth by examining cell cycle regulatory proteins.
MATERIALS AND METHODS
Materials.
The following reagents and chemicals were obtained from the respective suppliers: fisetin, anti-ß-actin, RIA-grade bovine serum albumin (BSA), and transferrin (Sigma); antibodies against CDC25C, phsopho-CDC2 (Tyr15), cyclin B1, and phospho-Rb (Ser807/811) (Cell Signaling); horse-radish peroxidase (HRP)-conjugated anti-rabbit and anti-mouse IgG (Amersham); [
-32P]ATP (NEN-Life Sciences); anti-cyclin D1 antibody (Neomarkers); and antibodies against p21CIP1/WAF1 (c-19), p27KIP1, cyclin A (c-19), cyclin E (M-20), CDK2 (M-2), CDK4 (c-22), p34CDC2, E2F-1 (C-20), Rb (c-15), and proliferating cell nuclear antigen (PCNA, PC10) (Santa Cruz Biotechnology).
Cell culture. The HT-29 cell line was obtained from the American Type Culture Collection and maintained in DMEM/F-12, containing 100 mL/L of fetal bovine serum (FBS), with 100,000 U/L of penicillin and 100 mg/L of streptomycin. HT-29 cells between passages 139 and 150 were used in these studies. To examine the effect of fisetin, cells were plated with DMEM/F-12 containing 10% FBS. Before the fisetin treatment, the cell monolayers were rinsed and serum-starved for 24 h, with DMEM/F-12 supplemented with 5 mg/L transferrin, 1 g/L BSA, and 5 µg/L selenium (serum-free medium). After serum starvation, fresh serum-free medium, with or without the indicated concentrations of fisetin, was replaced. The medium was changed every 2 d. Viable cell numbers were estimated 24, 48, and 72 h after the cells were exposed to fisetin using the 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) assay, as described previously (17). Fisetin was dissolved in dimethyl sulfoxide (DMSO), and all cells received DMSO to a final concentration of 0.1%.
To determine [3H]thymidine incorporation, HT-29 cells were plated in 96-well plates, at a density of 6000 cells/well, serum-starved, and treated with fisetin for 5 h, as described above. At this time, 0.5 µCi [3H]thymidine was added to each well, and the incubation continued for an additional 3 h at 37°C. The [3H]thymidine incorporation into the DNA of HT-29 cells was determined, as described previously (17).
Cell cycle analysis by flow cytometry. Cells were plated in 24-well plates, at 50,000 cells/well, in DMEM/F-12 containing 10% FBS. Cells were serum-starved and treated with 60 µmol/L fisetin, and were trypsinized. The nuclei were stained with propidium iodide as described previously (18) and subjected to fluorescence-activated cell sorting analysis utilizing FACScan (Becton Dickinson). The data were analyzed using Modfit version 1.2 software.
Immunoprecipitation and immunoblot analyses. Cell lysates were prepared as described previously (18), and the protein content determined using the bicinchoninic acid protein assay kit (Pierce). Cell lysates (750 µg protein) were precleared with 1.5 µg of normal rabbit IgG and 100 µL of protein A-Sepharose bead slurry (Amersham) on a rotating platform for 1 h and centrifuged at 14,000 x g for 10 min at 4°C. The supernatants were incubated with 1 µg of anti-CDK2, anti-CDK4, or anti-CDC2 antibody for 1 h at 4°C. The protein A-Sepharose was added and incubated for another hour at 4°C; the beads were then washed 4 times with lysis buffer by centrifugation at 600 x g for 5 min at 4°C. Total cell lysates (50 µg protein) or immunoprecipitated proteins were resolved on a SDS-PAGE (4–20% or 10–20%) and transferred onto a polyvinylidene fluoride membrane (Millipore). The blots were incubated for 1 h with anti-cyclin D1 (1:200), anti-cyclin A (1:1000), anti-cyclin E (1:1000), anti-CDC2 (1:1000), anti-CDK2 (1:1000), anti-CDK4 (1:1000), anti-p21CIP1/WAF1 (1:500), anti-p27KIP1, anti-phospho-Rb (1:1000), anti-Rb (1:1000), anti-PCNA (1:1000), or anti-ß-actin (1:2000) antibody. The blots were then incubated with anti-mouse or rabbit HRP-conjugated antibody. Signals were detected based on an enhanced chemiluminescence method, using the SuperSignal West Dura Extended Duration Substrate (Pierce). Densitometric analysis was carried out using the Bio-profile Bio-ID application (Vilber-Lourmat). Expression levels were normalized to ß-actin and the control levels were set at 100%.
RT-PCR. Total RNA was isolated using Tri reagent (Sigma), and cDNA synthesized using 2 µg of total RNA with SuperScriptTM II reverse transcriptase (Invitrogen), as described previously (19). For amplification of cDNA, primers for human p21CIP1/WAF1 (upstream primer; 5'-AAT AAG GAA GCG ACC TGC AA-3'; downstream primer, 5'-CCA ACG CTT TTA GAG GCA GA-3', annealing at 55°C for 1 min with 38 cycles) were used. The expressions of human ß-actin transcripts were examined as an internal control, as described previously (20). For each combination of primers, the kinetics of the PCR amplification was studied, the number of cycles corresponding to the plateau determined, and the PCR performed within the exponential range. The PCR products were separated on a 2% agarose gel and stained with ethidium bromide. Bands corresponding to each specific PCR product were quantified by densitometric scanning of the exposed film using the Bio-profile Bio-ID application (Vilber-Lourmat).
CDK kinase activity in fisetin-treated HT-29 cells.
Cell lysate (750 µg protein) was immunoprecipitated with polyclonal antibody against CDK2, CDC2, or CDK4, washed with either 20 mmol/L Tris-HCl, pH 7.5, and 4 mmol/L MgCl2 (CDK2 and CDC2 kinase buffer) or 50 mmol/L HEPES, 10 mmol/L ß-glycerophosphate, 1 mmol/L NaF, 0.1 mmol/L sodium orthovanadate, and 10 mmol/L MgCl2 (CDK4 kinase buffer). After washing, the beads were incubated in either 15 µL kinase buffer, containing 2 µg histone H1 (Roche) and 3 µCi [-32P]ATP, at 37°C for 30 min (CDK2 and CDC2 kinase assay) or 30 µL kinase buffer with 1 µg Rb (769, Santa Cruz) and 10 µCi [-32P]ATP at 30°C for 30 min (CDK4 kinase assay). The reaction was stopped by boiling the samples in SDS sample buffer for 5 min. The 32P-labeled histone H1 or Rb was resolved by SDS-PAGE; the gel was dried and subjected to autoradiography. Signals were quantified by densitometric scanning of the exposed film.
Assessment of fisetin effect on CDK4 activity in cell-free system.
Serum-starved HT-29 cells were lysed, and 750 µg total cellular protein used to immunoprecipitate the active CDK4 complexes using the anti CDK4 antibody. After capturing with protein A-Sepharose and subsequent washes, CDK4 activities of the active immune complexes were estimated in the presence of increasing concentrations of fisetin in CDK4 kinase buffer containing 10 µCi [-32P]ATP and 1 µg Rb. Reactions were incubated at 37°C for 30 min, terminated by the addition of SDS loading dye, resolved on SDS-PAGE, and the dried gels subjected to autoradiography.
Statistical analysis. For all studies, 3–6 independent experiments were performed with separate batches of cells. The results are expressed as means ± SEM. They were analyzed by 1-way, 2-way, or 2-factor repeated-measures ANOVA. Differences between the treatment groups were analyzed using Duncan’s multiple range or t tests. Different were considered significant at P < 0.05. Pearson’s correlation coefficients were calculated between CDK2 activity and p21 binding to CDK2. All statistical analyses were performed using the SAS System for Windows V8.1 (SAS Institute).
RESULTS
Effect of fisetin on growth and cell cycle progression in HT-29 cells. Fisetin decreased the viable HT-29 cell numbers, in a dose-dependent manner, with a 79 ± 1% decrease in cell numbers within 72 h of the addition of 60 µmol/L fisetin (Fig. 1). This decrease in cell numbers was attributable to the induction of cell cycle arrest. At 8 h after the addition of fisetin, the percentage of cells in the G1 phase increased, with a concomitant decline in the G2/M phase fraction. However, after 24 h, there was an accumulation of cells in the G2/M phase, with an equivalent decline in the G1 phase (Fig. 2). Consistent with the occurrence of cell cycle G1 arrest at 8 h, fisetin decreased the [3Hthymidine incorporation into the DNA of HT-29 cells (Fig. 3).
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, plays essential roles in DNA synthesis and repair (25). Western blot analysis revealed that fisetin did not affect PCNA protein levels (Fig. 8). Fisetin inhibits CDC2 activity. Because fisetin induced G2/M phase arrest at 24 h, the levels of G2/M regulatory proteins were examined in HT-29 cells treated with various concentrations of fisetin for 24 h. The protein levels of CDC2 and CDC25C decreased in cells treated with 40 µmol/L fisetin, whereas that of p-CDC2 (Tyr15) increased in cells treated with 20 µmol/L fisetin (P < 0.05). Fisetin had no effect on the cyclin B1 protein level (Fig. 9A). We next examined whether fisetin inhibited CDC2 activity. Cell lysates were incubated with the CDC2 antibody, and the immune complexes precipitated with protein A Sepharose. The results from an in vitro kinase assay using histone H1 as the substrate revealed that CDC2 activity decreased in cells treated with 40 and 60 µmol/L fisetin (Fig. 9B).
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DISCUSSION
One of the most common incidents required for human cancer development is deregulation of the cell cycle mechanism (26). Cell cycle regulatory proteins are the potential molecular targets for cancer therapy/prevention because their functions are well regulated in normal cells, but they are regulated differently in tumor cells. In recent years, considerable attention has been paid to the ability of dietary flavonoids to inhibit cell cycle progression. However, the effects of fisetin, which is present in fruits and vegetables, have not been studied extensively. In the present study, fisetin induced G1 and G2 arrests within 8 and 24 h, respectively, in HT-29 human colon cancer cells, and led to decreased CDK2, CDK4, and CDC2 activities. Because modulation of CDK activity is a very attractive target for the treatment and prevention of human cancers (27,28), future studies should investigate whether fisetin inhibits colon cancer development in animal models and human studies.
The levels of the cyclin proteins fluctuate temporally during the cell cycle, leading to activation of their respective CDKs. Treatment of cells with fisetin decreased cyclin E, but did not greatly affect CDK2 and cyclin A protein levels, suggesting that the decreased cyclin E contributed to the decreased CDK2 activities observed in fisetin-treated cells. Fisetin decreased the cyclin D1 protein levels, which might contribute to the decrease in CDK4 activity.
The CDK inhibitor p21CIP1/WAF1 binds and inhibits the cyclin D-, E- and A-dependent kinases, regulating the G1 to S phase transition of the cell cycle (29–31). p21CIP1/WAF1 also contributes to the maintenance of cell cycle arrest in the G2 phase (32). Overexpression of p21CIP1/WAF1 inhibits the proliferation of mammalian cells (30,33). In the present study, fisetin dose dependently increased the p21 protein levels and p21 binding to CDK2, indicating that the increased p21CIP1/WAF1 contributes to the fisetin-induced decrease in CDK activities due to binding with these enzymes. Transcription of the p21 gene is regulated by both p53-dependent and -independent mechanisms (34). We observed that fisetin induced p21 mRNA levels in a p53-independent manner in HT-29 cells because these cells lack functional p53 (35). Our results suggest that the increased p21CIP1/WAF1 contributes to fisetin-induced G1 arrest by binding to and inhibiting the activities of cyclin-dependent kinases.
Our in vitro kinase assay revealed that CDK4 activity was markedly decreased in fisetin-treated cells within 0.5 h, whereas CDK4 protein levels were moderately decreased and the cyclin D1 levels were not affected at this time point. In addition, when fisetin was added with immunoprecipitated CDK4 in a cell-free system, CDK4 activity was directly inhibited, indicating that fisetin directly inhibits the function of this enzyme. Indeed, it was shown recently that fisetin binds to the active form of the CDK4/6 subfamily, forming hydrogen bonds with the side chains of residues in the binding pocket, which go through large conformational changes during CDK activation by cyclin binding (36). These results indicate that the CDK4 inhibition was due to both direct and indirect actions of fisetin on this kinase.
CDKs phosphorylate members of the Rb family, which, in turn, promotes cell cycle progression ([reviewed in (37)]. Cyclin D-dependent kinases initiate Rb phosphorylation in the mid-G1 phase, after which cyclin E-CDK2 becomes active and completes the process by phosphorylating the Rb at additional sites (21,22,38). In quiescent cells, hypophosphorylated Rb associates with the E2F transcription factors (21,39,40). Phosphorylation of Rb proteins liberates the E2F proteins, allowing them to act as transcription activators. The E2F target genes that are upregulated include those necessary for the completion of the G1/S phase transition, as well as for DNA replication (41). In the present study, fisetin decreased the levels of phospho-Rb and increased hypophosphorylated Rb. These findings suggest that decreased phosphorylated Rb (or increased hypophosphorylated Rb), due to the decrease in CDK activity, may explain, at least in part, the effects of fisetin on cell cycle progression. In addition, the expression of E2F-1, a member of the E2F transcription factor family, was decreased in fisetin-treated HT-29 cells, suggesting that the decrease in E2F-1 levels may also contribute to cell cycle arrest at the G1 phase.
The G2 checkpoint prevents cells from entering mitosis when DNA is damaged, providing an opportunity for repair and stopping the proliferation of damaged cells. CDC2 is an important target of the pathways that mediate rapid G2 arrest in response to DNA damage. Inhibition of CDC2 activity takes place rapidly after DNA damage by blocking the dephosphorylation of CDC2 at tyrosine 15 and threonine 14 (42,43). In the present work, the treatment of cells with fisetin led to a decrease in CDC2 activity, which may be responsible for the G2/M arrest observed in fisetin-treated cells. Fisetin decreased CDC2 protein levels, but increased that of phospho-CDC2 (Tyr15), the inactive form of CDC2. Our results suggest that fisetin downregulates CDC25C, leading to decreased dephosphorylation of CDC2.
E2Fs regulate the expressions of genes required for the G1/S transition, including those encoding DNA replication proteins, the enzymes involved in nucleotide synthesis, and components of the origin recognition complex [reviewed in (44,45)]. In addition to this role of E2F, recent work showed that E2F induces genes that are normally regulated at the G2 phase of the cell cycle; they encode proteins such as cyclin B1 and CDC2 that function in mitosis (5,46–48). In the present study, fisetin downregulated the E2F-1 and CDC2 protein levels. It remains to be determined whether the decreased CDC2 levels in fisetin-treated cells were due to the decreased activity of E2F caused by the increased Rb hypophosphorylation.
To date, the absorption, metabolism, and blood concentrations of fisetin have not been well characterized. The present study utilized fisetin at concentrations of 20–60 µmol/L; other investigators (12–14) examined the effects of fisetin at slightly higher concentrations than those used in our studies. To determine whether the concentrations used in the cell culture studies are nutritionally relevant, the concentrations of fisetin in human serum as well as in a variety of foods should be determined in the future.
In summary, we showed that treating HT-29 cells with fisetin resulted in the arrest of the cell cycle at the G1 and G2 phases, which was due, at least in part, to the inhibition of CDKs. CDKs are promising anticancer drug targets because inhibitors of CDKs are cytotoxic to cancer cells in vitro and suppress tumor growth in animals (49–51). Future studies are warranted to investigate the effect of fisetin on colon cancer development in animal cancer models.
FOOTNOTES
1 Supported by Korea Research Foundation Grant (KRF-2003–041-C00408) and by the Research Grant from Hallym University, Korea. 
3 Abbreviations used: BSA, bovine serum albumin; CDC, cell division cycle; CDK, cyclin-dependent kinase; DMSO, dimethyl sulfoxide; FBS, fetal bovine serum; HRP, horse-radish peroxidase; MTT, 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide; PCNA, proliferating cell nuclear antigen; Rb, retinoblastoma protein.
Manuscript received 7 July 2005. Initial review completed 1 August 2005. Revision accepted 15 September 2005.
LITERATURE CITED
1. Lock K, Pomerleau J, Causer L, Altmann DR, McKee M. The global burden of disease attributable to low consumption of fruit and vegetables: implications for the global strategy on diet. Bull World Health Organ. 2005;83:100-108.[Medline]
2. Birt DF, Hendrich S, Wang W. Dietary agents in cancer prevention: flavonoids and isoflavonoids. Pharmacol Ther. 2001;90:157-177.[Medline]
3. Johnson DG, Walker CL. Cyclins and cell cycle checkpoints. Annu Rev Pharmacol Toxicol. 1999;39:295-312.[Medline]
4. Morgan DO. Cyclin-dependent kinases: engines, clocks, and microprocessors. Annu Rev Cell Dev Biol. 1997;13:261-291.[Medline]
5. Ishida S, Huang E, Zuzan H, Spang R, Leone G, West M, Nevins JR. Role for E2F in control of both DNA replication and mitotic functions as revealed from DNA microarray analysis. Mol Cell Biol. 2001;21:4684-4699.
6. Sherr CJ, Roberts JM. Inhibitors of mammalian G1 cyclin-dependent kinases. Genes Dev. 1995;9:1149-1163.
7. Poon RY, Yamashita K, Adamczewski JP, Hunt T, Shuttleworth J. The cdc2-related protein p40MO15 is the catalytic subunit of a protein kinase that can activate p33cdk2 and p34cdc2. EMBO J. 1993;12:3123-3132.[Medline]
8. Fesquet D, Labbe JC, Derancourt J, Capony JP, Galas S, Girard F, Lorca T, Shuttleworth J, Doree M, Cavadore JC. The MO15 gene encodes the catalytic subunit of a protein kinase that activates cdc2 and other cyclin-dependent kinases (CDKs) through phosphorylation of Thr161 and its homologues. EMBO J. 1993;12:3111-3121.[Medline]
9. Draetta G, Eckstein J. Cdc25 protein phosphatases in cell proliferation. Biochim Biophys Acta. 1997;1332:M53-M63.[Medline]
10. Arai Y, Watanabe S, Kimira M, Shimoi K, Mochizuki R, Kinae N. Dietary intakes of flavonols, flavones and isoflavones by Japanese women and the inverse correlation between quercetin intake and plasma LDL cholesterol concentration. J Nutr. 2000;130:2243-2250.
11. Fotsis T, Pepper MS, Montesano R, Aktas E, Breit S, Schweigerer L, Rasku S, Wähälä K, Adlercreutz H. Phytoestrogens and inhibition of angiogenesis. Baillieres Clin Endocrinol Metab. 1998;12:649-666.[Medline]
12. Lee WR, Shen SC, Lin HY, Hou WC, Yang LL, Chen YC. Wogonin and fisetin induce apoptosis in human promyeloleukemic cells, accompanied by a decrease of reactive oxygen species, and activation of caspase 3 and Ca(2+)-dependent endonuclease. Biochem Pharmacol. 2002;63:225-236.[Medline]
13. Stopper H, Schmitt E, Kobras K. Genotoxicity of phytoestrogens. Mutat Res. 2005;574:139-155.[Medline]
14. Kuntz S, Wenzel U, Daniel H. Comparative analysis of the effects of flavonoids on proliferation, cytotoxicity, and apoptosis in human colon cancer cell lines. Eur J Nutr. 1999;38:133-142.[Medline]
15. Potten CS. Epithelial cell growth and differentiation. II. Intestinal apoptosis. Am J Physiol Gastrointest Liver Physiol. 1997;273:G253-G257.
16. Gryfe R, Swallow C, Bapat B, Redston M, Gallinger S, Couture J. Molecular biology of colorectal cancer. Curr Probl Cancer. 1997;21:233-300.[Medline]
17. Kim EJ, Holthuizen PE, Park HS, Ha YL, Jung KC, Park JHY. Trans-10, cis-12 conjugated linoleic acid inhibits Caco-2 colon cancer cell growth. Am J Physiol Gastrointest Liver Physiol. 2002;283:G357-G367.
18. Lim do Y, Tyner AL, Park JB, Lee JY, Choi YH, Park JH. Inhibition of colon cancer cell proliferation by the dietary compound conjugated linoleic acid is mediated by the CDK inhibitor p21(CIP1/WAF1). J Cell Physiol. 2005;205:107-113.[Medline]
19. Kim EJ, Kang IJ, Cho HJ, Kim WK, Ha YL, Park JH. Conjugated linoleic acid downregulates insulin-like growth factor-I receptor levels in HT-29 human colon cancer cells. J Nutr. 2003;133:2675-2681.
20. Cho HJ, Kim WK, Kim EJ, Jung KC, Park S, Lee HS, Tyner AL, Park JH. Conjugated linoleic acid inhibits cell proliferation and ErbB3 signaling in HT-29 human colon cell line. Am J Physiol Gastrointest Liver Physiol. 2003;284:G996-G1005.
21. Sherr CJ. Cancer cell cycles. Science. 1996;274:1672-1677.
22. Weinberg RA. The retinoblastoma protein and cell cycle control. Cell. 1995;81:323-330.[Medline]
23. Helin K. Regulation of cell proliferation by the E2F transcription factors. Curr Opin Genet Dev. 1998;8:28-35.[Medline]
24. Garcia I, Murga M, Vicario A, Field SJ, Zubiaga AM. A role for E2F1 in the induction of apoptosis during thymic negative selection. Cell Growth Differ. 2000;11:91-98.
25. Waga S, Hannon GJ, Beach D, Stillman B. The p21 inhibitor of cyclin-dependent kinases controls DNA replication by interaction with PCNA. Nature. 1994;369:574-578.[Medline]
26. Senderowicz AM, Sausville EA. Preclinical and clinical development of cyclin-dependent kinase modulators. J Natl Cancer Inst. 2000;92:376-387.
27. Senderowicz AM. Novel direct and indirect cyclin-dependent kinase modulators for the prevention and treatment of human neoplasms. Cancer Chemother Pharmacol. 2003;52(Suppl 1):S61-S73.[Medline]
28. Senderowicz AM. Cyclin-dependent kinases as new targets for the prevention and treatment of cancer. Hematol Oncol Clin North Am. 2002;16:1229-1253.[Medline]
29. el-Deiry WS, Tokino T, Velculescu VE, Levy DB, Parsons R, Trent JM, Lin D, Mercer WE, Kinzler KW, Vogelstein B. WAF1, a potential mediator of p53 tumor suppression. Cell. 1993;75:817-825.[Medline]
30. Xiong Y, Hannon GJ, Zhang H, Casso D, Kobayashi R, Beach D. p21 is a universal inhibitor of cyclin kinases. Nature. 1993;366:701-704.[Medline]
31. Brugarolas J, Chandrasekaran C, Gordon JI, Beach D, Jacks T, Hannon GJ. Radiation-induced cell cycle arrest compromised by p21 deficiency. Nature. 1995;377:552-557.[Medline]
32. Niculescu AB, 3rd, Chen X, Smeets M, Hengst L, Prives C, Reed SI. Effects of p21(Cip1/Waf1) at both the G1/S and the G2/M cell cycle transitions: pRb is a critical determinant in blocking DNA replication and in preventing endoreduplication. Mol Cell Biol. 1998;18:629-643.
33. Lee MH, Reynisdottir I, Massague J. Cloning of p57KIP2, a cyclin-dependent kinase inhibitor with unique domain structure and tissue distribution. Genes Dev. 1995;9:639-649.
34. Gartel AL, Tyner AL. Transcriptional regulation of the p21(WAF1/CIP1) gene. Exp Cell Res. 1999;246:280-289.[Medline]
35. Gobert C, Skladanowski A, Larsen AK. The interaction between p53 and DNA topoisomerase I is regulated differently in cells with wild-type and mutant p53. Proc Natl Acad Sci U S A. 1999;96:10355-10360.
36. Lu H, Chang DJ, Baratte B, Meijer L, Schulze-Gahmen U. Crystal structure of a human cyclin-dependent kinase 6 complex with a flavonol inhibitor, fisetin. J Med Chem. 2005;48:737-743.[Medline]
37. Obaya AJ, Sedivy JM. Regulation of cyclin-Cdk activity in mammalian cells. Cell Mol Life Sci. 2002;59:126-142.[Medline]
38. Taya Y. RB kinases and RB-binding proteins: new points of view. Trends Biochem Sci. 1997;22:14-17.[Medline]
39. Ikeda MA, Jakoi L, Nevins JR. A unique role for the Rb protein in controlling E2F accumulation during cell growth and differentiation. Proc Natl Acad Sci U S A. 1996;93:3215-3220.
40. Moberg K, Starz MA, Lees JA. E2F-4 switches from p130 to p107 and pRB in response to cell cycle reentry. Mol Cell Biol. 1996;16:1436-1449.[Abstract]
41. Ohtani K. Implication of transcription factor E2F in regulation of DNA replication. Front Biosci. 1999;4:D793-D804.[Medline]
42. Lock RB, Ross WE. Inhibition of p34cdc2 kinase activity by etoposide or irradiation as a mechanism of G2 arrest in Chinese hamster ovary cells. Cancer Res. 1990;50:3761-3766.
43. Kharbanda S, Saleem A, Datta R, Yuan ZM, Weichselbaum R, Kufe D. Ionizing radiation induces rapid tyrosine phosphorylation of p34cdc2. Cancer Res. 1994;54:1412-1414.
44. Nevins JR. Toward an understanding of the functional complexity of the E2F and retinoblastoma families. Cell Growth Differ. 1998;9:585-593.[Medline]
45. Dyson N. The regulation of E2F by pRB-family proteins. Genes Dev. 1998;12:2245-2262.
46. Ren B, Cam H, Takahashi Y, Volkert T, Terragni J, Young RA, Dynlacht BD. E2F integrates cell cycle progression with DNA repair, replication, and G(2)/M checkpoints. Genes Dev. 2002;16:245-256.
47. Zhu W, Giangrande PH, Nevins JR. E2Fs link the control of G1/S and G2/M transcription. Embo J. 2004;23:4615-4626.[Medline]
48. Polager S, Kalma Y, Berkovich E, Ginsberg D. E2Fs up-regulate expression of genes involved in DNA replication, DNA repair and mitosis. Oncogene. 2002;21:437-446.[Medline]
49. Arguello F, Alexander M, Sterry JA, Tudor G, Smith EM, Kalavar NT, Greene JF, Jr, Koss W, Morgan CD, Stinson SF, Siford TJ, Alvord WG, Klabansky RL, Sausville EA. Flavopiridol induces apoptosis of normal lymphoid cells, causes immunosuppression, and has potent antitumor activity In vivo against human leukemia and lymphoma xenografts. Blood. 1998;91:2482-2490.
50. Patel V, Senderowicz AM, Pinto D, Jr, Igishi T, Raffeld M, Quintanilla-Martinez L, Ensley JF, Sausville EA, Gutkind JS. Flavopiridol, a novel cyclin-dependent kinase inhibitor, suppresses the growth of head and neck squamous cell carcinomas by inducing apoptosis. J Clin Invest. 1998;102:1674-1681.
51. Motwani M, Jung C, Sirotnak FM, She Y, Shah MA, Gonen M, Schwartz GK. Augmentation of apoptosis and tumor regression by flavopiridol in the presence of CPT-11 in Hct116 colon cancer monolayers and xenografts. Clin Cancer Res. 2001;7:4209-4219.
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