www.biomolther.org DOI: 10.4062/biomolther.2009.17.3.282
*Corresponding author
Tel: +82-2-3277-3023 Fax: +82-2-3277-3051 E-mail: sklee@ewha.ac.kr
Ochnaflavone, a Natural Biflavonoid, Induces Cell Cycle Arrest and Apoptosis in HCT-15 Human Colon Cancer Cells
You-Jin KANG1, Hye-Young MIN1, Ji-Young HONG1, Yeong Shik KIM2, Sam Sik KANG2, and Sang Kook LEE1,*
1Colloge of Pharmacy, Ewha Womans University, Seoul 120-750,
2Colloge of Pharmacy, Seoul National University, Seoul 151-742, Republic of Korea
(Received June 1, 2009; Revised June 12, 2009; Accepted June 17, 2009)
Abstract － Ochnaflavone is a natural biflavonoid and mainly found in the caulis of Lonicera japonica (Caprifoliaceae). Biological activities such as anti-inflammatory and anti-atherogenic effects have been previously reported. The anticancer activity of ochnaflavone, however, has been poorly elucidated yet. In the present study, we investigated the effect of ochnaflavone on the growth inhibitory activity in cultured human colon cancer cell line HCT-15. Ochnaflavone inhibited the proliferation of the cancer cells with an IC50 value of 4.1 μM. Flow cytometric analysis showed that ochnaflavone arrested cell cycle progression in the G2/M phase, and induced the increase of sub-G1 peak in a concentration-dependent manner. Induction of cell cycle arrest was correlated with the modulation of the expression of cell cycle regulating proteins including cdc2 (Tyr15), cyclin A, cyclin B1 and cyclin E. The increase of sub-G1 peak by the higher concentrations of ochnaflavone (over 20 μM) was closely related to the induction of apoptosis, which was evidenced by the induction of DNA fragmentation, activation of caspase-3, -8 and -9, and cleavage of poly-(ADP-ribose) polymerase. These findings suggest that the cell cycle arrest and induction of apoptosis might be one possible mechanism of actions for the anti-proliferative activity of ochnaflavone in human colon cancer cells.
Keywords: Ochnaflavone, Cell cycle arrest, Apoptosis, HCT15 human colon cancer cells
INTRODUCTION
Flavonoids which are a typical polyphenol have been re- ported to have a variety of biological activities including an- ti-oxidant, metal chelating, immunostimulatory, liver pro- tection, anti-inflammatory, and anti-carcinogenesis (Butenko et al., 1993; Grange et al., 1999; Ielpo et al., 2000; Sharon and Nora, 2000; Chowdhury et al., 2002; Molina et al., 2003).
Biflavonoid, a dimer of flavonoid, is generally found in the plants of gymnosperms, and the limited pharmaco- logical activities including inhibition of phosphodiestrase, aldose reductase of lens, and histamine release by mast cell were reported (Beretz et al., 1979; Amella et al., 1985;
Iwu et al., 1990).
Ochnaflavone, a naturally occurring biflavonoid, is main- ly found in the medicinal plant Lonicera japonica (Caprifoliaceae). Several biological activities mediated by ochnaflavone include the inhibition of phospholipase A2 in
rat platelet, lymphocyte proliferation, and arachidonate re- lease from rat peritoneal macrophage (Chang et al., 1994;
Lee et al., 1995; Lee et al., 1997). The anti-artherogenic activity of ochnaflavone was also revealed with the correla- tion of inhibition of human vascular smooth muscle cell proliferation induced by TNF-α via regulation of cell cycle related proteins, ERK and MMP-9 (Suh et al., 2006b).
Recent study also exhibited the inhibitory activity of CCl4-induced PE degradation in rat liver microsome (Moon et al., 2006) and anti-inflammatory effects with the down- regulation of inducible nitric oxide, cyclooxygenase-2 and 5-lipoxygenase through NF-κB and ERK pathway by och- naflavone (Son et al., 2006; Suh et al., 2006a). However, the anti-proliferative effect of ochnaflavone against human cancer cells has been poorly elucidated yet. We herein re- port for the first time that ochnaflavone exhibits the an- ti-proliferation of human colon cancer cells with the in- duction of cell cycle arrest and apoptosis.
Fig. 1. Chemical structure of ochna- flavone (A) and the growth inhibitory effects of chnaflavone on HCT-15 human colon cancer cells (B). Data are re- presented as the means ± S.E. (n=3).
MATERIALS AND METHODS Chemicals
Trichloroacetic acid (TCA), sulforhodamine B, ribonu- clease A (RNase A), propidium iodide (PI), bisbenzimide H 33258 (Hoechst 33258), and mouse monoclonal anti-β- actin primary antibody were purchased from Sigma (St.
Louis, MO, USA). Rosewell park memorial institute (RPMI) medium 1640, fetal bovine serum (FBS), non-essential amino acid solution (10 mM, 100×), trypsin-ethylenedia- minetetraacetic acid (EDTA) solution (1×) and anti- biotic-antimycotics solution (PSF) were from Invitrogen Co. (Grand Island, NY, USA). Rabbit anti-cyclin A, cyclin B1, horseradish peroxidase (HRP)-conjugated anti-mouse IgG, and horseradish peroxidase (HRP)-conjugated an- ti-rabbit IgG were purchased from Santa Cruz Biotechnol- ogy (Santa Cruz, CA, USA). Rabbit anti-caspase-3, an- ti-caspase-9, anti-phospho-cdc2 (Tyr15), anti-phospho-cy- clin B1, and mouse anti-caspase-8 primary antibody were obtained from Cell Signaling (Denver, MA, USA). Mouse monoclonal anti-PARP and anti-cyclin E were from BD Biosciences (San Diego, CA, USA). Ochnaflavone (Fig.
1A) isolated from the caulis of Lonicera japonica was pro- vided by Dr. Sam Sik Kang, a coauthor, College of Pharmacy, Seoul National University, Seoul, Korea.
Cell culture
Human colon cancer HCT-15 cells were obtained from the Korean Cell Line Bank (KCLB, Seoul, Korea), and the cells were cultured in RPMI supplemented with 10%
heat-inactivated fetal bovine serum, 100 units/ml penicillin, 100 μg/ml streptomycin, and 250 ng/ml amphotericin B.
The cells were maintained at 37oC in humidified atmos- phere with 5% CO2.
Evaluation of growth inhibitory potential
HCT-15 cells (5×104 cells/ml) were treated with various concentrations of ochnaflavone for 3 days. After incu- bation, cells were fixed with 10% TCA solution, and the cell viability was determined with sulforhodamine B (SRB) pro- tein staining method (Lee et al., 1998). The result was ex- pressed as a percentage, relative to solvent-treated con- trol incubations, and the IC50 values were calculated using non-linear regression analysis (percent survival versus concentration).
DNA fragmentation assay
HCT-15 cells were plated in 100 mm culture dish at a density of 1×106 cells/dish. Twenty-four hours later, fresh media containing test sample were added to cultured dishes. After treatment for 24 h, the cells were washed with PBS and lysed in buffer containing 50 mM Tris-HCl, pH 7.5, 20 mM EDTA, and 1.0% NP-40. After centrifugation, 1% SDS and RNase A (5 μg/μl) were added to the super- natants, and then incubated at 56oC for 2 h. Subsequently, proteinase K (2.5 μg/μl) was added and then incubated at 37oC for 2 h. DNA was precipitated with 0.5 volume of 10 M ammonium acetate and 2.5 volumes of cold ethanol at −20oC overnight. Precipitated DNA was dissolved in 50 μl of 10 mM Tris buffer (pH 8.0) containing 1 mM EDTA. DNA sam- ples (4 μg) were resolved by electrophoresis in 2% agar- ose gel, stained with SYBR Gold (Molecular Probes, Eugene, Oregon, USA), and visualized (Lee et al., 2002).
Analysis of cell cycle dynamics by flow cytometry Cell cycle analysis by flow cytometry was performed as previously described (Lee et al., 2002). Briefly, HCT-15 cells were plated at a density of 1×106 cells per 100-mm culture dish and incubated for 24 h. Fresh media contain- ing test samples were added to culture dishes. After 24 h,
Fig. 2. Effects of ochnaflavone on the cell cycle progression in HCT-15 cells.
HCT-15 cells were treated with vehicle or various concentrations of ochna- flavone (5, 10, 20 or 40 μM) for 24 h.
The cell cycle distribution was analyzed by FACScalibur and depicted with the histogram.
the floating and adherent cells were collected and washed with PBS twice. The cells were fixed with 70% ethanol, and incubated with a staining solution containing 0.2% NP-40, RNase A (30 μg/ml), and propidium iodide (50 μg/ml) in phosphate-citrate buffer (pH 7.2). Cellular DNA content was analyzed by flow cytometry using a Becton Dickinson laser-based flow cytometer (FACSCalibur, BD, Bioscien- ces). At least 20,000 cells were used for each analysis, and results were displayed as histograms. Cell cycle dis- tribution was analysed using the ModFit LT 2.0 program.
Evaluation on the expression of cell cycle regulating proteins and apoptosis-related proteins
Cells were treated with various concentrations of ochna- flavone for 24 h. After incubation, cells were lysed and pro- tein concentrations were determined by BCA method.
Each protein (30-50 μg) was subjected to 10% SDS-PAGE.
Proteins were transferred onto PVDF membranes by elec- troblotting, and membranes were treated for 1 h with block- ing buffer [5% non-fat dry milk in phosphate-buffered sal- ine-0.1% Tween 20 (PBST)]. Membranes were then in- cubated with indicated antibodies overnight at 4oC, wash- ed three times for 5 min with PBST. After washing, mem- branes were incubated with HRP-conjugated anti-mouse IgG diluted 1:1,500 in PBST for 3 h at room temperature, washed three times for 5 min with PBST, and detected us- ing ECL reagent (Lab Frontier, Seoul) (Lee et al., 2002).
Statistical analysis
Data were presented as the mean ± SE for the indicated
number of independently performed experiments. Figure data are shown as one representative of at least three in- dependent experiments.
RESULTS
Growth inhibitory effects of ochnaflavone on HCT-15 human colon cancer cells
The growth inhibitory potential of ochnaflavone was de- termined in cultured HCT-15 human colon cancer cells by a colorimetric SRB protein dye staining method. As a re- sult, ochnaflavone exhibited a remarkable growth inhibitory effect against HCT-15 human colon cancer cells with the IC50 value of approximately 4.1 μM (Fig. 1B). The concen- trations of the compound up to 10 μM exhibited mainly cell cycle arrest, but over 20 μM of the compound caused a cy- totoxic effect with the observation of floating dead cells.
Changes of cell cycle distribution by ochnaflavone HCT-15 cells were treated with ochnaflavone (0-40 μM) for 24 h, and the distribution of cells in various compart- ments of the cell cycle was analyzed by flow cytometry.
The cell cycle distribution by ochnaflavone seemed to be highly dependent on the concentration of the compound.
When treated with ochnaflavone of 5 μM cells were sig- nificantly accumulated in the G2/M phase, but 10 μM of the compound evoked the accumulation of S phase. In addi- tion, the higher concentrations of ochnaflavone with 20 and 40 μM increased in the sub-G1 phase, indicative of apoptotic peaks, during incubation time (Fig. 2).
Fig. 4. Effect of ochnaflavone on the expression of cell cycle regulatory pro- teins (A) and apoptosis-related pro- teins (B) in HCT-15 cells. HCT-15 cells were treated with various concentra- tions of ochnaflavone for 24 h. Cell lysates were subjected to western blot analysis as described in Materials and Methods. The level of protein was ana- lyzed by LAS-3000.
Fig. 3. Induction of DNA fragmentation by ochnaflavone in HCT-15 cells. HCT-15 cells were treated with vehicle or the indicated concentration of ochnaflavone (10, 20 and 40 μM) for 24 h. Extracted DNA was separated by agarose gel electro- phoresis and visualized under the UV transilluminator.
Induction of DNA fragmentation by ochnaflavone To further investigate whether the cytotoxic effect of ochnaflavone was associated with apoptosis, DNA was extracted from the cells exposed to the compound for 24 h and subjected to electrophoresis in agarose gel. DNA frag- mentation characteristic for apoptosis was clearly detected by exposure of ochnaflavone in a concentration-depend- ent manner as illustrated in Fig. 3.
Effects of ochnaflavone on the expression of cell cycle regulatory proteins and apoptosis related proteins Based on the cell cycle distribution data evoked by och- naflavone, in order to investigate whether ochnaflavone af- fects the expression of cell cycle regulatory proteins, the cells were treated with various concentrations of the com- pound for 24 h, and then the protein expressions were de- termined by western blot analysis. As depicted in Fig. 4A, the lower concentration of ochnaflavone (5 μM) increased the expression of phosphorylated cdc2 (Tyr-15) which is the inactive form of cdc2 (p-cdc2, Tyr-15) and this event seemed to be related with the interruption of the exit of G2/M phase of cell cycle. This might result in the accumu- lation of G2/M phase of cell distribution. In addition, apop- totic phenomena induced by the treatment of the higher concentrations of ochnaflavone (over 20 μM) were corre- lated with the activation of caspases. The expressions of procaspase-9, -8, and -3 were downregulated and thus the active forms of cleaved corresponding caspases were formed by the treatment of ochnaflavone (Fig. 4B). PARP cleavage which is the target of caspases was also in- duced, and these events subsequently affect to the in- duction of apoptosis.
DISCUSSION
Cancer is the main cause of death in the world.
Especially, colon cancer is the forth abundant causes of cancer mortality in Korea and the second in Western countries. One of the promising strategies in controlling colorectal cancer progression is considered cancer che- moprevention by taking dietary factors. Various com- pounds derived from natural products have been reported
to modulate the growth of colorectal cancer cells and thus are considered to be cancer chemopreventive agents (Bhanot and Möller, 2009). Ochnaflavone is a natural bi- flavonoid and has been demonstrated to have anti-in- flammatory and anti-arthreogenic activites (Lee et al., 1997; Moon et al., 2006; Son et al., 2006; Suh et al., 2006a; Suh et al., 2006b; Kim et al., 2008). Since chronic inflammation is highly correlated with colorectal carcino- genesis (Wada, 2009; Bhanot and Möller, 2009), anti-in- flammatory effect of ochnaflavone might suggest its possi- ble role in colon cancer chemoprevention. However, the di- rect effects of ochnaflavone against cancer cells have been poorly examined yet. The present study evaluated the effect of ochnaflavone on the proliferation of cultured human colorectal cancer HCT-15 cells and further inves- tigated the growth inhibitory mechanisms of action.
Primarily, the anti-proliferative activity of ochnaflavone in human colon cancer HCT-15 cells was examined. Ochna- flavone concentration-dependently inhibited the cell pro- liferation and the IC50 value for 3 days incubation was 4.1 μM (Fig. 1B). The IC50 value of ellipticine, a positive control, was 1.0 μM in the same culture condition. Based on the potential anti-proliferative activity of ochnaflavone we fur- ther investigated the mechanisms of action in terms of cell cycle analysis and expressions of cell-cycle regulatory pro- teins in HCT-15 cells. Flow cytometric analysis indicated that ochnaflavone significantly induced the accumulation of cell population in the G2/M phase at 5 μM (control;
28.3%, treatment; 59.35%), and the accumulation of S phase of cells was pronounced at 10 μM (control; 39.68%, treatment; G2/M 83.95%) during the incubation of the com- pound for 24 h. These results suggested that ochnaflavone differentially induced cell cycle arrest in a concen- tration-dependent manner. In addition, the higher concen- trations of ochnaflavone (20 or 40 μM) dramatically in- creased the sub-G1 peaks (34.83 and 49.19%, respec- tively), indicative of apoptotic phase. Therefore, the cell cy- cle arrest and induction of apoptosis might be one possible mechanisms of action in the growth inhibitory effect of och- naflavone in human colon cancer cells. To further confirm the cell cycle arrest and apoptosis induced by ochna- flavone we examined the cell cycle regulatory proteins in HCT-15 cells. Generally, cell cycle progression is regu- lated by cyclin/cyclin dependent kinase (CDK) complex.
Although CDK expression is constant during the cell cycle, expression of cyclin is changed according to the each phase of cell cycle. First, cyclin D is normally increased during mid/late G1 phase, and subsequent increase of cy- clin E is needed for entry to S phase. Up-regulation of cy- clin A and cyclin B leads up to S to G2/M phase entry. In
addition, cyclins/CDKs are regulated by several factors, such as kinase (WEE1, PLK) and phosphatase (CDC25C) (Russell and Nurse, 1987; Lundgren et al., 1991; Gu et al., 1992; Porter and Donoghue, 2003). When the times of G2 phase pass to M phase PLK transfers phosphate to the site of ser-147 of cyclin B, and then cyclin B moves into nu- cleus and combines with cdc2 (Toyoshima-Morimoto et al., 2001). Moreover, CDC25C removes the phosphate group at the site of cdc2 (Tyr-15), resulting in M phase progression. In this view, the induction of cell cycle arrest by ochnaflavone was highly correlated with the formation of inactive form of cdc2 (Tyr-15) (p-cdc2, phosphorylated cdc2 (Tyr-15)) (Fig. 4A). The increased inactive form of cdc2 interfered the cell cycle exit from G2/M phase and thus arrested in this phase of cell cycle. Increase of cyclin B1 at 5 μM of ochnaflavone was also thought to be in part in the process of inactivation of cdc-2. Although the accu- mulation of S phase was evoked by the treatment of 10 μM of ochnaflavone the cell cycle regulatory proteins were not dramatically changed. However, the cdc2 inactivation and the increase of cyclin A, B1 and E1 might be still in part re- lated to the arrest. The more detailed examination needs be remained. In addition, the growth inhibition by ochna- flavone was also related with the induction of apoptotic cell death with the treatment of over 10 μM. As depicted in Fig.
2, over 20 μM of ochnaflavone drastically increased the cell cycle distribution of the sub-G1 phase, indicative of apoptotic peaks. The induction of apoptosis was also con- firmed by the observation of DNA fragmentation (Fig. 3).
One of well-known phenomena of apoptosis is the activa- tion of caspases (Ahn et al., 2009). Ochnaflavone-mediated apoptosis seems to be explained the consequential activa- tion of caspase-8, -9 and caspase-3 and thus induces the cleavage of PARP, a target protein of caspases.
In summary, this study suggests that the growth in- hibitory effects of ochnaflavone against human colon can- cer cells are related to the cell cycle arrest and induction of apoptosis. The cell cycle arrest is closely correlated with cdc 2 inactivation and apoptosis is associated with the se- quential activation of caspases. Along with the potential cancer chemopreventive potential of ochnaflavone in vitro this study further suggests one plausible mechanism of ac- tions in the growth inhibitory effect of ochnaflavone in hu- man colon cancer cells.
ACKNOWLEDGMENTS
This study was supported by a grant Studies on the Identification of the Efficacy of Biologically Active Compo- nents from Oriental Herbal Medicines from the Korea Food
and Drug Administration (2007).
REFERENCES
Ahn, M. Y., Na, Y. J., Lee, J., Lee, B. M. and Kim, H. S. (2009).
Apicidin induces apoptosis via cytochrome c-mediated intrinsic pathway in human ovarian cancer cells. Biomol.
Ther. 17, 17-24.
Amella, M., Bronner, C., Briancon, F., Haag, M., Anton, R. and Landry, Y. (1985). Inhibition of mast cell histamine release by flavonoids and biflavonoids. Planta Med. 51, 16-20.
Beretz, A., Joly, M., Stoclet, J. C. and Anton, R. (1979).
Inhibition of 3’, 5’-AMP phosphodiesterase by biflavonoids and xanthones. Planta Med. 36, 193-195.
Bhanot, U. K. and Möller, P. (2009). Mechanisms of paren- chymal injury and signaling pathways in ectatic ducts of chronic pancreatitis: implications for pancreatic carcino- genesis. Lab. Invest. 89, 489-497.
Butenko, I. G., Gladtchenko, S. V. and Galushko, S. V. (1993).
Anti-inflammatory properties and inhibition of leukotriene C4 biosynthesis in vitro by flavonoid baicalein from Scutellaria baicalensis georgy roots. Agents Actions 39, 49-51.
Chang, H. W., Baek, S. H., Chung, K. W., Son, K. H., Kim, H. P.
and Kang, S. S. (1994). Inactivation of phospholipase A2 by naturally occurring biflavonoid, ochnaflavone. Biochem.
Biophys. Res. Commun. 205, 843-849.
Chowdhury, A. R., Sharma, S., Mandal, S., Goswami, A., Mukhopadhyay, S. and Majumder, H. K. (2002). Luteolin, an emerging anti-cancer flavonoid, poisons eukaryotic DNA topoisomerase I. Biochem. J. 366, 653-661.
Ielpo, M. T. L., Basile, A., Miranda, R., Moscatiello, V., Nappo, C., Sorbo, S., Laghi, E., Ricciardi, M. M., Ricciardi, L. and Vuotto, M. L. (2000). Immunopharmacological properties of flavonoids. Fitoterapia 71, 101-109.
Iwu, M. M. Igboko, O. A., Okunji, C. O. and Tempesta, M. S.
(1990). Antidiabetic and aldose reductase activities of biflavanones of Garcinia kola. J. Pharm. Pharmacol. 42, 290-292.
Grange, L. L., Wang, M., Watkins, R., Ortiz, D., Sanchez, M. E., Konst, J., Lee, C. and Reyes, E. (1999). Protective effects of the flavonoid mixture, silymarin, on fetal rat brain and liver. J.
Ethnopharmacol. 67, 53-62.
Gu, Y., Rosenblatt, J. and Morgan, D. O. (1992). Cell cycle regulation of CDK2 activity by phosphorylation of Thr160 and Tyr15. EMBO J. 11, 3995-4005.
Kim, H. P., Park, H., Son, K. H., Chang, H. W. and Kang, S. S.
(2008). Biochemical pharmacology of biflavonoids: Implica- tions for anti-inflammatory action. Arch. Pharm. Res. 31, 265-273.
Lee, S. K., Cui, B., Mehta, R. R., Kinghorn, A. D. and Pezzuto, J.
M. (1998). Cytostatic mechanism and antitumor potential of novel 1H-cyclopentea[b]benzofuran lignans isolated from Aglaia elliptica. Chem. Biol. Interact. 115, 215-228.
Lee, S. K., Heo, Y. H., Steele, V. E. and Pezzuto, J. M. (2002).
Induction of apoptosis by 1,4-phenylenebis(methylene)sele- nocyanate in cultured human colon cancer cells. Anticancer Res. 22, 97-102.
Lee, S. J., Choi, J. H., Son, K. H., Chang, H. W., Kang, S. S. and Kim, H. P. (1995). Suppression of mouse lymphocyte proli- feration in vitro by naturally-occurring biflavonoids. Life Sci.
57, 551-558.
Lee, S. J., Son, K. H., Chang, H. W., Kang, S. S. and Kim, H. P.
(1997). Inhibition of arachidonate release from rat peritoneal macrophage by biflavonoids. Arch. Pharm. Res. 20, 533-538.
Lundgren, K., Walworth, N., Booher, R., Dembski, M., Kirschner, M. and Beach, D. (1991). Mik1 and wee1 cooperate in the inhibitory tyrosine phosphorylation of cdc2. Cell 64, 1111- 1122.
Molina, M. F., Sanchez-Reus, I., Iglesias, I. and Benedi, J.
(2003). Quercetin, a flavonoid antioxidant, prevents and protects against ethanol-induced oxidative stress in mouse liver. Biol. Pharm. Bull. 26, 1398-1402.
Moon, T. C., Hwang, H. S., Quan, Z., Son, K. H., Kim, C. H., Kim, H. P., Kang, S. S., Son, J. K. and Chang, H. W. (2006).
Ochnaflavone, naturally occuring biflavonoid, inhibits pho- spholipase A2 dependent phosphatidylethanolamine degra- dation in a CCl4-induced rat liver. Biol. Pharm. Bull. 29, 2359-2361.
Porter, L. A. and Donoghue, D. J. (2003). Cyclin B1 and CDK1:
nuclear localization and upstream regulators. Prog. Cell Cycle Res. 5, 335-347.
Rashid, I. L., Liang, Q., Steven, J. S. and Basil, R. (1997).
Curcumin, a natural plant phenolic food additive, inhibits cell proliferation and induces cell cycle changes in colon adenocarcinoma cell lines by a prostaglndin-independent pathway. J. Lab. Clin. Med. 130, 576-584.
Russell, P. and Nurse, P. (1987). Negative regulation of mitosis by wee1+, a gene encoding a protein kinase homolog. Cell 49, 559-567.
Sharon, A. A. and Nora, M. O’B. (2000). Mechanism of protection by the flavonoids, quercetin and rutin, against tert-butylhydroperoxide-and menadione-induced DNA single strand breaks in Caco-2 cells. Free Radic. Biol. Med. 29, 507-514.
Shimizu, M., Deguchi, A., Hara, Y., Moriwaki, H. and Weinstein, I. B. (2005). EGCG inhibits activation of the insulin-like growth factor-1 receptor in human colon cancer cells.
Biochem. Biophys. Res. Commun. 334, 947-953.
Son, M. J., Moon, T. C., Lee, E. K., Son, K. H., Kim, H. P., Kang, S. S., Son, J. K., Lee, S. H. and Chang, H. W. (2006).
Naturally occurring biflavonoid, ochanflavone, inhibits cyclo-oxygenases-2 and 5-lipoxygenase in mouse bone marrow-derived mast cells. Arch. Pharm. Res. 29, 282-286.
Suh, S. J., Chung, T. W., Son, M. J., Kim, S. H., Moon, T. C., Son, K. H., Kim, H. P., Chang, H. W. and Kim, C. H. (2006a).
The naturally occurring biflavonoid, ochnaflavone, inhibits LPS-induced iNOS expression, which is mediated by ERK1/2 via NF-κB regulation, in RAW264.7 cells. Arch.
Biochem. Biophys. 447, 136-146.
Suh, S. J., Jin, U. H., Kim, S. H., Chang, H. W., Son, J. K., Lee, S. H., Son, K. H. and Kim, C. H. (2006b). Ochnaflavone inhibits TNF-α-induced human VSMC proliferation via regulation of cell cycle, ERK1/2, and MMP-9. J. Cell.
Biochem. 99, 1298-1307.
Toyoshima-Morimoto, F., Taniguchi, E., Shinya, N., Iwamatsu, A. and Nishida, E. (2001). Polo-like kinase 1 phosphorylates cyclin B1 and targets it to the nucleus during prophase.
Nature 410, 215-220.
Wada, R. (2009). Proposal of a new hypothesis on the development of colorectal epithelial neoplasia: nonspecific inflammation - colorectal paneth cell metaplasia - colorectal epithelial neoplasia. Digestion 79, 9-12.
