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Journal of Cancer Prevention

Original Article

JCP -0001; 19(3): 161-169

Published online November 30, -0001

https://doi.org/10.15430/JCP.2014.19.3.161

© Korean Society of Cancer Prevention

Kaempferol Downregulates Insulin-like Growth Factor-I Receptor and ErbB3 Signaling in HT-29 Human Colon Cancer Cells

Hyun Sook Lee1,*, Han Jin Cho2,3,*, Gyoo Taik Kwon2, and Jung Han Yoon Park2,4

1Department of Food Science and Nutrition, Dongseo University, Busan, Korea, 2Department of Food Science and Nutrition, Hallym University, Chuncheon, Korea, 3WCU Biomodulation Major, Department of Agricultural Biotechnology and Center for Food and Bioconvergence, Seoul National University, Seoul, Korea, 4Advanced Institutes of Convergence Technology, Seoul National University, Suwon, Korea

Correspondence to :
Jung Han Yoon Park, Department of Food Science and Nutrition, Hallym University, 1 Hallymdaehak-gil, Chuncheon 200-702, Korea, Tel: +82-33-248-2134, Fax: +82-33-256-0199, E-mail: jyoon@hallym.ac.kr, ORCID: Jung Han Yoon Park, http://orcid.org/0000-0002-5518-4279

Received: June 20, 2014; Revised: July 14, 2014; Accepted: July 14, 2014

This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/3.0) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

Background:

 Novel dietary agents for colon cancer prevention and therapy are desired. Kaempferol, a flavonol, has been reported to possess anticancer activity. However, little is known about the molecular mechanisms of the anticancer effects of kaempferol. The aim of this study was to determine the inhibitory effect of kaempferol on growth factor-induced proliferation and to elucidate its underlying mechanisms in the HT-29 human colon cancer cell line.


Methods:

 To assess the effects of kaempferol and/or growth factors [insulin-like growth factor (IGF)-I and heregulin (HRG)-β], cells were cultured with or without 60 μmol/L kaempferol and/or 10 nmol/L IGF-I or 20 μg/L HRG-β. Cell proliferation, DNA synthesis, and apoptosis were determined by a cell viability assay, a [3H]thymidine incorporation assay, and Annexin-V staining, respectively. Western blotting, immunoprecipitation, and an in vitro kinase assay were conducted to evaluate expression and activation of various signaling molecules involved in the IGF-I receptor (IGF-IR) and ErbB3 signaling pathways.


Results:

 IGF-I and HRG-β stimulated HT-29 cell growth but did not abrogate kaempferol-induced growth inhibition and apoptosis. Kaempferol reduced IGF-II secretion, HRG expression and phosphorylation of Akt and extracellular signal-regulated kinase (ERK)-1/2. Kaempferol reduced IGF-I- and HRG-β-induced phosphorylation of the IGF-IR and ErbB3, their association with p85, and phosphatidylinositol 3-kinase (PI3K) activity. Additionally, kaempferol inhibited IGF-I- and HRG-β-induced phosphorylation of Akt and ERK-1/2.


Conclusions:

 The results demonstrate that kaempferol downregulates activation of PI3K/Akt and ERK-1/2 pathways by inhibiting IGF-IR and ErbB3 signaling in HT-29 cells. We suggest that kaempferol could be a useful chemopreventive agent against colon cancer.

Keywords: Kaempferol, Insulin-like growth factor-I receptor, ErbB3, HT-29 human colon cancer

 Colon cancer is one of the leading causes of cancer-associated deaths worldwide.1,2 The incidence of colon cancer is strongly related to lifestyle, particularly dietary habits.3 Over the past several decades, there has been an increased interest in the utility of plant-derived polyphenols based upon their broad spectrum of biological properties, including antioxidative, anticarcinogenic, and cardioprotective activities.4 The important advantage with phytochemicals is that they are generally non-toxic and have wide human acceptance.3

 Flavonoids are polyphenolic compounds that are distributed widely in fruits and vegetables. The six major subgroups of flavonoids commonly found in the human diet are flavonols, flavones, anthocyanidins, catechins, flavonones and isoflavones.5 Their wide-ranging biological activities include antioxidant, anti-inflammatory and anti-cancer effects.6 Studies have shown that several flavonoids have potent anti-cancer activity against colon cancer cell lines.7?11 Individual intake of flavonoids varies depending upon the type of diet consumed. In Western populations, estimated daily intake of flavonoids is in the range of 20?50 mg per day.12

 Kaempferol (3,4’,5,7-tetrahydroxyflavone) is a flavonol (Fig. 1A) present in fruits and vegetables, including onions, kale, broccoli, apples, cherries, berries, tea and red wine.13,14 Kaempferol has broad biological properties including anticancer effects. For example, kaempferol induces apoptosis and cell cycle arrest in various cancer cell lines, including colon cancer cells.15?18 We have reported that kaempferol induces cell cycle arrest and apoptosis in HT-29 colon cancer cells.9,19 The gastrointestinal tract is the first line of contact with food and these studies suggest that kaempferol may have preventive/therapeutic benefits in colon cancer. Nevertheless, the underlying mechanisms of kaempferol in human colon cancer cells are not fully understood. It has been reported that various human colon cancer cell lines, including HT-29 cells, produce polypeptide growth factors such as transforming growth factor-α/epidermal growth factor (EGF) receptor, transforming growth factor-β, and platelet-derived growth factor.20 In addition, we have observed previously that HT-29 cells express other growth factors, including insulin-like growth factor (IGF)-II21 and heregulin (HRG).22 These observations suggest that colon cancer cells produce autocrine and/or paracrine growth factors that stimulate cell growth. Therefore, inhibiting the signaling growth factor may be a potential strategy for preventing colon cancer.

 The IGF system includes IGF-I, IGF-II, IGF-binding proteins and the IGF-I receptor (IGF-IR). This system plays an important role in the growth of various cancer cells, including colon cancer cells.23,24 The IGF-IR and its ligands IGF-I and IGF-II play critical roles in the regulation of cellular proliferation, apoptosis and transformation.25 Ligand binding to the IGF-IR triggers multiple signaling pathways, including the Ras/Raf/extracellular signal- regulated kinase (ERK) pathway implicated in receptor-mediated mitogenesis and transformation and the phosphatidylinositol 3-kinase (PI3K)/Akt pathway implicated in the transmission of cell survival signals.26,27

 The ErbB family of type I receptor tyrosine kinases has four members, including EGF receptor (EGFR; ErbB-1; HER1 in humans), ErbB2 (HER2; c-neu in rodents), ErbB3 (HER3) and ErbB4 (HER4).28 Activation of these receptors regulates a number of processes including cell proliferation, differentiation and survival. Excess ErbB signaling is associated with the development of a wide variety of human cancers including colon cancer.29?31 HRG is a potent mitogen of colon cancer cells and autocrine released HRG generates growth factor independence and prevents apoptosis.32,33

 Our recent study indicated that inhibiting the IGF-IR signaling pathway may be one of the mechanisms by which luteolin, 3’,4’,5,7-tetrahydroxyflavone, inhibits cell cycle progression and induces apoptosis in HT-29 human colon cancer cells.34,35 In the present study, we examined whether the growth inhibitory effects of kaempferol are related to changes in IGF-I-IGF-IR or HRG-ErbB3 signaling in the HT-29 human colon cancer cell line. We found that kaempferol markedly inhibited growth factor- induced cell proliferation. Kaempferol also inhibited activation of the PI3K/Akt and ERK-1/2 pathways.

Materials

 Kaempferol, essentially fatty acid-free bovine serum albumin, 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT), 7-amino-actinomycin D (7-AAD), phosphatidylinositol, transferrin and anti-β-actin antibody were obtained from Sigma (St. Louis, MO, USA); selenium and DMEM/Ham’s F-12 nutrient mixture (DMEM/F-12 were purchased from Gibco BRL (Gaithersburg, MD, USA)); [methyl-3H]thymidine, protein A-Sepharose and horse-radish peroxidase-conjugated anti-rabbit and anti-mouse IgG were from Amersham (Arlington Heights, IL, USA); antibodies against HRG, IGF-IRβ, p85 and ErbB3 were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA); antibodies against Akt, phospho (P)-Akt, ERK-1/2 and P-ERK-1/2 were from Cell Signaling Technology (Beverly, MA, USA); anti-phosphotyrosine-RC20 (PY20) antibody linked to horse-radish peroxidase was purchased from BD Transduction Laboratories (Palo Alto, CA, USA); anti-IGF-II antibody was from Amano International Enzyme (Troy, VA, USA); recombinant human HRG-β and recombinant human IGF-II were purchased from R&D Systems, (Minneapolis, MN, USA); phycoerythrin-conjugated Annexin V was from BD Pharmingen, (Franklin Lake, NJ, USA); and [γ-32P]ATP was obtained from PerkinElmer Life and Analytical Sciences, Inc. (Boston, MA, USA).


2. Cell culture


 HT-29 cells (American Type Culture Collection, Manassas, VA, USA) were maintained in DMEM/F12 supplemented with 100 mL/L fetal bovine serum, 100,000 U/L penicillin and 100 mg/L streptomycin. Cell monolayers were serum-starved in DMEM/F12 supplemented with 5 mg/L transferrin, 5 μg/L selenium and 0.1 g/L bovine serum albumin (serum-free medium) for 24 hours at 24 hour after plating. After serum starvation, the cells were incubated in serum-free medium containing various concentrations of kaempferol with or without growth factors (10 nmol/L IGF-I or 20 μg/L HRG-β) for the indicated time periods. Viable cell numbers were estimated by the MTT assay. Conditioned medium (24 hours) was collected and subjected to Western blot analysis to estimate IGF-II production.


3. [3H]Thymidine incorporation assay


 To determine DNA synthesis, HT-29 cells were plated in 96-well plates, serum-starved and treated with kaempferol and/or growth factors in the presence of [3H]thymidine. After 3 hours, the cells were harvested onto glass-fiber filters and the incorporated [3H]thymidine was quantified using a scintillation counter (Beckman Coulter Inc, Fullerton, CA, USA).


4. Fluorescence-activated cell sorting analysis


 HT-29 cells were plated in 24-well plates, serum-starved and treated with kaempferol and/or growth factors as described above. After 24 hours, the cells were trypsinized and loaded with phycoerythrin-conjugated Annexin V and 7-AAD. Annexin V- positive and 7-AAD-negative cells (early apoptotic cells) were counted by flow cytometry (Becton Dickinson, Franklin Lake, NJ, USA) and expressed as a percentage of the total cell number.


5. Western blot analysis and immunoprecipitation


 Total cell lysates were prepared, and immunoprecipitation studies were conducted as described previously.22 Briefly, total cell lysates were incubated with anti-IGF-IRβ antibody or anti-ErbB3 antibody overnight. The immune complexes were collected by adding protein A-Sepharose followed by centrifugation. Total cell lysates, 24-hour conditioned media, and the immunoprecipitated proteins were subjected to Western blot analysis with their relevant antibodies.


6. In vitro kinase assay


 ERK-1/2 activity was measured using a p44/p42 MAP Kinase Assay Kit (nonradioactive) in accordance with the manufacturer’s instruction (Cell Signaling Technology, Beverly, MA, USA). Briefly, total cell lysates were immunoprecipitated with immobilized phospho-p44/p42 mitogen activated protein kinases (MAPK) antibody (bead slurry). The immunoprecipitated proteins were incubated with ATP and Elk-1 fusion protein (an ERK-1/2 substrate). The resulting P-Elk-1 was analyzed by Western blotting with anti-P-Elk-1 antibody. The immunoprecipitated proteins (mentioned above) were incubated with phosphatidylinositol and [γ-32P]ATP as described previously to detect PI3K activity.22 The resulting 32P-labelled phosphatidylinositol 3-phosphate was separated by thin-layer chromatography and visualized by autoradiography.


7. Statistical analysis


 Results are expressed as means±SEM. Differences between groups were assessed via the Student’s t test, utilizing SAS statistical software ver. 9.2 (SAS Institute, Cary, NC, USA).

Materials

 Kaempferol, essentially fatty acid-free bovine serum albumin, 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT), 7-amino-actinomycin D (7-AAD), phosphatidylinositol, transferrin and anti-β-actin antibody were obtained from Sigma (St. Louis, MO, USA); selenium and DMEM/Ham’s F-12 nutrient mixture (DMEM/F-12 were purchased from Gibco BRL (Gaithersburg, MD, USA)); [methyl-3H]thymidine, protein A-Sepharose and horse-radish peroxidase-conjugated anti-rabbit and anti-mouse IgG were from Amersham (Arlington Heights, IL, USA); antibodies against HRG, IGF-IRβ, p85 and ErbB3 were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA); antibodies against Akt, phospho (P)-Akt, ERK-1/2 and P-ERK-1/2 were from Cell Signaling Technology (Beverly, MA, USA); anti-phosphotyrosine-RC20 (PY20) antibody linked to horse-radish peroxidase was purchased from BD Transduction Laboratories (Palo Alto, CA, USA); anti-IGF-II antibody was from Amano International Enzyme (Troy, VA, USA); recombinant human HRG-β and recombinant human IGF-II were purchased from R&D Systems, (Minneapolis, MN, USA); phycoerythrin-conjugated Annexin V was from BD Pharmingen, (Franklin Lake, NJ, USA); and [γ-32P]ATP was obtained from PerkinElmer Life and Analytical Sciences, Inc. (Boston, MA, USA).


2. Cell culture


 HT-29 cells (American Type Culture Collection, Manassas, VA, USA) were maintained in DMEM/F12 supplemented with 100 mL/L fetal bovine serum, 100,000 U/L penicillin and 100 mg/L streptomycin. Cell monolayers were serum-starved in DMEM/F12 supplemented with 5 mg/L transferrin, 5 μg/L selenium and 0.1 g/L bovine serum albumin (serum-free medium) for 24 hours at 24 hour after plating. After serum starvation, the cells were incubated in serum-free medium containing various concentrations of kaempferol with or without growth factors (10 nmol/L IGF-I or 20 μg/L HRG-β) for the indicated time periods. Viable cell numbers were estimated by the MTT assay. Conditioned medium (24 hours) was collected and subjected to Western blot analysis to estimate IGF-II production.


3. [3H]Thymidine incorporation assay


 To determine DNA synthesis, HT-29 cells were plated in 96-well plates, serum-starved and treated with kaempferol and/or growth factors in the presence of [3H]thymidine. After 3 hours, the cells were harvested onto glass-fiber filters and the incorporated [3H]thymidine was quantified using a scintillation counter (Beckman Coulter Inc, Fullerton, CA, USA).


4. Fluorescence-activated cell sorting analysis


 HT-29 cells were plated in 24-well plates, serum-starved and treated with kaempferol and/or growth factors as described above. After 24 hours, the cells were trypsinized and loaded with phycoerythrin-conjugated Annexin V and 7-AAD. Annexin V- positive and 7-AAD-negative cells (early apoptotic cells) were counted by flow cytometry (Becton Dickinson, Franklin Lake, NJ, USA) and expressed as a percentage of the total cell number.


5. Western blot analysis and immunoprecipitation


 Total cell lysates were prepared, and immunoprecipitation studies were conducted as described previously.22 Briefly, total cell lysates were incubated with anti-IGF-IRβ antibody or anti-ErbB3 antibody overnight. The immune complexes were collected by adding protein A-Sepharose followed by centrifugation. Total cell lysates, 24-hour conditioned media, and the immunoprecipitated proteins were subjected to Western blot analysis with their relevant antibodies.


6. In vitro kinase assay


 ERK-1/2 activity was measured using a p44/p42 MAP Kinase Assay Kit (nonradioactive) in accordance with the manufacturer’s instruction (Cell Signaling Technology, Beverly, MA, USA). Briefly, total cell lysates were immunoprecipitated with immobilized phospho-p44/p42 mitogen activated protein kinases (MAPK) antibody (bead slurry). The immunoprecipitated proteins were incubated with ATP and Elk-1 fusion protein (an ERK-1/2 substrate). The resulting P-Elk-1 was analyzed by Western blotting with anti-P-Elk-1 antibody. The immunoprecipitated proteins (mentioned above) were incubated with phosphatidylinositol and [γ-32P]ATP as described previously to detect PI3K activity.22 The resulting 32P-labelled phosphatidylinositol 3-phosphate was separated by thin-layer chromatography and visualized by autoradiography.


7. Statistical analysis


 Results are expressed as means±SEM. Differences between groups were assessed via the Student’s t test, utilizing SAS statistical software ver. 9.2 (SAS Institute, Cary, NC, USA).

 The increasing acceptability of phytochemicals, particularly those from dietary sources, as cancer chemopreventive agents in recent years may be attributed to their high potency, low toxicity and a relatively safe profile compared to synthetic anticancer agents.40 The anticarcinogenic effect of flavonoids has been the focus of many investigations. The flavonol kaempferol is abundant in onions, kale, broccoli, apples, cherries, berries, tea and red wine.13,14 We demonstrated previously that kaempferol induces cell cycle arrest and apoptosis in HT-29 colon cancer cells.9,19 Because human colon cancer cells produce autocrine and/or paracrine growth factors that stimulate cell growth,20 the present study was conducted to examine whether kaempferol could counteract the stimulated cell growth induced by growth factors and to identify the mechanisms. Our results indicate that kaempferol (1) abrogates the growth stimulatory effects of IGF-I and HRG-β; (2) reduces secretion of IGF-II and expression of HRG; (3) decreases phosphorylation of Akt and ERK-1/2 and the activity of ERK-1/2; (4) inhibits IGF-I- and HRG-β-induced tyrosine phosphorylation of IGF-IR or ErbB3, and the association of p85 with IGF-IR or ErbB3, and PI3K activity in HT-29 cells. These results indicate that inhibiting IGF-II and HRG secretion as well as downregulating IGF-IR and ErbB3 signaling may be important factors underlying the inhibitory effects of kaempferol on growth of HT-29 cells.

 PI3K is central to the coordinated control of multiple cell- signaling pathways leading to tumor development, including cell proliferation and apoptosis.41 We found that kaempferol decreased IGF-IR or ErbB3-associated PI3K activity (Fig. 4 and 5). Lee et al. reported that kaempferol binds to PI3K directly and inhibits PI3K activation in JB6 P+ mouse epidermal cells.42 Furthermore kaempferol decreased the IGF-I- and HRG-β-induced association of p85 with IGF-IR or ErbB3 (Fig. 4A and 5A). Taken together, these results suggest that kaempferol inhibits PI3K activity directly by binding to PI3K and indirectly by decreasing the association between p85 and growth factor receptors.

Akt, a downstream target of PI3K, regulates a variety of cellular process (including cell-cycle regulation and apoptosis) by phosphorylating multiple substrates.43 Activating of ErbB3 and IGF-IR signaling leads to activation of Akt.21,22,38 We have reported previously that kaempferol reduces phosphorylation and activity of Akt in HT-29 cells.19 Consistent with our results, several studies have indicated that kaempferol inhibits phosphorylation of Akt in human leukemia cells44 and A549 lung cancer cells.15 Deregulation of the PI3K/Akt signaling pathway can lead to an altered aspects of cell physiology that comprise the hallmarks of cancer.44 Therefore inhibiting PI3K and subsequently inactivating Akt may contribute to the anti-cancer effect of kaempferol.

 The MAPK family, which includes ERK-1/2, c-Jun N-terminal kinase, p38 and ERK5 subgroups, regulates proliferation and apoptosis.45 Each MAPK is activated through a specific phosphorylation cascade. Activation of ERK-1/2 has been implicated in mitogenic signaling by IGF-I- and HRG-induced activation of IGF-IR and ErbB3, respectively.38,39 The ERK-1/2 cascade is associated with cell differentiation, proliferation and survival. In the present study, we observed that kaempferol reduced phosphorylation and activity of ERK-1/2 (Fig. 3). It has also been reported that kaempferol inhibits phosphorylation of ERK-1/2 in SCC4 oral cancer cells.46 In contrast, Nguyen et al. reported that activating ERK-1/2 is a requirement for kaempferol-induced apoptosis in A549 lung cancer cells.15 Because it is possible to induce apoptosis through the activation of ERK-1/2,47 future studies are needed to explore the detailed mechanisms between kaempferol-induced apoptosis and ERK-1/2 activation in various cell lines. In the present study, kaempferol decreased both basal and IGF-I- and HRG-β-induced phosphorylation of ERK-1/2 in HT-29 cells (Fig. 4B and5B), indicating that inhibiting HT-29 cell growth by kaempferol may be mediated, at least in part, by the decrease in ERK-1/2 activation.

In summary, we demonstrated that IGF-I and HRG-β stimulated DNA synthesis and inhibited apoptosis but did not abrogate the growth inhibitory effect of kaempferol. Kaempferol inhibited IGF-II secretion and HRG protein expression as well as growth factor-stimulated phosphorylation of Akt and ERK-1/2. These results suggest that the growth inhibitory effect of kaempferol may be mediated by decreasing IGF-I/IGF-IR and HRG-β/ErbB3, PI3K/Akt pathway and ERK-1/2 signaling. However, other pathways may also play roles that require an in-depth investigation. Based on these observations, future in vivo studies using animal tumor models are needed to examine whether kaempferol could be a useful chemopreventive agent against colon cancer.

Fig. 1. Effect of kaempferol on cell viability in growth factor-treated HT-29 cells. (A) Chemical structure of kaempferol. (B, C) HT-29 cells were plated and serum-starved. After serum starvation, the cells were treated with 0 or 60 μmol/L kaempferol with or without growth factors [B: 10 nmol/L insulin-like growth factor-I (IGF-I); C: 20 μg/L heregulin (HRG)-β]. Viable cell numbers were estimated by the 3-[4,5-dimethylth-iazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) assay. Each bar represents the mean±SEM (n=6). *P < 0.05 as compared with the untreated cells. P < 0.05 as compared with the cells treated with kaempferol.
Fig. 2. Effect of kaempferol and/or growth factors on DNA synthesis and apoptosis in HT-29 cells. (A, B) HT-29 cells were plated in 96-well plates, serum-starved and treated with 0 or 60 μmol/L kaempferol and/or growth factors [A: 10 nmol/L insulin-like growth factor-I (IGF-I); B: 20 μg/L heregulin (HRG)-β)] in the presence of [3H]thymidine. The cells were incubated for 3 hours and then harvested, and DNA synthesis was measured by incorporation of [3H]thymidine into DNA. (C, D) HT-29 cells were plated in 24-well plates, serum-starved, and treated with 0 or 60 μmol/L kaempferol and/or growth factors (C: 10 nmol/L IGF-I; D: 20 μg/L HRG-β). At 24 hours after treatment, the cells were trypsinized and loaded with Annexin V and 7-amino-actinomycin D (7-AAD). The percentages of early apoptotic cells (Annexin V+/7-AAD?) were quantified by flow cytometry. Each bar represents the mean±SEM (n=6). *P < 0.05 as compared with the untreated cells.
Fig. 3. Effect of kaempferol on the expression of insulin-like growth factor (IGF)-II and heregulin (HRG) and the phosphorylation of Akt and extracellular regulated kinase (ERK)- 1/2. HT-29 cells were serum-starved and treated with various concentrations (0, 20, 40, or 60 μmol/L) of kaempferol. (A) At 24 hours after treatment, 24 hour-conditioned media were collected and subjected to Western blot analysis with anti-IGF-II antibody. Media loaded onto the gel was adjusted for equivalent cell numbers. (B) At the indicated time point after kaempferol treatment, total cell lysates were prepared and subjected to Western blot analysis with their relevant antibodies. (C, D) At 2 hours after kaempferol treatment, total cell ly-sates were prepared. (C) Total cell lysates were subjected to Western blot analysis with the relevant antibodies. (D) Total cell lysates were incubated with immobilized P-ERK- 1/2 antibody overnight (4°C). After centrifugation, the immunoprecipitated proteins were incubated with Elk-1 (an ERK-1/2 substrate) and ATP for 30 minutes (30°C). The resulting P-Elk-1 was analyzed by Western blot analysis with an anti-P-Elk antibody. IP, immunoprecipitation; WB, western blot analysis.
Fig. 4. Effect of kaempferol on the insulin-like growth factor-I receptor (IGF-IR) signaling pathway. HT-29 cells were treated with 0 or 60 μmol/L kaempferol for 2 hours and lysed after 0, 1, or 5 minutes of stimulation with 10 nmol/L insulin-like growth factor-I (IGF-I). (A) Total cell lysates were incubated with anti-IGF-IRβ antibody and the immune complexes were precipitated with protein A-Sepharose. The immunoprecipitated proteins were subjected to Western blot analysis with the relevant antibodies. The immunoprecipitated proteins were incubated with phosphatidylinositol and [γ-32P]ATP to detect phosphatidylinositol 3-kinase (PI3K) activity. The resulting 32P-labelled phosphatidylinositol 3-phosphate (PIP) was separated by thin-layer chromatography and visualized by autoradiography. (B) Total cell lysates were subjected to Western blot analysis with their relevant antibodies. ERK, extracellular regulated kinase; IP, immunoprecipitation; WB, western blot analysis.
Fig. 5. Effect of kaempferol on the ErbB3 signaling pathway. HT-29 cells were treated with 0 or 60 μmol/L kaempferol for 2 hours and lysed after 0, 1, or 5 minutes of stimulation with 20 μg/L heregulin (HRG)-β. (A) Total cell lysates were incubated with anti-ErbB3 antibody and the immune complexes were precipitated with protein A-Sepharose. The immunoprecipitated proteins were subjected to Western blot analysis and in vitro phosphatidylinositol 3-kinase (PI3K) assay as described in . (B) Total cell lysates were subjected to Western blot analysis with their relevant antibodies. ERK, extracellular regulated kinase; IP, immunoprecipitation; PIP, phosphatidylinositol 3-phosphate; WB, western blot analysis.
  1. Jemal A, Bray F, Center MM, Ferlay J, Ward E, Forman D. Global cancer statistics. CA Cancer J Clin 2011;61:69-90.
    Pubmed CrossRef
  2. Sung JJ, Lau JY, Goh KL, Leung WK. Asia Pacific Working Group on Colorectal Cancer. Increasing incidence of colorectal cancer in Asia: implications for screening. Lancet Oncol 2005;6:871-6.
    CrossRef
  3. Surh YJ. Cancer chemoprevention with dietary phytochemicals. Nat Rev Cancer 2003;3:768-80.
    Pubmed CrossRef
  4. Han X, Shen T, Lou H. Dietary polyphenols and their biological significance. Int J Mol Sci 2007;8:950-88.
    CrossRef
  5. Ross JA, Kasum CM. Dietary flavonoids: bioavailability, metabolic effects, and safety. Annu Rev Nutr 2002;22:19-34.
    Pubmed CrossRef
  6. Jin H, Leng Q, Li C. Dietary flavonoid for preventing colorectal neoplasms. Cochrane Database Syst Rev 2012;8:CD009350.
    Pubmed
  7. Wenzel U, Kuntz S, Brendel MD, Daniel H. Dietary flavone is a potent apoptosis inducer in human colon carcinoma cells. Cancer Res 2000;60:3823-31.
    Pubmed
  8. Hoensch HP, Kirch W. Potential role of flavonoids in the prevention of intestinal neoplasia: a review of their mode of action and their clinical perspectives. Int J Gastrointest Cancer 2005;35:187-95.
    CrossRef
  9. Cho HJ, Park JHY. Kaempferol induces cell cycle arrest in HT-29 human colon cancer cells. J Cancer Prev 2013;18:257-63.
  10. Lu X, Jung J, Cho HJ, Lim do Y, Lee HS, Chun HS, et al. Fisetin inhibits the activities of cyclin-dependent kinases leading to cell cycle arrest in HT-29 human colon cancer cells. J Nutr 2005;135:2884-90.
    Pubmed
  11. Lim do Y, Park JH. Induction of p53 contributes to apoptosis of HCT-116 human colon cancer cells induced by the dietary compound fisetin. Am J Physiol Gastrointest Liver Physiol 2009;296:G1060-8.
    Pubmed CrossRef
  12. Cermak R, Wolffram S. The potential of flavonoids to influence drug metabolism and pharmacokinetics by local gastrointestinal mechanisms. Curr Drug Metab 2006;7:729-44.
    CrossRef
  13. Hollman PC, Katan MB. Dietary flavonoids: intake, health effects and bioavailability. Food Chem Toxicol 1999;37:937-42.
    CrossRef
  14. 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;130:2243-50.
    Pubmed
  15. Nguyen TT, Tran E, Ong CK, Lee SK, Do PT, Huynh TT, et al. Kaempferol-induced growth inhibition and apoptosis in A549 lung cancer cells is mediated by activation of MEK-MAPK. J Cell Physiol 2003;197:110-21.
    Pubmed CrossRef
  16. Choi EJ, Ahn WS. Kaempferol induced the apoptosis via cell cycle arrest in human breast cancer MDA-MB-453 cells. Nutr Res Pract 2008;2:322-5.
    Pubmed CrossRef
  17. Li W, Du B, Wang T, Wang S, Zhang J. Kaempferol induces apoptosis in human HCT116 colon cancer cells via the Ataxia-Telangiectasia Mutated-p53 pathway with the involvement of p53 upregulated modulator of apoptosis. Chem Biol Interact 2009;177:121-7.
    Pubmed CrossRef
  18. Kang JW, Kim JH, Song K, Kim SH, Yoon JH, Kim KS. Kaempferol and quercetin, components of Ginkgo biloba extract (EGb 761), induce caspase-3-dependent apoptosis in oral cavity cancer cells. Phytother Res 2010;24(suppl 1):S77-82.
    Pubmed CrossRef
  19. Lee HS, Cho HJ, Yu R, Lee KW, Chun HS, Park JH. Mechanisms underlying apoptosis-inducing effects of Kaempferol in HT-29 human colon cancer cells. Int J Mol Sci 2014;15:2722-37.
    Pubmed CrossRef
  20. Anzano MA, Rieman D, Prichett W, Bowen-Pope DF, Greig R. Growth factor production by human colon carcinoma cell lines. Cancer Res 1989;49:2898-904.
    Pubmed
  21. 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;133:267581.
    Pubmed
  22. Cho HJ, Kim WK, Kim EJ, Jung KC, Park S, Lee HS, et al. Conjugated linoleic acid inhibits cell proliferation and ErbB3 signaling in HT-29 human colon cell line. Am J Physiol Gastrointest Liver Physiol 2003;284:G996-1005.
    Pubmed CrossRef
  23. Frasca F, Pandini G, Sciacca L, Pezzino V, Squatrito S, Belfiore A, et al. The role of insulin receptors and IGF-I receptors in cancer and other diseases. Arch Physiol Biochem 2008;114:23-37.
    Pubmed CrossRef
  24. Jung JI, Cho HJ, Kim J, Kwon DY, Park JH. trans-10,cis-12 conjugated linoleic acid inhibits insulin-like growth factor-I receptor signaling in TSU-Pr1 human bladder cancer cells. J Med Food 2010;13:13-9.
    Pubmed CrossRef
  25. Durai R, Yang W, Gupta S, Seifalian AM, Winslet MC. The role of the insulin-like growth factor system in colorectal cancer: review of current knowledge. Int J Colorectal Dis 2005;20:203-20.
    CrossRef
  26. Samani AA, Fallavollita L, Jaalouk DE, Galipeau J, Brodt P. Inhibition of carcinoma cell growth and metastasis by a vesicular stomatitis virus G-pseudotyped retrovector expressing type I insulinlike growth factor receptor antisense. Hum Gene Ther 2001;12:1969-77.
    Pubmed CrossRef
  27. Hanahan D, Weinberg RA. The hallmarks of cancer. Cell 2000;100:57-70.
    Pubmed CrossRef
  28. Yarden Y, Sliwkowski MX. Untangling the ErbB signalling network. Nat Rev Mol Cell Biol 2001;2:127-37.
    CrossRef
  29. Roskoski R Jr. The ErbB/HER family of protein-tyrosine kinases and cancer. Pharmacol Res 2014;79:34-74.
    Pubmed CrossRef
  30. Bublil EM, Yarden Y. The EGF receptor family: spearheading a merger of signaling and therapeutics. Curr Opin Cell Biol 2007;19:124-34.
    Pubmed CrossRef
  31. Porebska I, Harlozi?ska A, Bojarowski T. Expression of the tyrosine kinase activity growth factor receptors (EGFR, ERB B2, ERB B3) in colorectal adenocarcinomas and adenomas. Tumour Biol 2000;21:105-15.
    Pubmed CrossRef
  32. Venkateswarlu S, Dawson DM, St Clair P, Gupta A, Willson JK, Brattain MG. Autocrine heregulin generates growth factor independence and blocks apoptosis in colon cancer cells. Oncogene 2002;21:78-86.
    Pubmed CrossRef
  33. Jackson JG, St Clair P, Sliwkowski MX, Brattain MG. Blockade of epidermal growth factor- or heregulin-dependent ErbB2 activation with the anti-ErbB2 monoclonal antibody 2C4 has divergent downstream signaling and growth effects. Cancer Res 2004;64:2601-9.
    Pubmed CrossRef
  34. Lim DY, Jeong Y, Tyner AL, Park JH. Induction of cell cycle arrest and apoptosis in HT-29 human colon cancer cells by the dietary compound luteolin. Am J Physiol Gastrointest Liver Physiol 2007;292:G66-75.
    Pubmed CrossRef
  35. Lim do Y, Cho HJ, Kim J, Nho CW, Lee KW, Park JH. Luteolin decreases IGF-II production and downregulates insulin-like growth factor-I receptor signaling in HT-29 human colon cancer cells. BMC Gastroenterol 2012;12:9.
    Pubmed CrossRef
  36. Ichimatsu D, Nomura M, Nakamura S, Moritani S, Yokogawa K, Kobayashi S, et al. Structure-activity relationship of flavonoids for inhibition of epidermal growth factor-induced transformation of JB6 Cl 41 cells. Mol Carcinog 2007;46:436-45.
    Pubmed CrossRef
  37. Williams RJ, Spencer JP, Rice-Evans C. Flavonoids: antioxidants or signalling molecules? Free Radic Biol Med 2004;36:838-49.
    CrossRef
  38. Pollak MN, Schernhammer ES, Hankinson SE. Insulin-like growth factors and neoplasia. Nat Rev Cancer 2004;4:505-18.
  39. Vijapurkar U, Cheng K, Koland JG. Mutation of a Shc binding site tyrosine residue in ErbB3/HER3 blocks heregulin-dependent activation of mitogen-activated protein kinase. J Biol Chem 1998;273:20996-1002.
    Pubmed CrossRef
  40. Priyadarsini RV, Nagini S. Cancer chemoprevention by dietary phytochemicals: promises and pitfalls. Curr Pharm Biotechnol 2012;13:125-36.
    Pubmed CrossRef
  41. Krasilnikov MA. Phosphatidylinositol-3 kinase dependent pathways:the role in control of cell growth, survival, and malignant transformation. Biochemistry (Mosc) 2000;65:59-67.
    Pubmed CrossRef
  42. Lee KM, Lee DE, Seo SK, Hwang MK, Heo YS, Lee KW, et al. Phosphatidylinositol 3-kinase, a novel target molecule for the inhibitory effects of kaempferol on neoplastic cell transformation. Carcinogenesis 2010;31:1338-43.
    Pubmed CrossRef
  43. Brazil DP, Yang ZZ, Hemmings BA. Advances in protein kinase B signalling: AKTion on multiple fronts. Trends Biochem Sci 2004;29:233-42.
    Pubmed CrossRef
  44. Marfe G, Tafani M, Indelicato M, Sinibaldi-Salimei P, Reali V, Pucci B, et al. Kaempferol induces apoptosis in two different cell lines via Akt inactivation, Bax and SIRT3 activation, and mitochondrial dysfunction. J Cell Biochem 2009;106:643-50.
    Pubmed CrossRef
  45. Plotnikov A, Zehorai E, Procaccia S, Seger R. The MAPK cascades:signaling components, nuclear roles and mechanisms of nuclear translocation. Biochim Biophys Acta 2011;1813:1619-33.
    Pubmed CrossRef
  46. Lin CW, Chen PN, Chen MK, Yang WE, Tang CH, Yang SF, et al. Kaempferol reduces matrix metalloproteinase-2 expression by down-regulating ERK1/2 and the activator protein-1 signaling pathways in oral cancer cells. PLoS One 2013;8:e80883.
    Pubmed KoreaMed CrossRef
  47. Cagnol S, Van Obberghen-Schilling E, Chambard JC. Prolonged activation of ERK1,2 induces FADD-independent caspase 8 activation and cell death. Apoptosis 2006;11:337-46.
    Pubmed CrossRef

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