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Journal of Cancer Prevention

Original Article

JCP 2014; 19(3): 161-169

Published online October 1, 2014

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.
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