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

Abstract

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 [(3)H]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: ErbB3; HT-29 human colon cancer; Insulin-like growth factor-I receptor; Kaempferol.

Figure 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.

Figure 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.

Figure 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.

Figure 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.

Figure 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 Figure 4. (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.