Luteolin decreases IGF-II production and downregulates insulin-like growth factor-I receptor signaling in HT-29 human colon cancer cells
© Lim et al; licensee BioMed Central Ltd. 2012
Received: 24 June 2011
Accepted: 23 January 2012
Published: 23 January 2012
Luteolin is a 3',4',5,7-tetrahydroxyflavone found in various fruits and vegetables. We have shown previously that luteolin reduces HT-29 cell growth by inducing apoptosis and cell cycle arrest. The objective of this study was to examine whether luteolin downregulates the insulin-like growth factor-I receptor (IGF-IR) signaling pathway in HT-29 cells.
In order to assess the effects of luteolin and/or IGF-I on the IGF-IR signaling pathway, cells were cultured with or without 60 μmol/L luteolin and/or 10 nmol/L IGF-I. Cell proliferation, DNA synthesis, and IGF-IR mRNA levels were evaluated by a cell viability assay, [3H]thymidine incorporation assays, and real-time polymerase chain reaction, respectively. Western blot analyses, immunoprecipitation, and in vitro kinase assays were conducted to evaluate the secretion of IGF-II, the protein expression and activation of IGF-IR, and the association of the p85 subunit of phophatidylinositol-3 kinase (PI3K) with IGF-IR, the phosphorylation of Akt and extracellular signal-regulated kinase (ERK)1/2, and cell division cycle 25c (CDC25c), and PI3K activity.
Luteolin (0 - 60 μmol/L) dose-dependently reduced the IGF-II secretion of HT-29 cells. IGF-I stimulated HT-29 cell growth but did not abrogate luteolin-induced growth inhibition. Luteolin reduced the levels of the IGF-IR precursor protein and IGF-IR transcripts. Luteolin reduced the IGF-I-induced tyrosine phosphorylation of IGF-IR and the association of p85 with IGF-IR. Additionally, luteolin inhibited the activity of PI3K activity as well as the phosphorylation of Akt, ERK1/2, and CDC25c in the presence and absence of IGF-I stimulation.
The present results demonstrate that luteolin downregulates the activation of the PI3K/Akt and ERK1/2 pathways via a reduction in IGF-IR signaling in HT-29 cells; this may be one of the mechanisms responsible for the observed luteolin-induced apoptosis and cell cycle arrest.
Colon cancer is the second most frequent cause of cancer-related death in the Western world . Dietary patterns and lifestyle are the principal determining factors for colorectal cancer risk. The results of epidemiological studies have shown that the consumption of fruits and vegetables can reduce or prevent the risk of colon cancer . Flavonoids are polyphenols, which are abundantly present in fruits and vegetables, and have been shown to have a variety of biological effects, including cancer prevention.
Insulin-like growth factors (IGFs) are polypeptides that stimulate the growth of a variety of mammalian cells . These effects are mediated through the insulin-like growth factor I receptor (IGF-IR), and IGF-I and IGF-II are well-known ligands of IGF-IR. The binding of these ligands to IGF-IR results in the autophosphorylation of the receptor at the intracellular domain of β-subunits, resulting in the activation of the intrinsic tyrosine kinase of the IGF-IR. Subsequently, several adaptor molecules are recruited and activated via phosphorylation. Two distinct signaling pathways are activated by IGF-IR. The recruitment and activation of growth factor receptor-bound protein-2/son of sevenless or Shc can lead to the recruitment and activation of the Ras/Raf/mitogen activated protein kinase (MAPK) cascade, ultimately resulting in the activation of extracellular signal-regulated kinase (ERK)1/2. Alternatively, insulin receptor substrate-1 can be recruited and phosphorylated on multiple tyrosine residues that function as docking sites for the p85 subunit of phosphatidylinositol 3-kinase (PI3K) and activate the PI3K/Akt signaling pathway (reviewed in [4, 5]). The activation of these pathways induces cell cycle progression and prevents apoptosis [6, 7]. IGFs are also strong mitogens and survival factors for a variety of cancer cells, including prostate and colon cancer cells (Reviewed in ), and IGF-I and IGF-II mRNA levels were reported to be highly elevated in colon cancer . We have previously reported that, in human colon cancer cells, including HT-29 cells and Caco-2 cells, IGF-II is synthesized and secreted, and an IGF-II autocrine loop stimulates the growth of these cancer cells [10, 11].
Luteolin, 3',4',5,7-tetrahydroxyflavone, is found in a variety of vegetables, fruits, and medicinal herbs. Luteolin has been shown to function as an anti-oxidant, anti-inflammatory, and anti-cancer agent [12–15]. Additionally, luteolin induces cell cycle arrest and apoptosis in the liver and lung cancer and leukemia cell lines [16–20]. Our previous results indicated that luteolin inhibited HT-29 cell proliferation by inducing cell cycle arrest and apoptosis . Therefore, in this study, we attempted to determine whether luteolin downregulates IGF-IR signaling in HT-29 cells.
Human colon cancer cells (HT-29 and Caco-2 cells) and rat intestinal epithelial cell line-6 (IEC-6 cells) were purchased from the American Type Culture Collection (Manassas, VA) and maintained in DMEM/F12 containing 100 mL/L of fetal bovine serum (FBS), with 100,000 U/L of penicillin and 100 mg/L of streptomycin. In order to determine the effects of luteolin and/or IGF-I on cell growth, we plated the cells with DMEM/F12 containing 10% FBS. Prior to luteolin treatment, the cell monolayers were subjected to 24 h of serum starvation with DMEM/F12 supplemented with 5 mg/L transferrin, 1 g/L BSA, and 5 μg/L selenium (serum-free medium). The cells were then incubated in serum-free medium with or without 60 μmol/L of luteolin (Sigma, St. Louis, MO, USA) and/or 10 nmol/L IGF-I (R & D System, Minneapolis, MN, USA) for 24, 48 or 72 h. Viable cell numbers were estimated via MTT assays. Luteolin was dissolved in DMSO and all cells were treated with DMSO at a final concentration of 0.1%.
[3H]Thymidine incorporation assay
To determine DNA synthesis, the cells were plated at a density of 6,000 cells per well in 96-well plates and serum-starved as described above. After serum starvation, the cells were incubated for 2 h in serum-free medium containing 0 or 60 μmol/L of luteolin with or without IGF-I. 0.5 μCi [3H]thymidine was then added, and the incubation was continued for another 1 h. The incorporation of [3H]thymidine into DNA was estimated as previously described .
For the determination of IGF-II, HT-29 cells were plated in 100 mm dishes at a concentration of 2 × 106 cells/dish and after 24 h, the monolayers were serum-starved and treated with various concentrations of luteolin (0 - 60 μmol/L) for 24 h. Conditioned media were collected and concentrated 20-fold, and immunoblot analysis was conducted using anti-IGF-II clone S1F2 (Upstate Biotechnology, Inc., Lake Placid, NY, USA) as previously described .
Immunoprecipitation and immunoblot analyses
Cells were incubated for 2 h with 0 or 60 μmol/L of luteolin, and 10 nmol/L of IGF-I was added. At 0, 1, or 30 min after the addition of IGF-I, the cell lysates were prepared and immunoprecipitated with indicated antibodies. Immunoblot analyses were conducted as described previously . Signals were detected via the enhanced chemiluminescence method using SuperSignal West Dura Extended Duration Substrate (Pierce, Rockford, IL, USA). The relative abundance of each protein band was analyzed via densitometric scanning of the exposed films. Immunoblots were probed with an antibody for β-actin as a protein loading control. The following antibodies were purchased from the indicated suppliers: anti-IGF-IRβ (C-20) (Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA); anti-phospho-tyrosine-RC20 antibody (PY20) linked to horseradish peroxidase (Transduction Laboratories, Palo Alto, CA, USA); anti-PI3K p85 antibody (Upstate Biotechnology, Inc.); anti-phospho-IGF-IR (P-IGF-IR, Abcam, Cambridge, MA, USA); and anti-ERK-1/2, anti-P-ERK-1/2 (Thr202/Tyr203), anti-cell division cycle 25c (CDC25c), anti-P-CDC25c, anti-Akt, and anti-P-Akt Ser473 (Cell Signaling Technology, Inc., Beverly, MA, USA).
Real-time-polymerase chain reaction (RT-PCR)
Total RNA was isolated using RNeasy Plus Mini Kit (Qiagen, Valencia, CA, USA) and cDNA was synthesized using 3 μg of total RNA with SuperScript II reverse transcriptase (Invitrogen, Carlsbad, CA, USA). Real time-PCR was conducted as described previously . Sequences used for primer sets were as follows: IGF-IR; forward-TGG AGT GCT GTA TGC CTC TG, backward-TGA TGA CCA GTG TTG GCT GG, β-actin; forward-GTT TGA GAC CTT CAA CAC CCC, backward-GTG GCC ATC TCC TGC TCG AAG TC. The levels of mRNA were normalized to β-actin and the control (0 μmol/L luteolin) levels were set to 100%.
PI3K activity was estimated as described previously . Cell lysates (1 mg protein) were immunoprecipitated with a polyclonal antibody against IGF-IRβ followed by incubation with protein A-Sepharose beads. After washing, the beads were resuspended in 20 μL of kinase buffer containing 4 μg of phosphatidylinositol (Sigma, St. Louis, MO, USA), 10 μmol/L of ATP, 5 mmol/L of MnCl2, and 10 μCi of [γ-32P]ATP and incubated for 20 min at 30°C. In order to determine whether luteolin directly inhibits the kinase activity of PI3K, active PI3Kα (100 ng, Millipore, Billerica, MA, USA) was incubated for 10 min in the absence or presence of 20 μmol/L of luteolin at 30°C in 20 μL of kinase buffer. Phosphatidylinositol (25 μg) was added and the incubation was continued for another 5 min at room temperature. 10 μCi of [γ-32P]ATP was then added and reactions were incubated for 10 min at 30°C. The resultant 32P-labeled phosphatidylinositol 3-phosphate (PIP) lipids were separated from reaction products by thin layer chromatography (TLC) and visualized by autoradiography. The radioactive PIP signals were quantitated via densitometry using the Bio-profile Bio-1D application (Vilber-Lourmat, France) .
Data were expressed as means ± SEM values and analyzed via analysis of variance. Differences between treatment groups were analyzed by Duncan's multiple range test or Student's t-test. The means were considered significantly different at P < 0.05. All statistical analyses were conducted using the SAS system for Windows, version 8.12 (SAS, Inc., Cary, NC, USA).
Luteolin reduces IGF-II secretion in HT-29 cells
Luteolin abrogates the growth stimulatory effects of exogenous IGF-I on HT-29 cells
Luteolin reduces the levels of the IGF-IR precursor protein and IGF-IR transcripts in HT-29 cells
Luteolin inhibits IGF-I-induced activation of IGF-IR, Akt, and ERK1/2 in HT-29 cells
In order to determine whether luteolin down-regulates IGF-I-induced tyrosine phosphorylation of the IGF-IR, cells were treated for 2 h with 0 or 60 μmol/L of luteolin, and IGF-IR was stimulated with 10 nmol/L IGF-I for 0, 1, or 30 minutes. Total cell lysates were prepared and immunoprecipitated using an IGF-IRβ antibody. The immune complexes were used for Western blot analysis with an anti-P-tyrosine antibody (PY20). IGF-I induced tyrosine phosphorylation of IGF-IR at 1 min; tyrosine phosphorylation levels were slightly reduced at 30 min. Luteolin significantly inhibited the phosphorylation of IGF-IRβ at 1 min after IGF-I treatment. At 30 min, the phosphorylation status of IGF-IR did not differ between the control and luteolin-treated cells.
The IGF system (IGF-I, IGF-II, IGF-binding protein, and IGF-IR) performs an important role in the growth of various cancer cells, including colon cancer cells [8, 27]. We have reported previously that luteolin inhibited the proliferation of HT-29 human colon cancer cells by inducing cell cycle arrest and apoptosis . The results of a previous study revealed that luteolin reduced the expression of cyclin D1 and cyclin B1 and inhibited the activities of CDKs, thereby suppressing HT-29 cell cycle progression. Additionally, luteolin induced the activation of caspases and reduced the levels of proteins involved in the suppression of apoptosis, including Bcl-xL and Mdm-2 . Thus, in the present study, we explored the upstream signals that are important for the regulation of cell cycle progression and apoptosis in HT-29 cells. Our previous data demonstrated that HT-29 cells synthesized and secreted IGF-II and expressed IGF-IR, and that IGF-II stimulated HT-29 cell growth via an autocrine mechanism [10, 28]. Kim et al. also reported that the reduction of IGF-II secretion in Caco-2 colon cancer cells inhibited cell growth . Using PC-3 and DU145 human prostate cancer cells, Fang et al.  have demonstrated that luteolin inhibits the IGF-I-induced activation of IGF-IR and AKT as well as the downstream targets of AKT, p70S6K1, GSK-3β, and FKHR/FKHRL1. In the present study, we demonstrate that, in HT-29 human colon carcinoma cells, luteolin 1) reduces IGF-II secretion; 2) inhibits the growth-stimulatory effects of IGF-I; 3) reduces the levels of IGF-IR transcripts and the IGF-IR precursor protein; 4) reduces the IGF-I-induced tyrosine phosphorylation of IGF-IRβ and the association of p85 with IGF-IRβ; 5) inhibits IGF-I-induced PI3K activity 6) inhibits IGF-I-induced Akt activation; and 7) inhibits the IGF-I-induced phosphorylation of ERK1/2 and CDC25c. These results indicate that the reduction in IGF-II secretion and changes in IGF-IR signaling by luteolin may be important factors underlying the growth-inhibitory effects of HT-29 cells. Additionally, we have demonstrated that luteolin directly inhibits the activity of PI3K in a cell-free system.
When HT-29 cells were treated with exogenous IGF-I, IGF-I did not abrogate the growth-inhibitory effects of luteolin (Figure 2), although luteolin reduced IGF-II secretion (Figure 1). These results indicated that luteolin inhibits IGF-IR signaling in HT-29 cells. IGF-IR consists of two extracellular α-subunits and two transmembrane β-subunits, and IGF-I and IGF-II bind to the α-subunits of IGF-IR, thus resulting in the activation of the intrinsic tyrosine kinase in the intracellular domain of the β-subunits . In this study, luteolin reduced the levels of the IGF-IR precursor but did not reduce the levels of IGF-IR β-subunits; this suggests that the levels of IGF-IR α-subunits may have been reduced by luteolin treatment. The finding that IGF-IR mRNA levels were continuously decreased during 24 h of luteolin treatment (Figure 3C) indicates that the expression of IGF-IR protein is regulated by luteolin, at least in part, at an RNA level. The effects of luteolin on IGF-IR mRNA and protein stability will require further study in the future.
Fang et al. demonstrated that prostate cancer cells in which the IGF-IR gene is knocked down grew at a slower rate relative to that in control cells, and the inhibition of cell growth by luteolin treatment was similar to that observed in IGF-IR-depleted cells . In this study, we demonstrate that luteolin inhibits IGF-II secretion, and that IGF-I-stimulated HT-29 cell proliferation was inhibited by luteolin (Figure 2). These results suggest that the inhibition of the IGF/IGF-IR signaling pathway by luteolin might be one of the mechanisms for the suppression of proliferation and apoptosis in HT-29 cells. In 1994, Lahm et al. demonstrated that Alpha IR3, a neutralizing monoclonal antibody directed against human IGF-IR, inhibited proliferation in HT-29 cells . It has also been demonstrated that the blockade of IGF-IR with IGF-IR monoclonal antibodies inhibited proliferation, arresting the cell cycle and inducing the apoptosis of HT-29 cells . Additionally, an anti-human/mouse IGF-II-neutralizing antibody effectively inhibited the hepatic metastasis of HT-29 cells . In vitro experiments have also demonstrated that IGF-II-neutralizing antibody treatment completely blocked IGF-IR phosphorylation in serum-starved HT-29 cells . These results indicate that IGF-II is an autocrine growth factor of HT-29 cells and that the inhibition of IGF-II secretion and/or IGF-IR signaling inhibits HT-29 cell proliferation.
In our HT-29 cells, it is possible that the luteolin-induced downregulation of the IGF-IR α-subunit results in reduced phosphorylation of the β-subunit. It is also possible that luteolin directly interferes with the binding of IGF-I to IGF-IR, which would consequently inhibit the phosphorylation of the β-subunit. This reduced IGF-I-induced tyrosine phosphorylation of IGF-IRβ by luteolin led to the reduced association of p85 with IGF-IRβ and the subsequent activation of PI3K/Akt and ERK1/2 (Figures 4, 5 and 6). Additionally, luteolin inhibited PI3K activity in a cell-free system (Figure 4D), thereby indicating that luteolin can also modulate the activity of this enzyme via direct interaction with this kinase. As the activation of Akt and ERK1/2 induces cell proliferation and inhibits apoptosis in various cancers [34, 35], the PI3K/Akt and ERK1/2 pathways may be important targets in cancer therapies involving natural bioactive compounds [6, 23, 28, 29, 36–38]. Akt regulates the expression and activity of proteins involved in the regulation of apoptosis and cell cycle progression, including Bad, p21, cyclin D1, and Mdm-2 (Reviewed in ). Previously, we have demonstrated that luteolin downregulates the expression of Mdm-2 and cyclin D1 . Fang et al. also reported that luteolin treatment induced a reduction in the levels of P-IGF-IR, P-Akt, and cyclin D1 in PC3 prostate cancer cells . The results of previous studies and of the present study indicate that the inhibition of Akt activation by luteolin may result in the downregulation of Mdm-2 and cyclin D1, which may contribute to the induction of apoptosis and cell cycle arrest in colon and prostate cancer cells. Collectively, these results indicate that the downregulation of IGF-IR/PI3K/Akt by luteolin is one of the principal signaling pathways for the induction of cell cycle arrest and apoptosis in HT-29 cells.
ERK-MAP kinases also regulate cell cycle- and apoptosis-related proteins. ERK1/2 activation leads to the phosphorylation of the protein phosphatase CDC25c during the G2/M transition of cell cycle progression . Phosphorylated CDC25c dephosphorylates CDC2, which results in the activation of the CDC2/cyclin B1 complex. Luteolin has been reported to reduce the levels of the CDC25c, CDC2, and cyclin B1 proteins and induces G2/M phase arrest in human gastric cancer cell lines . In our previous study, luteolin reduced cyclin B1 levels, markedly inhibited CDC2 activity, and promoted G2/M phase arrest in HT-29 cells . In the present study, we determined that luteolin reduced the levels of P-CDC25c in HT-29 cells (Figure 6C). Together, these results indicate that the attenuated ERK1/2 activation contributed to the reduction of P-CDC25c levels in luteolin-treated cells. The reduction in CDC25c activation may have contributed to the induction of G2/M arrest in HT-29 cells.
insulin-like growth factor-1 receptor
insulin-like growth factor-II
extracellular signal-regulated kinase-1/2
thin layer chromatography
cell division cycle.
This study was supported by the Regional Core Research Program/Medical & Bio-Material Research Center, the Mid-career Researcher Program (2010-0006923), and the SRC program (Center for Food & Nutritional Genomics: grant number 2010-0001886) of the National Research Foundation (NRF) of Korea, funded by the Ministry of Education, Science, and Technology.
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