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.