Luteolin Inhibits Proliferation Induced by IGF-1 Pathway

Breast cancer is one of the most serious problems in oncology and is also the second leading cause of cancerrelated death in women in the Western world, after lung cancer (Jemal et al., 2011), with about 132,000 deaths each year and a 5-year overall survival of approximately 79% (Berrino et al., 2007). The etiology of breast cancer is not understood clearly. There is accumulating evidence implicating IGF-1 system in development and/ or progression of breast cancer (Sachdev et al., 2007). The insulin-like growth factor-1 (IGF-1) system (also called IGF-1 axis), including IGFs (IGF-1and IGF-2), IGF-1R (IGF-1receptor) and IGFBPs (IGF binding proteins), has been shown to play an important role in regulating normal and malignant cell growth (Sachdev et al., 2001). Molecular targeting drugs are hotspot in cancer therapy. Because there is clear risk to treat breast cancer with chemotherapy, researchers worldwide started to search for natural products that have better anticancer activity and reduced side effect. Luteolin is


Introduction
Breast cancer is one of the most serious problems in oncology and is also the second leading cause of cancerrelated death in women in the Western world, after lung cancer (Jemal et al., 2011), with about 132,000 deaths each year and a 5-year overall survival of approximately 79% (Berrino et al., 2007).The etiology of breast cancer is not understood clearly.There is accumulating evidence implicating IGF-1 system in development and/ or progression of breast cancer (Sachdev et al., 2007).The insulin-like growth factor-1 (IGF-1) system (also called IGF-1 axis), including IGFs (IGF-1and IGF-2), IGF-1R (IGF-1receptor) and IGFBPs (IGF binding proteins), has been shown to play an important role in regulating normal and malignant cell growth (Sachdev et al., 2001).
Molecular targeting drugs are hotspot in cancer therapy.Because there is clear risk to treat breast cancer with chemotherapy, researchers worldwide started to search for natural products that have better anticancer activity and reduced side effect.Luteolin is natural herbs, vegetables and fruits.Recent studies have against breast cancer (Ren et al., 2003;Du et al., 2008).

Abstract
The growth of many breast tumors is stimulated by IGF-1, which activates signal transduction pathways in vitro results showed that luteolin could effectively block IGF-1-stimulated MCF-7 cell proliferation in a dosecytometric detection of sub-G1DNA content.Luteolin markedly decreased IGF-1-dependent IGF-1R and Akt inhibitory effect of luteolin on the growth of MCF-7 cells is via inhibiting IGF-1 mediated PI3K-Akt pathway Keywords:

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This effect is mainly exerted through blocking signal transduction in cancer cells (Lin et al., 2008;Dong et al., signaling pathways have been recently shown also to be involved in the metastatic cascade in breast cancer cells (Zhu et al., 2011).During tumorigenesis, IGF-1 mediated mitogen-activated protein kinase (MAPK) pathway and phosphatidylinositol 3-kinase (PI3K) pathway are important (Sachdev et al., 2001).For example, Fang et al. (2007) demonstrated that luteolin inhibited prostate cancer through inhibiting IGF-1 mediated MAPK and PI3K pathways.Xia et al. (2007) demonstrated that curcumin exhibited a potent ability to reverse the IGF-1induced cell growth and apoptosis resistance involving IGF-1 mediated MAPK and PI3K pathway.In addition, it was previously believed that IGF-1 mediated signal transduction pathway is linear, as do most growth factor studies revealed that IGF-1 pathway is also regulated by hormones and intracellular proteins.For example, estrogen positively regulates IGF-1R by promoting IGF-1R and IRS-1 expression, resulting in the downstream activation al. (2001) reported that estrogen stimulates IGF-1R expression, which activates PI3K-Akt pathway, leading to increased mastocarcinoma mitosis.The biological effect of estrogen is primarily mediated through estrogen and auto-regulate the activity of IGF/IGF-1R pathway in breast cancer cells depending on the estrogen receptor status (Sarfstein et al., 2012).Estrogen binds with estrogen of multiple estrogen responsive genes (for example IGF-1R), leading to the change of related protein levels in between the inhibitory effect of luteolin on the growth systematically studied.To elucidate the target of the our current study investigated relationships among IGF-1
2 incubator.During test, cells in log phase were used.plate at 4×10 3 cells/well, cultured for 24 h, then switched to serum-free medium for 24 h.After that, cells were treated respectively with luteolin (0, 10, 20, 40 µmol/L), 5 mg/ml) was added to each well and culture continued reference wavelength at 570 nm.The assay was repeated 3 times (n=3) unless noted otherwise.at 2×10 5 cells/well using complete medium overnight for attachment, then switched to serum-free medium for 24 h.After that, cells were treated respectively with of free medium for 24 h.Adherent cells were released by trypsinization, combined with nonadherent cells, and When cells became attached in 6-well plate, medium was replaced with serum-free medium and the cells were cultured for 24 h.After treatment with luteolin for 5 h, cells were treated with inhibitors (LY-294002 for 1 h.Finally, cells were treated with IGF-1 for 30 min.After luteolin treatment growth medium was removed and cells were washed with ice-cold PBS and lysed in deoxycholate, 10 µg/mL each of aprotinin, leupeptin, supernatant was collected and regarded as whole cell and then transferred onto nitrocellulose membranes (0.45µm, Bio-Rad).Blots were incubated overnight at 4 and scanned with scanner.Transillumination light density was measured with gel image system.
Transfection was conducted using Lipofectamine TM2000 according to the manufacturer's recommendation.seeded in 6-well plate.Transfection was done at 90% control (cells without any treatment); (2) negative control Excel XP were used for t-test.Differences with p<0.05

Results
At 24 h, cells survial rate in serum-free medium complete medium (containing 10% newborn calf serum).When IGF-1 was added, cell survial rate was enhanced in a dose-dependent manner.After stimulation with IGF-1 at observed in complete medium, respectively.When the cell growth between the cultures for 24 h and 48 h.When cells were treated with different concentrations of IGF-1 for 48 h in serum-free medium, cell survial rate 1).We further observed that the increasing rate of cell proliferation was slowed down when high concentrations conditions for the subsequent experiments.

Flow cytometric analysis showed that cell cycle
Western blot analysis showed that the amount of p-Akt in IGF-1 group was 1.15 fold that of serum-free control (p<0.05).The amount of p-Erk1/2 was increased, though 88.93% (p<0.01)without an effect on p-Erk1/2 level (p Akt phosphorylation.When PI3K inhibitor LY-294002 was added, the level of p-Akt was declined by 50.10% when compared with treatment with luteolin and IGF-1 combined (p<0.05).Yet when MEK inhibitor PD-98059 5).This suggests that the inhibitory effect of luteolin on IGF-1 induced breast cancer cell proliferation was mainly mediated by PI3K-Akt signal transduction pathway, not by MAPK/Erk1/2 pathway.dependent manner.When treated for 48 h, compared with IGF-1 treatment, IGF-1 and luteolin treatment decreased cell survival rate by 46.18% (p<0.01),IGF-1 and treatment decreased cell survival rate by 47.64% (p<0.01), the inhibitory effect of luteolin on IGF-1 induced breast cell growth inhibitory effect of luteolin, we tested the knockdown was verified with Western blot analysis (Figure 8A).Before transfection, treatment with luteolin decreased p-Akt level by 73.07%(p<0.01),compared with IGF-1 treatment.After transfection, treatment with cells were more resistant to luteolin inhibition.These suggest that that the inhibitory effect of luteolin on the expression.

Discussion
targets with central role in breast carcinogenesis represent a rational approach forprevention and treatment (Sogno et Data published during the last decade has implicated insulin-like growth factor (IGF) and its signaling cascade in the development and progression of breast cancer (Pollak 2008).Studies recently demonstrated that overexpression of IGF-1 in cancer cells is associated with tumor growth (Werner et al., 2000;Bustin et al., 2002).The current experiment showed that when cells enhanced, compared with serum-free control group.our results indicated that IGF-1 was actually one of the results are consistent with previous reports (Kappel et al., 1994;Resnicoff et al., 1995).
Recently, researchers worldwide started to search for many natural herbs.Recent studies revealed the inhibitory effect of most flavonoids is mainly exerted through blocking IGF-1system in cancer cells.Luteolin, the 3', 4', and has been found to have a wide spectrum of anti-tumor activities.As expected, we documented that luteolin did proliferation and inhibitory effect on apoptosis.These inhibitory effects were associated with a S cell cycle blockage and a sub-G1 apoptotic peak induction.This data suggests that luteolin inhibited IGF-1-induced cell cycle progression and resistance of breast cancer cells studies that showed luteolin exerted its anticancer effects via proliferation inhibition and apoptosis induction in prostate cancer cells.IGF-1 mediated signal transduction pathway is critical in oncogenesis and tumor development (Surmacz primarily mediated through MAPK/Erk1/2 and PI3K-Akt pathways (Dong et al., 2007).The binding of IGF-1 with the extracellular domain of IGF-1R causes phosphorylation of IGF-1R and downstream substrate, ultimately phosphorylate MAPK and Akt.Thus IGF-1 signal is transmitted to nucleus, initiating gene expression to promote cell proliferation and to inhibit apoptosis.IGF-IR was required for oncogenic transformation and has an established role in breast cancer tumorigenesis (Buck et al., 2010).Because of its critical importance, the IGF-IR pathway has been intensively studied as a cancer therapeutic target and multiple agents are in clinical development (Yuen et al., 2008;Baserga 2009;Gualberto et al., 2009;Buck et al., 2011).The current experiment demonstrated that luteolin markedly decreased IGF-1R and Akt phosphorylation without affecting Erk1/2 phosphorylation.The group treated with PI3K inhibitor LY-294002 had lower p-Akt level, compared with the group treated with luteolin.Yet, the group treated with in p-Akt level compared with the groups treated with luteolin.This suggests that the inhibitory effect of luteolin on IGF-1 induced breast cancer cell proliferation was mainly mediated by PI3K-Akt signal transduction pathway, not by MAPK/Erk1/2 pathway.
It was previously believed that IGF-1 mediated signal transduction pathway is linear, as do most growth factor studies revealed that IGF-1 pathway is also regulated by hormones and intracellular proteins.IGF-IR expression level has been associated with ER positivity in both breast  (Surmacz et al., 2004;Weroha et al., 2008; could associates with IGF-1R leading to activation of downstream PI3K-Akt and MAPK signaling cascades.sequence, it was found to co-immunoprecipitate with IGF-1R in the membrane.Klotz et al. (2002) demonstrated that showed that a nanomolar level IGF-1 could not stimulate proliferation effect was recovered.In our experiment, When cells were treated for 48 h, cell survival rates were directly involved in luteolin inhibiting IGF-1 induced we determined inhibition effects of luteolin on IGF-1 treatment.The current experiment showed that blocking p-Akt expression.Before transfection, compared with IGF-1 control, treatment with luteolin markedly reduced p-Akt level.After transfection, luteolin treatment had a p-Akt level similar to the control group.In other words, the inhibitory effect of luteolin on IGF-1 disappeared cells via inhibiting IGF-1 mediated PI3K-Akt pathway the targets of luteolin.
Although IGF-IR signaling activation has been detected in both ER+and ER-breast cancers with associated poor clinical outcome (Law et al., 2008), multiple studies have demonstrated a functional relationship and extensive cross-talk between IGF-IR and powerful predictor of breast cancer prognosis as well as an important contributor to the biology of carcinogenesis.and in multiple fashions, ultimately resulting in increased receptor superfamily and is one of the transcription factors.
Taken together, data presented here demonstrate that luteolin exhibited a potent ability to blunt IGFis possibly one of the targets of luteolin.Therefore, we suggest luteolin as a novel drug that may be effective in the treatment of breast cancer.

Figure 1 .
Figure 1.The Effect of IGF-1 on MCF-7 Cell Proliferation in Vitro.determined using the MTT assay.Each graph represents data

Figure 4 .
Figure 4. Effects of Luteolin on IGF-1 Induced IGF-1R Phosphorylation.IGF-1 as described in Materials and methods.The amount of proteins was assessed by immunoblotting.The intensity of the different bands were determined by densitometry and plotted