Aflatoxin B1 Up-Regulates Insulin Receptor Substrate 2 and Stimulates Hepatoma Cell Migration

<div><p>Aflatoxin B1 (AFB1) is a potent carcinogen that can induce hepatocellular carcinoma. AFB1-8,9-exo-epoxide, one of AFB1 metabolites, acts as a mutagen to react with DNA and induce gene mutations, including the tumor suppressor <em>p53</em>. In addition, AFB1 reportedly stimulates IGF receptor activation. Aberrant activation of IGF-I receptor (IGF-IR) signaling is tightly associated with various types of human tumors. In the current study, we investigated the effects of AFB1 on key elements in IGF-IR signaling pathway, and the effects of AFB1 on hepatoma cell migration. The results demonstrated that AFB1 induced IGF-IR, Akt, and Erk1/2 phosphorylation in hepatoma cell lines HepG2 and SMMC-7721, and an immortalized human liver cell line Chang liver. AFB1 also down-regulated insulin receptor substrate (IRS) 1 but paradoxically up-regulated IRS2 through preventing proteasomal degradation. Treatment of hepatoma cells and Chang liver cells with IGF-IR inhibitor abrogated AFB1-induced Akt and Erk1/2 phosphorylation. In addition, IRS2 knockdown suppressed AFB1-induced Akt and Erk1/2 phosphorylation. Finally, AFB1 stimulated hepatoma cell migration. IGF-IR inhibitor or IRS2 knockdown suppressed AFB1-induced hepatoma cell migration. These data demonstrate that AFB1 stimulates hepatoma cell migration through IGF-IR/IRS2 axis.</p> </div

AFB1 stimulates hepatoma cell migration through IGF-IR/IRS2 axis.

<p>(<b>A</b>) SMMC-7721 cells were seeded into 6-well plates. Upon confluency, scratches were made in cell cultures. To inhibit cell proliferation, the cells were treated with 2 µg/ml mitomycin C. Also, the cells were treated with or without 2.5 µM AFB1 and 10 µM IGF-IR inhibitor AG1024 for 4 days. <i>Bar</i>, 1000 µm. (<b>B</b>) SMMC-7721 cells were transfected with siCtrl or siIRS2. Twenty-four hours later scratches were made in cell cultures. The cells were treated with 2 µg/ml mitomycin C, and treated with or without 2.5 µM AFB1 for 4 days. <i>Bar</i>, 1000 µm. Cell lysates from siCtrl- or siIRS2-transfected cells were harvested and subjected to Western blot analysis of IRS2 expression.</p

AFB1 induces IGF-IR phosphorylation, down-regulates IRS1 but up-regulates IRS2.

<p>(<b>A</b>) HepG2 cells were treated with 2.5 µM AFB1 for 1, 3, or 5 days, or treated with 1, 2.5, and 5 µM AFB1 for 3 days, followed by western blot analysis of IGF-IR and phosphorylated IGF-IR, IRS1, and IRS2. (<b>B</b>) SMMC-7721 cells were treated with 2.5 µM AFB1 for 1, 3, or 5 days, or treated with 1, 2.5, and 5 µM AFB1 for 3 days, followed by western blot analysis of IGF-IR and phosphorylated IGF-IR, IRS1, and IRS2. (<b>C</b>) Chang liver cells were treated with 2.5 µM AFB1 for 3 days, followed by western blot analysis of IGF-IR and phosphorylated IGF-IR, IRS1, and IRS2. All blots were subjected to densitometric analysis. The relative levels of IGF-IR, phosphorylated IGF-IR, IRS1, and IRS2 after normalization to actin were plotted. The relative levels of target proteins in un-treated group were set as 1. A statistical analysis of densitometric quantification of immunoblots from individual experiments was shown. *, <i>p</i><0.05.</p

AFB1 induces Akt and Erk1/2 phosphorylation.

<p>(<b>A</b>) HepG2 cells were treated with 2.5 µM AFB1 for 1, 3, or 5 days, or treated with 1, 2.5, and 5 µM AFB1 for 3 days, followed by western blot analysis of Akt and phosphorylated Akt, Erk1/2 and phosphorylated Erk1/2. (<b>B</b>) SMMC-7721 cells were treated with 2.5 µM AFB1 for 1, 3, or 5 days, or treated with 1, 2.5, and 5 µM AFB1 for 3 days, followed by western blot analysis of Akt and phosphorylated Akt, Erk1/2 and phosphorylated Erk1/2. (<b>C</b>) Chang liver cells were treated with 2.5 µM AFB1 for 3 days, followed by western blot analysis of Akt and phosphorylated Akt, Erk1/2 and phosphorylated Erk1/2. Immunoblots were subjected to densitometric analysis. The relative levels of Akt and phosphorylated Akt, Erk1/2 and phosphorylated Erk1/2 after normalization to actin were plotted. The relative levels of target proteins in cells treated without AFB1 were set as 1. A statistical analysis of densitometric quantification of immunoblots from individual experiments was shown. *, <i>p</i><0.05.</p

Inhibition of IGF-IR and IRS2 suppresses AFB1-induced Akt and Erk1/2 phosphorylation.

<p>(<b>A</b>) HepG2, SMMC-7721, and Chang liver cells were treated with or without 2.5 µM AFB1 and 10 µM IGF-IR inhibitor AG1024 for 3 days, followed by western blot analysis of Akt and phosphorylated Akt, Erk1/2 and phosphorylated Erk1/2, IGF-IR and phosphorylated IGF-IR. (<b>B</b>) HepG2, SMMC-7721, and Chang liver cells were transfected with control siRNA (siCtrl) or IGF-IR siRNA (siIGFIR). Twenty-four hours later, the cells were treated with or without 2.5 µM AFB1 for 3 days. Cell lysates were subjected to western blot analysis of Akt and phosphorylated Akt, Erk1/2 and phosphorylated Erk1/2, IGF-IR and phosphorylated IGF-IR. (<b>C</b>) HepG2, SMMC-7721, and Chang liver cells were transfected with control siRNA (siCtrl) or IRS2 siRNA (siIRS2). Twenty-four hours later, the cells were treated with or without 2.5 µM AFB1 for 3 days. Cell lysates were subjected to western blot analysis of IRS2, Akt and phosphorylated Akt, Erk1/2 and phosphorylated Erk1/2.</p

AFB1 stimulates hepatoma cell growth and down-regulates IRS1 in a dose-dependent manner.

<p>(<b>A</b>) HepG2 and SMMC-7721 cells were plated into 96-well plates, and treated with or without AFB1 at indicated dose for 5 days. Cell growth was detected by CCK-8 reagent. The relative cell growth was plotted. <i>Bars</i>, SE. *, <i>p</i><0.05, compared with vehicle-treated cells. (<b>B</b>) HepG2 and SMMC-7721 cells were treated with or without AFB1 at indicated dose for 5 days. Cell lysates were subjected to western blot analysis of IRS1, IRS2 and phosphorylated IGF-IR.</p