Aflatoxin B1 up-regulates insulin receptor substrate 2 and stimulates hepatoma cell migration

Abstract

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 p53. 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.

Figure 1. AFB1 induces IGF-IR phosphorylation, down-regulates IRS1 but up-regulates IRS2.

(A) 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) 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. (C) 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. *, p<0.05.

Figure 2. AFB1 does not affect IRS1 and IRS2 transcription but regulates IRS1 and IRS2 degradation.

(A) SMMC-7721 cells were treated with or without AFB1 at indicated dose for 3 days, followed by RT-PCR analysis of IRS1 and IRS2 transcription. GAPDH transcription was also detected as internal control. (B) SMMC-7721 cells were treated with or without AFB1 at indicated dose for 3 days, followed by real-time RT-PCR analysis of IRS1 and IRS2 transcription. The relative IRS1 or IRS2 levels were plotted. (C) HepG2 and SMMC-7721 cells were treated with 25 µg/ml CHX to inhibit new protein synthesis for the times indicated. In parallel, the cells were treated with combination of AFB1 and CHX. Total proteins were harvested and subjected to western blotting for IRS1, IRS2 and β-actin to control for loading. The blots were subjected to densitometric analysis. The relative levels of IRS1 and IRS2 after normalization to actin were plotted. The relative levels of IRS1 and IRS2 in cells treated without CHX were set as 1. The statistical analysis of densitometric quantification of immunoblots from individual experiments were shown. *, p<0.05. (D) HepG2 and SMMC-7721 cells were treated with or without 2.5 µM AFB1 and 2 µM proteasome inhibitor MG132 for 3 days, followed by western blot analysis of IRS1 and IRS2 levels. All blots were subjected to densitometric analysis. The relative levels of IRS1 and IRS2 after normalization to actin were plotted. The relative levels of IRS1 and IRS2 in cells treated without AFB1 and MG132 were set as 1. A statistical analysis of densitometric quantification of immunoblots from individual experiments was shown. *, p<0.05.

Figure 3. AFB1 induces Akt and Erk1/2 phosphorylation.

(A) 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) 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. (C) 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. *, p<0.05.

Figure 4. Inhibition of IGF-IR and IRS2 suppresses AFB1-induced Akt and Erk1/2 phosphorylation.

(A) 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) 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. (C) 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.

Figure 5. AFB1 stimulates hepatoma cell growth and down-regulates IRS1 in a dose-dependent manner.

(A) 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. Bars, SE. *, p<0.05, compared with vehicle-treated cells. (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.

Figure 6. AFB1 stimulates hepatoma cell migration through IGF-IR/IRS2 axis.

(A) 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. Bar, 1000 µm. (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. Bar, 1000 µm. Cell lysates from siCtrl- or siIRS2-transfected cells were harvested and subjected to Western blot analysis of IRS2 expression.