gamma-Tocotrienol inhibits ErbB3-dependent PI3K/Akt mitogenic signalling in neoplastic mammary epithelial cells

Abstract

The antiproliferative effects of gamma-tocotrienol are associated with suppression in epidermal growth factor (EGF)-dependent phosphatidylinositol-3-kinase (PI3K)/PI3K-dependent kinase-1 (PDK-1)/Akt mitogenic signalling in neoplastic mammary epithelial cells. Studies were conducted to investigate the direct effects of gamma-tocotrienol treatment on specific components within the PI3K/PDK-1/Akt mitogenic pathway. +SA cells were grown in culture and maintained in serum-free media containing 10 ng/ml EGF as a mitogen. Treatment with 0-8 microm gamma-tocotrienol resulted in a dose-responsive decrease in the +SA cell growth and a corresponding decrease in phospho-Akt (active) levels. However, gamma-tocotrienol treatment had no direct inhibitory effect on Akt or PI3K enzymatic activity, suggesting that the inhibitory effects of gamma-tocotrienol occur upstream of PI3K, possibly at the level of the EGF-receptor (ErbB1). Additional studies were conducted to determine the effects of gamma-tocotrienol on ErbB receptor activation. Results showed that gamma-tocotrienol treatment had little or no effect on ErbB1 or ErbB2 receptor tyrosine phosphorylation, a prerequisite for substrate interaction and signal transduction, but did cause a significant and progressive decrease in the ErbB3 tyrosine phosphorylation. Because ErbB1 or ErbB2 receptors form heterodimers with the ErbB3 receptor, and ErbB3 heterodimers have been shown to be the most potent activators of PI3K, these findings strongly suggest that the antiproliferative effects of gamma-tocotrienol in neoplastic +SA mouse mammary epithelial cells are mediated by a suppression in ErbB3-receptor tyrosine phosphorylation and subsequent reduction in PI3K/PDK-1/Akt mitogenic signalling.

Figure 1

Effects of 0–8 µmγ‐tocotrienol (top) and 0–400 µmα‐tocopherol (bottom) on +SA malignant mammary epithelial cell growth over a 72 h treatment period. Cells in all treatment groups were initially plated at a density of 5 × 10⁴ cells/well in 24‐well culture plates. Vertical bars indicate the mean viable cell number/well ± SEM for six replicates in each treatment group. *P < 0.05, as compared to untreated controls.

Figure 2

Western blot and scanning densitometric analysis of PI3K p85 (regulatory subunit), PI3K p110α (catalytic subunit), phospho‐PDK1 (active form), phospho‐Akt (active form) and total Akt levels in malignant +SA mammary epithelial cells following a 0–72 h treatment exposure to 7 µmγ‐tocotrienol. Cells in each treatment group were initially plated at a density of 1 × 10⁶ cells/100 mm culture dish. Following treatment exposure, whole cell lysates were prepared for subsequent fractionation by SDS‐PAGE (30 µg/lane), followed by Western blot analysis. Each Western blot is a representative example of data obtained for experiments that were repeated at least three times. Scanning densitometric analysis was performed for each blot and is shown next to their respective Western blot. Vertical bars represent the integrated optical density of bands visualized in each lane.

Figure 3

Direct effects of 0–10 µmγ‐tocotrienol (top) and 0–100 µmα‐tocopherol (bottom) on Akt kinase activity. Various doses of γ‐tocotrienol or α‐tocopherol were dissolved in 100% ethanol, and then incubated with 25 ng of activated Akt and excess ATP as described in the instructions provided by the manufacturer in the assay kit. Akt activity is expressed as optical density units at 450 nm. Vertical bars represent the mean ± SD of duplicates in each treatment group. Each experiment was repeated at two to three times.

Figure 4

Direct effects of 0–10 µmγ‐tocotrienol (top) and 0–100 µmα‐tocopherol (bottom) on PI3K activity. Various doses of γ‐tocotrienol or α‐tocopherol were dissolved in 100% ethanol, and then incubated with 25 ng of activated PI3K, excess ATP, and substrate (PIP2) following the instructions provided by the manufacturer in the assay kit. PI3K activity is expressed as PI3K‐dependent PIP3 formation in pmol. Vertical bars represent the mean ± SD of duplicates in each treatment group. Each experiment was repeated at two to three times.

Figure 5

Western blot and scanning densitometric analysis of phosphoErbB1 (Tyr992), phosphoErbB1 (Tyr1068), phosphoErbB2 (Tyr877), phosphoErbB2 (Tyr1248) and phosphoErbB3 (Tyr1289) in neoplastic +SA mammary epithelial cells following a 0–72‐h treatment exposure to 7 µmγ‐tocotrienol. Cells in each treatment group were initially plated at a density of 1 × 10⁶ cells/100 mm culture dish. Following treatment exposure, whole cell lysates were prepared for subsequent fractionation by SDS‐PAGE (30 µg/lane), followed by Western blot analysis. Each Western blot is a representative example of data obtained for experiments that were repeated at least three times. Scanning densitometric analysis was performed for each blot and is shown next to their respective Western blot. Vertical bars represent the integrated optical density of bands visualized in each lane.