Anti-proliferative effects of gamma-tocotrienol on mammary tumour cells are associated with suppression of cell cycle progression

Abstract

Objectives: Previous studies have shown that gamma-tocotrienol induces potent anti-proliferative effects on +SA mammary tumour cells in culture; here, investigations have been conducted to determine its effects on intracellular signalling proteins involved in regulating cell cycle progression.

Materials and methods: +SA cells were maintained in mitogen-free defined media containing 0 or 4 micromgamma-tocotrienol, for 48 h to synchronize cell cycle in G(0) phase, and then they were exposed to 100 ng/ml EGF to initiate cell cycle progression. Whole cell lysates were collected at various time points from each treatment group and were prepared for Western blot analysis.

Results and conclusions: Treatment with 4 micromgamma-tocotrienol significantly inhibited +SA cell proliferation over a 4-day culture period. Moreover, this treatment resulted in a relatively large reduction in cyclin D1, cyclin dependent kinase (CDK)4, CDK2 and CDK6 levels, between 4 and 24 h after EGF exposure. Tocotrienol treatment also resulted in a relatively large increase in CDK inhibitor (CKI) p27, prior to and after EGF exposure, but had little effect on levels of CKIs, p21 and p15. Tocotrienol treatment also induced a large relative reduction in retinoblastoma (Rb) protein phosphorylation at ser780 and ser807/811. These findings strongly suggest that anti-proliferative effects of gamma-tocotrienol are associated with reduction in cell cycle progression from G(1) to S, as evidenced by increased p27 levels, and a corresponding decrease in cyclin D1, CDK2, CDK4, CDK6 and phosphorylated Rb levels.

Figure 2

Effects of γ‐tocotrienol on cyclin D1 and CDK levels. +SA cells were plated at a density of 1 × 10⁶ cells/100 mm culture dishes in serum‐free defined control media, to allow cells to attach. The following day they were divided into control and treatment groups, media were removed, cells were washed and then fed fresh mitogen‐free media containing 0 or 4 μmγ‐tocotrienol (γT³), to establish cell cycle synchronization in all groups. After 48 h incubation period, media were removed and cells were fed their respective control or treatment media containing 100 ng/ml EGF. Cells were then collected at 0, 2, 4, 6, 12 and 24 h after mitogen stimulation and whole cell lysates were prepared for Western blot analysis of intracellular levels of cyclin D1, CDK2, CDK4, CDK6 and actin (α, β, γ) proteins. For each experiment, samples were electrophoresed through SDS–polyacrylamide minigels run simultaneously and proteins from two minigels were transblotted on to a single PVDF membrane, followed by Western blot analysis. Each Western blot image is a representative example of data obtained from experiments that were repeated at least three times. Scanning densitometric analysis was performed for each blot and net intensity of each band was normalized with corresponding actin values, as shown in bar graphs adjacent to their respective Western blot images. Vertical bars in the graph indicate relative protein expression in control and treatment groups over 24 h after mitogen (EGF) stimulation.

Figure 3

Effects of γ‐tocotrienol on CKI levels. +SA cells were plated at a density of 1 × 10⁶ cells/100 mm culture dishes and fed serum‐free defined control media to allow cells to attach. The following day, cells were divided into control and treatment groups, media were removed, cells were washed and then fed fresh mitogen‐free media containing 0 or 4 μmγ‐tocotrienol (γT³), to establish cell cycle synchronization between all groups. After 48 h incubation, media were removed and cells in all groups were fed their respective control or treatment media containing 100 ng/ml EGF. Cells were collected at various time points over the next 24 h period and whole cell lysates were prepared for Western blot analysis of p21, p27, p15 and actin (α, β, γ) proteins. For each experiment, samples were electrophoresed through SDS–polyacrylamide minigels run simultaneously and proteins from two minigels were transblotted on to a single PVDF membrane, followed by Western blot analysis. Each Western blot image is a representative example of data obtained from experiments that were repeated at least three times. Scanning densitometric analysis was performed for each blot and net intensity of each band was normalized to corresponding actin values, as shown in bar graphs adjacent to their respective Western blot images. Vertical bars in the graph indicate relative protein expression in control and treatment groups over 24 h after mitogen (EGF) stimulation.

Figure 4

Effects of γ‐tocotrienol on retinoblastoma (Rb) phosphorylation. +SA cells were plated at a density of 1 × 10⁶ cells/100 mm culture dishes and fed serum‐free defined control media to allow cells to attach. The following day, cells were divided into control and treatment groups, media were removed, cells washed, and then fed fresh mitogen‐free media containing 0 or 4 μmγ‐tocotrienol (γT³), to establish cell cycle synchronization in all groups. After 48 h incubation, media were removed and cells in all groups were fed their respective control or treatment media containing 100 ng/ml EGF. Cells were collected at specific times over the next 24 h period and whole cell lysates were prepared for Western blot analysis of intracellular levels of phospho‐Rb (ser780), phospho‐Rb (ser807/811) and actin (α, β, γ) proteins. For each experiment, samples were electrophoresed through SDS–polyacrylamide minigels run simultaneously and proteins from two minigels were transblotted on to a single PVDF membrane, followed by Western blot analysis. Each Western blot image is a representative example of data obtained from experiments that were repeated at least three times. Scanning densitometric analysis was performed for each blot and net intensity of each band was normalized with corresponding actin values, as shown in bar graphs adjacent to their respective Western blot images. Vertical bars in the graph indicate relative protein expression in control and treatment groups over 24 h after mitogen (EGF) stimulation.

Figure 1

Effects of 0–4 μmγ‐tocotrienol on the highly malignant +SA mammary epithelial cell proliferation over a 96 h treatment period. Cells in all treatment groups were initially plated (day 0) at a density of 5 × 10⁴ cells/well in 24‐well culture plates. Vertical bars indicate mean viable cell number/well ± SEM for six replicates in each treatment group. *P < 0.05, as compared to vehicle‐treated cells in the control group.