δ-Tocopherol inhibits the development of prostate adenocarcinoma in prostate specific Pten-/- mice

Abstract

The PTEN/PI3K/AKT axis plays a critical role in regulating cell growth, differentiation and survival. Activation of this signaling pathway is frequently found in human cancers. Our previous studies demonstrated that δ-tocopherol (δ-T) attenuates the activation of AKT by growth factor in prostate cancer cell lines, leading to inhibition of proliferation and induction of apoptosis. Herein, we investigated whether δ-T inhibits the development of prostate adenocarcinoma in prostate-specific Pten-/- (Ptenp-/-) mice in which the activation of AKT is the major driving force for tumorigenesis. By feeding Ptenp-/- mice with AIN93M or 0.2% δ-T supplemented diet starting at the age of 6 or 12 weeks, we found that δ-T treatment reduced prostate adenocarcinoma multiplicity at the age of 40 weeks by 53.3 and 42.7%, respectively. Immunohistochemical (IHC) analysis demonstrated that the phosphorylation of AKT (T308) was reduced in the prostate of the mice administered the δ-T diet. Consistently, proliferation was reduced and apoptosis was increased in prostate lesions of mice on the δ-T diet. Oxidative stress, as determined by IHC staining of 8-OH-dG, was not altered during prostate tumorigenesis, nor was it affected by administration of δ-T. In contrast, α-tocopherol (α-T) at 0.2% in the diet did not affect prostate adenocarcinoma multiplicity in the Ptenp-/- mice. This finding is consistent with data from our previous study that δ-T, but not α-T, inhibits the activation of AKT and the growth of prostate cancer cells. Together, these results demonstrate that δ-T inhibits the development of prostate adenocarcinoma in Ptenp-/- mice, mainly through inhibition of AKT activation.

Figure 1.

The percentage of prostate glands developing adenocarcinoma in 40-week-old Pten^p−/−mice fed AIN93M, diet supplemented with 0.2% δ-T or 0.2% α-T starting at the age of 12 weeks. (A) Illustration of the experimental procedure for mice fed different diets starting from the age of 12 weeks. (B) The adenocarcinoma multiplicity (glands developed adenocarcinoma/100 glands counted) in dorsal, ventral and lateral lobes in mice at the age of 40 weeks fed AIN93M (n = 8), 0.2% δ-T diet (n = 10) or 0.2% α-T diet (n = 8). Data are presented as mean ± SD. One-way ANOVA followed by Tukey’s post hoc test is used to compare the percentages of prostate glands developing adenocarcinoma among the three groups (*P = 0.001).

Figure 2.

Dietary δ-T reduced pAKT, suppressed proliferation and increased apoptosis in prostate tissues. Pten^p−/− mice fed either an AIN93M (n = 8), a 0.2% δ-T diet (n = 10) or a 0.2% α-T diet (n = 8) and the WT mice fed an AIN93M diet (n = 7) were analyzed for the active AKT, proliferation and apoptosis using IHC staining for pAKT (T308), Ki67 and cleaved-Caspase 3 (C-Caspase 3), respectively. Images of representative IHC staining for these mouse prostate samples are shown in (A–D, pAKT; E–H, Ki67; I–L, C-Caspase 3). The scale bar in the figures represents 50 µm. Quantified results of pAKT (T308), Ki67 and C-Caspase 3 IHC staining for the four groups of mice were determined using the Aperio ScanScope and summarized in (M), (N) and (O), respectively. Data are presented as mean ± SD. One-way ANOVA followed by Tukey’s post hoc test is used to compare the IHC staining results in the groups (*P-value = 0.037, 0.018 and 0.031 in M, N and O, respectively).

Figure 3.

The percentage of prostate glands developed HG-PIN and adenocarcinoma in Pten^p−/−mice fed experimental diet at the age of 6 weeks. (A) At the age of 25 weeks, Pten^p−/− mice fed an AIN93M (n = 8) and a 0.2% δ-T diet (n = 8) from the age of 6 weeks were sacrificed to collect prostate tissues for histopathological characterization. The percentages of prostate glands developing HG-PIN in each lobe of the two groups of mice were scored. Data are presented as mean ± SD. There is no statistical significant difference in the percentage of HG-PIN glands in each lobe between the mice fed an AIN93M and a 0.2% δ-T diet (two-tailed Student’s t-test). (B) At the age of 40 weeks, Pten^p−/− mice fed either an AIN93M (n = 9) or a 0.2% δ-T diet (n = 10) were sacrificed to collect prostate tissues for histopathological characterization. The adenocarcinoma multiplicity (glands developed adenocarcinoma/100 glands counted) in dorsal, ventral and lateral lobes of the two groups of mice are presented in the figure. Data are presented as mean ± SD (*P = 0.0045).

Figure 4.

Dietary δ-T reduced the levels of pAKT in prostate of Pten^p−/− mice. Prostates of the WT mice fed an AIN93M and Pten^p−/− mice fed either an AIN93M or a 0.2% δ-T diet were analyzed for pAKT (T308) levels using IHC staining. Images of representative IHC staining for these mouse prostate samples at the age of 12, 25 and 40 weeks are shown (A–I). The scale bar represents 50 µm. The quantified results of pAKT (T308) levels for the three groups of mice were determined using the Aperio ScanScope and are summarized in (J). Data are presented as mean ± SD (the number of WT mice is six for each time point; the number of Pten^p−/− mice on the AIN93M diet is 7, 7 or 9 for 12, 25 or 40 weeks, respectively and the number of Pten^p−/− mice on the 0.2% δ-T diet is 7, 7 or 10 for 12, 25 or 40 weeks, respectively). *P = 0.033, **P = 0.017 and ***P = 0.023.

Figure 5.

Dietary δ-T reduced cell proliferation in prostate of Pten^p−/− mice. Prostates of the WT mice fed an AIN93M and Pten^p−/− mice fed either an AIN93M or a 0.2% δ-T diet were analyzed for cellular proliferation using IHC stainingfor Ki67. Images of representative IHC staining for the mouse prostate samples at the ages of 12, 25 and 40 weeks are shown (A–I). The scale bar represents 50 µm. The quantified results of positive Ki67 stained cells in the three groups of mice were determined using the Aperio ScanScope and are summarized in (J). Data are presented as mean ± SD (Experimental conditions are the same as Figure 4). *P = 0.029, **P = 0.013 and ***P = 0.01.

Figure 6.

Dietary δ-T increased apoptosis in prostate of Pten^p−/− mice. Prostates of the WT mice fed an AIN93M and Pten^p−/− mice fed either an AIN93M or a 0.2% δ-T diet were analyzed for cellular apoptosis using C-Caspase 3 IHC staining. Images of representative IHC staining for the mouse prostate samples at the ages of 12, 25 and 40 weeks are shown (A–I). The scale bar represents 50 µm. The quantified results of the numbers of cells positively stained for C-Caspase 3 in the three groups of mice were determined using the Aperio ScanScope and are summarized in (J). Data are presented as mean ± SD (Experimental conditions are the same as Figure 4). *P = 0.011.