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. 2022 Apr 1;15(4):233-245.
doi: 10.1158/1940-6207.CAPR-21-0508.

δ-Tocotrienol is the Most Potent Vitamin E Form in Inhibiting Prostate Cancer Cell Growth and Inhibits Prostate Carcinogenesis in Ptenp-/- Mice

Hong Wang  1 , William Yan  1 , Yuhai Sun  1 , Chung S Yang  1
Affiliations

δ-Tocotrienol is the Most Potent Vitamin E Form in Inhibiting Prostate Cancer Cell Growth and Inhibits Prostate Carcinogenesis in Ptenp-/- Mice

Hong Wang et al. Cancer Prev Res (Phila). .

Abstract

Vitamin E compounds, consisting of α, β, γ, and δ forms of tocopherols and tocotrienols, display different cancer preventive activities in experimental models. Tocotrienols may have higher potential for clinical use due to their lower effective doses in laboratory studies. However, most studies on tocotrienols have been carried out using cancer cell lines. Strong data from animal studies may encourage the use of tocotrienols for human cancer prevention research. To examine the cancer inhibitory activity of different vitamin E forms, we first investigated their inhibitory activities of different vitamin E forms in prostate cancer cell lines. We found that δ-tocotrienol (δT3) was the most effective form in inhibiting cell growth at equivalent doses. Because of this in vitro potency, δT3 was further studied using prostate-specific Pten-/- (Ptenp-/-) mice. We found that 0.05% δT3 in diet reduced prostate adenocarcinoma multiplicity by 32.7%, featuring increased apoptosis and reduced cell proliferation. The inhibitory effect of 0.05% δT3 in diet was similar to that of 0.2% δ-tocopherol (δT) in diet reported previously. Our further study on the δT3-induced transcriptome changes indicated that δT3 inhibited genes in blood vessel development in the prostate of Ptenp-/- mice, which was confirmed by IHC. Together, our results demonstrate that δT3 effectively inhibits the development of prostate adenocarcinoma in Ptenp-/- mice, which involves inhibition of proliferation and angiogenesis and promotion of apoptosis.

Prevention relevance: We demonstrated that δ-tocotrienol is the most active vitamin E form in inhibiting the growth of several prostate cancer cell lines. In transgenic Ptenp-/- mice, δ-tocotrienol inhibited the formation of prostate cancer. This result would encourage and help design clinical studies for the application of δ-tocotrienol for prostate cancer prevention.

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Conflict of interest statement

Conflicts of interest:

The authors declare no potential conflicts of interest.

Figures

Figure 1.
Figure 1.. δT3 is the most potent vitamin E form in inhibiting the growth of prostate cancer cells.
(A-B) DU145 and Pten-CaP8 cells were treated with different concentrations of tocopherol or tocotrienol for 48 hours, and the percentage of viable cells were determined by the MTT assay. The error bar represents SD (n=8). (C) DU145 and Pten-CaP8 cells treated with tocopherol or tocotrienol for 24 hours were analysed for apoptosis using the western blot analysis of C-Casp3. The sample loading was monitored using the β-actin levels. The uncropped blot images were provided in Supplementary Figure 2A. (D-E) DU145 cells, treated with DMSO, 24 μM δT or 12 μM δT3 for 12 hours, were challenged with 10 ng/ml IGF1 and then collected for the western blot analyses of pAkt and Akt. Representative results are shown in D. The fold increases of the ratio of pAkt to Akt at 5, 10, 15, and 20 minutes (from the zero time), in three individual experiments, were summarized in E (the uncropped blot images of three experiments were provided in Supplementary Figure 2B). The error bar represents SD. a and b indicate the difference among all groups with statistical significance (ANOVA; P-value < 0.05) at the same time point.
Figure 2.
Figure 2.. Feeding diet supplemented with 0.05% δT3 reduced the multiplicity of adenocarcinoma in the prostate of Ptenp−/− mice.
(A) The adenocarcinoma multiplicity in the prostate of Ptenp−/− mice fed either AIN93M (n=15) or 0.05% δT3 diet (n=13) at 40 weeks of age. Data are presented as mean±SD. * indicates the difference with statistical significance (P-value = 0.001). (B-C) The prostates of Ptenp−/− mice on AIN93M or 0.05% δT3 diet were analyzed for cell proliferation and apoptosis by the IHC staining for Ki67 and C-Casp3. The prostates (n=5) of the same aged Wt mice were used as the controls. The percentages of gland luminal epithelial or tumor cells positively stained for Ki67 in the nucleus or C-Casp3 were determined by Aperio’s IHC Nuclear Image Analysis algorithm. a, b and c indicate the difference among all groups with statistical significance (ANOVA; P-value < 0.05). (D) Representative images of the Ki67 and C-Casp3 stained prostate samples of Wt and Ptenp−/− mice. The scale bar represents 50 μm.
Figure 3.
Figure 3.. Top 20 pathways and processes enriched by δT3-regulated genes in the prostate of Ptenp−/− mice.
(A) Top 20 pathways and processes that were enriched by 189 up-regulated and 1033 down-regulated genes by δT3 were identified by Metascape. Log10(P) is the P-value in log base 10. (B) The transcription regulatory networks enriched by δT3-regulated genes (P-value ≤ 0.01) were revealed by the TRRUST analysis in Metascape.
Figure 4.
Figure 4.. The expression levels of endothelial cell markers and angiogenesis factors in the prostates of Ptenp−/− mice on AIN93M and δT3 diets at 20 weeks.
(A) The expression levels of β-actin, Gaphd, CD31/Pecam1, Vwf, Vegfa, Vegfb, Vegfc, and Vegfd in the prostates of Ptenp−/− mice on AIN93M and δT3 diets were obtained from the normalized results of the quantification RNA-sequencing. *, **, *** indicate the difference between δT3 and AIN93M groups with statistical significance (n=6 in each group; P-value = 0.0175, 0.0180 and 0.0234, respectively). (B-D) The expression levels of CD31/Pecam1, Vwf and Vegfd were validated by qPCR. The data presented in this figure are normalized by the levels of β-actin. * indicates the difference between δT3 and AIN93M groups with statistical significance (n=6 in each group; P-value < 0.001).
Figure 5.
Figure 5.. The IHC staining of CD31/Pecam1 in the prostates of Ptenp−/− mice on AIN93M and δT3 diets at 40 weeks.
The blood vessels in the prostate of Wt and Ptenp−/− mice at 40 weeks age were identified by the IHC staining of C31/Pecam1. Representative images of the prostate of Wt (A) and Ptenp−/− (B) mice were shown in this figure (S-stromal area; G-lesioned gland area). Representative images of the blood vessel-rich stroma (C and E) and lesioned glands (D and F) in the prostate of Ptenp−/− mice on AIN93M and δT3 diet were also shown in this figure. Microvessel densities (the averages of blood vessel counts) in the stroma and lesioned glands of the mice on AIN93M (n=15) and δT3 (n=13) diet were summarized in G. The scale bar represents 50 μm. * indicates the difference between δT3 and AIN93M groups with statistical significance (P-value < 0.001).
Figure 6.
Figure 6.. δT3 attenuated the activation of Erk1/2.
(A-B) DU145 cells were challenged with IGF1 (10 ng/ml) after cultured in the medium with δT3 (5 μM), αT (20 μM), or DMSO for 12 hours. The activation of Erk1/2 was determined by western blot analyses using rabbit antibody against pErk1/2 and mouse antibody against Erk1/2 plus IRDye 680RD-labbeled donkey anti-rabbit and IRDye 800CW-labbeled donkey anti-mouse antibodies. Representative results are shown in A. The fold increases of the ratio of pErk1/2 to Erk1/2 at the peak time points (2 and 5 min), compared to the control (0 min), in three individual experiments (the uncropped blot images were provided in Supplementary Figure 8) using cells treated with δT3 or DMSO was summarized in B (* and ** indicate the differences between two groups with statistical significance; P-value = 0.0311 and 0.0093, respectively). (C-D) The western blot results of pErk1/2 and Erk1/2 in the prostates collected at 20 weeks from Ptenp−/− mice on AIM83M or 0.05% δT3 diet are shown in C. The ratios of pERK1/2 to ERK1/2 were summarized in D. (E-F) The activation of Erk1/2 was determined in the prostates collected at 40 weeks by IHC staining of pErk1/2. The average staining intensities were quantified and are presented in E (n=15 and 13 for AIN93M and δT3 groups, respectively; * indicates the differences between two groups with statistical significance; P-value = 0.0479). Representative images of pErk1/2 stained prostate samples of Wt and Ptenp−/− mice were shown in F. The scale bars in F represent 50 μm.
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