Skip to main page content
Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2020 May 15;20(1):428.
doi: 10.1186/s12885-020-06947-6.

Upregulation of pERK and c-JUN by γ-Tocotrienol and Not α-Tocopherol Are Essential to the Differential Effect on Apoptosis in Prostate Cancer Cells

Affiliations
Free PMC article

Upregulation of pERK and c-JUN by γ-Tocotrienol and Not α-Tocopherol Are Essential to the Differential Effect on Apoptosis in Prostate Cancer Cells

Christine Moore et al. BMC Cancer. .
Free PMC article

Abstract

Background: α-tocopherol (AT) and γ-tocotrienol (GT3) are vitamin E isoforms considered to have potential chemopreventive properties. AT has been widely studied in vitro and in clinical trials with mixed results. The latest clinical study (SELECT trial) tested AT in prostate cancer patients, determined that AT provided no benefit, and could promote cancer. Conversely, GT3 has shown antineoplastic properties in several in vitro studies, with no clinical studies published to date. GT3 causes apoptosis via upregulation of the JNK pathway; however, inhibition results in a partial block of cell death. We compared side by side the mechanistic differences in these cells in response to AT and GT3.

Methods: The effects of GT3 and AT were studied on androgen sensitive LNCaP and androgen independent PC-3 prostate cancer cells. Their cytotoxic effects were analyzed via MTT and confirmed by metabolic assays measuring ATP. Cellular pathways were studied by immunoblot. Quantitative analysis and the determination of relationships between cell signaling events were analyzed for both agents tested. Non-cancerous prostate RWPE-1 cells were also included as a control.

Results: The RAF/RAS/ERK pathway was significantly activated by GT3 in LNCaP and PC-3 cells but not by AT. This activation is essential for the apoptotic affect by GT3 as demonstrated the complete inhibition of apoptosis by MEK1 inhibitor U0126. Phospho-c-JUN was upregulated by GT3 but not AT. No changes were observed on AKT for either agent, and no release of cytochrome c into the cytoplasm was detected. Caspases 9 and 3 were efficiently activated by GT3 on both cell lines irrespective of androgen sensitivity, but not in cells dosed with AT. Cell viability of non-cancerous RWPE-1 cells was affected neither by GT3 nor AT.

Conclusions: c-JUN is a recognized master regulator of apoptosis as shown previously in prostate cancer. However, the mechanism of action of GT3 in these cells also include a significant activation of ERK which is essential for the apoptotic effect of GT3. The activation of both, ERK and c-JUN, is required for apoptosis and may suggest a relevant step in ensuring circumvention of mechanisms of resistance related to the constitutive activation of MEK1.

Keywords: Differential effect; Prevention; Prostate cancer; Vitamin E isoforms; α-Tocopherol; γ-Tocotrienol.

Conflict of interest statement

The authors declare that they have no competing interests.

Figures

Fig. 1
Fig. 1
Effect of AT and GT3 on prostate cancer cells. a and b: LNCaP and PC-3 were treated with AT or GT3 at doses ranging from 10 to 80 μM. After 6 h of treatment, cell viability was determined via MTT. c, d and e: LNCaP, PC-3, and non-tumorigenic RWPE-1 cells underwent the same treatment as described above for 12 h. All graphs shown correspond to data obtained by analysis of metabolic activity from at least three independent experiments. For quantification spectrophotometric data were calculated as percentages of the value for the untreated cells (100%) ± standard deviations n = 3, *p < 0.05, **p < 0.01
Fig. 2
Fig. 2
Involvement of the pERK, pc-JUN and pAKT in the response to treatment with GT3 or AT of LNCaP prostate cancer cells. a, c, and e: LNCaP cells were grown on 6-wellplates and treated with GT3 for 6 h at doses ranging from 10 to 80 μM. Immmunoblots from SDS total extracts were obtained using antibodies against total and phosphorylated forms of ERK (a) (c) c-JUN, and (e) AKT. b, d, and F: Similarly, LNCaP cells treated with AT for 6 h at the same dose range as described above. Immunoblots from total SDS extracts using antibodies against the total and phosphorylated forms of ERK (b), c-JUN (d) and AKT (f) are shown above. The membranes were reprobed for β-actin as a loading control. The results presented are representative of three separate experiments. The values are averages ± standard deviations from three independent experiments;*p < 0.05, **p < 0.01. Autoradiographs of the original blots are available in the Supplementary Section Figure S5.
Fig. 3
Fig. 3
Involvement of the pERK, pc-JUN and pAKT in the response to treatment with GT3 or AT of PC3 prostate cancer cells. a, c, and e: PC3 cells were grown on 6-well plates and treated with GT3 in the same manner as described above. Immunoblots were obtained from SDS total extracts by using antibodies against the total and phosphorylated forms of ERK (a), c-JUN (c), and AKT (e). b, d, and f: Immunoblots of SDS total extracts obtained from PC3 cells treated with AT as described above, were obtained using antibodies against the phosphorylated and total forms of ERK (b) c-JUN (d), and AKT (f). Cells that were treated with neither GT3 nor AT, only with dissolution vehicle, were run as controls for each experiment. The membranes were reprobed for β-actin as a loading control. The results presented are representative of three separate experiments. The values are averages ± standard deviations from three independent experiments;*p < 0.05. Autoradiographs of the original blots are available in the Supplementary Section Figure S6.
Fig. 4
Fig. 4
GT3 and AT have a similar effect on non-tumerigenic prostate cells. a and b: RWPE-1 cells grown on 6-wellplates and treated with (a) GT3 or (b) AT for 6 h at doses ranging from 10 to 80 μM were analyzed by immunoblot using antibodies against the total and phosphorylated forms of ERK. c and d: RWPE-1 cells were treated as described above, and analyzed via immunoblot. SDS total extracts of (c) GT3 or (d) AT treated cells were probed using antibodies against total and activated c-JUN. e and f: The expression levels of total and activated AKT were analyzed via immunoblot in RWPE-1 cells treated with (e) GT3 or (f) AT. The membranes were reprobed for β-actin as al loading control. The values are averages ± standard deviations from at least three independent experiments; *p < 0.05. Autoradiographs of the original blots are available in the Supplementary Section Figure S7
Fig. 5
Fig. 5
Effect on mitochondrial function of GT3 and AT on prostate cancer cells. a and b: LNCaP cells grown on 6-well plates and treated with (a) GT3 or (b) AT for 6 h at doses ranging from 10 to 80 μM were analyzed by immunoblot of SDS total extracts using antibodies against pBAD serine 112. c and d: Cytoplasmic (CF) and mitochondrial fractions (MF) were obtained from LNCaP cells treated with (c) GT3 or (d) AT as described above, and analyzed for the presence of cytochrome C by immunoblot. COX IV was used as a mitochondrial marker. e and f: PC3 cells were grown on 6-well plates and dosed as described above with (e) GT3 or (f) AT. The SDS total extracts were analyzed for the presence of pBAD serine 112 via immunoblot. g and h: PC3 cells dosed with (g) GT3 or (h) AT as described above were processed to obtain cytoplasmic (CF) and mitochondrial fractions (MF) and probed for the presence of cytochrome C and mitochondrial marker COX IV via immunoblot. The values of each analyzed protein were normalized to β-actin. The values are averages ± standard deviations from at least three independent experiments,*p < 0.05. Autoradiographs of the original blots are available in the Supplementary Section Fig. S8.
Fig. 6
Fig. 6
Apoptotic effect of GT3 and AT on prostate cancer cells. a, b, c, and d: SDS total extracts of LNCaP cells treated with (a and c) GT3 or (b and d) AT at doses ranging from 10 to 80 μM were by immunoblot analyzed for the presence of cleaved caspase 9 (a and b) and caspase 3 (c and d). e, f, g, and h: In the same manner as described above, PC3 cells treated with (e and g) GT3 or (f and h) AT, were processed for immunoblot analysis with antibodies against cleaved caspases 9 and 3. The values of each analyzed protein were normalized to β-actin. The values are averages ± standard deviations from at least three independent experiments; *p < 0.05. Autoradiographs of the original blots are available in the Supplementary Section Fig. S9
Fig. 7
Fig. 7
Effect of GT3 and AT on prostate cancer cells in the presence of inhibitor U0126.a, b and c: Prostate cancer cells (a) LNCaP, b PC-3, and (c) non-tumorigenic RWPE-1 cells were grown in 96-well plates. Two plates were used per cell line; plate one was pre-treated with inhibitor U0126 for 1 h, after which, plates one and two were dosed for 24 h with GT3 at 0, 20, 40 and 80 μM. Cell viability was analyzed by MTT or metabolic assays. For quantification spectrophotometric data were calculated as percentages of the value for the untreated cells (100%) ± standard deviations from at least three independent experiments, *p < 0.05, ***p < 0.001. Statistical analysis using ANOVA and Bonferroni test indicates a significant difference between cells treated with and without inhibitor

Similar articles

See all similar articles

References

    1. Siegel RL, Miller KD, Jemal A. Cancer statistics, 2018. CA Cancer J Clin. 2018;68(1):7–30. - PubMed
    1. Cancer Genome Atlas Research N The molecular taxonomy of primary prostate Cancer. Cell. 2015;163(4):1011–1025. - PMC - PubMed
    1. Fair WR, Fleshner NE, Heston W. Cancer of the prostate: a nutritional disease? Urology. 1997;50(6):840–848. - PubMed
    1. Tsao AS, Kim ES, Hong WK. Chemoprevention of cancer. CA Cancer J Clin. 2004;54(3):150–180. - PubMed
    1. Kayden HJ, Traber MG. Absorption, lipoprotein transport, and regulation of plasma concentrations of vitamin E in humans. J Lipid Res. 1993;34(3):343–358. - PubMed
Feedback