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Research article
Koyamangalath Krishnan
Corresponding Author
ORCiD: https://orcid.org/0000-0002-6342-4412
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This work is licensed under a CC BY 4.0 License. Read Full License
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
γ-tocotrienol, α-tocopherol, prostate cancer, prevention, differential effect, vitamin E isoforms
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View the published article at BMC Cancer.
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On 13 Apr, 2020
Review #2 received
Received 11 Apr, 2020
Review #1 received
Received 08 Apr, 2020
Reviewer #2 agreed
On 01 Apr, 2020
Reviewers invited
Invitations sent on 30 Mar, 2020
Reviewer #1 agreed
On 30 Mar, 2020
Editor assigned
On 27 Mar, 2020
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Editor invited
On 26 Mar, 2020
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Editorial decision: Major revision
On 05 Nov, 2019
Review #3 received
Received 31 Oct, 2019
Review #2 received
Received 31 Oct, 2019
Reviewer #3 agreed
On 21 Oct, 2019
Reviewer #2 agreed
On 20 Oct, 2019
Review #1 received
Received 20 Oct, 2019
Reviewer #1 agreed
On 08 Oct, 2019
Reviewers invited
Invitations sent on 07 Oct, 2019
Submission checks complete
On 24 Sep, 2019
Editor assigned
On 18 Sep, 2019
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See the published version of this preprint at BMC Cancer.
This is a preprint, a preliminary version of a manuscript that has not completed peer review at a journal. Research Square does not conduct peer review prior to posting preprints. The posting of a preprint on this server should not be interpreted as an endorsement of its validity or suitability for dissemination as established information or for guiding clinical practice.
Learn more about In Review
Research article
Koyamangalath Krishnan
Corresponding Author
ORCiD: https://orcid.org/0000-0002-6342-4412
Badges
Prescreen
This manuscript passed our Prescreen assessment
Complete author information
Appropriate declaration statements
Potential risks to human health
Prescreen
Peer Review Timeline
CURRENT STATUS: Published
View the published article at BMC Cancer.
Version 3
Posted 29 Apr, 2020
No community comments so far
Editorial decision: Minor revision
On 21 Apr, 2020
Editor assigned
On 20 Apr, 2020
Submission checks complete
On 19 Apr, 2020
Editor invited
On 19 Apr, 2020
No comments provided
Editorial decision: Minor revision
On 13 Apr, 2020
Review #2 received
Received 11 Apr, 2020
Review #1 received
Received 08 Apr, 2020
Reviewer #2 agreed
On 01 Apr, 2020
Reviewers invited
Invitations sent on 30 Mar, 2020
Reviewer #1 agreed
On 30 Mar, 2020
Editor assigned
On 27 Mar, 2020
Submission checks complete
On 26 Mar, 2020
Editor invited
On 26 Mar, 2020
No comments provided
Editorial decision: Major revision
On 05 Nov, 2019
Review #3 received
Received 31 Oct, 2019
Review #2 received
Received 31 Oct, 2019
Reviewer #3 agreed
On 21 Oct, 2019
Reviewer #2 agreed
On 20 Oct, 2019
Review #1 received
Received 20 Oct, 2019
Reviewer #1 agreed
On 08 Oct, 2019
Reviewers invited
Invitations sent on 07 Oct, 2019
Submission checks complete
On 24 Sep, 2019
Editor assigned
On 18 Sep, 2019
First submitted
On 17 Sep, 2019
Editor invited
On 17 Sep, 2019
Metrics
Comments: 0
PDF Downloads: ...
HTML Views: ...
License:
This work is licensed under a CC BY 4.0 License. Read Full License
View the published article at BMC Cancer.
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.
Figure 1
Figure 2
Figure 3
Figure 4
Figure 5
Figure 6
Figure 7
This is a list of supplementary files associated with this preprint. Click to download.
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