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Nelfinavir and nelfinavir analogs block site-2 protease cleavage to inhibit castration-resistant prostate cancer.

Guan M, Su L, Yuan YC, Li H, Chow WA - Sci Rep (2015)

Bottom Line: Nelfinavir and its analogs are more potent inhibitors of S2P cleavage activity than 1,10-phenanthroline, a metalloprotease-specific inhibitor.Further, cluster analysis of gene expression from treated DU145 and PC3 cell lines demonstrate a close similarity of nelfinavir, its analogs, and 1,10-phenanthroline.This leads to accumulation of precursor SREBP-1 and ATF6, and development of insufficient reserves of their transcriptionally-active forms.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Pharmacology, Beckman Research Institute of the City of Hope, Duarte, CA, USA.

ABSTRACT
Nelfinavir and its analogs inhibit proliferation and induce apoptosis of castration-resistant prostate cancer through inhibition of site-2 protease (S2P) activity, which leads to suppression of regulated intramembrane proteolysis. Western blotting in nelfinavir and its analog treated cells confirms accumulation of precursor SREBP-1 and ATF6. Nelfinavir and its analogs inhibit human homolog M. jannaschii S2P cleavage of an artificial protein substrate CED-9 in an in vitro proteolysis assay in a dose-dependent manner. Nelfinavir and its analogs are more potent inhibitors of S2P cleavage activity than 1,10-phenanthroline, a metalloprotease-specific inhibitor. Further, cluster analysis of gene expression from treated DU145 and PC3 cell lines demonstrate a close similarity of nelfinavir, its analogs, and 1,10-phenanthroline. These results show nelfinavir and its analogs inhibit castration-resistant prostate cancer proliferation by blocking regulated intramembrane proteolysis through suppression of S2P cleavage activity. This leads to accumulation of precursor SREBP-1 and ATF6, and development of insufficient reserves of their transcriptionally-active forms. The present results validate S2P and regulated intramembrane proteolysis as novel therapeutic targets for castration-resistant prostate cancer therapeutics. A clinical trial of nelfinavir or its analogs should be developed for castration-resistant prostate cancer.

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Nelfinavir and its analogs increase precursor SREBP-1 and ATF6 protein accumulation, and decrease FAS and increase GRP78 expression.DU145 and PC-3 cells were treated with nelfinavir or analogs (10 μM) for 24 hr and lysate was harvested for Western blot analysis of SREBP-1, FAS and GRP78. pATF6-EGFP (or mock) was transfected 24 hr prior to the treatment of nelfinavir or analogs for 24 hr for analysis of ATF6. (A–B) Nelfinavir analogs increase precursor SREBP-1 and ATF6 accumulation. (C) Nelfinavir analogs decrease FAS expression. (D) Nelfinavir increases GRP78 expression. The images are representative of at least three experiments. Cropped blots are from gels run under same condition. Quantification of Western blot was by Quantity One (Bio-Rad, Hercules, CA) and normalized to control GAPGH.
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f3: Nelfinavir and its analogs increase precursor SREBP-1 and ATF6 protein accumulation, and decrease FAS and increase GRP78 expression.DU145 and PC-3 cells were treated with nelfinavir or analogs (10 μM) for 24 hr and lysate was harvested for Western blot analysis of SREBP-1, FAS and GRP78. pATF6-EGFP (or mock) was transfected 24 hr prior to the treatment of nelfinavir or analogs for 24 hr for analysis of ATF6. (A–B) Nelfinavir analogs increase precursor SREBP-1 and ATF6 accumulation. (C) Nelfinavir analogs decrease FAS expression. (D) Nelfinavir increases GRP78 expression. The images are representative of at least three experiments. Cropped blots are from gels run under same condition. Quantification of Western blot was by Quantity One (Bio-Rad, Hercules, CA) and normalized to control GAPGH.

Mentions: Our previous data showed that nelfinavir inhibited CRPC proliferation through inhibition of RIP-mediated processing of precursor SREBP-1 and ATF624. Accordingly, both precursor and mature SREBP-1 and ATF6 detection (Fig. 3 A and 3B) were quantified by Western blot in CRPC cells treated with nelfinavir or its analogs. As shown in Fig. 3A and B, nelfinavir and and all five analogs increase the precursor level of ATF6 whereas only nelfinavir, #6, #31 increase SREBP-1 precursor in DU145 cells. Nelfinavir, and all analogs with the exception of #8 increase detection of precursor SREBP-1 and ATF6 in PC-3 cells. As a transcriptional target of SREBP-1, FAS expression was examined in nelfinavir and nelfinavir analog-treated DU145 cells. The immunoblot demonstrates reduced FAS expression (Fig. 3C).


Nelfinavir and nelfinavir analogs block site-2 protease cleavage to inhibit castration-resistant prostate cancer.

Guan M, Su L, Yuan YC, Li H, Chow WA - Sci Rep (2015)

Nelfinavir and its analogs increase precursor SREBP-1 and ATF6 protein accumulation, and decrease FAS and increase GRP78 expression.DU145 and PC-3 cells were treated with nelfinavir or analogs (10 μM) for 24 hr and lysate was harvested for Western blot analysis of SREBP-1, FAS and GRP78. pATF6-EGFP (or mock) was transfected 24 hr prior to the treatment of nelfinavir or analogs for 24 hr for analysis of ATF6. (A–B) Nelfinavir analogs increase precursor SREBP-1 and ATF6 accumulation. (C) Nelfinavir analogs decrease FAS expression. (D) Nelfinavir increases GRP78 expression. The images are representative of at least three experiments. Cropped blots are from gels run under same condition. Quantification of Western blot was by Quantity One (Bio-Rad, Hercules, CA) and normalized to control GAPGH.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4816264&req=5

f3: Nelfinavir and its analogs increase precursor SREBP-1 and ATF6 protein accumulation, and decrease FAS and increase GRP78 expression.DU145 and PC-3 cells were treated with nelfinavir or analogs (10 μM) for 24 hr and lysate was harvested for Western blot analysis of SREBP-1, FAS and GRP78. pATF6-EGFP (or mock) was transfected 24 hr prior to the treatment of nelfinavir or analogs for 24 hr for analysis of ATF6. (A–B) Nelfinavir analogs increase precursor SREBP-1 and ATF6 accumulation. (C) Nelfinavir analogs decrease FAS expression. (D) Nelfinavir increases GRP78 expression. The images are representative of at least three experiments. Cropped blots are from gels run under same condition. Quantification of Western blot was by Quantity One (Bio-Rad, Hercules, CA) and normalized to control GAPGH.
Mentions: Our previous data showed that nelfinavir inhibited CRPC proliferation through inhibition of RIP-mediated processing of precursor SREBP-1 and ATF624. Accordingly, both precursor and mature SREBP-1 and ATF6 detection (Fig. 3 A and 3B) were quantified by Western blot in CRPC cells treated with nelfinavir or its analogs. As shown in Fig. 3A and B, nelfinavir and and all five analogs increase the precursor level of ATF6 whereas only nelfinavir, #6, #31 increase SREBP-1 precursor in DU145 cells. Nelfinavir, and all analogs with the exception of #8 increase detection of precursor SREBP-1 and ATF6 in PC-3 cells. As a transcriptional target of SREBP-1, FAS expression was examined in nelfinavir and nelfinavir analog-treated DU145 cells. The immunoblot demonstrates reduced FAS expression (Fig. 3C).

Bottom Line: Nelfinavir and its analogs are more potent inhibitors of S2P cleavage activity than 1,10-phenanthroline, a metalloprotease-specific inhibitor.Further, cluster analysis of gene expression from treated DU145 and PC3 cell lines demonstrate a close similarity of nelfinavir, its analogs, and 1,10-phenanthroline.This leads to accumulation of precursor SREBP-1 and ATF6, and development of insufficient reserves of their transcriptionally-active forms.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Pharmacology, Beckman Research Institute of the City of Hope, Duarte, CA, USA.

ABSTRACT
Nelfinavir and its analogs inhibit proliferation and induce apoptosis of castration-resistant prostate cancer through inhibition of site-2 protease (S2P) activity, which leads to suppression of regulated intramembrane proteolysis. Western blotting in nelfinavir and its analog treated cells confirms accumulation of precursor SREBP-1 and ATF6. Nelfinavir and its analogs inhibit human homolog M. jannaschii S2P cleavage of an artificial protein substrate CED-9 in an in vitro proteolysis assay in a dose-dependent manner. Nelfinavir and its analogs are more potent inhibitors of S2P cleavage activity than 1,10-phenanthroline, a metalloprotease-specific inhibitor. Further, cluster analysis of gene expression from treated DU145 and PC3 cell lines demonstrate a close similarity of nelfinavir, its analogs, and 1,10-phenanthroline. These results show nelfinavir and its analogs inhibit castration-resistant prostate cancer proliferation by blocking regulated intramembrane proteolysis through suppression of S2P cleavage activity. This leads to accumulation of precursor SREBP-1 and ATF6, and development of insufficient reserves of their transcriptionally-active forms. The present results validate S2P and regulated intramembrane proteolysis as novel therapeutic targets for castration-resistant prostate cancer therapeutics. A clinical trial of nelfinavir or its analogs should be developed for castration-resistant prostate cancer.

Show MeSH
Related in: MedlinePlus