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The C-terminal region Mesd peptide mimics full-length Mesd and acts as an inhibitor of Wnt/β-catenin signaling in cancer cells.

Lin C, Lu W, Zhang W, Londoño-Joshi AI, Buchsbaum DJ, Bu G, Li Y - PLoS ONE (2013)

Bottom Line: In our previous study, we found that the C-terminal region of Mesd, which is absent in sequences from invertebrates, is necessary and sufficient for binding to mature LRP6 on the cell surface.We found that Mesd C-terminal region-derived peptides block Mesd binding to LRP5 at the cell surface too.Finally, we found that treatment of the full-length Mesd protein and its C-terminal region peptide significantly increased chemotherapy agent Adriamycin-induced cytotoxicity in HS578T and PC-3 cells.

View Article: PubMed Central - PubMed

Affiliation: Drug Discovery Division, Southern Research Institute, Birmingham, Alabama, United States of America.

ABSTRACT
While Mesd was discovered as a specialized molecular endoplasmic reticulum chaperone for the Wnt co-receptors LRP5 and LRP6, recombinant Mesd protein is able to bind to mature LRP5 and LRP6 on the cell surface and acts as a universal antagonist of LRP5/6 modulators. In our previous study, we found that the C-terminal region of Mesd, which is absent in sequences from invertebrates, is necessary and sufficient for binding to mature LRP6 on the cell surface. In the present studies, we further characterized the interaction between the C-terminal region Mesd peptide and LRP5/6. We found that Mesd C-terminal region-derived peptides block Mesd binding to LRP5 at the cell surface too. We also showed that there are two LRP5/6 binding sites within Mesd C-terminal region which contain several positively charged residues. Moreover, we demonstrated that the Mesd C-terminal region peptide, like the full-length Mesd protein, blocked Wnt 3A- and Rspodin1-induced Wnt/β-catenin signaling in LRP5- and LRP6- expressing cells, suppressed Wnt/β-catenin signaling in human breast HS578T cells and prostate cancer PC-3 cells, and inhibited cancer cell proliferation, although the full-length Mesd protein is more potent than its peptide. Finally, we found that treatment of the full-length Mesd protein and its C-terminal region peptide significantly increased chemotherapy agent Adriamycin-induced cytotoxicity in HS578T and PC-3 cells. Together, our results suggest that Mesd C-terminal region constitutes the major LRP5/6-binding domain, and that Mesd protein and its C-terminal region peptide have a potential therapeutic value in cancer.

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Mesd C-terminal region peptide blocks Wnt/β-catenin signaling in prostate cancer PC-3 and breast cancer HS578T cells.(A) PC-3 and HT578T cells in 24-well plates were transiently transfected with the Super8XTOPFlash luciferase construct and β-galactosidase-expressing vector in each well. After 24 h incubation, cells were treated with mouse Mesd (mMesd), human Mesd peptide hMesd (160–197) or control peptide at the indicated concentrations. The luciferase activity was then measured 24 h later with normalization to the activity of the β-galactosidase. Values are the average of triple determinations with the s.d. indicated by error bars. **P<0.01 compared to the control cells without Mesd or Mesd peptide treatment. (B) PC-3 and HS578T cells in 6-well plates were treated with mMesd, hMesd (160–197) or control peptide at the indicated concentrations for 24 h. The levels of cytosolic free β-catenin, and total cellular β-catenin, LRP6, Axin2, cyclin D1 and phosphorylated LRP6 were then analyzed by Western blotting. Samples were also probed with the anti-actin antibody to verify equal loading.
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pone-0058102-g005: Mesd C-terminal region peptide blocks Wnt/β-catenin signaling in prostate cancer PC-3 and breast cancer HS578T cells.(A) PC-3 and HT578T cells in 24-well plates were transiently transfected with the Super8XTOPFlash luciferase construct and β-galactosidase-expressing vector in each well. After 24 h incubation, cells were treated with mouse Mesd (mMesd), human Mesd peptide hMesd (160–197) or control peptide at the indicated concentrations. The luciferase activity was then measured 24 h later with normalization to the activity of the β-galactosidase. Values are the average of triple determinations with the s.d. indicated by error bars. **P<0.01 compared to the control cells without Mesd or Mesd peptide treatment. (B) PC-3 and HS578T cells in 6-well plates were treated with mMesd, hMesd (160–197) or control peptide at the indicated concentrations for 24 h. The levels of cytosolic free β-catenin, and total cellular β-catenin, LRP6, Axin2, cyclin D1 and phosphorylated LRP6 were then analyzed by Western blotting. Samples were also probed with the anti-actin antibody to verify equal loading.

Mentions: Accumulating evidence indicates that Wnt co-receptor LRP6 play a critical role in the Wnt/β-catenin signaling activation in human prostate and breast cancer cells and is important in the development and progression of these two types of cancer [8], [13], [14], [29]–[33]. To test whether the Mesd C-terminal region peptide is able to suppress Wnt/β-catenin signaling in human prostate and breast cancer cells, we prepared a human Mesd C-terminal region peptide hMesd (160–197) which is corresponding to mouse Mesd C-terminal region peptide mMesd (155–191). We also prepared a negative control peptide based on the sequence of mMesd (155–164). Similar to mMesd (155–191), hMesd (160–197) displayed inhibitory effects on Wnt/β-catenin signaling induced by LRP6, Wnt3A, Rspo1, Wnt1 and Wnt10b (Figure S1 and S2). We found that hMesd (160–197), but not the control peptide, mimicked Mesd protein and significantly repressed the Wnt reporter luciferase activity in prostate cancer PC3 cells and breast cancer HS578T cells (Figure 5A). Accordingly, hMesd (160–197) greatly decreased the levels of cytosolic free β-catenin, LRP6 phosphorylation and Wnt/β-catenin signaling targets Axin2 and cyclin D1 expression in PC-3 and HS578T cells (Figure 5B).


The C-terminal region Mesd peptide mimics full-length Mesd and acts as an inhibitor of Wnt/β-catenin signaling in cancer cells.

Lin C, Lu W, Zhang W, Londoño-Joshi AI, Buchsbaum DJ, Bu G, Li Y - PLoS ONE (2013)

Mesd C-terminal region peptide blocks Wnt/β-catenin signaling in prostate cancer PC-3 and breast cancer HS578T cells.(A) PC-3 and HT578T cells in 24-well plates were transiently transfected with the Super8XTOPFlash luciferase construct and β-galactosidase-expressing vector in each well. After 24 h incubation, cells were treated with mouse Mesd (mMesd), human Mesd peptide hMesd (160–197) or control peptide at the indicated concentrations. The luciferase activity was then measured 24 h later with normalization to the activity of the β-galactosidase. Values are the average of triple determinations with the s.d. indicated by error bars. **P<0.01 compared to the control cells without Mesd or Mesd peptide treatment. (B) PC-3 and HS578T cells in 6-well plates were treated with mMesd, hMesd (160–197) or control peptide at the indicated concentrations for 24 h. The levels of cytosolic free β-catenin, and total cellular β-catenin, LRP6, Axin2, cyclin D1 and phosphorylated LRP6 were then analyzed by Western blotting. Samples were also probed with the anti-actin antibody to verify equal loading.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3585277&req=5

pone-0058102-g005: Mesd C-terminal region peptide blocks Wnt/β-catenin signaling in prostate cancer PC-3 and breast cancer HS578T cells.(A) PC-3 and HT578T cells in 24-well plates were transiently transfected with the Super8XTOPFlash luciferase construct and β-galactosidase-expressing vector in each well. After 24 h incubation, cells were treated with mouse Mesd (mMesd), human Mesd peptide hMesd (160–197) or control peptide at the indicated concentrations. The luciferase activity was then measured 24 h later with normalization to the activity of the β-galactosidase. Values are the average of triple determinations with the s.d. indicated by error bars. **P<0.01 compared to the control cells without Mesd or Mesd peptide treatment. (B) PC-3 and HS578T cells in 6-well plates were treated with mMesd, hMesd (160–197) or control peptide at the indicated concentrations for 24 h. The levels of cytosolic free β-catenin, and total cellular β-catenin, LRP6, Axin2, cyclin D1 and phosphorylated LRP6 were then analyzed by Western blotting. Samples were also probed with the anti-actin antibody to verify equal loading.
Mentions: Accumulating evidence indicates that Wnt co-receptor LRP6 play a critical role in the Wnt/β-catenin signaling activation in human prostate and breast cancer cells and is important in the development and progression of these two types of cancer [8], [13], [14], [29]–[33]. To test whether the Mesd C-terminal region peptide is able to suppress Wnt/β-catenin signaling in human prostate and breast cancer cells, we prepared a human Mesd C-terminal region peptide hMesd (160–197) which is corresponding to mouse Mesd C-terminal region peptide mMesd (155–191). We also prepared a negative control peptide based on the sequence of mMesd (155–164). Similar to mMesd (155–191), hMesd (160–197) displayed inhibitory effects on Wnt/β-catenin signaling induced by LRP6, Wnt3A, Rspo1, Wnt1 and Wnt10b (Figure S1 and S2). We found that hMesd (160–197), but not the control peptide, mimicked Mesd protein and significantly repressed the Wnt reporter luciferase activity in prostate cancer PC3 cells and breast cancer HS578T cells (Figure 5A). Accordingly, hMesd (160–197) greatly decreased the levels of cytosolic free β-catenin, LRP6 phosphorylation and Wnt/β-catenin signaling targets Axin2 and cyclin D1 expression in PC-3 and HS578T cells (Figure 5B).

Bottom Line: In our previous study, we found that the C-terminal region of Mesd, which is absent in sequences from invertebrates, is necessary and sufficient for binding to mature LRP6 on the cell surface.We found that Mesd C-terminal region-derived peptides block Mesd binding to LRP5 at the cell surface too.Finally, we found that treatment of the full-length Mesd protein and its C-terminal region peptide significantly increased chemotherapy agent Adriamycin-induced cytotoxicity in HS578T and PC-3 cells.

View Article: PubMed Central - PubMed

Affiliation: Drug Discovery Division, Southern Research Institute, Birmingham, Alabama, United States of America.

ABSTRACT
While Mesd was discovered as a specialized molecular endoplasmic reticulum chaperone for the Wnt co-receptors LRP5 and LRP6, recombinant Mesd protein is able to bind to mature LRP5 and LRP6 on the cell surface and acts as a universal antagonist of LRP5/6 modulators. In our previous study, we found that the C-terminal region of Mesd, which is absent in sequences from invertebrates, is necessary and sufficient for binding to mature LRP6 on the cell surface. In the present studies, we further characterized the interaction between the C-terminal region Mesd peptide and LRP5/6. We found that Mesd C-terminal region-derived peptides block Mesd binding to LRP5 at the cell surface too. We also showed that there are two LRP5/6 binding sites within Mesd C-terminal region which contain several positively charged residues. Moreover, we demonstrated that the Mesd C-terminal region peptide, like the full-length Mesd protein, blocked Wnt 3A- and Rspodin1-induced Wnt/β-catenin signaling in LRP5- and LRP6- expressing cells, suppressed Wnt/β-catenin signaling in human breast HS578T cells and prostate cancer PC-3 cells, and inhibited cancer cell proliferation, although the full-length Mesd protein is more potent than its peptide. Finally, we found that treatment of the full-length Mesd protein and its C-terminal region peptide significantly increased chemotherapy agent Adriamycin-induced cytotoxicity in HS578T and PC-3 cells. Together, our results suggest that Mesd C-terminal region constitutes the major LRP5/6-binding domain, and that Mesd protein and its C-terminal region peptide have a potential therapeutic value in cancer.

Show MeSH
Related in: MedlinePlus