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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 Wnt1-induced 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 Wnt1 plasmid along 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 transiently transfected with Wnt1 plasmid or the corresponding control vector. After being incubated for 24 h, cells 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 Wnt1, 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-g006: Mesd C-terminal region peptide blocks Wnt1-induced 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 Wnt1 plasmid along 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 transiently transfected with Wnt1 plasmid or the corresponding control vector. After being incubated for 24 h, cells 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 Wnt1, 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: Wnt1 regulates cellular differentiation and proliferation of the mammary epithelium, and Wnt1 up-regulation in the mammary gland has been shown to induce mammary hyperplasia and adenocarcinoma [34], [35]. Overexpression of Wnt1 was found in the majority of prostate carcinoma specimens [36]. To further characterize the inhibitory effect of the Mesd C-terminal region peptide in prostate and breast cancer cells, we transiently transfected PC-3 and HS578T cells with Wnt1 and Wnt reporter plasmids and treated with Mesd protein or human Mesd peptide hMesd (160–197). We found again that hMesd (160–197), but not the control peptide, mimicked Mesd protein to significantly repress Wnt reporter luciferase activity induced by Wnt1 in PC3 and HS578T cells (Figure 6A). Moreover, hMesd (160–197) greatly decreased the levels of cytosolic free β-catenin, LRP6 phosphorylation and Wnt/β-catenin signaling targets Axin2 and cyclin D1 in Wnt1-expressing PC-3 and HS578T cells (Figure 6B).


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 Wnt1-induced 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 Wnt1 plasmid along 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 transiently transfected with Wnt1 plasmid or the corresponding control vector. After being incubated for 24 h, cells 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 Wnt1, 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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pone-0058102-g006: Mesd C-terminal region peptide blocks Wnt1-induced 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 Wnt1 plasmid along 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 transiently transfected with Wnt1 plasmid or the corresponding control vector. After being incubated for 24 h, cells 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 Wnt1, 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: Wnt1 regulates cellular differentiation and proliferation of the mammary epithelium, and Wnt1 up-regulation in the mammary gland has been shown to induce mammary hyperplasia and adenocarcinoma [34], [35]. Overexpression of Wnt1 was found in the majority of prostate carcinoma specimens [36]. To further characterize the inhibitory effect of the Mesd C-terminal region peptide in prostate and breast cancer cells, we transiently transfected PC-3 and HS578T cells with Wnt1 and Wnt reporter plasmids and treated with Mesd protein or human Mesd peptide hMesd (160–197). We found again that hMesd (160–197), but not the control peptide, mimicked Mesd protein to significantly repress Wnt reporter luciferase activity induced by Wnt1 in PC3 and HS578T cells (Figure 6A). Moreover, hMesd (160–197) greatly decreased the levels of cytosolic free β-catenin, LRP6 phosphorylation and Wnt/β-catenin signaling targets Axin2 and cyclin D1 in Wnt1-expressing PC-3 and HS578T cells (Figure 6B).

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