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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 inhibits prostate cancer PC-3 and breast cancer HS578T cell proliferation.(A) Cancer cells in 6-well plates were treated with mouse Mesd (mMesd, 2 µM), human Mesd peptide hMesd (160–197) (4 µM) or control peptide (4 µM) in RPMI-1640 medium containing 2% FBS for PC-3 cells or DMEM medium containing 2% FBS for HS578T for 10 days. The media were changed every other day, and cells were harvested and counted using the trypan blue exclusion assay. Values are averages of three determinations with the standard deviations indicated by error bars. ** P<0.01 compared to the cells treated with PBS or control peptide. (B) Cancer cells in T-25 flasks were treated with human Mesd peptide hMesd (160–197) (2 µM) or control peptide (2 µM) in the culture medium containing 2% FBS for 7 days. The media were changed every other day. The cells were then harvested and seeded into 96-well tissue culture plates at a density of 5000 cells/well with hMesd (160–197) (2 µM) or control peptide (2 µM) in the culture medium containing 10% FBS for 2 days. Cell proliferation was measured by BrdU proliferation ELISA. All the values are the average of six determinations with the s.d. indicated by error bars. **P<0.01 compared to cells treated with control peptide.
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pone-0058102-g007: Mesd C-terminal region peptide inhibits prostate cancer PC-3 and breast cancer HS578T cell proliferation.(A) Cancer cells in 6-well plates were treated with mouse Mesd (mMesd, 2 µM), human Mesd peptide hMesd (160–197) (4 µM) or control peptide (4 µM) in RPMI-1640 medium containing 2% FBS for PC-3 cells or DMEM medium containing 2% FBS for HS578T for 10 days. The media were changed every other day, and cells were harvested and counted using the trypan blue exclusion assay. Values are averages of three determinations with the standard deviations indicated by error bars. ** P<0.01 compared to the cells treated with PBS or control peptide. (B) Cancer cells in T-25 flasks were treated with human Mesd peptide hMesd (160–197) (2 µM) or control peptide (2 µM) in the culture medium containing 2% FBS for 7 days. The media were changed every other day. The cells were then harvested and seeded into 96-well tissue culture plates at a density of 5000 cells/well with hMesd (160–197) (2 µM) or control peptide (2 µM) in the culture medium containing 10% FBS for 2 days. Cell proliferation was measured by BrdU proliferation ELISA. All the values are the average of six determinations with the s.d. indicated by error bars. **P<0.01 compared to cells treated with control peptide.

Mentions: Having established that the Mesd C-terminal region peptide suppresses Wnt/β-catenin signaling in PC-3 and HS578T cells, we then examined the effect of the Mesd C-terminal region peptide on cancer cell proliferation. As seen in Figure 7, hMesd (160–197), but not the control peptide, mimicked Mesd protein and displayed inhibitory effect on PC-3 and HS578T cell proliferation in a time-dependent manner (Figure 7A). To confirm the inhibitory effect of the Mesd peptide on cancer cell proliferation, we performed BrdU incorporation assay after the cells were treated with peptides. It was found that the Mesd C-terminal region peptide decreased BrdU incorporation into PC-3 and HS578T cells (Figure 7B).


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 inhibits prostate cancer PC-3 and breast cancer HS578T cell proliferation.(A) Cancer cells in 6-well plates were treated with mouse Mesd (mMesd, 2 µM), human Mesd peptide hMesd (160–197) (4 µM) or control peptide (4 µM) in RPMI-1640 medium containing 2% FBS for PC-3 cells or DMEM medium containing 2% FBS for HS578T for 10 days. The media were changed every other day, and cells were harvested and counted using the trypan blue exclusion assay. Values are averages of three determinations with the standard deviations indicated by error bars. ** P<0.01 compared to the cells treated with PBS or control peptide. (B) Cancer cells in T-25 flasks were treated with human Mesd peptide hMesd (160–197) (2 µM) or control peptide (2 µM) in the culture medium containing 2% FBS for 7 days. The media were changed every other day. The cells were then harvested and seeded into 96-well tissue culture plates at a density of 5000 cells/well with hMesd (160–197) (2 µM) or control peptide (2 µM) in the culture medium containing 10% FBS for 2 days. Cell proliferation was measured by BrdU proliferation ELISA. All the values are the average of six determinations with the s.d. indicated by error bars. **P<0.01 compared to cells treated with control peptide.
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pone-0058102-g007: Mesd C-terminal region peptide inhibits prostate cancer PC-3 and breast cancer HS578T cell proliferation.(A) Cancer cells in 6-well plates were treated with mouse Mesd (mMesd, 2 µM), human Mesd peptide hMesd (160–197) (4 µM) or control peptide (4 µM) in RPMI-1640 medium containing 2% FBS for PC-3 cells or DMEM medium containing 2% FBS for HS578T for 10 days. The media were changed every other day, and cells were harvested and counted using the trypan blue exclusion assay. Values are averages of three determinations with the standard deviations indicated by error bars. ** P<0.01 compared to the cells treated with PBS or control peptide. (B) Cancer cells in T-25 flasks were treated with human Mesd peptide hMesd (160–197) (2 µM) or control peptide (2 µM) in the culture medium containing 2% FBS for 7 days. The media were changed every other day. The cells were then harvested and seeded into 96-well tissue culture plates at a density of 5000 cells/well with hMesd (160–197) (2 µM) or control peptide (2 µM) in the culture medium containing 10% FBS for 2 days. Cell proliferation was measured by BrdU proliferation ELISA. All the values are the average of six determinations with the s.d. indicated by error bars. **P<0.01 compared to cells treated with control peptide.
Mentions: Having established that the Mesd C-terminal region peptide suppresses Wnt/β-catenin signaling in PC-3 and HS578T cells, we then examined the effect of the Mesd C-terminal region peptide on cancer cell proliferation. As seen in Figure 7, hMesd (160–197), but not the control peptide, mimicked Mesd protein and displayed inhibitory effect on PC-3 and HS578T cell proliferation in a time-dependent manner (Figure 7A). To confirm the inhibitory effect of the Mesd peptide on cancer cell proliferation, we performed BrdU incorporation assay after the cells were treated with peptides. It was found that the Mesd C-terminal region peptide decreased BrdU incorporation into PC-3 and HS578T cells (Figure 7B).

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