Limits...
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.

Show MeSH

Related in: MedlinePlus

Mesd protein and its C-terminal region peptide potentiate chemotherapy agent Adriamycin-induced cytotoxicity in PC-3 and HS578T cells.(A) Cancer cells in T-25 flasks were treated with mouse Mesd (2 µM) in RPMI-1640 medium containing 2% FBS for PC-3 cells or DMEM medium containing 2% FBS for HS578T for 4 days. The media were changed every other day, and the cells were harvested and seeded into 96-well tissue culture plates at a density of 5000 cells/well with Mesd (2 µM) and/or Adriamycin (0.5 µM) in RPMI-1640 medium containing 10% FBS for PC-3 cells or DMEM medium containing 10% FBS for HS578T for 2 days. Cell viability was then measured by the Cell Titer Glo Assay system. (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 cells were harvested and seeded into 96-well tissue culture plates at a density of 5000 cells/well with hMesd (160–197) (2 µM), control peptide (2 µM) and Adriamycin (0.5 µM) in the culture medium containing 10% FBS for 2 days. Cell viability was then measured by the Cell Titer Glo Assay system. All the values are the average of quadruple determinations with the s.d. indicated by error bars. *P<0.05, **P<0.01 compared to untreated cells or cells treated with control peptide. #P<0.05, ##P<0.01 compared to cells treated with Mesd alone, hMesd (160–197) alone or Adriamycin alone.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC3585277&req=5

pone-0058102-g008: Mesd protein and its C-terminal region peptide potentiate chemotherapy agent Adriamycin-induced cytotoxicity in PC-3 and HS578T cells.(A) Cancer cells in T-25 flasks were treated with mouse Mesd (2 µM) in RPMI-1640 medium containing 2% FBS for PC-3 cells or DMEM medium containing 2% FBS for HS578T for 4 days. The media were changed every other day, and the cells were harvested and seeded into 96-well tissue culture plates at a density of 5000 cells/well with Mesd (2 µM) and/or Adriamycin (0.5 µM) in RPMI-1640 medium containing 10% FBS for PC-3 cells or DMEM medium containing 10% FBS for HS578T for 2 days. Cell viability was then measured by the Cell Titer Glo Assay system. (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 cells were harvested and seeded into 96-well tissue culture plates at a density of 5000 cells/well with hMesd (160–197) (2 µM), control peptide (2 µM) and Adriamycin (0.5 µM) in the culture medium containing 10% FBS for 2 days. Cell viability was then measured by the Cell Titer Glo Assay system. All the values are the average of quadruple determinations with the s.d. indicated by error bars. *P<0.05, **P<0.01 compared to untreated cells or cells treated with control peptide. #P<0.05, ##P<0.01 compared to cells treated with Mesd alone, hMesd (160–197) alone or Adriamycin alone.

Mentions: Adriamycin is a common chemotherapy agent. We then tested whether Mesd protein and its C-terminal region peptide can increase chemotherapy agent Adriamycin-induced cytotoxicity in cancer cells. As seen in Figure 8, combination treatment caused more cytotoxicity in HS578T and PC-3 cells than individual agent treatment. For example, treatment of HS578T cells with Mesd protein (2 µM) alone and Adriamycin (0.5 µM) alone resulted in 25% and 69% inhibition of cell viability, respectively. However, when treated with Mesd protein plus Adriamycin, the cell viability of HS578T cells was reduced to 8% (Figure 8).


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 protein and its C-terminal region peptide potentiate chemotherapy agent Adriamycin-induced cytotoxicity in PC-3 and HS578T cells.(A) Cancer cells in T-25 flasks were treated with mouse Mesd (2 µM) in RPMI-1640 medium containing 2% FBS for PC-3 cells or DMEM medium containing 2% FBS for HS578T for 4 days. The media were changed every other day, and the cells were harvested and seeded into 96-well tissue culture plates at a density of 5000 cells/well with Mesd (2 µM) and/or Adriamycin (0.5 µM) in RPMI-1640 medium containing 10% FBS for PC-3 cells or DMEM medium containing 10% FBS for HS578T for 2 days. Cell viability was then measured by the Cell Titer Glo Assay system. (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 cells were harvested and seeded into 96-well tissue culture plates at a density of 5000 cells/well with hMesd (160–197) (2 µM), control peptide (2 µM) and Adriamycin (0.5 µM) in the culture medium containing 10% FBS for 2 days. Cell viability was then measured by the Cell Titer Glo Assay system. All the values are the average of quadruple determinations with the s.d. indicated by error bars. *P<0.05, **P<0.01 compared to untreated cells or cells treated with control peptide. #P<0.05, ##P<0.01 compared to cells treated with Mesd alone, hMesd (160–197) alone or Adriamycin alone.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3585277&req=5

pone-0058102-g008: Mesd protein and its C-terminal region peptide potentiate chemotherapy agent Adriamycin-induced cytotoxicity in PC-3 and HS578T cells.(A) Cancer cells in T-25 flasks were treated with mouse Mesd (2 µM) in RPMI-1640 medium containing 2% FBS for PC-3 cells or DMEM medium containing 2% FBS for HS578T for 4 days. The media were changed every other day, and the cells were harvested and seeded into 96-well tissue culture plates at a density of 5000 cells/well with Mesd (2 µM) and/or Adriamycin (0.5 µM) in RPMI-1640 medium containing 10% FBS for PC-3 cells or DMEM medium containing 10% FBS for HS578T for 2 days. Cell viability was then measured by the Cell Titer Glo Assay system. (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 cells were harvested and seeded into 96-well tissue culture plates at a density of 5000 cells/well with hMesd (160–197) (2 µM), control peptide (2 µM) and Adriamycin (0.5 µM) in the culture medium containing 10% FBS for 2 days. Cell viability was then measured by the Cell Titer Glo Assay system. All the values are the average of quadruple determinations with the s.d. indicated by error bars. *P<0.05, **P<0.01 compared to untreated cells or cells treated with control peptide. #P<0.05, ##P<0.01 compared to cells treated with Mesd alone, hMesd (160–197) alone or Adriamycin alone.
Mentions: Adriamycin is a common chemotherapy agent. We then tested whether Mesd protein and its C-terminal region peptide can increase chemotherapy agent Adriamycin-induced cytotoxicity in cancer cells. As seen in Figure 8, combination treatment caused more cytotoxicity in HS578T and PC-3 cells than individual agent treatment. For example, treatment of HS578T cells with Mesd protein (2 µM) alone and Adriamycin (0.5 µM) alone resulted in 25% and 69% inhibition of cell viability, respectively. However, when treated with Mesd protein plus Adriamycin, the cell viability of HS578T cells was reduced to 8% (Figure 8).

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