Limits...
Serglycin is implicated in the promotion of aggressive phenotype of breast cancer cells.

Korpetinou A, Skandalis SS, Moustakas A, Happonen KE, Tveit H, Prydz K, Labropoulou VT, Giannopoulou E, Kalofonos HP, Blom AM, Karamanos NK, Theocharis AD - PLoS ONE (2013)

Bottom Line: Serglycin exhibited a strong cytoplasmic staining in these cells, observable at the cell periphery in a thread of filaments near the cell membrane, but also in filopodia-like structures.Serglycin was purified from conditioned medium of MDA-MB-231 cells, and represented the major proteoglycan secreted by these cells, having a molecular size of ~ 250 kDa and carrying chondroitin sulfate side chains, mainly composed of 4-sulfated (~ 87%), 6-sulfated (~ 10%) and non-sulfated (~ 3%) disaccharides.Interestingly, over-expression of serglycin lacking the glycosaminoglycan attachment sites failed to promote these cellular functions, suggesting that glycanation of serglycin is a pre-requisite for its oncogenic properties.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Biochemistry, Department of Chemistry, University of Patras, Patras, Greece.

ABSTRACT
Serglycin is a proteoglycan expressed by some malignant cells. It promotes metastasis and protects some tumor cells from complement system attack. In the present study, we show for the first time the in situ expression of serglycin by breast cancer cells by immunohistochemistry in patients' material. Moreover, we demonstrate high expression and constitutive secretion of serglycin in the aggressive MDA-MB-231 breast cancer cell line. Serglycin exhibited a strong cytoplasmic staining in these cells, observable at the cell periphery in a thread of filaments near the cell membrane, but also in filopodia-like structures. Serglycin was purified from conditioned medium of MDA-MB-231 cells, and represented the major proteoglycan secreted by these cells, having a molecular size of ~ 250 kDa and carrying chondroitin sulfate side chains, mainly composed of 4-sulfated (~ 87%), 6-sulfated (~ 10%) and non-sulfated (~ 3%) disaccharides. Purified serglycin inhibited early steps of both the classical and the lectin pathways of complement by binding to C1q and mannose-binding lectin. Stable expression of serglycin in less aggressive MCF-7 breast cancer cells induced their proliferation, anchorage-independent growth, migration and invasion. Interestingly, over-expression of serglycin lacking the glycosaminoglycan attachment sites failed to promote these cellular functions, suggesting that glycanation of serglycin is a pre-requisite for its oncogenic properties. Our findings suggest that serglycin promotes a more aggressive cancer cell phenotype and may protect breast cancer cells from complement attack supporting their survival and expansion.

Show MeSH

Related in: MedlinePlus

Over-expression of glycanated serglycin increases breast cancer cell migration.Cells (5×105) were plated in triplicates in 12 well plates and cultured until confluence. Then wounds were made using a sterile pipette tip, debris was removed and fresh culture medium was added. The cells were monitored at 0, 24 and 48 h and were photographed (A). Wound areas were quantified at various time intervals using Image J software (B). Data are given as means and SD of three independent experiments. Statistical significance of variances was calculated using a one-way ANOVA test. Asterisk (*) indicates statistically significant differences (p<0.05). (C) Migratory properties of the cells were also evaluated by Transwell migration assay. 1×105 cells were suspended in culture medium supplemented with 0.5% FBS and loaded onto the top of Transwell chambers. Cells were then maintained in Transwell chambers for 48 h with 10% FBS as chemotactic stimuli in the bottom chamber. Transmigrating cells were stained with Giemsa and counted. Data are given as means and SD of three independent experiments. Statistical significance of variances was calculated using a one-way ANOVA test. Asterisk (*) indicates statistically significant differences (p<0.05). (D) In a set of experiments, cells were cultured on glass coverslips. After wounding with a sterile pipette tip, debris was removed, and cells were either immediately fixed in 3% formaldehyde in PBS (t = 0 h) or cultured for 24 h and then fixed. Immunofluorescence staining for serglycin (green), nuclei (blue) and F-actin (red) in MCF-7VSG cells was performed. Bars, 25 µm.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC3815026&req=5

pone-0078157-g008: Over-expression of glycanated serglycin increases breast cancer cell migration.Cells (5×105) were plated in triplicates in 12 well plates and cultured until confluence. Then wounds were made using a sterile pipette tip, debris was removed and fresh culture medium was added. The cells were monitored at 0, 24 and 48 h and were photographed (A). Wound areas were quantified at various time intervals using Image J software (B). Data are given as means and SD of three independent experiments. Statistical significance of variances was calculated using a one-way ANOVA test. Asterisk (*) indicates statistically significant differences (p<0.05). (C) Migratory properties of the cells were also evaluated by Transwell migration assay. 1×105 cells were suspended in culture medium supplemented with 0.5% FBS and loaded onto the top of Transwell chambers. Cells were then maintained in Transwell chambers for 48 h with 10% FBS as chemotactic stimuli in the bottom chamber. Transmigrating cells were stained with Giemsa and counted. Data are given as means and SD of three independent experiments. Statistical significance of variances was calculated using a one-way ANOVA test. Asterisk (*) indicates statistically significant differences (p<0.05). (D) In a set of experiments, cells were cultured on glass coverslips. After wounding with a sterile pipette tip, debris was removed, and cells were either immediately fixed in 3% formaldehyde in PBS (t = 0 h) or cultured for 24 h and then fixed. Immunofluorescence staining for serglycin (green), nuclei (blue) and F-actin (red) in MCF-7VSG cells was performed. Bars, 25 µm.

Mentions: To evaluate the migratory capacity of breast cancer cells over-expressing serglycin, we performed a wound healing assay (Fig. 8A). We found that over-expression of intact serglycin (MCF-7VSG) markedly induced cell migration 24 h after wounding, and closure of the wound was achieved in 48 h (Fig. 8B). Mock-transfected MCF cells (MCF-7V) and MCF-7VSG/−GAG exhibited much lower migration 24 h post-wounding (approximately 50% less), and closure was not achieved at 48 h after wounding (Fig. 8A and B). We also measured the motility of cancer cells using a transwell assay. We found that MCF-7VSG cells expressing intact serglycin migrated significantly faster than mock transfected MCF-7V and MCF-7VSG/−GAG cells (Fig. 8C). In parallel wound healing assay experiments, MCF-7VSG cells that were at the edge of the wound at time 0 h as well as migratory cells at time 24 h were stained for serglycin and F-actin (Fig. 8D). Serglycin showed a cytoplasmic and pericellular membrane associated localization in MCF-7VSG grown at confluence and co-localized with the cortical F-actin on cell-cell adhesions (Fig. 8D, t = 0 h). In migratory cells, serglycin showed a filamentous cytoplasmic staining, but was also present in filopodia-like structures, co-localizing with actin (Fig. 8D, t = 24 h). The potential of cancer cells to invade through matrigel was also examined. Cells over-expressing serglycin (MCF-7VSG) exhibited significantly higher capability to invade through matrigel compared to mock transfected MCF-7V cells and cells expressing non-glycanated serglycin (MCF-7VSG/−GAG) (Fig. 9A and B).


Serglycin is implicated in the promotion of aggressive phenotype of breast cancer cells.

Korpetinou A, Skandalis SS, Moustakas A, Happonen KE, Tveit H, Prydz K, Labropoulou VT, Giannopoulou E, Kalofonos HP, Blom AM, Karamanos NK, Theocharis AD - PLoS ONE (2013)

Over-expression of glycanated serglycin increases breast cancer cell migration.Cells (5×105) were plated in triplicates in 12 well plates and cultured until confluence. Then wounds were made using a sterile pipette tip, debris was removed and fresh culture medium was added. The cells were monitored at 0, 24 and 48 h and were photographed (A). Wound areas were quantified at various time intervals using Image J software (B). Data are given as means and SD of three independent experiments. Statistical significance of variances was calculated using a one-way ANOVA test. Asterisk (*) indicates statistically significant differences (p<0.05). (C) Migratory properties of the cells were also evaluated by Transwell migration assay. 1×105 cells were suspended in culture medium supplemented with 0.5% FBS and loaded onto the top of Transwell chambers. Cells were then maintained in Transwell chambers for 48 h with 10% FBS as chemotactic stimuli in the bottom chamber. Transmigrating cells were stained with Giemsa and counted. Data are given as means and SD of three independent experiments. Statistical significance of variances was calculated using a one-way ANOVA test. Asterisk (*) indicates statistically significant differences (p<0.05). (D) In a set of experiments, cells were cultured on glass coverslips. After wounding with a sterile pipette tip, debris was removed, and cells were either immediately fixed in 3% formaldehyde in PBS (t = 0 h) or cultured for 24 h and then fixed. Immunofluorescence staining for serglycin (green), nuclei (blue) and F-actin (red) in MCF-7VSG cells was performed. Bars, 25 µm.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3815026&req=5

pone-0078157-g008: Over-expression of glycanated serglycin increases breast cancer cell migration.Cells (5×105) were plated in triplicates in 12 well plates and cultured until confluence. Then wounds were made using a sterile pipette tip, debris was removed and fresh culture medium was added. The cells were monitored at 0, 24 and 48 h and were photographed (A). Wound areas were quantified at various time intervals using Image J software (B). Data are given as means and SD of three independent experiments. Statistical significance of variances was calculated using a one-way ANOVA test. Asterisk (*) indicates statistically significant differences (p<0.05). (C) Migratory properties of the cells were also evaluated by Transwell migration assay. 1×105 cells were suspended in culture medium supplemented with 0.5% FBS and loaded onto the top of Transwell chambers. Cells were then maintained in Transwell chambers for 48 h with 10% FBS as chemotactic stimuli in the bottom chamber. Transmigrating cells were stained with Giemsa and counted. Data are given as means and SD of three independent experiments. Statistical significance of variances was calculated using a one-way ANOVA test. Asterisk (*) indicates statistically significant differences (p<0.05). (D) In a set of experiments, cells were cultured on glass coverslips. After wounding with a sterile pipette tip, debris was removed, and cells were either immediately fixed in 3% formaldehyde in PBS (t = 0 h) or cultured for 24 h and then fixed. Immunofluorescence staining for serglycin (green), nuclei (blue) and F-actin (red) in MCF-7VSG cells was performed. Bars, 25 µm.
Mentions: To evaluate the migratory capacity of breast cancer cells over-expressing serglycin, we performed a wound healing assay (Fig. 8A). We found that over-expression of intact serglycin (MCF-7VSG) markedly induced cell migration 24 h after wounding, and closure of the wound was achieved in 48 h (Fig. 8B). Mock-transfected MCF cells (MCF-7V) and MCF-7VSG/−GAG exhibited much lower migration 24 h post-wounding (approximately 50% less), and closure was not achieved at 48 h after wounding (Fig. 8A and B). We also measured the motility of cancer cells using a transwell assay. We found that MCF-7VSG cells expressing intact serglycin migrated significantly faster than mock transfected MCF-7V and MCF-7VSG/−GAG cells (Fig. 8C). In parallel wound healing assay experiments, MCF-7VSG cells that were at the edge of the wound at time 0 h as well as migratory cells at time 24 h were stained for serglycin and F-actin (Fig. 8D). Serglycin showed a cytoplasmic and pericellular membrane associated localization in MCF-7VSG grown at confluence and co-localized with the cortical F-actin on cell-cell adhesions (Fig. 8D, t = 0 h). In migratory cells, serglycin showed a filamentous cytoplasmic staining, but was also present in filopodia-like structures, co-localizing with actin (Fig. 8D, t = 24 h). The potential of cancer cells to invade through matrigel was also examined. Cells over-expressing serglycin (MCF-7VSG) exhibited significantly higher capability to invade through matrigel compared to mock transfected MCF-7V cells and cells expressing non-glycanated serglycin (MCF-7VSG/−GAG) (Fig. 9A and B).

Bottom Line: Serglycin exhibited a strong cytoplasmic staining in these cells, observable at the cell periphery in a thread of filaments near the cell membrane, but also in filopodia-like structures.Serglycin was purified from conditioned medium of MDA-MB-231 cells, and represented the major proteoglycan secreted by these cells, having a molecular size of ~ 250 kDa and carrying chondroitin sulfate side chains, mainly composed of 4-sulfated (~ 87%), 6-sulfated (~ 10%) and non-sulfated (~ 3%) disaccharides.Interestingly, over-expression of serglycin lacking the glycosaminoglycan attachment sites failed to promote these cellular functions, suggesting that glycanation of serglycin is a pre-requisite for its oncogenic properties.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Biochemistry, Department of Chemistry, University of Patras, Patras, Greece.

ABSTRACT
Serglycin is a proteoglycan expressed by some malignant cells. It promotes metastasis and protects some tumor cells from complement system attack. In the present study, we show for the first time the in situ expression of serglycin by breast cancer cells by immunohistochemistry in patients' material. Moreover, we demonstrate high expression and constitutive secretion of serglycin in the aggressive MDA-MB-231 breast cancer cell line. Serglycin exhibited a strong cytoplasmic staining in these cells, observable at the cell periphery in a thread of filaments near the cell membrane, but also in filopodia-like structures. Serglycin was purified from conditioned medium of MDA-MB-231 cells, and represented the major proteoglycan secreted by these cells, having a molecular size of ~ 250 kDa and carrying chondroitin sulfate side chains, mainly composed of 4-sulfated (~ 87%), 6-sulfated (~ 10%) and non-sulfated (~ 3%) disaccharides. Purified serglycin inhibited early steps of both the classical and the lectin pathways of complement by binding to C1q and mannose-binding lectin. Stable expression of serglycin in less aggressive MCF-7 breast cancer cells induced their proliferation, anchorage-independent growth, migration and invasion. Interestingly, over-expression of serglycin lacking the glycosaminoglycan attachment sites failed to promote these cellular functions, suggesting that glycanation of serglycin is a pre-requisite for its oncogenic properties. Our findings suggest that serglycin promotes a more aggressive cancer cell phenotype and may protect breast cancer cells from complement attack supporting their survival and expansion.

Show MeSH
Related in: MedlinePlus