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SULF2 overexpression positively regulates tumorigenicity of human prostate cancer cells.

Vicente CM, Lima MA, Nader HB, Toma L - J. Exp. Clin. Cancer Res. (2015)

Bottom Line: Transfection of DU-145 and PC3 prostate cancer cells with SULF2 resulted in increased viability, which did not occur with normal prostate cells.The effect was reverted by the knockdown of SULF2 using specific siRNAs.Detailed structural analysis of HS from cells overexpressing SULF2 showed a reduction of the trisulfated disaccharide UA(2S)-GlcNS(6S).

View Article: PubMed Central - PubMed

Affiliation: Departamento de Bioquímica, Disciplina de Biologia Molecular, Universidade Federal de São Paulo, UNIFESP, Rua Três de Maio, 100 - 4° andar, Vila Clementino, CEP 04044-020, São Paulo, SP, Brazil. carolmv@yahoo.com.

ABSTRACT

Background: SULF2 is a 6-O-endosulfatase which removes 6-O sulfate residues from N-glucosamine present on heparan sulfate (HS). The sulfation pattern of HS influences signaling events mediated by heparan sulfate proteoglycans (HSPGs) located on cell surface, which are critical for the interactions with growth factors and their receptors. Alterations in SULF2 expression have been identified in the context of several cancer types but its function in cancer is still unclear where the precise molecular mechanism involved has not been fully deciphered. To further investigate SULF2 role in tumorigenesis, we overexpressed such gene in prostate cancer cell lines.

Methods: The normal prostate epithelial cell line RWPE-1 and the prostate cancer cells DU-145, and PC3 were transfected with SULF2-expressing plasmid pcDNA3.1/Myc-His(-)-Hsulf-2. Transfected cells were then submitted to viability, migration and colony formation assays.

Results: Transfection of DU-145 and PC3 prostate cancer cells with SULF2 resulted in increased viability, which did not occur with normal prostate cells. The effect was reverted by the knockdown of SULF2 using specific siRNAs. Furthermore, forced expression of SULF2 augmented cell migration and colony formation in both prostate cell lines. Detailed structural analysis of HS from cells overexpressing SULF2 showed a reduction of the trisulfated disaccharide UA(2S)-GlcNS(6S). There was an increase in epithelial-mesenchymal transition markers and an increase in WNT signaling pathway.

Conclusions: These results indicate that SULF2 have a pro-tumorigenic effect in DU-145 and PC3 cancer cells, suggesting an important role of this enzyme in prostatic cancer metastasis.

No MeSH data available.


Related in: MedlinePlus

SULF2 enzymatic activity in prostate cancer cells. GAGs labeled with [35S]Na2SO4. were purified from the culture medium (MEDIUM), the cancer cells (CELL) extracted with EDTA, and the matrix (MATRIX) produced by cells. The content of GAGs from these compartments was analyzed through agarose gel electrophoresis. The gel was exposed to a radiosensitive screen and quantification was performed by densitometry with Opti-Quanti Software (A). Each sample was digested using a mixture of heparin lyases and analyzed on a PhenoSphere™ 5 μm SAX 80 Å LC Column 150 × 4.6 mm. Δ-disaccharide were eluted with a linear gradient of NaCl 0–1 M over a 30-min period at a flow rate of 1 ml.min-1. Individual fractions (0.5 ml) were collected and counted using a micro-beta counter (B). The bars indicate the average of three independent experiments. The arrows show the trisulfated disaccharide UA(2S)-GlcNS(6S). *P ≤ 0.05.
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Fig2: SULF2 enzymatic activity in prostate cancer cells. GAGs labeled with [35S]Na2SO4. were purified from the culture medium (MEDIUM), the cancer cells (CELL) extracted with EDTA, and the matrix (MATRIX) produced by cells. The content of GAGs from these compartments was analyzed through agarose gel electrophoresis. The gel was exposed to a radiosensitive screen and quantification was performed by densitometry with Opti-Quanti Software (A). Each sample was digested using a mixture of heparin lyases and analyzed on a PhenoSphere™ 5 μm SAX 80 Å LC Column 150 × 4.6 mm. Δ-disaccharide were eluted with a linear gradient of NaCl 0–1 M over a 30-min period at a flow rate of 1 ml.min-1. Individual fractions (0.5 ml) were collected and counted using a micro-beta counter (B). The bars indicate the average of three independent experiments. The arrows show the trisulfated disaccharide UA(2S)-GlcNS(6S). *P ≤ 0.05.

Mentions: In order to analyze whether the forced overexpression of SULF2 gene resulted in up-regulation of the active enzyme, we verified the content of sulfated HS in PC3 and DU-145 transfected cells. Indeed, we observed a decrease of approximately 50% of sulfated HS in all of the compartments studied, medium, cell, and ECM, in both cells (Figure 2A). We also performed the analyses of HS disaccharides from PC3 and DU-145 transfected cells, using a strong anion-exchange (SAX) column. It was possible to observe an expressive decrease of the trisulfated disaccharide UA(2S)-GlcNS(6S) (Figure 2B). Our result is consistent with previous data from the literature, which describes that the trisulfated disaccharide from HS is the main substrate for both SULF1 and SULF2 [24,26,37,38].Figure 2


SULF2 overexpression positively regulates tumorigenicity of human prostate cancer cells.

Vicente CM, Lima MA, Nader HB, Toma L - J. Exp. Clin. Cancer Res. (2015)

SULF2 enzymatic activity in prostate cancer cells. GAGs labeled with [35S]Na2SO4. were purified from the culture medium (MEDIUM), the cancer cells (CELL) extracted with EDTA, and the matrix (MATRIX) produced by cells. The content of GAGs from these compartments was analyzed through agarose gel electrophoresis. The gel was exposed to a radiosensitive screen and quantification was performed by densitometry with Opti-Quanti Software (A). Each sample was digested using a mixture of heparin lyases and analyzed on a PhenoSphere™ 5 μm SAX 80 Å LC Column 150 × 4.6 mm. Δ-disaccharide were eluted with a linear gradient of NaCl 0–1 M over a 30-min period at a flow rate of 1 ml.min-1. Individual fractions (0.5 ml) were collected and counted using a micro-beta counter (B). The bars indicate the average of three independent experiments. The arrows show the trisulfated disaccharide UA(2S)-GlcNS(6S). *P ≤ 0.05.
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4374423&req=5

Fig2: SULF2 enzymatic activity in prostate cancer cells. GAGs labeled with [35S]Na2SO4. were purified from the culture medium (MEDIUM), the cancer cells (CELL) extracted with EDTA, and the matrix (MATRIX) produced by cells. The content of GAGs from these compartments was analyzed through agarose gel electrophoresis. The gel was exposed to a radiosensitive screen and quantification was performed by densitometry with Opti-Quanti Software (A). Each sample was digested using a mixture of heparin lyases and analyzed on a PhenoSphere™ 5 μm SAX 80 Å LC Column 150 × 4.6 mm. Δ-disaccharide were eluted with a linear gradient of NaCl 0–1 M over a 30-min period at a flow rate of 1 ml.min-1. Individual fractions (0.5 ml) were collected and counted using a micro-beta counter (B). The bars indicate the average of three independent experiments. The arrows show the trisulfated disaccharide UA(2S)-GlcNS(6S). *P ≤ 0.05.
Mentions: In order to analyze whether the forced overexpression of SULF2 gene resulted in up-regulation of the active enzyme, we verified the content of sulfated HS in PC3 and DU-145 transfected cells. Indeed, we observed a decrease of approximately 50% of sulfated HS in all of the compartments studied, medium, cell, and ECM, in both cells (Figure 2A). We also performed the analyses of HS disaccharides from PC3 and DU-145 transfected cells, using a strong anion-exchange (SAX) column. It was possible to observe an expressive decrease of the trisulfated disaccharide UA(2S)-GlcNS(6S) (Figure 2B). Our result is consistent with previous data from the literature, which describes that the trisulfated disaccharide from HS is the main substrate for both SULF1 and SULF2 [24,26,37,38].Figure 2

Bottom Line: Transfection of DU-145 and PC3 prostate cancer cells with SULF2 resulted in increased viability, which did not occur with normal prostate cells.The effect was reverted by the knockdown of SULF2 using specific siRNAs.Detailed structural analysis of HS from cells overexpressing SULF2 showed a reduction of the trisulfated disaccharide UA(2S)-GlcNS(6S).

View Article: PubMed Central - PubMed

Affiliation: Departamento de Bioquímica, Disciplina de Biologia Molecular, Universidade Federal de São Paulo, UNIFESP, Rua Três de Maio, 100 - 4° andar, Vila Clementino, CEP 04044-020, São Paulo, SP, Brazil. carolmv@yahoo.com.

ABSTRACT

Background: SULF2 is a 6-O-endosulfatase which removes 6-O sulfate residues from N-glucosamine present on heparan sulfate (HS). The sulfation pattern of HS influences signaling events mediated by heparan sulfate proteoglycans (HSPGs) located on cell surface, which are critical for the interactions with growth factors and their receptors. Alterations in SULF2 expression have been identified in the context of several cancer types but its function in cancer is still unclear where the precise molecular mechanism involved has not been fully deciphered. To further investigate SULF2 role in tumorigenesis, we overexpressed such gene in prostate cancer cell lines.

Methods: The normal prostate epithelial cell line RWPE-1 and the prostate cancer cells DU-145, and PC3 were transfected with SULF2-expressing plasmid pcDNA3.1/Myc-His(-)-Hsulf-2. Transfected cells were then submitted to viability, migration and colony formation assays.

Results: Transfection of DU-145 and PC3 prostate cancer cells with SULF2 resulted in increased viability, which did not occur with normal prostate cells. The effect was reverted by the knockdown of SULF2 using specific siRNAs. Furthermore, forced expression of SULF2 augmented cell migration and colony formation in both prostate cell lines. Detailed structural analysis of HS from cells overexpressing SULF2 showed a reduction of the trisulfated disaccharide UA(2S)-GlcNS(6S). There was an increase in epithelial-mesenchymal transition markers and an increase in WNT signaling pathway.

Conclusions: These results indicate that SULF2 have a pro-tumorigenic effect in DU-145 and PC3 cancer cells, suggesting an important role of this enzyme in prostatic cancer metastasis.

No MeSH data available.


Related in: MedlinePlus