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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

WNT signaling pathway in SULF2 ovexpressing prostate cancer cells. DU-145 and PC3 cells were immunostained with WNT 3A and β-catenin antibodies (R&D) and analyzed by flow cytometry, as described in methods. The graphics represent relative quantity of positive cells (A). Representative pictures are shown, indicating the percent of double-stained cells (B). Prostate cancer cells were immunostained for β-catenin and analyzed by confocal microscopy. The nuclei (blue) were stained with DAPI. (C). (VECTOR: cells transfected with empty vector, SULF2: cells transfected with SULF2 expressing plasmid). *P ≤ 0.05.
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Fig7: WNT signaling pathway in SULF2 ovexpressing prostate cancer cells. DU-145 and PC3 cells were immunostained with WNT 3A and β-catenin antibodies (R&D) and analyzed by flow cytometry, as described in methods. The graphics represent relative quantity of positive cells (A). Representative pictures are shown, indicating the percent of double-stained cells (B). Prostate cancer cells were immunostained for β-catenin and analyzed by confocal microscopy. The nuclei (blue) were stained with DAPI. (C). (VECTOR: cells transfected with empty vector, SULF2: cells transfected with SULF2 expressing plasmid). *P ≤ 0.05.

Mentions: Previous studies have shown that SULFs regulate WNT signaling pathway [25,37]. Therefore, we analyzed the presence of WNT 3A and β-catenin in DU-145 and PC3 cells overexpressing SULF2. By flow cytomery, we observed an increase of active unphosphorylated β-catenin in both SULF2 transfected cells (Figure 7A). Moreover, the proportion of cells presenting both WNT 3A and β-catenin (Figure 7B) also increased. DU-145 cancer cells overexpressing SULF2 showed 33.7% of cells double stained for WNT 3A and β-catenin respectively, in comparison to 20.7% in control cells transfected with empty vector. PC3 cells overexpressing SULF2 showed 40.2% of cells double stained for WNT 3A and β-catenin respectively, compared to 26.0% in control cells transfected with empty vector. Finally, by confocal microscopy, we could detect β-catenin located close to the cells membrane in control cells, while cells with forced expression of SULF2, presented a nuclear staining for β-catenin (arrows, Figure 7C).Figure 7


SULF2 overexpression positively regulates tumorigenicity of human prostate cancer cells.

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

WNT signaling pathway in SULF2 ovexpressing prostate cancer cells. DU-145 and PC3 cells were immunostained with WNT 3A and β-catenin antibodies (R&D) and analyzed by flow cytometry, as described in methods. The graphics represent relative quantity of positive cells (A). Representative pictures are shown, indicating the percent of double-stained cells (B). Prostate cancer cells were immunostained for β-catenin and analyzed by confocal microscopy. The nuclei (blue) were stained with DAPI. (C). (VECTOR: cells transfected with empty vector, SULF2: cells transfected with SULF2 expressing plasmid). *P ≤ 0.05.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Fig7: WNT signaling pathway in SULF2 ovexpressing prostate cancer cells. DU-145 and PC3 cells were immunostained with WNT 3A and β-catenin antibodies (R&D) and analyzed by flow cytometry, as described in methods. The graphics represent relative quantity of positive cells (A). Representative pictures are shown, indicating the percent of double-stained cells (B). Prostate cancer cells were immunostained for β-catenin and analyzed by confocal microscopy. The nuclei (blue) were stained with DAPI. (C). (VECTOR: cells transfected with empty vector, SULF2: cells transfected with SULF2 expressing plasmid). *P ≤ 0.05.
Mentions: Previous studies have shown that SULFs regulate WNT signaling pathway [25,37]. Therefore, we analyzed the presence of WNT 3A and β-catenin in DU-145 and PC3 cells overexpressing SULF2. By flow cytomery, we observed an increase of active unphosphorylated β-catenin in both SULF2 transfected cells (Figure 7A). Moreover, the proportion of cells presenting both WNT 3A and β-catenin (Figure 7B) also increased. DU-145 cancer cells overexpressing SULF2 showed 33.7% of cells double stained for WNT 3A and β-catenin respectively, in comparison to 20.7% in control cells transfected with empty vector. PC3 cells overexpressing SULF2 showed 40.2% of cells double stained for WNT 3A and β-catenin respectively, compared to 26.0% in control cells transfected with empty vector. Finally, by confocal microscopy, we could detect β-catenin located close to the cells membrane in control cells, while cells with forced expression of SULF2, presented a nuclear staining for β-catenin (arrows, Figure 7C).Figure 7

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