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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

Cocultures of prostate cancer cells overexpressing SULF2 and stromal cells. Prostate cancer cells (PC3 or DU-145) were seeded inside the O-ring and fibroblasts stromal cells were seeded around the O-ring. The O-ring was removed and the cultures maintained in the same conditions until the cells filled the O-ring area. The cells were firstly visualized in phase contrast in optical microscope in bright field. Immunolocalization of actin, SULF2, vimentin, and fibronectin were performed after fixation of the cells with 2% formaldehyde. The nuclei (blue) were stained with DAPI. F: fibroblasts, P: prostate cancer cells. Scale bar represents 100 μm.
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Fig8: Cocultures of prostate cancer cells overexpressing SULF2 and stromal cells. Prostate cancer cells (PC3 or DU-145) were seeded inside the O-ring and fibroblasts stromal cells were seeded around the O-ring. The O-ring was removed and the cultures maintained in the same conditions until the cells filled the O-ring area. The cells were firstly visualized in phase contrast in optical microscope in bright field. Immunolocalization of actin, SULF2, vimentin, and fibronectin were performed after fixation of the cells with 2% formaldehyde. The nuclei (blue) were stained with DAPI. F: fibroblasts, P: prostate cancer cells. Scale bar represents 100 μm.

Mentions: The O-ring co-culture system is an attempt to mimic a tumor microenvironment. The stromal cells are seeded and cultured immediately around the tumor cell line, allowing cell–cell contact besides establishing a gradient of soluble factors throughout the stromal cells, similar to that found in tissues [35]. As expected, after 2 days of culture, PC3 and DU-145 prostate cancer cells overexpressing SULF2 had already connected to fibroblasts, whereas the control cells transfected with empty vectors remained distant from fibroblasts (Figure 8). This probably occurred due to the increased migration presented by PC3 and DU-145 with forced expression of SULF2. Moreover, by using this system coupled to immunocytochemistry, we analyze the region of intersection between stromal cells and tumor cells (Figure 8). We observed an apparent increase in vimentin in the intersection area between cancer cells and fibroblasts for both transfected cells.Figure 8


SULF2 overexpression positively regulates tumorigenicity of human prostate cancer cells.

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

Cocultures of prostate cancer cells overexpressing SULF2 and stromal cells. Prostate cancer cells (PC3 or DU-145) were seeded inside the O-ring and fibroblasts stromal cells were seeded around the O-ring. The O-ring was removed and the cultures maintained in the same conditions until the cells filled the O-ring area. The cells were firstly visualized in phase contrast in optical microscope in bright field. Immunolocalization of actin, SULF2, vimentin, and fibronectin were performed after fixation of the cells with 2% formaldehyde. The nuclei (blue) were stained with DAPI. F: fibroblasts, P: prostate cancer cells. Scale bar represents 100 μm.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Fig8: Cocultures of prostate cancer cells overexpressing SULF2 and stromal cells. Prostate cancer cells (PC3 or DU-145) were seeded inside the O-ring and fibroblasts stromal cells were seeded around the O-ring. The O-ring was removed and the cultures maintained in the same conditions until the cells filled the O-ring area. The cells were firstly visualized in phase contrast in optical microscope in bright field. Immunolocalization of actin, SULF2, vimentin, and fibronectin were performed after fixation of the cells with 2% formaldehyde. The nuclei (blue) were stained with DAPI. F: fibroblasts, P: prostate cancer cells. Scale bar represents 100 μm.
Mentions: The O-ring co-culture system is an attempt to mimic a tumor microenvironment. The stromal cells are seeded and cultured immediately around the tumor cell line, allowing cell–cell contact besides establishing a gradient of soluble factors throughout the stromal cells, similar to that found in tissues [35]. As expected, after 2 days of culture, PC3 and DU-145 prostate cancer cells overexpressing SULF2 had already connected to fibroblasts, whereas the control cells transfected with empty vectors remained distant from fibroblasts (Figure 8). This probably occurred due to the increased migration presented by PC3 and DU-145 with forced expression of SULF2. Moreover, by using this system coupled to immunocytochemistry, we analyze the region of intersection between stromal cells and tumor cells (Figure 8). We observed an apparent increase in vimentin in the intersection area between cancer cells and fibroblasts for both transfected cells.Figure 8

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