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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 overexpression increased prostate cancer cells invasiveness and colony formation. For transwell migration assay, cells were plated on the top chamber of transwell membranes (8 μm pore size). Migrating cells were fixed with 4% formaldehyde and stained with crystal violet (A). The graphics represent the relative migration (B). For colony formation assay cells were diluted in medium containing 0.35% agar and colonies were photographed after 20 days (C). Tables indicate the number and the size of colonies formed by prostate cancer cells (D). (VECTOR: cells transfected with the empty vector; SULF2: cells transfected with sulfatase 2 containing vector). *P ≤ 0.05.
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Fig5: SULF2 overexpression increased prostate cancer cells invasiveness and colony formation. For transwell migration assay, cells were plated on the top chamber of transwell membranes (8 μm pore size). Migrating cells were fixed with 4% formaldehyde and stained with crystal violet (A). The graphics represent the relative migration (B). For colony formation assay cells were diluted in medium containing 0.35% agar and colonies were photographed after 20 days (C). Tables indicate the number and the size of colonies formed by prostate cancer cells (D). (VECTOR: cells transfected with the empty vector; SULF2: cells transfected with sulfatase 2 containing vector). *P ≤ 0.05.

Mentions: In order to determine whether SULF2 increase was able to exacerbate the tumorigenic phenotype of prostate cancer cells in vitro, PC3 and DU-145 cells were submitted to colony formation and transmigration assays. For transmigration assay, cancer cells were plated on the top of membranes with pore diameter of 8 μm and allowed to migrate for 24 h. DU-145 cancer cells transfected with SULF2 presented a 3-fold increase on migration through the membrane, and PC3 cancer cells transfected with SULF2 presented a 2-fold increase on migration (Figure 5A). For colony formation assay, the cancer cells were embedded in soft agar and the colonies growth was followed for 20 days. DU-145 and PC3 prostate cancer cells overexpressing SULF2 presented an increase of 3-fold on the size of the colonies formed, compared to cells transfected with empty vector (Figure 5B). Equally important, the SULF2 overexpressing cells also formed more colonies on soft agar (Figure 5B).Figure 5


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 overexpression increased prostate cancer cells invasiveness and colony formation. For transwell migration assay, cells were plated on the top chamber of transwell membranes (8 μm pore size). Migrating cells were fixed with 4% formaldehyde and stained with crystal violet (A). The graphics represent the relative migration (B). For colony formation assay cells were diluted in medium containing 0.35% agar and colonies were photographed after 20 days (C). Tables indicate the number and the size of colonies formed by prostate cancer cells (D). (VECTOR: cells transfected with the empty vector; SULF2: cells transfected with sulfatase 2 containing vector). *P ≤ 0.05.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Fig5: SULF2 overexpression increased prostate cancer cells invasiveness and colony formation. For transwell migration assay, cells were plated on the top chamber of transwell membranes (8 μm pore size). Migrating cells were fixed with 4% formaldehyde and stained with crystal violet (A). The graphics represent the relative migration (B). For colony formation assay cells were diluted in medium containing 0.35% agar and colonies were photographed after 20 days (C). Tables indicate the number and the size of colonies formed by prostate cancer cells (D). (VECTOR: cells transfected with the empty vector; SULF2: cells transfected with sulfatase 2 containing vector). *P ≤ 0.05.
Mentions: In order to determine whether SULF2 increase was able to exacerbate the tumorigenic phenotype of prostate cancer cells in vitro, PC3 and DU-145 cells were submitted to colony formation and transmigration assays. For transmigration assay, cancer cells were plated on the top of membranes with pore diameter of 8 μm and allowed to migrate for 24 h. DU-145 cancer cells transfected with SULF2 presented a 3-fold increase on migration through the membrane, and PC3 cancer cells transfected with SULF2 presented a 2-fold increase on migration (Figure 5A). For colony formation assay, the cancer cells were embedded in soft agar and the colonies growth was followed for 20 days. DU-145 and PC3 prostate cancer cells overexpressing SULF2 presented an increase of 3-fold on the size of the colonies formed, compared to cells transfected with empty vector (Figure 5B). Equally important, the SULF2 overexpressing cells also formed more colonies on soft agar (Figure 5B).Figure 5

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