Limits...
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

Forced expression of SULF2 increased prostate cancer cells viability and migration. For MTT assays, cells were plated into 96-well plates at 3000 cells per well and incubated in 10% FBS for 24 and 48 hours, lysed in DMSO and absorbance was measured in 540 nm (A). Scratch wounds were made in confluent cell culture monolayers with a 200-μL pipette tip; photomicrographs of the wounds were taken at 0 and 24 hours thereafter (B). (VECTOR: cells transfected with the empty vector; SULF2: cells transfected with SULF2). *P ≤ 0.05.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Fig3: Forced expression of SULF2 increased prostate cancer cells viability and migration. For MTT assays, cells were plated into 96-well plates at 3000 cells per well and incubated in 10% FBS for 24 and 48 hours, lysed in DMSO and absorbance was measured in 540 nm (A). Scratch wounds were made in confluent cell culture monolayers with a 200-μL pipette tip; photomicrographs of the wounds were taken at 0 and 24 hours thereafter (B). (VECTOR: cells transfected with the empty vector; SULF2: cells transfected with SULF2). *P ≤ 0.05.

Mentions: After analyzing the effects of SULF2 forced expression on the structure of HS from prostate cancer cells, we studied the differences on cell viability and migration. Initially, cell viability was measured by MTT colorimetric assay. The overexpression of SULF2 had no effect on the viability of RWPE-1 cells (Figure 3A). However, both prostate cancer cells, PC3 and DU-145 presented increase on cell viability. The migration was also analyzed by wound healing assay. A scratch was performed on confluent cell cultures and the cells were allowed to migrate for 24 h. Interestingly, the normal prostate epithelial cell line RWPE-1 transfected with SULF2, did not present any increase on cell migration (Figure 3B). However, prostate cancer cells showed a robust migratory phenotype. These results indicate that forced expression of SULF2 increased cell viability and migration solely on prostate cancer cells, but did not enhance these characteristics on normal cells.Figure 3


SULF2 overexpression positively regulates tumorigenicity of human prostate cancer cells.

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

Forced expression of SULF2 increased prostate cancer cells viability and migration. For MTT assays, cells were plated into 96-well plates at 3000 cells per well and incubated in 10% FBS for 24 and 48 hours, lysed in DMSO and absorbance was measured in 540 nm (A). Scratch wounds were made in confluent cell culture monolayers with a 200-μL pipette tip; photomicrographs of the wounds were taken at 0 and 24 hours thereafter (B). (VECTOR: cells transfected with the empty vector; SULF2: cells transfected with SULF2). *P ≤ 0.05.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Fig3: Forced expression of SULF2 increased prostate cancer cells viability and migration. For MTT assays, cells were plated into 96-well plates at 3000 cells per well and incubated in 10% FBS for 24 and 48 hours, lysed in DMSO and absorbance was measured in 540 nm (A). Scratch wounds were made in confluent cell culture monolayers with a 200-μL pipette tip; photomicrographs of the wounds were taken at 0 and 24 hours thereafter (B). (VECTOR: cells transfected with the empty vector; SULF2: cells transfected with SULF2). *P ≤ 0.05.
Mentions: After analyzing the effects of SULF2 forced expression on the structure of HS from prostate cancer cells, we studied the differences on cell viability and migration. Initially, cell viability was measured by MTT colorimetric assay. The overexpression of SULF2 had no effect on the viability of RWPE-1 cells (Figure 3A). However, both prostate cancer cells, PC3 and DU-145 presented increase on cell viability. The migration was also analyzed by wound healing assay. A scratch was performed on confluent cell cultures and the cells were allowed to migrate for 24 h. Interestingly, the normal prostate epithelial cell line RWPE-1 transfected with SULF2, did not present any increase on cell migration (Figure 3B). However, prostate cancer cells showed a robust migratory phenotype. These results indicate that forced expression of SULF2 increased cell viability and migration solely on prostate cancer cells, but did not enhance these characteristics on normal cells.Figure 3

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