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

Knockdown of SULF2 decreased viability and migration of prostate cells. RWPE-1 epithelial prostate cells and DU-145 and PC3 prostate cancer cells were transfected with siRNA (Life Technologies) targeting SULF2. To silence SULF2 gene, trials with siRNA preset by the manufacturer (Life Technologies, CA, USA) were performed as described in Methods. Three shRNAs were used to each gene, in addition to the positive (GAPDH) and negative (scramble sequence) controls. The gene silencing was confirmed by Real Time PCR 48 h after transfection (A). MTT viability assay (B) and wound healing assay (C) were performed as described previously. (CTRL NEG: scramble siRNA sequence). *P ≤ 0.05.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Fig4: Knockdown of SULF2 decreased viability and migration of prostate cells. RWPE-1 epithelial prostate cells and DU-145 and PC3 prostate cancer cells were transfected with siRNA (Life Technologies) targeting SULF2. To silence SULF2 gene, trials with siRNA preset by the manufacturer (Life Technologies, CA, USA) were performed as described in Methods. Three shRNAs were used to each gene, in addition to the positive (GAPDH) and negative (scramble sequence) controls. The gene silencing was confirmed by Real Time PCR 48 h after transfection (A). MTT viability assay (B) and wound healing assay (C) were performed as described previously. (CTRL NEG: scramble siRNA sequence). *P ≤ 0.05.

Mentions: In order to confirm that the previous cell behaviors were indeed acquired due to the overexpression of SULF2, we studied the consequences of SULF2 knockdown on the same prostate cells. For this purpose, prostate cells were transfected with siRNAs targeting SULF2 mRNA. Gene silencing was confirmed by quantitative RT-PCR (Figure 4A), and the levels of SULF2 mRNA were reduced in at least 95%. After that, the cells were submitted to viability and migration assays as previously described. Interestingly, the knockdown of SULF2 reduced the cell viability of RWPE-1 normal prostate cells, as well as reduced the cell viability of DU-145 and PC3 prostate cancer cells (Figure 4B). In addition, SULF2 silencing impaired cell migration (Figure 4C). Apparently, the overexpression of SULF2 was not sufficient to increase normal epithelial prostate cells growth and migration. However, the enzyme must be important for these cell properties, since its knockdown also decreased normal prostate cells migration and viability.Figure 4


SULF2 overexpression positively regulates tumorigenicity of human prostate cancer cells.

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

Knockdown of SULF2 decreased viability and migration of prostate cells. RWPE-1 epithelial prostate cells and DU-145 and PC3 prostate cancer cells were transfected with siRNA (Life Technologies) targeting SULF2. To silence SULF2 gene, trials with siRNA preset by the manufacturer (Life Technologies, CA, USA) were performed as described in Methods. Three shRNAs were used to each gene, in addition to the positive (GAPDH) and negative (scramble sequence) controls. The gene silencing was confirmed by Real Time PCR 48 h after transfection (A). MTT viability assay (B) and wound healing assay (C) were performed as described previously. (CTRL NEG: scramble siRNA sequence). *P ≤ 0.05.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Fig4: Knockdown of SULF2 decreased viability and migration of prostate cells. RWPE-1 epithelial prostate cells and DU-145 and PC3 prostate cancer cells were transfected with siRNA (Life Technologies) targeting SULF2. To silence SULF2 gene, trials with siRNA preset by the manufacturer (Life Technologies, CA, USA) were performed as described in Methods. Three shRNAs were used to each gene, in addition to the positive (GAPDH) and negative (scramble sequence) controls. The gene silencing was confirmed by Real Time PCR 48 h after transfection (A). MTT viability assay (B) and wound healing assay (C) were performed as described previously. (CTRL NEG: scramble siRNA sequence). *P ≤ 0.05.
Mentions: In order to confirm that the previous cell behaviors were indeed acquired due to the overexpression of SULF2, we studied the consequences of SULF2 knockdown on the same prostate cells. For this purpose, prostate cells were transfected with siRNAs targeting SULF2 mRNA. Gene silencing was confirmed by quantitative RT-PCR (Figure 4A), and the levels of SULF2 mRNA were reduced in at least 95%. After that, the cells were submitted to viability and migration assays as previously described. Interestingly, the knockdown of SULF2 reduced the cell viability of RWPE-1 normal prostate cells, as well as reduced the cell viability of DU-145 and PC3 prostate cancer cells (Figure 4B). In addition, SULF2 silencing impaired cell migration (Figure 4C). Apparently, the overexpression of SULF2 was not sufficient to increase normal epithelial prostate cells growth and migration. However, the enzyme must be important for these cell properties, since its knockdown also decreased normal prostate cells migration and viability.Figure 4

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