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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 expression in prostate cancer cells. Normal prostate epithelial cell line RWPE-1, and prostate cancer cell lines LNCap, PC3 and DU-145 SULF2 mRNA level was analyzed by RT-PCR in agarose gel (A). RWPE-1, PC3 and DU-145 cells were transfected with either SULF2 expressing plasmid pcDNA3.1/Myc-His(−)Hsulf-2 (Addgene plasmid 13004) or empty vector using Fugene reagent (Promega). The control cells (CTRL) were not transfected. SULF2 mRNA expression was confirmed with quantitative real-time PCR. The expression level of each gene was normalized by GAPDH expression (B). The overexpression of SULF2 was also confirmed by protein western blotting under non-reducing conditions (C) and immunofluorescence analyzed in confocal microscope (D). The data from each experiment was obtained in triplicate and are represented as the average ± standard deviation. (CTRL: not transfected cells; VECTOR: cells transfected with the empty vector; SULF2: cells transfected with SULF2 containing vector). Scale bars 20 μm. *P ≤ 0.05.
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Fig1: SULF2 expression in prostate cancer cells. Normal prostate epithelial cell line RWPE-1, and prostate cancer cell lines LNCap, PC3 and DU-145 SULF2 mRNA level was analyzed by RT-PCR in agarose gel (A). RWPE-1, PC3 and DU-145 cells were transfected with either SULF2 expressing plasmid pcDNA3.1/Myc-His(−)Hsulf-2 (Addgene plasmid 13004) or empty vector using Fugene reagent (Promega). The control cells (CTRL) were not transfected. SULF2 mRNA expression was confirmed with quantitative real-time PCR. The expression level of each gene was normalized by GAPDH expression (B). The overexpression of SULF2 was also confirmed by protein western blotting under non-reducing conditions (C) and immunofluorescence analyzed in confocal microscope (D). The data from each experiment was obtained in triplicate and are represented as the average ± standard deviation. (CTRL: not transfected cells; VECTOR: cells transfected with the empty vector; SULF2: cells transfected with SULF2 containing vector). Scale bars 20 μm. *P ≤ 0.05.

Mentions: SULF2 gene expression was analyzed in RWPE-1 normal prostate epithelial cells and in LNCap, PC3 and DU-145 prostate cancer cells. Total RNA was extracted from the cells, and gene expression was analyzed by semi-quantitative electrophoresis after PCR. Our result showed that SULF2 is similarly expressed in normal and prostate cancer cells (Figure 1A). Subsequently, we transfected RWPE-1 normal cell, PC-3 and DU-145 cancer cells with the expression plasmid pcDNA 3.1 containing the SULF 2 gene. As control, we transfected the same cells with the empty vector. The cells were clonally selected and the transfection efficiency confirmed by quantitative RT-PCR, western blotting and immunofluorescence. We observed a significant increase (50-fold) of SULF2 gene expression in the transfected cells (Figure 1B). The overexpression of SULF2 did not affect the mRNA levels of SULF1 in all the transfected cells (data not shown). Moreover, western blotting analyzes demonstrated that SULF2 was increased in cells extracts, but mainly in the medium (Figure 1C). The increased expression of SULF2 was also observed by immunofluorescence (Figure 1D).Figure 1


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 expression in prostate cancer cells. Normal prostate epithelial cell line RWPE-1, and prostate cancer cell lines LNCap, PC3 and DU-145 SULF2 mRNA level was analyzed by RT-PCR in agarose gel (A). RWPE-1, PC3 and DU-145 cells were transfected with either SULF2 expressing plasmid pcDNA3.1/Myc-His(−)Hsulf-2 (Addgene plasmid 13004) or empty vector using Fugene reagent (Promega). The control cells (CTRL) were not transfected. SULF2 mRNA expression was confirmed with quantitative real-time PCR. The expression level of each gene was normalized by GAPDH expression (B). The overexpression of SULF2 was also confirmed by protein western blotting under non-reducing conditions (C) and immunofluorescence analyzed in confocal microscope (D). The data from each experiment was obtained in triplicate and are represented as the average ± standard deviation. (CTRL: not transfected cells; VECTOR: cells transfected with the empty vector; SULF2: cells transfected with SULF2 containing vector). Scale bars 20 μm. *P ≤ 0.05.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4374423&req=5

Fig1: SULF2 expression in prostate cancer cells. Normal prostate epithelial cell line RWPE-1, and prostate cancer cell lines LNCap, PC3 and DU-145 SULF2 mRNA level was analyzed by RT-PCR in agarose gel (A). RWPE-1, PC3 and DU-145 cells were transfected with either SULF2 expressing plasmid pcDNA3.1/Myc-His(−)Hsulf-2 (Addgene plasmid 13004) or empty vector using Fugene reagent (Promega). The control cells (CTRL) were not transfected. SULF2 mRNA expression was confirmed with quantitative real-time PCR. The expression level of each gene was normalized by GAPDH expression (B). The overexpression of SULF2 was also confirmed by protein western blotting under non-reducing conditions (C) and immunofluorescence analyzed in confocal microscope (D). The data from each experiment was obtained in triplicate and are represented as the average ± standard deviation. (CTRL: not transfected cells; VECTOR: cells transfected with the empty vector; SULF2: cells transfected with SULF2 containing vector). Scale bars 20 μm. *P ≤ 0.05.
Mentions: SULF2 gene expression was analyzed in RWPE-1 normal prostate epithelial cells and in LNCap, PC3 and DU-145 prostate cancer cells. Total RNA was extracted from the cells, and gene expression was analyzed by semi-quantitative electrophoresis after PCR. Our result showed that SULF2 is similarly expressed in normal and prostate cancer cells (Figure 1A). Subsequently, we transfected RWPE-1 normal cell, PC-3 and DU-145 cancer cells with the expression plasmid pcDNA 3.1 containing the SULF 2 gene. As control, we transfected the same cells with the empty vector. The cells were clonally selected and the transfection efficiency confirmed by quantitative RT-PCR, western blotting and immunofluorescence. We observed a significant increase (50-fold) of SULF2 gene expression in the transfected cells (Figure 1B). The overexpression of SULF2 did not affect the mRNA levels of SULF1 in all the transfected cells (data not shown). Moreover, western blotting analyzes demonstrated that SULF2 was increased in cells extracts, but mainly in the medium (Figure 1C). The increased expression of SULF2 was also observed by immunofluorescence (Figure 1D).Figure 1

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