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Heparanase activity in alveolar and embryonal rhabdomyosarcoma: implications for tumor invasion.

Masola V, Maran C, Tassone E, Zin A, Rosolen A, Onisto M - BMC Cancer (2009)

Bottom Line: Stable HPSE silencing by shRNA technique determined a significant knockdown of gene expression equal to 76% and 58% in RH30 and RD cell lines respectively and induced a less invasive behaviour compared to untreated cells.Finally, we observed that HPSE mRNA expression in biopsies was higher than in foetal skeletal muscle and that plasma from RMS patients displayed significantly more elevated HPSE levels than healthy subjects with a trend to higher levels in ARMS.Moreover, HPSE expression in RMS patients is significantly higher with respect to healthy subjects.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Experimental Biomedical Sciences, University of Padova, 35121 Padova, Italy. valentina.masola@unipd.it

ABSTRACT

Background: Rhabdomyosarcoma (RMS) is a malignant soft tissue sarcoma of childhood including two major histological subtypes, alveolar (ARMS) and embryonal (ERMS) RMS. Like other human malignancies RMS possesses high metastatic potential, more pronounced in ARMS than in ERMS. This feature is influenced by several biological molecules, including soluble factors secreted by tumor cells, such as heparanase (HPSE). HPSE is an endo-beta-D-glucuronidase that cleaves heparan sulphate proteoglycans.

Methods: We determined HPSE expression by Western blot analysis in ARMS and ERMS cells lines and activity in supernatants by an ELISA assay. Stable HPSE silencing has been performed by shRNA technique in RH30 and RD cell lines and their invasiveness has been evaluated by Matrigel-invasion assay. HPSE activity and mRNA expression have also been quantified in plasma and biopsies from RMS patients.

Results: HPSE expression and activity have been detected in all RMS cell lines. Stable HPSE silencing by shRNA technique determined a significant knockdown of gene expression equal to 76% and 58% in RH30 and RD cell lines respectively and induced a less invasive behaviour compared to untreated cells. Finally, we observed that HPSE mRNA expression in biopsies was higher than in foetal skeletal muscle and that plasma from RMS patients displayed significantly more elevated HPSE levels than healthy subjects with a trend to higher levels in ARMS.

Conclusion: In conclusion, our data demonstrate for the first time HPSE expression and activity in RMS and highlight its involvement in tumor cell invasion as revealed by shRNA silencing. Moreover, HPSE expression in RMS patients is significantly higher with respect to healthy subjects. Further studies are warranted to assess possible relationships between HPSE and clinical behaviour in RMS.

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Stable heparanase silencing of RH30 and RD cell lines with shRNA. (A) HPSE mRNA expression levels in control (black bars) and silenced (hatched bars) RH30 and RD cell lines were determined by Real-time PCR. The results were normalized using GAPDH as internal control and represent the mean ± standard deviation of three samples performed in duplicate. (B) Western blot analysis was performed to demonstrate HPSE silencing between control and silenced RH30 and RD cell lines. GAPDH was included as loading control. (C) Heparanase activity in serum-free conditioned media obtained from control (black bars) and silenced (hatched bars) RH30 and RD cell lines. The enzymatic activity is expressed as nanograms of heparan sulphate (HS) removed per minute. The results represent the mean ± standard deviation of three independent experiments performed in duplicate. Platelet extract was used as positive control (PC, white bar). An asterisk (*) indicates a significant difference from control (P < 0.05).
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Figure 3: Stable heparanase silencing of RH30 and RD cell lines with shRNA. (A) HPSE mRNA expression levels in control (black bars) and silenced (hatched bars) RH30 and RD cell lines were determined by Real-time PCR. The results were normalized using GAPDH as internal control and represent the mean ± standard deviation of three samples performed in duplicate. (B) Western blot analysis was performed to demonstrate HPSE silencing between control and silenced RH30 and RD cell lines. GAPDH was included as loading control. (C) Heparanase activity in serum-free conditioned media obtained from control (black bars) and silenced (hatched bars) RH30 and RD cell lines. The enzymatic activity is expressed as nanograms of heparan sulphate (HS) removed per minute. The results represent the mean ± standard deviation of three independent experiments performed in duplicate. Platelet extract was used as positive control (PC, white bar). An asterisk (*) indicates a significant difference from control (P < 0.05).

Mentions: To further investigate the role of HPSE expression in RMS, we downregulated HPSE in RH30 and RD cell lines. In particular, from each 29 mer-shRNA targeting HPSE and the relative negative controls, we obtained three clones under puromycin selection. We evaluated HPSE mRNA expression in all clones by Real-time PCR analysis (data not shown) and we chose the pHPSE-3 sequence that showed the best silencing rate in both cell lines. In this way we obtained a significant knockdown of gene expression equal to 76% and 58% for RH30 and RD, respectively (P < 0.05) (Figure 3A). A clear reduction in HPSE protein expression was observed in total cell lysate (Figure 3B). As regards with HPSE activity in serum-free conditioned media from untreated and silenced RH30 and RD cells, we detected a remarkable reduction equal to 70% and 87%, respectively (P < 0.05) (Figure 3C). HPSE expression was comparable in wild type cell lines and in negative control clones (data not shown).


Heparanase activity in alveolar and embryonal rhabdomyosarcoma: implications for tumor invasion.

Masola V, Maran C, Tassone E, Zin A, Rosolen A, Onisto M - BMC Cancer (2009)

Stable heparanase silencing of RH30 and RD cell lines with shRNA. (A) HPSE mRNA expression levels in control (black bars) and silenced (hatched bars) RH30 and RD cell lines were determined by Real-time PCR. The results were normalized using GAPDH as internal control and represent the mean ± standard deviation of three samples performed in duplicate. (B) Western blot analysis was performed to demonstrate HPSE silencing between control and silenced RH30 and RD cell lines. GAPDH was included as loading control. (C) Heparanase activity in serum-free conditioned media obtained from control (black bars) and silenced (hatched bars) RH30 and RD cell lines. The enzymatic activity is expressed as nanograms of heparan sulphate (HS) removed per minute. The results represent the mean ± standard deviation of three independent experiments performed in duplicate. Platelet extract was used as positive control (PC, white bar). An asterisk (*) indicates a significant difference from control (P < 0.05).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2743710&req=5

Figure 3: Stable heparanase silencing of RH30 and RD cell lines with shRNA. (A) HPSE mRNA expression levels in control (black bars) and silenced (hatched bars) RH30 and RD cell lines were determined by Real-time PCR. The results were normalized using GAPDH as internal control and represent the mean ± standard deviation of three samples performed in duplicate. (B) Western blot analysis was performed to demonstrate HPSE silencing between control and silenced RH30 and RD cell lines. GAPDH was included as loading control. (C) Heparanase activity in serum-free conditioned media obtained from control (black bars) and silenced (hatched bars) RH30 and RD cell lines. The enzymatic activity is expressed as nanograms of heparan sulphate (HS) removed per minute. The results represent the mean ± standard deviation of three independent experiments performed in duplicate. Platelet extract was used as positive control (PC, white bar). An asterisk (*) indicates a significant difference from control (P < 0.05).
Mentions: To further investigate the role of HPSE expression in RMS, we downregulated HPSE in RH30 and RD cell lines. In particular, from each 29 mer-shRNA targeting HPSE and the relative negative controls, we obtained three clones under puromycin selection. We evaluated HPSE mRNA expression in all clones by Real-time PCR analysis (data not shown) and we chose the pHPSE-3 sequence that showed the best silencing rate in both cell lines. In this way we obtained a significant knockdown of gene expression equal to 76% and 58% for RH30 and RD, respectively (P < 0.05) (Figure 3A). A clear reduction in HPSE protein expression was observed in total cell lysate (Figure 3B). As regards with HPSE activity in serum-free conditioned media from untreated and silenced RH30 and RD cells, we detected a remarkable reduction equal to 70% and 87%, respectively (P < 0.05) (Figure 3C). HPSE expression was comparable in wild type cell lines and in negative control clones (data not shown).

Bottom Line: Stable HPSE silencing by shRNA technique determined a significant knockdown of gene expression equal to 76% and 58% in RH30 and RD cell lines respectively and induced a less invasive behaviour compared to untreated cells.Finally, we observed that HPSE mRNA expression in biopsies was higher than in foetal skeletal muscle and that plasma from RMS patients displayed significantly more elevated HPSE levels than healthy subjects with a trend to higher levels in ARMS.Moreover, HPSE expression in RMS patients is significantly higher with respect to healthy subjects.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Experimental Biomedical Sciences, University of Padova, 35121 Padova, Italy. valentina.masola@unipd.it

ABSTRACT

Background: Rhabdomyosarcoma (RMS) is a malignant soft tissue sarcoma of childhood including two major histological subtypes, alveolar (ARMS) and embryonal (ERMS) RMS. Like other human malignancies RMS possesses high metastatic potential, more pronounced in ARMS than in ERMS. This feature is influenced by several biological molecules, including soluble factors secreted by tumor cells, such as heparanase (HPSE). HPSE is an endo-beta-D-glucuronidase that cleaves heparan sulphate proteoglycans.

Methods: We determined HPSE expression by Western blot analysis in ARMS and ERMS cells lines and activity in supernatants by an ELISA assay. Stable HPSE silencing has been performed by shRNA technique in RH30 and RD cell lines and their invasiveness has been evaluated by Matrigel-invasion assay. HPSE activity and mRNA expression have also been quantified in plasma and biopsies from RMS patients.

Results: HPSE expression and activity have been detected in all RMS cell lines. Stable HPSE silencing by shRNA technique determined a significant knockdown of gene expression equal to 76% and 58% in RH30 and RD cell lines respectively and induced a less invasive behaviour compared to untreated cells. Finally, we observed that HPSE mRNA expression in biopsies was higher than in foetal skeletal muscle and that plasma from RMS patients displayed significantly more elevated HPSE levels than healthy subjects with a trend to higher levels in ARMS.

Conclusion: In conclusion, our data demonstrate for the first time HPSE expression and activity in RMS and highlight its involvement in tumor cell invasion as revealed by shRNA silencing. Moreover, HPSE expression in RMS patients is significantly higher with respect to healthy subjects. Further studies are warranted to assess possible relationships between HPSE and clinical behaviour in RMS.

Show MeSH
Related in: MedlinePlus