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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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Related in: MedlinePlus

Heparanase expression in rhabdomyosarcoma cell lines. Western blot analysis was performed to detect HPSE in total cell lysates. Platelet extract and GAPDH were included as positive and loading controls respectively. One of three independent experiments is reported.
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Figure 1: Heparanase expression in rhabdomyosarcoma cell lines. Western blot analysis was performed to detect HPSE in total cell lysates. Platelet extract and GAPDH were included as positive and loading controls respectively. One of three independent experiments is reported.

Mentions: In order to define whether heparanase was expressed in the two major RMS histotypes and whether gene expression correlated with their different metastatic potential, HPSE expression was evaluated in ARMS and ERMS cell lines. Results from absolute quantitative Real-time PCR assay revealed a similar mRNA expression among all cell lines analyzed (data not shown). Subsequently, we performed Western blot analysis to detect HPSE protein in total cell lysates of the same cell lines. Primary polyclonal HPSE antibody was able to recognize both the active (50 kDa) and inactive (65 kDa) isoforms of the enzyme. As shown in Figure 1, both HPSE isoforms were detected in all cell lines under investigation. In particular, ARMS cell lines displayed two different patterns: RH30 and RH4 cell lines were characterized by high levels of the active isoform and low levels of the inactive one, whereas RH18 and RH28 showed a low expression of the active enzyme. Similarly, all ERMS cell lines displayed low levels of active HPSE isoform.


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)

Heparanase expression in rhabdomyosarcoma cell lines. Western blot analysis was performed to detect HPSE in total cell lysates. Platelet extract and GAPDH were included as positive and loading controls respectively. One of three independent experiments is reported.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 1: Heparanase expression in rhabdomyosarcoma cell lines. Western blot analysis was performed to detect HPSE in total cell lysates. Platelet extract and GAPDH were included as positive and loading controls respectively. One of three independent experiments is reported.
Mentions: In order to define whether heparanase was expressed in the two major RMS histotypes and whether gene expression correlated with their different metastatic potential, HPSE expression was evaluated in ARMS and ERMS cell lines. Results from absolute quantitative Real-time PCR assay revealed a similar mRNA expression among all cell lines analyzed (data not shown). Subsequently, we performed Western blot analysis to detect HPSE protein in total cell lysates of the same cell lines. Primary polyclonal HPSE antibody was able to recognize both the active (50 kDa) and inactive (65 kDa) isoforms of the enzyme. As shown in Figure 1, both HPSE isoforms were detected in all cell lines under investigation. In particular, ARMS cell lines displayed two different patterns: RH30 and RH4 cell lines were characterized by high levels of the active isoform and low levels of the inactive one, whereas RH18 and RH28 showed a low expression of the active enzyme. Similarly, all ERMS cell lines displayed low levels of active HPSE isoform.

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