Limits...
Non-caveolar caveolin-1 expression in prostate cancer cells promotes lymphangiogenesis.

Nassar ZD, Hill MM, Parton RG, Francois M, Parat MO - Oncoscience (2015)

Bottom Line: The effect of PCa cell conditioned media on lymphatic endothelial cell (LEC) viability, chemotaxis, chemokinesis and differentiation was assessed.In contrast, caveolar Cav-1 (in DU145 cells) did not significantly affect PCa cell lymphangiogenic potential.The effect of non-caveolar Cav-1 on LECs was mediated by increased expression of VEGF-A as demonstrated by neutralization by anti-VEGF-A antibody.

View Article: PubMed Central - PubMed

Affiliation: The University of Queensland, School of Pharmacy, QLD, Australia.

ABSTRACT
Lymphangiogenesis allows prostate cancer (PCa) lymphatic metastasis, which is associated with poor prognosis and short survival rates. Caveolin-1 (Cav-1) is a membrane protein localized in caveolae, but also exists in non-caveolar, cellular or extracellular forms. Cav-1 is overexpressed in PCa, promotes prostate tumour progression and metastasis. We investigated the effect of caveolar and non-caveolar Cav-1 on PCa lymphangiogenic potential. Cav-1 was down-regulated in PC3 and DU145, and ectopically expressed in LNCaP cells. The effect of PCa cell conditioned media on lymphatic endothelial cell (LEC) viability, chemotaxis, chemokinesis and differentiation was assessed. The effect of Cav-1 on PCa cell expression of lymphangiogenesis-modulators VEGF-A and VEGF-C was assessed using qPCR and ELISA of the conditioned medium. Non-caveolar Cav-1, whether exogenous or endogenous (in LNCaP and PC3 cells, respectively) enhanced LEC proliferation, migration and differentiation. In contrast, caveolar Cav-1 (in DU145 cells) did not significantly affect PCa cell lymphangiogenic potential. The effect of non-caveolar Cav-1 on LECs was mediated by increased expression of VEGF-A as demonstrated by neutralization by anti-VEGF-A antibody. This study unveils for the first time a crucial role for non-caveolar Cav-1 in modulating PCa cell expression of VEGF-A and subsequent LEC proliferation, migration and tube formation.

No MeSH data available.


Related in: MedlinePlus

Effect of anti-VEGF-A antibody on prostate cancer cell-conditioned media-induced LEC migrationChemotaxis towards PCa cell-conditioned media was tested using the Boyden chamber assay in the presence or absence of anti-VEGF-A antibody as indicated. Results are reported as percent of the migration to control cell conditioned medium and shown as mean ± S.E.M. (n=3 separate experiment), *p<0.05, **p<0.01, ***p<0.001, ns no statistical significance.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4549361&req=5

Figure 6: Effect of anti-VEGF-A antibody on prostate cancer cell-conditioned media-induced LEC migrationChemotaxis towards PCa cell-conditioned media was tested using the Boyden chamber assay in the presence or absence of anti-VEGF-A antibody as indicated. Results are reported as percent of the migration to control cell conditioned medium and shown as mean ± S.E.M. (n=3 separate experiment), *p<0.05, **p<0.01, ***p<0.001, ns no statistical significance.

Mentions: To establish a causality link between the positive effects of Cav-1 expression in prostate cancer on VEGF-A, on the one hand, and functional in vitro assays indicating increased lymphangiogenesis, on the other hand, we tested whether VEGF-A antibody could neutralize the effect of Cav-1 expression on the pro-lymphangiogenic effects of the conditioned media of PCa cells. To that extent, we measured chemotaxis of LEC towards conditioned media from PCa cells added with anti-VEGF-A antibody (for cells that exhibited increased production of VEGF-A, namely LNCaP and PC3). The anti-VEGF-A neutralizing antibody reversed the effect of Cav-1 expression on PCa pro-lymphangiogenic potential: there was no statistically significant difference between sh-Cont and sh-Cav-1 medium or between GFP or Cav-1-GFP LNCaP medium in the presence of anti-VEGF-A (Figure 6), and there was statistically significant differences between IgG- and anti VEGF-A-added conditioned medium of Cav-1-expressing cells (sh-Cont PC3 or Cav-1-GFP LNCaP). These results indicate that VEGF-A at least partly mediates the pro-lymphangiogenic effect of Cav-1 expression in PCa. We did not test whether adding anti-VEGF-C antibody to the conditioned medium would abolish the difference between LEC migration elicited by Cav-1 down-regulated and control PC3 cells, because the concentration found in their conditioned medium (~0.75 ng/ml) is below the minimal concentration of VEGF-C that elicits LEC chemotaxis in our model (10 ng/ml, data not shown). Together with the fact that Cav-1 expression alters the pro-lymphangiogenic potential of LNCaP cells despite their lack of VEGF-C expression, these results suggest that the effect of Cav-1 on PCa lymphangiogenesis in our experiments is likely not VEGF-C mediated.


Non-caveolar caveolin-1 expression in prostate cancer cells promotes lymphangiogenesis.

Nassar ZD, Hill MM, Parton RG, Francois M, Parat MO - Oncoscience (2015)

Effect of anti-VEGF-A antibody on prostate cancer cell-conditioned media-induced LEC migrationChemotaxis towards PCa cell-conditioned media was tested using the Boyden chamber assay in the presence or absence of anti-VEGF-A antibody as indicated. Results are reported as percent of the migration to control cell conditioned medium and shown as mean ± S.E.M. (n=3 separate experiment), *p<0.05, **p<0.01, ***p<0.001, ns no statistical significance.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4549361&req=5

Figure 6: Effect of anti-VEGF-A antibody on prostate cancer cell-conditioned media-induced LEC migrationChemotaxis towards PCa cell-conditioned media was tested using the Boyden chamber assay in the presence or absence of anti-VEGF-A antibody as indicated. Results are reported as percent of the migration to control cell conditioned medium and shown as mean ± S.E.M. (n=3 separate experiment), *p<0.05, **p<0.01, ***p<0.001, ns no statistical significance.
Mentions: To establish a causality link between the positive effects of Cav-1 expression in prostate cancer on VEGF-A, on the one hand, and functional in vitro assays indicating increased lymphangiogenesis, on the other hand, we tested whether VEGF-A antibody could neutralize the effect of Cav-1 expression on the pro-lymphangiogenic effects of the conditioned media of PCa cells. To that extent, we measured chemotaxis of LEC towards conditioned media from PCa cells added with anti-VEGF-A antibody (for cells that exhibited increased production of VEGF-A, namely LNCaP and PC3). The anti-VEGF-A neutralizing antibody reversed the effect of Cav-1 expression on PCa pro-lymphangiogenic potential: there was no statistically significant difference between sh-Cont and sh-Cav-1 medium or between GFP or Cav-1-GFP LNCaP medium in the presence of anti-VEGF-A (Figure 6), and there was statistically significant differences between IgG- and anti VEGF-A-added conditioned medium of Cav-1-expressing cells (sh-Cont PC3 or Cav-1-GFP LNCaP). These results indicate that VEGF-A at least partly mediates the pro-lymphangiogenic effect of Cav-1 expression in PCa. We did not test whether adding anti-VEGF-C antibody to the conditioned medium would abolish the difference between LEC migration elicited by Cav-1 down-regulated and control PC3 cells, because the concentration found in their conditioned medium (~0.75 ng/ml) is below the minimal concentration of VEGF-C that elicits LEC chemotaxis in our model (10 ng/ml, data not shown). Together with the fact that Cav-1 expression alters the pro-lymphangiogenic potential of LNCaP cells despite their lack of VEGF-C expression, these results suggest that the effect of Cav-1 on PCa lymphangiogenesis in our experiments is likely not VEGF-C mediated.

Bottom Line: The effect of PCa cell conditioned media on lymphatic endothelial cell (LEC) viability, chemotaxis, chemokinesis and differentiation was assessed.In contrast, caveolar Cav-1 (in DU145 cells) did not significantly affect PCa cell lymphangiogenic potential.The effect of non-caveolar Cav-1 on LECs was mediated by increased expression of VEGF-A as demonstrated by neutralization by anti-VEGF-A antibody.

View Article: PubMed Central - PubMed

Affiliation: The University of Queensland, School of Pharmacy, QLD, Australia.

ABSTRACT
Lymphangiogenesis allows prostate cancer (PCa) lymphatic metastasis, which is associated with poor prognosis and short survival rates. Caveolin-1 (Cav-1) is a membrane protein localized in caveolae, but also exists in non-caveolar, cellular or extracellular forms. Cav-1 is overexpressed in PCa, promotes prostate tumour progression and metastasis. We investigated the effect of caveolar and non-caveolar Cav-1 on PCa lymphangiogenic potential. Cav-1 was down-regulated in PC3 and DU145, and ectopically expressed in LNCaP cells. The effect of PCa cell conditioned media on lymphatic endothelial cell (LEC) viability, chemotaxis, chemokinesis and differentiation was assessed. The effect of Cav-1 on PCa cell expression of lymphangiogenesis-modulators VEGF-A and VEGF-C was assessed using qPCR and ELISA of the conditioned medium. Non-caveolar Cav-1, whether exogenous or endogenous (in LNCaP and PC3 cells, respectively) enhanced LEC proliferation, migration and differentiation. In contrast, caveolar Cav-1 (in DU145 cells) did not significantly affect PCa cell lymphangiogenic potential. The effect of non-caveolar Cav-1 on LECs was mediated by increased expression of VEGF-A as demonstrated by neutralization by anti-VEGF-A antibody. This study unveils for the first time a crucial role for non-caveolar Cav-1 in modulating PCa cell expression of VEGF-A and subsequent LEC proliferation, migration and tube formation.

No MeSH data available.


Related in: MedlinePlus