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Mechanisms of osteopontin and CD44 as metastatic principles in prostate cancer cells.

Desai B, Rogers MJ, Chellaiah MA - Mol. Cancer (2007)

Bottom Line: BPs inhibits the mevalonate pathway, which in turn, prevents the prenylation of a number of small GTPases.Attenuation of Rho GTPase activation by BPs may have contributed to the down regulation of cell surface CD44/MMP-9 interaction, MMP-9 activation/secretion, and cell migration.The various steps involved in the above mentioned signaling pathway and/or the molecules regulating the activation of MMP-9 are potential therapeutic target.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Biomedical Sciences, Dental School, University of Maryland, Baltimore, MD 21201, USA. bdesa002@umaryland.edu

ABSTRACT

Background: The expression level of osteopontin correlates with the metastatic potential of several tumors. Osteopontin is a well-characterized ligand for the alphavbeta3 integrin. The present study was undertaken to elucidate the possible role of osteopontin/alphavbeta3 signaling in prostate cancer cell migration.

Results: We generated stable prostate cancer cell (PC3) lines that over-express osteopontin (PC3/OPN), mutant OPN in the integrin binding-site (PC3/RGDDeltaRGA), and for OPN (PC3/SiRNA). The following observations were made in PC3/OPN cells as compared with PC3 cells: 1) an increase in multinucleated giant cells and RANKL expression; 2) an increase in CD44 surface expression, interaction of CD44/MMP-9 on the cell surface, MMP-9 activity in the conditioned medium, and cell migration; 3) western blot analysis of concentrated conditioned medium exhibited equal levels of MMP-9 protein in all PC3 cells. However, zymography analysis demonstrated that the levels of MMP-9 activity in the conditioned media reflect the CD44 surface expression pattern of the PC3 cell lines; 4) although MMP-9 and MMP-2 are secreted by PC3 cells, only the secretion of MMP-9 is regulated by OPN expression. A strong down regulation of the above-mentioned processes was observed in PC3/OPN (RGA) and PC3/SiRNA cells. PC3/OPN cells treated with bisphosphonate (BP) reproduce the down-regulation observed in PC3/OPN (RGA) and PC3/SiRNA cells.

Conclusion: Rho signaling plays a crucial role in CD44 surface expression. BPs inhibits the mevalonate pathway, which in turn, prevents the prenylation of a number of small GTPases. Attenuation of Rho GTPase activation by BPs may have contributed to the down regulation of cell surface CD44/MMP-9 interaction, MMP-9 activation/secretion, and cell migration. Taken together, these observations suggest that CD44 surface expression is an important event in the activation of MMP-9 and migration of prostate cancer cells. The various steps involved in the above mentioned signaling pathway and/or the molecules regulating the activation of MMP-9 are potential therapeutic target.

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Analysis of the effects of OPN over expression on cell morphology and RANKL expression in PC3 cell lines. A. Indicated PC3 cell lines were photographed using a phase contrast microscope. An increase in multinucleated giant cells was observed in PC3/OPN cells (magnification × 200). B. Immunoblotting analysis of RANKL expression in PC3 cell lines Immunoblotting analysis in protein lysates made from the indicated cell lines was performed with an antibody to RANKL. PC3/OPN cells express greater level of RANKL that PC3 cells. Results shown are a representation of three independent experiments. Bottom panel shows normalization with GAPDH.
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Figure 6: Analysis of the effects of OPN over expression on cell morphology and RANKL expression in PC3 cell lines. A. Indicated PC3 cell lines were photographed using a phase contrast microscope. An increase in multinucleated giant cells was observed in PC3/OPN cells (magnification × 200). B. Immunoblotting analysis of RANKL expression in PC3 cell lines Immunoblotting analysis in protein lysates made from the indicated cell lines was performed with an antibody to RANKL. PC3/OPN cells express greater level of RANKL that PC3 cells. Results shown are a representation of three independent experiments. Bottom panel shows normalization with GAPDH.

Mentions: Analysis of morphology of live PC3 cells by phase contrast microscopy: Over-expression of OPN has been shown to augment the occurrence of multinucleated giant cells in pancreatic adenocarcinoma [35] and macrophages of rat glomerulonephritis [36]. These observations prompted investigation on cell morphology in PC3 cell lines that have been used for the above-mentioned studies (Figure 6A). We demonstrated here that the number of multinucleated giant cells was increased in PC3/OPN cells. PC3/RGA cells have multinucleated giant cells at a level comparable to that of PC3 cells. PC3/SiRNA cells failed to display multinucleated giant cells (Figure 6A). In our observations, repeated passaging of PC3/OPN has no effect on the survival of multinucleated giant cells, indicating the genomic stability of this cell line.


Mechanisms of osteopontin and CD44 as metastatic principles in prostate cancer cells.

Desai B, Rogers MJ, Chellaiah MA - Mol. Cancer (2007)

Analysis of the effects of OPN over expression on cell morphology and RANKL expression in PC3 cell lines. A. Indicated PC3 cell lines were photographed using a phase contrast microscope. An increase in multinucleated giant cells was observed in PC3/OPN cells (magnification × 200). B. Immunoblotting analysis of RANKL expression in PC3 cell lines Immunoblotting analysis in protein lysates made from the indicated cell lines was performed with an antibody to RANKL. PC3/OPN cells express greater level of RANKL that PC3 cells. Results shown are a representation of three independent experiments. Bottom panel shows normalization with GAPDH.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC1828067&req=5

Figure 6: Analysis of the effects of OPN over expression on cell morphology and RANKL expression in PC3 cell lines. A. Indicated PC3 cell lines were photographed using a phase contrast microscope. An increase in multinucleated giant cells was observed in PC3/OPN cells (magnification × 200). B. Immunoblotting analysis of RANKL expression in PC3 cell lines Immunoblotting analysis in protein lysates made from the indicated cell lines was performed with an antibody to RANKL. PC3/OPN cells express greater level of RANKL that PC3 cells. Results shown are a representation of three independent experiments. Bottom panel shows normalization with GAPDH.
Mentions: Analysis of morphology of live PC3 cells by phase contrast microscopy: Over-expression of OPN has been shown to augment the occurrence of multinucleated giant cells in pancreatic adenocarcinoma [35] and macrophages of rat glomerulonephritis [36]. These observations prompted investigation on cell morphology in PC3 cell lines that have been used for the above-mentioned studies (Figure 6A). We demonstrated here that the number of multinucleated giant cells was increased in PC3/OPN cells. PC3/RGA cells have multinucleated giant cells at a level comparable to that of PC3 cells. PC3/SiRNA cells failed to display multinucleated giant cells (Figure 6A). In our observations, repeated passaging of PC3/OPN has no effect on the survival of multinucleated giant cells, indicating the genomic stability of this cell line.

Bottom Line: BPs inhibits the mevalonate pathway, which in turn, prevents the prenylation of a number of small GTPases.Attenuation of Rho GTPase activation by BPs may have contributed to the down regulation of cell surface CD44/MMP-9 interaction, MMP-9 activation/secretion, and cell migration.The various steps involved in the above mentioned signaling pathway and/or the molecules regulating the activation of MMP-9 are potential therapeutic target.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Biomedical Sciences, Dental School, University of Maryland, Baltimore, MD 21201, USA. bdesa002@umaryland.edu

ABSTRACT

Background: The expression level of osteopontin correlates with the metastatic potential of several tumors. Osteopontin is a well-characterized ligand for the alphavbeta3 integrin. The present study was undertaken to elucidate the possible role of osteopontin/alphavbeta3 signaling in prostate cancer cell migration.

Results: We generated stable prostate cancer cell (PC3) lines that over-express osteopontin (PC3/OPN), mutant OPN in the integrin binding-site (PC3/RGDDeltaRGA), and for OPN (PC3/SiRNA). The following observations were made in PC3/OPN cells as compared with PC3 cells: 1) an increase in multinucleated giant cells and RANKL expression; 2) an increase in CD44 surface expression, interaction of CD44/MMP-9 on the cell surface, MMP-9 activity in the conditioned medium, and cell migration; 3) western blot analysis of concentrated conditioned medium exhibited equal levels of MMP-9 protein in all PC3 cells. However, zymography analysis demonstrated that the levels of MMP-9 activity in the conditioned media reflect the CD44 surface expression pattern of the PC3 cell lines; 4) although MMP-9 and MMP-2 are secreted by PC3 cells, only the secretion of MMP-9 is regulated by OPN expression. A strong down regulation of the above-mentioned processes was observed in PC3/OPN (RGA) and PC3/SiRNA cells. PC3/OPN cells treated with bisphosphonate (BP) reproduce the down-regulation observed in PC3/OPN (RGA) and PC3/SiRNA cells.

Conclusion: Rho signaling plays a crucial role in CD44 surface expression. BPs inhibits the mevalonate pathway, which in turn, prevents the prenylation of a number of small GTPases. Attenuation of Rho GTPase activation by BPs may have contributed to the down regulation of cell surface CD44/MMP-9 interaction, MMP-9 activation/secretion, and cell migration. Taken together, these observations suggest that CD44 surface expression is an important event in the activation of MMP-9 and migration of prostate cancer cells. The various steps involved in the above mentioned signaling pathway and/or the molecules regulating the activation of MMP-9 are potential therapeutic target.

Show MeSH
Related in: MedlinePlus