Limits...
Novel anti-metastatic action of cidofovir mediated by inhibition of E6/E7, CXCR4 and Rho/ROCK signaling in HPV tumor cells.

Amine A, Rivera S, Opolon P, Dekkal M, Biard DS, Bouamar H, Louache F, McKay MJ, Bourhis J, Deutsch E, Vozenin-Brotons MC - PLoS ONE (2009)

Bottom Line: E6 and E7 silencing using stable knock-down and the approved anti-viral agent, Cidofovir decreased CXCR4 gene expression as well as both, constitutive and SDF-1alpha-induced cell invasion.The contribution of the Rho/ROCK pathway was suggested by the inhibitory effect triggered by Cidofovir and further confirmed using Y-27632 (a small molecule ROCK inhibitor).These data suggest a novel and highly translatable therapeutic approach to cervix cancer, by inhibition of adhesion and invasion of circulating HPV-positive tumor cells, using Cidofovir and/or ROCK inhibition.

View Article: PubMed Central - PubMed

Affiliation: Laboratoire UPRES EA 27-10 Radiosensibilité des Tumeurs et Tissus Sains, Institut Gustave Roussy/Institut de Radioprotection et de Sureté Nucléaire, Villejuif, France.

ABSTRACT
Cervical cancer is frequently associated with HPV infection. The expression of E6 and E7 HPV oncoproteins is a key factor in its carcinogenicity and might also influence its virulence, including metastatic conversion. The cellular mechanisms involved in metastatic spread remain elusive, but pro-adhesive receptors and their ligands, such as SDF-1alpha and CXCR4 are implicated. In the present study, we assessed the possible relationship between SDF-1alpha/CXCR4 signaling, E6/E7 status and the metastatic process. We found that SDF-1alpha stimulated the invasion of E6/E7-positive cancer cell lines (HeLa and TC-1) in Matrigel though CXCR4 and subsequent Rho/ROCK activation. In pulmonary metastatic foci generated by TC-1 cells IV injection a high proportion of cells expressed membrane-associated CXCR4. In both cases models (in vitro and in vivo) cell adhesion and invasion was abrogated by CXCR4 immunological blockade supporting a contribution of SDF-1alpha/CXCR4 to the metastatic process. E6 and E7 silencing using stable knock-down and the approved anti-viral agent, Cidofovir decreased CXCR4 gene expression as well as both, constitutive and SDF-1alpha-induced cell invasion. In addition, Cidofovir inhibited lung metastasis (both adhesion and invasion) supporting contribution of E6 and E7 oncoproteins to the metastatic process. Finally, potential signals activated downstream SDF-1alpha/CXCR4 and involved in lung homing of E6/E7-expressing tumor cells were investigated. The contribution of the Rho/ROCK pathway was suggested by the inhibitory effect triggered by Cidofovir and further confirmed using Y-27632 (a small molecule ROCK inhibitor). These data suggest a novel and highly translatable therapeutic approach to cervix cancer, by inhibition of adhesion and invasion of circulating HPV-positive tumor cells, using Cidofovir and/or ROCK inhibition.

Show MeSH

Related in: MedlinePlus

Cidofovir inhibits Rho/p160ROCK activation induced by SDF-1/CXCR4.(A) Active Rho was isolated by pull-down assay from HeLa and TC-1 cells after treatment with 100 ng/mL SDF-1 and 10 µM Cidofovir. Densitometric quantification was performed using ImageJ software. **P<0.05, ***P<0.01. (B) Membrane-associated Rho was studied by confocal microscopy. (C) p160ROCK activity was indirectly assessed by measuring myosin light chain phosphorylation (MLC-p). Densitometric analyses were performed using ImageJ software. ***P<0.01.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2657827&req=5

pone-0005018-g006: Cidofovir inhibits Rho/p160ROCK activation induced by SDF-1/CXCR4.(A) Active Rho was isolated by pull-down assay from HeLa and TC-1 cells after treatment with 100 ng/mL SDF-1 and 10 µM Cidofovir. Densitometric quantification was performed using ImageJ software. **P<0.05, ***P<0.01. (B) Membrane-associated Rho was studied by confocal microscopy. (C) p160ROCK activity was indirectly assessed by measuring myosin light chain phosphorylation (MLC-p). Densitometric analyses were performed using ImageJ software. ***P<0.01.

Mentions: Because Rho/ROCK activation is involved in CXCR4 signaling, we examined its' possible activation by the SDF-1α ligand. In E6/E7 positive cell lines HeLa and TC-1, pre-incubation with SDF-1α resulted in clear activation of the Rho protein, as quantified by pull-down assay (Figure 6A, ** P<0.01). In contrast, cells exposed to Cidofovir demonstrated a significant decrease in both baseline and SDF-1α-activated levels of the Rho-GTP active form (Figure 6A, *** P<0.01). Although not quantitative, immunofluorescence assays confirmed pull-down result: SDF-1α-treated HeLa cells exhibited a membrane-associated Rho location, consistent with Rho's activation status; Cidofovir treatement decreased the fluorescence brightness (Figure 6B). We also indirectly examined ROCK activation by assaying the phosphorylation status of the myosin light chain (MLC), confirming that Cidofovir inhibited ROCK activity in both cell types (Figure 6C) *** P<0.01).


Novel anti-metastatic action of cidofovir mediated by inhibition of E6/E7, CXCR4 and Rho/ROCK signaling in HPV tumor cells.

Amine A, Rivera S, Opolon P, Dekkal M, Biard DS, Bouamar H, Louache F, McKay MJ, Bourhis J, Deutsch E, Vozenin-Brotons MC - PLoS ONE (2009)

Cidofovir inhibits Rho/p160ROCK activation induced by SDF-1/CXCR4.(A) Active Rho was isolated by pull-down assay from HeLa and TC-1 cells after treatment with 100 ng/mL SDF-1 and 10 µM Cidofovir. Densitometric quantification was performed using ImageJ software. **P<0.05, ***P<0.01. (B) Membrane-associated Rho was studied by confocal microscopy. (C) p160ROCK activity was indirectly assessed by measuring myosin light chain phosphorylation (MLC-p). Densitometric analyses were performed using ImageJ software. ***P<0.01.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2657827&req=5

pone-0005018-g006: Cidofovir inhibits Rho/p160ROCK activation induced by SDF-1/CXCR4.(A) Active Rho was isolated by pull-down assay from HeLa and TC-1 cells after treatment with 100 ng/mL SDF-1 and 10 µM Cidofovir. Densitometric quantification was performed using ImageJ software. **P<0.05, ***P<0.01. (B) Membrane-associated Rho was studied by confocal microscopy. (C) p160ROCK activity was indirectly assessed by measuring myosin light chain phosphorylation (MLC-p). Densitometric analyses were performed using ImageJ software. ***P<0.01.
Mentions: Because Rho/ROCK activation is involved in CXCR4 signaling, we examined its' possible activation by the SDF-1α ligand. In E6/E7 positive cell lines HeLa and TC-1, pre-incubation with SDF-1α resulted in clear activation of the Rho protein, as quantified by pull-down assay (Figure 6A, ** P<0.01). In contrast, cells exposed to Cidofovir demonstrated a significant decrease in both baseline and SDF-1α-activated levels of the Rho-GTP active form (Figure 6A, *** P<0.01). Although not quantitative, immunofluorescence assays confirmed pull-down result: SDF-1α-treated HeLa cells exhibited a membrane-associated Rho location, consistent with Rho's activation status; Cidofovir treatement decreased the fluorescence brightness (Figure 6B). We also indirectly examined ROCK activation by assaying the phosphorylation status of the myosin light chain (MLC), confirming that Cidofovir inhibited ROCK activity in both cell types (Figure 6C) *** P<0.01).

Bottom Line: E6 and E7 silencing using stable knock-down and the approved anti-viral agent, Cidofovir decreased CXCR4 gene expression as well as both, constitutive and SDF-1alpha-induced cell invasion.The contribution of the Rho/ROCK pathway was suggested by the inhibitory effect triggered by Cidofovir and further confirmed using Y-27632 (a small molecule ROCK inhibitor).These data suggest a novel and highly translatable therapeutic approach to cervix cancer, by inhibition of adhesion and invasion of circulating HPV-positive tumor cells, using Cidofovir and/or ROCK inhibition.

View Article: PubMed Central - PubMed

Affiliation: Laboratoire UPRES EA 27-10 Radiosensibilité des Tumeurs et Tissus Sains, Institut Gustave Roussy/Institut de Radioprotection et de Sureté Nucléaire, Villejuif, France.

ABSTRACT
Cervical cancer is frequently associated with HPV infection. The expression of E6 and E7 HPV oncoproteins is a key factor in its carcinogenicity and might also influence its virulence, including metastatic conversion. The cellular mechanisms involved in metastatic spread remain elusive, but pro-adhesive receptors and their ligands, such as SDF-1alpha and CXCR4 are implicated. In the present study, we assessed the possible relationship between SDF-1alpha/CXCR4 signaling, E6/E7 status and the metastatic process. We found that SDF-1alpha stimulated the invasion of E6/E7-positive cancer cell lines (HeLa and TC-1) in Matrigel though CXCR4 and subsequent Rho/ROCK activation. In pulmonary metastatic foci generated by TC-1 cells IV injection a high proportion of cells expressed membrane-associated CXCR4. In both cases models (in vitro and in vivo) cell adhesion and invasion was abrogated by CXCR4 immunological blockade supporting a contribution of SDF-1alpha/CXCR4 to the metastatic process. E6 and E7 silencing using stable knock-down and the approved anti-viral agent, Cidofovir decreased CXCR4 gene expression as well as both, constitutive and SDF-1alpha-induced cell invasion. In addition, Cidofovir inhibited lung metastasis (both adhesion and invasion) supporting contribution of E6 and E7 oncoproteins to the metastatic process. Finally, potential signals activated downstream SDF-1alpha/CXCR4 and involved in lung homing of E6/E7-expressing tumor cells were investigated. The contribution of the Rho/ROCK pathway was suggested by the inhibitory effect triggered by Cidofovir and further confirmed using Y-27632 (a small molecule ROCK inhibitor). These data suggest a novel and highly translatable therapeutic approach to cervix cancer, by inhibition of adhesion and invasion of circulating HPV-positive tumor cells, using Cidofovir and/or ROCK inhibition.

Show MeSH
Related in: MedlinePlus