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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.

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Cidofovir alters CXCR4 gene and cell surface receptor expression.(A) Quantitative RT-PCR analysis of CXCR4 mRNA level in HeLa cells treated or not with SDF-1 and CXCR4- specific probe. **P<0.05. (B, C) FACS analysis of the cell surface expression of the receptor CXCR4 on HeLa (105 cells/mL) (B) and (C) TC-1 cells (105 cells/mL) treated or not with Cidofovir. Purple area = isotype control. (D) Subcellular localization of the CXCR4 receptor in HeLa studied by confocal microscopy using de MAB173 antibody from R&D System) at 1 µg/mL.
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pone-0005018-g004: Cidofovir alters CXCR4 gene and cell surface receptor expression.(A) Quantitative RT-PCR analysis of CXCR4 mRNA level in HeLa cells treated or not with SDF-1 and CXCR4- specific probe. **P<0.05. (B, C) FACS analysis of the cell surface expression of the receptor CXCR4 on HeLa (105 cells/mL) (B) and (C) TC-1 cells (105 cells/mL) treated or not with Cidofovir. Purple area = isotype control. (D) Subcellular localization of the CXCR4 receptor in HeLa studied by confocal microscopy using de MAB173 antibody from R&D System) at 1 µg/mL.

Mentions: Because Cidofovir treatment inhibits cell invasion in vitro, we reasoned that Cidofovir might alter the abundance of CXCR4 mRNA and cell surface protein level. Q-RT-PCR analysis confirmed this assumption, showing a two-fold decrease in CXCR4 mRNA levels after TC-1 cellular incubation with Cidofovir (Figure 4A, ** P<0.05), whereas in the negative control, SDF-1α did not alter CXCR4 mRNA levels. FACS analysis confirmed that Cidofovir reduced CXCR4 receptors on the surface of both HeLa and TC-1 cells (Figure 4B: P<0.05 and Figure 4C: P<0.01). Immunofluorescence analysis confirmed the aforementioned data, showing both the expected exclusive membrane location of CXCR4 and its' reduced abundance on the cell surface after Cidofovir treatment (Figure 4D)


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 alters CXCR4 gene and cell surface receptor expression.(A) Quantitative RT-PCR analysis of CXCR4 mRNA level in HeLa cells treated or not with SDF-1 and CXCR4- specific probe. **P<0.05. (B, C) FACS analysis of the cell surface expression of the receptor CXCR4 on HeLa (105 cells/mL) (B) and (C) TC-1 cells (105 cells/mL) treated or not with Cidofovir. Purple area = isotype control. (D) Subcellular localization of the CXCR4 receptor in HeLa studied by confocal microscopy using de MAB173 antibody from R&D System) at 1 µg/mL.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0005018-g004: Cidofovir alters CXCR4 gene and cell surface receptor expression.(A) Quantitative RT-PCR analysis of CXCR4 mRNA level in HeLa cells treated or not with SDF-1 and CXCR4- specific probe. **P<0.05. (B, C) FACS analysis of the cell surface expression of the receptor CXCR4 on HeLa (105 cells/mL) (B) and (C) TC-1 cells (105 cells/mL) treated or not with Cidofovir. Purple area = isotype control. (D) Subcellular localization of the CXCR4 receptor in HeLa studied by confocal microscopy using de MAB173 antibody from R&D System) at 1 µg/mL.
Mentions: Because Cidofovir treatment inhibits cell invasion in vitro, we reasoned that Cidofovir might alter the abundance of CXCR4 mRNA and cell surface protein level. Q-RT-PCR analysis confirmed this assumption, showing a two-fold decrease in CXCR4 mRNA levels after TC-1 cellular incubation with Cidofovir (Figure 4A, ** P<0.05), whereas in the negative control, SDF-1α did not alter CXCR4 mRNA levels. FACS analysis confirmed that Cidofovir reduced CXCR4 receptors on the surface of both HeLa and TC-1 cells (Figure 4B: P<0.05 and Figure 4C: P<0.01). Immunofluorescence analysis confirmed the aforementioned data, showing both the expected exclusive membrane location of CXCR4 and its' reduced abundance on the cell surface after Cidofovir treatment (Figure 4D)

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