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Inhibition of cell growth and invasion by epidermal growth factor-targeted phagemid particles carrying siRNA against focal adhesion kinase in the presence of hydroxycamptothecin.

Cai XM, Xie HL, Liu MZ, Zha XL - BMC Biotechnol. (2008)

Bottom Line: However, no significant cell growth inhibition was obtained.Moreover, we also observed that the particles could potently suppress cell growth and cell invasion.These results indicated that EGF-targeted phagemid particles might be a promising tool for anti-cancer siRNA delivery in the presence of HCPT.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Biochemistry and Molecular Biology, Shanghai Medical College, Fudan University, Shanghai, PR China. caixm06@gmail.com

ABSTRACT

Background: Previous studies demonstrated the EGF-targeted phagemid particles carrying siRNA against Akt could be expressed efficiently in the presence of hydroxycamptothecin (HCPT). However, no significant cell growth inhibition was obtained. This study was to further investigate whether the EGF-targeted phagemid particles carrying siRNA would be a promising tool for anti-cancer siRNA delivery.

Results: We found that pSi4.1-siFAK phagemid particles could significantly inhibit the expression of focal adhesion kinase in the HCPT-treated cells. Moreover, we also observed that the particles could potently suppress cell growth and cell invasion.

Conclusion: These results indicated that EGF-targeted phagemid particles might be a promising tool for anti-cancer siRNA delivery in the presence of HCPT.

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Related in: MedlinePlus

Immunocytochemistry assay of EGFR expression and Western-blot analysis of the specific FAK gene silencing by EGF-targeted phagemid particles mediated RNA interference. A: In the immunocytochemical assay, H1299 cells showed a strong positive EGFR immunoreactivity, while very light immunostaining was observed in the U87 cells that were used as negative controls. 1, H1299; 2, U87. B: H1299 cells were infected with pSi4.1-siFAK phagemid particles. In HCPT treated-cells, the pSi4.1-siFAK phagemid particles could inhibit FAK expression to a great extent. 1, H1299; 2, H1299 infected with pSi4.1-simock phagemid particles; 3, H1299 infected with pSi4.1-simock phagemid particles in the presence of 2.5 μM HCPT; 4, H1299 infected with pSi4.1-siFAK phagemid particles; 5, H1299 infected with pSi4.1-siFAK phagemid particles in the presence of 2.5 μM HCPT; 6, H1299 transfected with pSi4.1-siFAK using Lipofectamine 2000.
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Figure 2: Immunocytochemistry assay of EGFR expression and Western-blot analysis of the specific FAK gene silencing by EGF-targeted phagemid particles mediated RNA interference. A: In the immunocytochemical assay, H1299 cells showed a strong positive EGFR immunoreactivity, while very light immunostaining was observed in the U87 cells that were used as negative controls. 1, H1299; 2, U87. B: H1299 cells were infected with pSi4.1-siFAK phagemid particles. In HCPT treated-cells, the pSi4.1-siFAK phagemid particles could inhibit FAK expression to a great extent. 1, H1299; 2, H1299 infected with pSi4.1-simock phagemid particles; 3, H1299 infected with pSi4.1-simock phagemid particles in the presence of 2.5 μM HCPT; 4, H1299 infected with pSi4.1-siFAK phagemid particles; 5, H1299 infected with pSi4.1-siFAK phagemid particles in the presence of 2.5 μM HCPT; 6, H1299 transfected with pSi4.1-siFAK using Lipofectamine 2000.

Mentions: Previous studies showed that the cell-targeted phagemid particles were efficient siRNA delivery vectors in the presence of HCPT and they could efficiently deliver siRNA against Akt into targeted cells in the presence of HCPT [10]. But, no significant growth inhibition was observed. Thus, to be an effective anti-cancer siRNA delivery vector, more studies should be performed, such as carrying siRNA against other oncogenes. In this study, we made phagemid particles carrying siRNA against FAK to infect H1299 cells and examined the therapeutic potential of this approach. First, the short hairpin RNA (shRNA) against FAK was subcloned into pSi4.1CMV-f1, thus forming pSilencer4.1-siFAK (pSi4.1-siFAK) (Fig. 1A). Then, we purified ssDNA from phagemid particles to analyze the ratio of phagemids to helper phage genomes packaged in the phagemid particles. The results indicated that almost all the DNA packaged comprised phagemids (Fig. 1B). Previously, the modified helper phage genome (plasmid) M13EGFKO7CT was created to produce EGF-targeted phagemid particles [8,10]. The M13EGFKO7CT plasmid was used to package pSi4.1-siFAK phagemid particles, following which the phagemid particles displayed the EGF ligand. In the immunocytochemical assay, we found that H1299 cells showed a strong positive EGFR immunoreactivity, while very light immunostaining was observed in the U87 cells that were used as negative controls (Fig. 2A). Therefore, we infected H1299 cells with pSi4.1-siFAK phagemid particles. Western blotting assay showed that the pSi4.1-siFAK plasmid transfected by lipofectamine could significantly block the expression of FAK. This was not observed in cells transduced with pSi4.1-siFAK phagemid particles without HCPT treatment. Surprisingly, in HCPT treated-cells, the pSi4.1-siFAK phagemid particles could inhibit FAK expression to a great extent. Inhibition of FAK expression was not found in the cells infected with mock phagemid particles (Fig. 2B). Taken together, the vectors could deliver siRNA to human carcinoma cells efficiently in the presence of HCPT. HCPT had been shown to increase the efficiency of transduction of the phagemid vectors [1,9,16]. However, the mechanism by which HCPT increased transgene expression was not fully understood [1,9]. It was thought to involve the activation of the host cell repair machinery in response to DNA damage [1,16,17]; however, further studies are required to confirm this.


Inhibition of cell growth and invasion by epidermal growth factor-targeted phagemid particles carrying siRNA against focal adhesion kinase in the presence of hydroxycamptothecin.

Cai XM, Xie HL, Liu MZ, Zha XL - BMC Biotechnol. (2008)

Immunocytochemistry assay of EGFR expression and Western-blot analysis of the specific FAK gene silencing by EGF-targeted phagemid particles mediated RNA interference. A: In the immunocytochemical assay, H1299 cells showed a strong positive EGFR immunoreactivity, while very light immunostaining was observed in the U87 cells that were used as negative controls. 1, H1299; 2, U87. B: H1299 cells were infected with pSi4.1-siFAK phagemid particles. In HCPT treated-cells, the pSi4.1-siFAK phagemid particles could inhibit FAK expression to a great extent. 1, H1299; 2, H1299 infected with pSi4.1-simock phagemid particles; 3, H1299 infected with pSi4.1-simock phagemid particles in the presence of 2.5 μM HCPT; 4, H1299 infected with pSi4.1-siFAK phagemid particles; 5, H1299 infected with pSi4.1-siFAK phagemid particles in the presence of 2.5 μM HCPT; 6, H1299 transfected with pSi4.1-siFAK using Lipofectamine 2000.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
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Figure 2: Immunocytochemistry assay of EGFR expression and Western-blot analysis of the specific FAK gene silencing by EGF-targeted phagemid particles mediated RNA interference. A: In the immunocytochemical assay, H1299 cells showed a strong positive EGFR immunoreactivity, while very light immunostaining was observed in the U87 cells that were used as negative controls. 1, H1299; 2, U87. B: H1299 cells were infected with pSi4.1-siFAK phagemid particles. In HCPT treated-cells, the pSi4.1-siFAK phagemid particles could inhibit FAK expression to a great extent. 1, H1299; 2, H1299 infected with pSi4.1-simock phagemid particles; 3, H1299 infected with pSi4.1-simock phagemid particles in the presence of 2.5 μM HCPT; 4, H1299 infected with pSi4.1-siFAK phagemid particles; 5, H1299 infected with pSi4.1-siFAK phagemid particles in the presence of 2.5 μM HCPT; 6, H1299 transfected with pSi4.1-siFAK using Lipofectamine 2000.
Mentions: Previous studies showed that the cell-targeted phagemid particles were efficient siRNA delivery vectors in the presence of HCPT and they could efficiently deliver siRNA against Akt into targeted cells in the presence of HCPT [10]. But, no significant growth inhibition was observed. Thus, to be an effective anti-cancer siRNA delivery vector, more studies should be performed, such as carrying siRNA against other oncogenes. In this study, we made phagemid particles carrying siRNA against FAK to infect H1299 cells and examined the therapeutic potential of this approach. First, the short hairpin RNA (shRNA) against FAK was subcloned into pSi4.1CMV-f1, thus forming pSilencer4.1-siFAK (pSi4.1-siFAK) (Fig. 1A). Then, we purified ssDNA from phagemid particles to analyze the ratio of phagemids to helper phage genomes packaged in the phagemid particles. The results indicated that almost all the DNA packaged comprised phagemids (Fig. 1B). Previously, the modified helper phage genome (plasmid) M13EGFKO7CT was created to produce EGF-targeted phagemid particles [8,10]. The M13EGFKO7CT plasmid was used to package pSi4.1-siFAK phagemid particles, following which the phagemid particles displayed the EGF ligand. In the immunocytochemical assay, we found that H1299 cells showed a strong positive EGFR immunoreactivity, while very light immunostaining was observed in the U87 cells that were used as negative controls (Fig. 2A). Therefore, we infected H1299 cells with pSi4.1-siFAK phagemid particles. Western blotting assay showed that the pSi4.1-siFAK plasmid transfected by lipofectamine could significantly block the expression of FAK. This was not observed in cells transduced with pSi4.1-siFAK phagemid particles without HCPT treatment. Surprisingly, in HCPT treated-cells, the pSi4.1-siFAK phagemid particles could inhibit FAK expression to a great extent. Inhibition of FAK expression was not found in the cells infected with mock phagemid particles (Fig. 2B). Taken together, the vectors could deliver siRNA to human carcinoma cells efficiently in the presence of HCPT. HCPT had been shown to increase the efficiency of transduction of the phagemid vectors [1,9,16]. However, the mechanism by which HCPT increased transgene expression was not fully understood [1,9]. It was thought to involve the activation of the host cell repair machinery in response to DNA damage [1,16,17]; however, further studies are required to confirm this.

Bottom Line: However, no significant cell growth inhibition was obtained.Moreover, we also observed that the particles could potently suppress cell growth and cell invasion.These results indicated that EGF-targeted phagemid particles might be a promising tool for anti-cancer siRNA delivery in the presence of HCPT.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Biochemistry and Molecular Biology, Shanghai Medical College, Fudan University, Shanghai, PR China. caixm06@gmail.com

ABSTRACT

Background: Previous studies demonstrated the EGF-targeted phagemid particles carrying siRNA against Akt could be expressed efficiently in the presence of hydroxycamptothecin (HCPT). However, no significant cell growth inhibition was obtained. This study was to further investigate whether the EGF-targeted phagemid particles carrying siRNA would be a promising tool for anti-cancer siRNA delivery.

Results: We found that pSi4.1-siFAK phagemid particles could significantly inhibit the expression of focal adhesion kinase in the HCPT-treated cells. Moreover, we also observed that the particles could potently suppress cell growth and cell invasion.

Conclusion: These results indicated that EGF-targeted phagemid particles might be a promising tool for anti-cancer siRNA delivery in the presence of HCPT.

Show MeSH
Related in: MedlinePlus