Inhibition of cell proliferation and migration through nucleobase-modified polyamidoamine-mediated p53 delivery

Abstract

Introduction: The nucleobase 2-amino-6-chloropurine-modified polyamidoamine (AP-PAMAM) was used as a carrier for p53 gene delivery to achieve the antitumor effects.

Methods and materials: The condensation of p53 plasmid was studied through gel retardation assay, and the transfection efficiency was evaluated through the transfection assay of pEGFP-N3 and pGL-3 plasmids. Using human cervical carcinoma cell line HeLa as a model, the inhibition of cell proliferation and migration was studied through flow cytometry, wound healing and Transwell migration assays, respectively. The p53 expression level was detected through quantitative polymerase chain reaction and Western blotting analyses.

Results: The carrier could condense p53 plasmid into stable nanoparticles at N/P ratios of 2.0, and higher transfection efficiency than polyamidoamine (PAMAM) could be obtained at all the N/P ratios studied. AP-PAMAM-mediated p53 delivery could achieve stronger antiproliferative effect than PAMAM/p53. The antiproliferative effect was identified to be triggered by the induction of cell apoptosis (apoptotic ratio of 26.17%) and cell cycle arrest at S phase. Additionally, AP-PAMAM/p53 transfection has been found to suppress the cell migration and invasion of cancer cells. Finally, the enhanced p53 expression level could be detected after p53 transfection at mRNA and protein levels.

Conclusion: The PAMAM derivative-mediated p53 delivery could be a promising strategy for achieving tumor gene therapy.

Keywords: cell migration; cell proliferation; nucleobase; p53 delivery; polyamidoamine.

Figure 1

Gel retardation assay of AP-PAMAM (A) and PAMAM (B) with the plasmid p3XFLAG-CMV-p53. Abbreviations: AP-PAMAM, 2-amino-6-chloropurine-modified PAMAM; PAMAM, polyamidoamine.

Figure 2

Cytotoxicity analysis of derivative AP-PAMAM at various concentrations in HeLa cells (A) and inhibition of cell proliferation after the p53 transfection mediated by different carriers (B). Notes: The data were presented as the mean ± SD of triplicate experiments *P<0.05; **P<0.01. Abbreviations: AP-PAMAM, 2-amino-6-chloropurine-modified PAMAM; PAMAM, polyamidoamine.

Figure 3

Live/dead assays of HeLa cells after p53 transfection mediated by different carriers. Note: The scale bar is 200 μm. Abbreviations: AP-PAMAM, 2-amino-6-chloropurine-modified PAMAM; PAMAM, polyamidoamine.

Figure 4

Induction of cell apoptosis through the carriers mediated p53 delivery: (A) control; (B) AP-PAMAM; (C) PAMAM/p53; and (D) AP-PAMAM/p53. Note: The cell apoptosis was detected through Annexin V-FITC/PI double staining followed by flow cytometer analysis. Abbreviations: AP-PAMAM, 2-amino-6-chloropurine-modified PAMAM; FITC, fluoresceine isothiocyanate; PI, propidium iodide; PAMAM, polyamidoamine.

Figure 5

RT-PCR (A) and qPCR (B) analyses of p53 mRNA level after p53 transfection mediated by different carriers. Note: **P<0.01. Abbreviations: AP-PAMAM, 2-amino-6-chloropurine-modified PAMAM; PAMAM, polyamidoamine; RT-PCR, real-time polymerase chain reaction; qPCR, quantitative polymerase chain reaction.

Figure 6

Western blotting analysis for the expression levels of p53 and apoptosis-associated proteins. Abbreviations: AP-PAMAM, 2-amino-6-chloropurine-modified PAMAM; PAMAM, polyamidoamine.

Figure 7

The mitochondrial membrane potential analysis of HeLa cells after p53 transfection mediated by different carriers. Abbreviations: AP-PAMAM, 2-amino-6-chloropurine-modified PAMAM; PAMAM, polyamidoamine.

Figure 8

Cell cycle analysis through PI staining and following flow cytometry for the cells after different carriers mediated p53 transfection (A) and the quantitative measurement of cell cycle phase (B). (a) control; (b) AP-PAMAM; (c) PAMAM/p53; and (d) AP-PAMAM/p53. Abbreviations: AP-PAMAM, 2-amino-6-chloropurine-modified PAMAM; PI, propidium iodide; PAMAM, polyamidoamine.

Figure 9

Wound healing assay of HeLa cells after p53 transfection for different time (A) and the quantitative analysis of wound size (B). Note: The scale bar is 400 μm. Abbreviations: AP-PAMAM, 2-amino-6-chloropurine-modified PAMAM; PAMAM, polyamidoamine.

Figure 10

Transwell migration assay of HeLa cells after p53 transfection by different carriers: (A) control; (B) AP-PAMAM; (C) PAMAM/p53; and (D) AP-PAMAM/p53. Note: The scale bar is 100 μm. Abbreviations: AP-PAMAM, 2-amino-6-chloropurine-modified PAMAM; PAMAM, polyamidoamine.

Scheme 1

Synthesis of AP-PAMAM through the modification of 2-amino-6-chloropurine on PAMAM dendrimer. Abbreviations: AP-PAMAM, 2-amino-6-chloropurine-modified PAMAM; PAMAM, polyamidoamine.