5-Fluorouracil-loaded poly(ε-caprolactone) nanoparticles combined with phage E gene therapy as a new strategy against colon cancer

Abstract

This work aimed to develop a new therapeutic approach to increase the efficacy of 5-fluorouracil (5-FU) in the treatment of advanced or recurrent colon cancer. 5-FU-loaded biodegradable poly(ε-caprolactone) nanoparticles (PCL NPs) were combined with the cytotoxic suicide gene E (combined therapy). The SW480 human cancer cell line was used to assay the combined therapeutic strategy. This cell line was established from a primary adenocarcinoma of the colon and is characterized by an intrinsically high resistance to apoptosis that correlates with its resistance to 5-FU. 5-FU was absorbed into the matrix of the PCL NPs during synthesis using the interfacial polymer disposition method. The antitumor activity of gene E from the phage ϕX174 was tested by generating a stable clone (SW480/12/E). In addition, the localization of E protein and its activity in mitochondria were analyzed. We found that the incorporation of 5-FU into PCL NPs (which show no cytotoxicity alone), significantly improved the drug's anticancer activity, reducing the proliferation rate of colon cancer cells by up to 40-fold when compared with the nonincorporated drug alone. Furthermore, E gene expression sensitized colon cancer cells to the cytotoxic action of the 5-FU-based nanomedicine. Our findings demonstrate that despite the inherent resistance of SW480 to apoptosis, E gene activity is mediated by an apoptotic phenomenon that includes modulation of caspase-9 and caspase-3 expression and intense mitochondrial damage. Finally, a strongly synergistic antiproliferative effect was observed in colon cancer cells when E gene expression was combined with the activity of the 5-FU-loaded PCL NPs, thereby indicating the potential therapeutic value of the combined therapy.

Keywords: 5-fluorouracil; E gene; colon cancer; combined therapy; gene therapy; poly (ε-caprolactone).

Figure 1

Scanning electron microscope (A) and high resolution transmission electron microscope (B) images of PCL NPs. Note: All scale bars shown in the figure are 150 nm. Abbreviations: NPs, nanoparticles; PCL, poly(ε-caprolactone).

Figure 2

5-FU loading and in vitro release studies. (A) Efficiency of 5-FU entrapment (%) into PCL NPs as a function of antitumor drug concentration (inset: corresponding 5-FU loading values, %). The lines are guides for the eye and have no other significance. (B) 5-FU release (%) from PCL NPs at 37.0°C ± 0.5°C as a function of incubation time in PBS (pH 7.4 ± 0.1). Abbreviations: 5-FU, 5-fluorouracil; NPs, nanoparticles; PBS, phosphate-buffered saline; PCL, poly(ε-caprolactone).

Figure 3

In vitro cytotoxicity of PCL NPs over a range of concentrations (10–200 mM) (A) and cytotoxicity of 5-FU-loaded PCL NPs in comparison to 5-FU (B) in colon cancer SW480 cells after 3 days of incubation. Data are represented as means ± SD of quadruplicate cultures. To calculate the %RCV (see Materials and methods) SW480 cells without treatment were used as control. Abbreviations: 5-FU, 5-fluorouracil; NPs, nanoparticles; PBS, phosphate-buffered saline; PCL, poly(ε-caprolactone); %RCV, percentage of relative cell viability; SD, standard deviation.

Figure 4

Growth arrest by E gene expression in SW480 cells. (A) RT-PCR detection of E gene expression in SW480 transfected cells (SW480/12/E) before and after Dox exposure. Amplified RT-PCR products of E and β-actin mRNA at different time periods were separated by 2% agarose gel electrophoresis and visualized with ethidium bromide. RT-PCR of gene E: Lane 1: SW480/12/E cells in the absence of Dox; lanes 2–4: SW480/12/E cells 24, 48, and 72 hours after Dox induction, respectively. RT-PCR of β-actin: Lane 5: SW480/12/E cells in absence of Dox; lanes 6–8: SW480/12/E cells 24, 48. and 72 hours after Dox induction, respectively. Lane 9: DNA ladder; lane 10: pTRE-E (positive control); lane 11: parental SW480 cells (negative control). (B) MTT assay of SW480/12/E cells induced with Dox showed a significantly higher rate of cell death than for SW480/12/E cells in the absence of Dox, parental cells and cells transfected with empty vector (P < 0.05). To calculate the %RCV (see Materials and methods) SW480 cells without transfect reagents were used as control. Data are represented as means ± SD of quadruplicate cultures. Abbreviations: Dox, doxorubicin; 5-FU, 5-fluorouracil; NPs, nanoparticles; PCL, poly(ε-caprolactone); %RCV, percentage of relative cell viability; SD, standard deviation.

Figure 5

Apoptosis signals after E gene expression in SW480 cells. (A) SW480/12/E cells before and after Dox induction at times indicated were analyzed by FACScan to determine apoptotic cell death. The apoptosis was assessed after propidium iodine staining by calculating the percentage of cells in the sub-G1 fraction. SW480/12/E cells treated with zVAD-fmk were also analyzed at 72 hours. SW480 parental cells were used as the control. Data shown are representative results from four independent experiments. (B) Activated (cleaved) caspase-3, caspase-9, and pro-caspase-8 were detected by Western blot analysis using specific antibodies at the indicated time points after Dox treatment. zVAD-fmk was applied to determine whether caspases were involved in the apoptotic process in SW480/12/E cells induced with Dox (72 hours). The filter was probed with a β-actin antibody to determine whether the amounts of protein in each lane were comparable. Immunoblots were visualized using an enhanced chemiluminescence detection system. Abbreviation: Dox, doxorubicin.

Figure 6

Mitochondrial localization of the GFP/E fusion protein in SW480 cells. Representative fluorescent microscopy images of transfected SW480 cells expressing fusion protein E-GFP at 24 (×10), 48 (×40), and 72 hours (×40). The dotted pattern of GFP-E fluorescence is shown in green. The majority of expressed GFP-E was found to be colocalized with MitoFluor (shown in red color). Colocalization is shown in yellow. Cell nuclei were counterstained with DAPI.

Figure 7

Mitochondrial damage caused by E protein in SW480 cells. (A) SW480 parental cells and SW480/12/E before and 24, 48, and 72 hours after Dox induction were stained with DiOC6, and analyzed by flow cytometry to determine the mitochondrial membrane potential (ΔΨm) disruption caused by E gene expression. Data shown are representative results from four independent experiments. (B) Ultrastructural analysis showed that the morphology of SW480/12/E cells not induced by Dox was similar to that of SW480 parental cells, with a typical presence of a large nucleus and light cytoplasmic complexion containing well-preserved organelles including mitochondria (a, 2000×). In contrast, E gene expression in SW480/12/E after Dox exposure generated a large number of altered mitochondria with disrupted cristae (b, 6000×). These cells eventually presented noticeably dilated mitochondria (c, 12,000×). Furthermore, mitochondrial changes in some cells were accompanied by the presence of dilated smooth endoplasmic reticulum (d, 9000×), and the presence of clusters of intermediate filaments (e, 4000×).

Figure 8

Synergistic effect of 5-FU-loaded PCL NPs associated with E gene expression in SW480 cancer cells. The combined therapy showed (A) a synergistic effect on growth arrest in the SW480/12/E colon cancer cells, and (B) a significant increase in apoptosis compared to single treatments. To calculate the %RCV (see Materials and methods) SW480 cells without treatment were used as control. Data are represented as means ± SD of quadruplicate cultures. Abbreviations: Dox, doxorubicin; 5-FU, 5-fluorouracil; NPs, nanoparticles; PCL, poly(ε-caprolactone); %RCV, percentage of relative cell viability; SD, standard deviation.