Colloids and Surfaces B: Biointerfaces 129 (2015) 54–62
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N-Isopropylacrylamide-modified polyethylenimine-mediated p53 gene delivery to prevent the proliferation of cancer cells Jianxu Zhang 1 , Di Wu 1 , Zhen Xing, Shaojun Liang, Haobo Han, Hui Shi, Yan Zhang, Yan Yang ∗ , Quanshun Li ∗ Key Laboratory for Molecular Enzymology and Engineering of Ministry of Education, School of Life Sciences, Jilin University, Changchun 130012, China
a r t i c l e
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Article history: Received 16 December 2014 Received in revised form 16 February 2015 Accepted 10 March 2015 Available online 18 March 2015 Keywords: N-Isopropylacrylamide Polyethylenimine p53 gene Gene therapy Cell apoptosis Cell cycle arrest
a b s t r a c t In this paper, N-isopropylacrylamide-modified polyethylenimine (PEN) was constructed through Michael addition and employed as a carrier to achieve the p53 gene delivery, using HeLa (p53wt) and PC-3 cells (p53null) as models. After PEN-mediated p53 transfection, expression level of p53 in HeLa and PC3 cells was up-regulated at both mRNA and protein levels. Due to the exogenous p53 expression, the inhibition of cell proliferation was observed through MTT analysis, attributing to the activation of apoptosis and cell cycle arrest. Using flow cytometric analysis, early apoptotic ratios of 54.95% and 27.06% after PENmediated p53 transfection were detected in PC-3 and HeLa cells, respectively, indicating that PC-3 cells were more sensitive to the exogenous p53 transfection than HeLa cells. Meanwhile, G1 phase arrest was detected in PC-3 cells while a unique G2 phase arrest was identified in HeLa cells after p53 transfection. Through Western blotting, activity analysis of caspase-3, caspase-8 and caspase-9 and mitochondrial membrane potential measurement, the apoptosis induced by PEN-mediated p53 transfection was conducted in a mitochondria-dependent apoptosis pathway. These results demonstrated that PEN could successfully mediate the p53 gene delivery and up-regulate the cellular p53 expression level, triggering a significant p53-dependent anti-proliferative effect on tumor cells. © 2015 Elsevier B.V. All rights reserved.
1. Introduction The p53 gene has been widely studied and is considered to be an effective tumor suppressor that plays key roles in apoptosis induction and cell cycle regulation [1]. The processes are mainly dependent on the DNA repair proteins and on pro-apoptotic proteins in the mitochondrial pathway induced by the p53 protein [2]. Nearly 50% of human tumors have been identified to possess mutations or deletions in the p53 gene [3,4], and thus, it is regarded as an important target in anticancer gene therapy. Previous reports have shown that transfection of exogenous p53 gene into p53null and p53mut cell lines could induce cell apoptosis and cell cycle arrest [5,6] and even improve the efficiency of conventional chemo/radiotherapy [7,8]. Meanwhile, enhancing the activity of wild-type p53 in p53wt cells can also trigger a variety of anti-proliferative effects (e.g., activation of apoptotic cell death) [9]. Thus, inducing the expression of wild-type p53 through gene transfer techniques or carriers could be an effective strategy for
∗ Corresponding authors. Tel.: +86 431 85155381; fax: +86 431 85155200. E-mail addresses: [email protected] (Y. Yang), [email protected] (Q. Li). 1 These authors contributed equally to the work. http://dx.doi.org/10.1016/j.colsurfb.2015.03.032 0927-7765/© 2015 Elsevier B.V. All rights reserved.
cancer treatment. However, constructing safe and efficient gene delivery carriers is still a key issue to be addressed in p53-based gene therapy. Non-viral carriers, and especially cationic polymers, have been widely used as gene delivery systems due to their low immunogenicity, high gene-loading capacity, flexible designability, potential safety and low production cost [10–12]. Among these carriers, branched polyethylenimine (PEI) with a weight-average molecular weight of 25 kDa (PEI25K) has exhibited superior gene transfection efficiency both in vitro and in vivo, because its high positive charge and proton-buffering capacity can condense and protect nucleic acids in serum and facilitate their internalization into cells and release from endosomes [13–17]. However, high positive charge density can lead to membrane-disruptive properties and nonspecific interaction with negatively charged proteins in the serum and thus cause severe problems [18,19], e.g., high cytotoxicity and hemolysis, and serum instability of the nanoparticles. To overcome these disadvantages, chemical modification of PEI25K to improve its transfection efficiency and/or to decrease its cytotoxicity has been actively investigated, especially grafting of hydrophobic groups [20–23]. These PEI derivatives exhibited amphiphilic characteristics, similar to the phospholipid layers in the cell membrane, leading to enhanced biocompatibility
J. Zhang et al. / Colloids and Surfaces B: Biointerfaces 129 (2015) 54–62
and transfection efficiency. Meanwhile, the grafted hydrophobic moieties were favorable for reducing the cationic density, which would not only reduce the cytotoxicity but also facilitate gene dissociation from polyplexes in the cytosol to further improve the transfection efficiency. Recently, Tian et al. [24] constructed a PEI derivative (namely PEN) with a higher transfection efficiency and lower cytotoxicity than PEI25K through modification of PEI25K with N-isopropylacrylamide via Michael addition. Compared with previous modification strategies, this approach did not reduce the content of protonated amines, and would not influence the formation of stable nanocomplexes between the carrier and pDNA through electrostatic interaction. The improved hydrophobicity would be favorable for the complexes’ interaction with cell membrane and gene dissociation in the cytosol, leading to enhanced endocytosis and transfection. Notably, this derivative showed an improved ability of nuclear uptake, despite no conjugation with targeting ligands or nuclear localization signals. Thus, the derivative PEN is of great potential in the delivery of plasmid harboring a tumor suppressor gene to cell nuclei to achieve gene expression. In the present research, the derivative PEN was employed as a carrier to achieve p53 gene delivery using the human cervical carcinoma cell line HeLa (p53wt) and the prostate tumor cell line PC-3 (p53null) as models. The performance of the gene transfection was evaluated at the mRNA and protein expression levels, and the inhibition of cancer cell proliferation after p53 gene transfection was investigated. Finally, the detailed mechanism of the therapeutic effect induced by cell apoptosis and cell cycle arrest was systematically elucidated. 2. Materials and methods
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36 h, 20 L of MTT solution (5 mg/mL in PBS) was added to each well, and the plates were incubated for an additional 4 h. The MTT solution was then removed, and 150 L of DMSO was added to dissolve the formazan crystals. The plates were incubated for an additional 10 min, after which the absorbance at 492 nm was recorded using a GF-M3000 microplate reader (Shandong, China). The cell viability (%) was calculated as Asample /Acontrol , where Asample and Acontrol were the absorbance values of the treated and untreated cells, respectively. Additionally, after treatment with polymer/p53 nanocomplex (2:1, wt/wt) with a plasmid mass of 2.5 g for 36 h, the cells in a 6-well plate were harvested, washed with ice-cold PBS and fixed with 70% ice-cold ethanol at 4 ◦ C for 5 min. The fixed cells were then washed with PBS, and stained with DAPI solution (1 g/mL) for 10 min at room temperature. The cellular morphology was observed through an Olympus IX71 fluorescence microscope (Japan).
2.3. Induction of cell apoptosis by p53 transfection HeLa and PC-3 cells were seeded at a density of 5 × 105 cells per well in 6-well plates and incubated for 24 h before transfection. As described above, the p53 transfection was performed using different polymer/p53 samples (2:1, wt/wt) with a plasmid mass of 2.5 g. After 36 h, the cells were harvested, washed with cold PBS twice, and re-suspended in binding buffer according to the manufacturer’s protocols. The cells were then incubated with annexin-V-FITC and PI, mixed and incubated at room temperature for 10 min in the dark. Apoptosis was determined by analyzing 15,000 ungated cells using FACSCalibur (BD Biosciences, Mountain View, USA) and CellQuest software (Becton-Dickinson, USA).
2.1. Materials 2.4. Cell cycle analysis after p53 transfection The plasmid p3XFLAG-CMV-p53 encoding wild-type p53 protein was kindly provided by Dr. Yuhang Zhang (Institute of Biochemistry and Cell Biology, CAS, China), amplified in Escherichia coli DH5␣ and purified using Plasmid Maxi kit (Axygen). Branched PEI25K, N-isopropylacrylamide and heparin were purchased from Aldrich and used as received. 3-(4,5-Dimethylthiazol-2yl)-2,5-diphenyltetrazolium bromide (MTT) and 4,6-diamidino-2phenylindile (DAPI) were purchased from Amersco (Solon, USA). Dulbecco’s modified Eagle’s medium (DMEM) and fetal bovine serum (FBS) were purchased from Gibco (Grand Island, USA). Lipofectamine2000 was purchased from Invitrogen, and polyvinylidene fluoride (PVDF) membrane was obtained from Millipore. Annexin-V-FITC/PI apoptosis detection kit, cell cycle detection kit, and caspase-3, 8 and 9 activity assay kits were purchased from Bestbio (Beijing, China). The mitochondrial membrane potential assay kit with JC-1 and BCA protein assay kit were obtained from Beyotime (Jiangsu, China). DNase I, PrimeScript TMRT Master Mix, RNAIso Plus and ExTaq reverse transcriptase were obtained from TaKaRa (Dalian, China). All the other chemicals were of the highest reagent grade commercially available and used as received. 2.2. Cell proliferation assay HeLa (p53wt) and PC-3 (p53null) cells were propagated to confluence in DMEM supplemented with 100 IU/mL penicillin, 100 mg/mL streptomycin and 10% FBS and were maintained at 37 ◦ C in a humidified atmosphere of 5% CO2 . The inhibition of cell proliferation induced by p53 gene transfection was evaluated by MTT assay. The cells were seeded onto 96-well plates at a density of 8.0 × 103 cells per well, pre-incubated in FBS-free DMEM for 24 h, and further incubated with polymer/p53 nanocomplex (2:1, wt/wt) with a plasmid mass of 0.1 g. After
For cell cycle analysis, HeLa and PC-3 cells were seeded at a density of 5 × 105 cells per well in 6-well plates and incubated for 24 h and then subjected to p53 transfection using different polymer/p53 samples (2:1, wt/wt) with a plasmid mass of 2.5 g. After 36 h, the cells were harvested, fixed in ice-cold 70% ethanol and then stored at 4 ◦ C overnight. Prior to analysis, the cells were washed twice with PBS, suspended in 0.5 mL of cold PI solution containing 10 L of RNase A (25 g/mL) and 10 L of PI (50 g/mL), and incubated at 37 ◦ C for an additional 30 min in the dark. The cell cycle was determined by analyzing 15,000 ungated cells using FACSCalibur (BD Biosciences, Mountain View, USA) and CellQuest software (Becton-Dickinson, USA).
2.5. Evaluation of p53 gene transfection at mRNA level The p53 transfection at the mRNA level was evaluated using the reverse transcription PCR (RT-PCR) technique, with -actin as a reference housekeeping gene. Total RNA was extracted from cells using RNAiso Plus, as recommended by the manufacturer. For reverse transcription, first-strand cDNA was synthesized from 1 g of total RNA, and the obtained cDNA was used as a template for classical PCR amplification, as follows: 1 cycle at 94 ◦ C for 2 min, followed by 30 cycles at 94 ◦ C for 30 s, 55 ◦ C for 30 s and 72 ◦ C for 1 min. The PCR products were detected by 2% agarose gel electrophoresis. The primers were as follows: p53 forward: 5 -GGCTCTGACTGTACCACCATCCA-3 ; reverse: 5 -GGCACAAACACGCACCTCAAAG-3 ; -actin forward: 5 -TCTGGCACCACACCTTCTACAATG-3 ; reverse: 5 -GGATAGCACAGCCTGGATAGCAA-3 .
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2.6. Western blotting analysis The p53 transfection at the protein level and the expression of other proteins associated with apoptosis were evaluated by Western blotting analysis, and the protein expression was normalized against -actin. After p53 transfection (polymer/p53 of 2:1, wt/wt) for 36 h, HeLa and PC-3 cells were harvested, washed with ice-cold PBS twice and lysed with RIPA lysis buffer on ice for 2 h. The lysates were centrifuged at 12,000 rpm for 10 min. The supernatants were collected, and the concentrations of protein were measured by BCA protein assay kit. Equal amounts of protein were subjected to electrophoresis by SDS-PAGE and transferred to PVDF membranes by electroblotting. The membranes were blocked in PBS solution containing 10% non-fat milk and 0.1% Tween-20 for 2 h and subsequently incubated with the desired antibodies at 4 ◦ C overnight. The membranes were then incubated with appropriate HRP-labeled secondary antibody for 1 h, and specific proteins were detected by enhanced chemical luminescence (Amersham, UK). 2.7. Caspase-3, 8 and 9 activity assay The caspase-3, 8 and 9 activities were determined through corresponding activity assay kits according to the manufacturer’s protocols. Briefly, HeLa and PC-3 cells after p53 transfection (polymer/p53 of 2:1, wt/wt) for 36 h were harvested, washed twice with PBS, and lysed in 100 mL lysis buffer supplied in the kit. The resulting suspension was centrifuged at 10,000 × g for 10 min (4 ◦ C), and 10 mL supernatant was used for detecting the caspase-3, 8 and 9 activities. The activities were monitored by measuring the changes of absorbance at 405 nm using a GF-M3000 microplate reader (Shandong, China). 2.8. Mitochondrial membrane potential measurement Mitochondrial membrane potential changes were detected using the fluorescent probe JC-1 according to a previous report [25]. Briefly, HeLa and PC-3 cells were seeded at a density of 5 × 105 cells per well in 6-well plates and incubated for 24 h before transfection. After p53 transfection (polymer/p53 of 2:1, wt/wt) for 36 h, the cells were rinsed with PBS, harvested with trypsin-EDTA solution and collected by centrifugation at 2000 × g for 2 min. The cell pellets were then stained with fluorescent probe JC-1 provided by the mitochondrial membrane potential assay kit, washed twice with PBS and examined through an Olympus IX71 fluorescence microscope (Japan). 3. Results and discussion 3.1. Synthesis and characterization of PEN The derivative PEN was synthesized through the modification of PEI25K with N-isopropylacrylamide via Michael addition according to the previous report [24]. Typical 1 H NMR spectra of PEI25K and PEN are shown in Fig. S1, and the number of grafted N-isopropylacrylamide units could be determined based on the integral ratio of the peak at 4.05 ppm to the peak groups of PEI25K (2.50–2.70 ppm). Based on a previous report on optimization of the grafting ratio [24], PEN175 was selected and employed in the present research. The binding affinity of PEN to plasmid pEGFP-N3 was examined by agarose gel retardation assay (Fig. S2), and complete pDNA retardation by PEN could be induced at a critical mass ratio of 0.4, with a similar binding ability as PEI25K (critical mass ratio of 0.3). Additionally, PEN could efficiently protect the plasmid from degradation of DNase I, indicating the formation of stable PEN/pDNA nanocomplex in the presence of DNase I (Fig. S3). The particle size and zeta potential of PEI25K/pDNA and PEN/pDNA at
Fig. 1. The cell viability assay of PC-3 (A) and HeLa cells (B) after the p53 transfection mediated by different carriers. The data were expressed as mean value ± SD of three experiments.
different mass ratios were then determined by Malvern Nano ZS90 Zetasizer. As shown in Fig. S4, the particle size values of nanocomplexes were in the range of 70–175 nm in the mass ratios studied, and the zeta potential exhibited an increasing tendency with the increasing ratio of carrier. For PEN/pDNA nanocomplex, the particle size and zeta potential were measured to be 108 nm and +15.3 mV at a mass ratio of 2.0, and thus it will be suitable for further transfection investigations especially for potential in vivo application. To study the interaction between the carriers and proteins, BSA was used as a model protein to simulate the carriers’ electrostatic adsorption of serum proteins. As shown in Fig. S5, the adsorption ability was slightly decreased after the modification, as the modification of N-isopropylacrylamide did not introduce the hydrophilic segments to resist the adsorption of negatively charged proteins. The hemoglobin release from erythrocytes after treatment with different concentration of carriers was then monitored to further investigate the nonspecific interactions of polymers with blood components (Fig. S6). Compared with PEI25K, PEN exhibited relatively lower hemolysis and less negative effects on the erythrocyte membranes, which meant an improved hemocompatibility.
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Fig. 2. Classical RT-PCR analysis of p53 expression in PC-3 and HeLa cells after the p53 transfection mediated by different carriers. Lane 1: control; lane 2: Lipofectamine2000 /p53 transfection; lane 3: PEI25K/p53 transfection; lane 4: PEN/p53 transfection.
Fig. 3. Western blotting analysis of p53 expression in PC-3 and HeLa cells after the p53 transfection mediated by different carriers. Lane 1: control; lane 2: Lipofectamine2000 /p53 transfection; lane 3: PEI25K/p53 transfection; lane 4: PEN/p53 transfection.
Fig. 4. Induction of apoptosis in PC-3 cells treated with p53 transfection determined by FACS: (a) control; (b) transfected with p3XFLAG-CMV-p53 plasmid only; (c) treated with PEN; (d) Lipofectamine2000 /p53 transfection; (e) PEI25K/p53 transfection; (f) PEN/p53 transfection.
The in vitro cytotoxicity of PEN was assessed in HeLa cells by MTT assay (Fig. S7), and the cell viabilities at various PEN concentrations were higher than those for PEI25K as anticipated, due to the shielding of positive charges after modification. Remarkably, the cell viability was nearly 70% when PEN was used at a concentration of 20 g/mL, whereas the value was found to be only 30% for PEI25K at the same concentration. Meanwhile, the transfection
of non-therapeutic plasmid pEGFP-N3 had almost no influences on the viability of cancer cells. Finally, the in vitro transfection efficiency of PEN was assessed in HeLa cells in the presence or absence of 10% FBS, using the plasmid pEGFP-N3 as a model. As shown in Fig. S8, the transfection efficiency exhibited an obviously improved tendency with an increasing mass ratio of PEN/pEGFPN3. At a mass ratio of 2.0, PEN exhibited slightly higher transfection
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Fig. 5. Induction of cell cycle arrest in PC-3 cells treated with p53 transfection determined by FACS. (A) Histograms of cell cycle distribution of control (a), Lipofectamine2000 /p53 transfection (b), PEI25K/p53 transfection (c) and PEN/p53 transfection (d) and (B) relative distribution of cell population in the cell cycle phases.
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Fig. 6. Western blotting analysis for the expression level of procaspase-3, procaspase-8, procaspase-9 and PARP. Lane 1: control; lane 2: Lipofectamine2000 /p53 transfection; lane 3: PEI25K/p53 transfection; lane 4: PEN/p53 transfection.
efficiency than Lipofectamine2000 did, and thus, a mass ratio of 2.0 was employed for further p53 gene delivery. In addition, in the presence of 10% FBS, the derivative PEN still possessed high transfection efficiency. In sum, these characteristics made the derivative PEN a potentially efficient carrier for p53 gene delivery. 3.2. Inhibition of cell proliferation induced by PEN-mediated p53 transfection The inhibition of cell proliferation after p53 transfection was detected by MTT assay (Fig. 1). In the present study, all of the carriers showed few effects on the cell viabilities of both PC-3 and HeLa cells, with values of more than 80%. In PC-3 cells, the commercial transfection agent Lipofectamine2000 exhibited relatively higher cytotoxicity, with a cell viability of 83% after 36 h of treatment. Compared with proliferation with the carriers alone, cell proliferation was greatly inhibited in the Lipofectamine2000 /p53, PEI25K/p53 and PEN/p53 groups. The inhibition of growth in these groups was maintained at a similar level in PC-3 cells, although PEN-mediated p53 transfection could inhibit proliferation more obviously than the other transfections could in HeLa cells. Moreover, among PC-3 cells, all of the p53 transfection groups exhibited stronger inhibitory effects than the corresponding groups of HeLa cells did. The phenomenon was probably caused by the fact that HeLa cells possess the wild-type p53 gene, so exogenous p53 expression will lead to less perturbation of the protein’s intrinsic signaling pathway; in contrast, PC-3 is a p53-deficient cell line, so exogenous p53 expression produces a significant inhibitory effect on the cells’ proliferation. The morphological analysis using DAPI staining revealed that compared with the control, the presence of nuclei with chromatin condensation and the formation of apoptotic bodies in cells were clearly observed in the PEI25K/p53 and PEN/p53 groups, and these phenomena were much more obvious in the PEN/p53 group (Fig. S9). However, in the Lipofectamine2000 /p53 group, many cell fragments were detected, indicating that the commercial transfection agent Lipofectamine2000 was of higher cytotoxicity and not suitable for gene delivery, especially at higher concentrations. To elucidate whether the inhibition of cell proliferation was induced by p53 transfection, the expression level of the p53 gene was evaluated at the mRNA and protein levels. As shown in Fig. 2,
no expression of p53 mRNA was detected in PC-3 cells, which was consistent with the cells’ p53 gene expression status, and p53 mRNA expression (549 bp) was markedly observed in cells transfected with p53 using Lipofectamine2000 , PEI25K or PEN. In HeLa cells, relatively lower expression of p53 mRNA was detected, and the intensity of the p53 bands was dramatically enhanced after p53 transfection. In addition, there were no significant differences in p53 expression at the mRNA level in Lipofectamine2000 /p53, PEI25K/p53 and PEN/p53 groups. In the protein-level analysis, the tendency was identical to that in the classical RT-PCR analysis, as shown in Fig. 3. In PC-3 cells, the control appeared not to express the p53 protein, and obvious p53 expression was observed in all of the p53 transfection groups. Similarly, after p53 transfection, HeLa cells showed considerably enhanced expression of the p53 protein compared with the control. The Western blotting analysis also validated that the expression level of p53 showed no remarkable differences between Lipofectamine2000 , PEI25K and PEN use, which indicated that PEN still possessed a stable and efficient transfection capability after modification. In this case, due to the lower cytotoxicity of PEN, it possessed superior potential to be used as a biocompatible gene carrier to attain the efficient delivery of exogenous p53 gene. 3.3. Induction of apoptosis and cell cycle arrest by PEN-mediated p53 transfection After the confirmation of successful p53 delivery and expression in HeLa and PC-3 cells, the anti-proliferation routes, including apoptosis induction and cell cycle arrest, were analyzed. Apoptosis induction was studied by flow cytometry using annexin V-FITC labeling for the detection of phosphatidylserine externalization, occurring as an early step during apoptosis. The proportions of early apoptotic cells (annexin V-FITC positive and PI negative) were determined. As shown in Fig. 4, compared with the control (0.27%), Lipofectamine2000 /p53, PEI25K/p53 and PEN/p53 transfection could efficiently induce cell apoptosis, with early apoptosis ratios of 50.88%, 50.81% and 54.95%, respectively. In contrast, the plasmid and the carrier PEN could only produce slight cell apoptosis, with early apoptosis ratios lower than 10%. A similar tendency was observed in HeLa cells, with PEN-mediated p53 transfection resulted in the highest apoptotic ratio (27.06%), as shown in
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Fig. S10. These results demonstrated that p53 transfection could induce early apoptosis, which is a key route for anti-proliferative effects. Although all of these carriers could achieve successful p53 transfection and further trigger cell apoptosis, PEN-mediated p53 transfection exhibited remarkably higher efficiency in inducing cell apoptosis than Lipofectamine2000 /p53 and PEI25K/p53 did in both PC-3 and HeLa cells. In addition, PC-3 cells were more sensitive to p53 transfection than HeLa cells were, which was consistent with MTT analysis and could be attributed to the difference in the p53 gene status of these two tumor cell lines. Additionally, cell cycle analysis via flow cytometry was performed to further elucidate whether the anti-proliferative effect after p53 transfection was associated with cell cycle arrest. As shown in Fig. 5, after transfection of the exogenous p53 gene using PEI25K or PEN, the cell cycle of PC-3 revealed a significant increase in cells in the G1 phase, and PEN-mediated p53 transfection could lead to obvious G1 phase arrest (66.89%), with much higher frequency than in the PEI25K/p53 group (60.13%). The results were consistent with the fact that p53 is generally known to arrest cell growth by holding the cell cycle at the G1/S phase [9]. Compared with the control, increased sub-G1 population values were observed in the Lipofectamine2000 /p53, PEI25K/p53 and PEN/p53 groups, indicating that p53 transfection induced significant cell cycle perturbation prior to apoptosis. Interestingly, in HeLa cells, p53 transfection yielded a different phenomenon in PC-3 cells (Fig. S11). Although a minor increase in cells in the G1 phase was caused in the PEI25K/p53 and PEN/p53 groups, a significant increase in cells in the G2 phase was detected indicating that p53 transfection mediated by PEI25K or PEN would lead to G2 phase arrest. As HeLa cells express the wild-type p53 gene, exogenous p53 expression did not yield obvious perturbation in the cell cycle at an early stage. The cells continuously divided until the G2 phase, which was then interrupted by induction of exogenous p53 protein. For Lipofectamine2000 /p53 transfection group, the increased proportion of cells in the S phase was probably caused by its intrinsic cytotoxicity. However, the precise mechanism of unique cell cycle arrest in p53wt cell lines is still yet to be elucidated. Besides the anti-proliferative effects mediated by p53 transfection, the wound healing assay was performed to check whether the p53 transfection could inhibit the cell migration. Compared with the control, obvious inhibition in cell migration was observed in PC-3 and HeLa cells treated by PEN or PEN/p53, indicating the inhibition of cell migration could be induced by both the carrier and p53 transfection (Figs. S12 and S13). Similar to cell apoptosis, PC-3 cells exhibited a higher sensitive response to exogenous p53 transfection than HeLa cells did. Thus, the PEN/p53 delivery system could be potentially employed to prevent or treat the metastasis-related death in clinic, and the detailed mechanism for p53 transfectionmediated anti-migration is yet to be elucidated in future. 3.4. Molecular mechanism analysis of inhibition of cell proliferation After p53 transfection, the expression level of proteins associated with cell apoptosis was detected. In cell apoptosis, an important feature is the cleavage of the DNA repair enzyme poly(ADP-ribose) polymerase (PARP), which will result in an enhancement in the level of cleaved PARP [26]. As shown in Fig. 6, after p53 transfection using Lipofectamine2000 , PEI25K or PEN, the expression levels of PARP (116 kDa) were obviously decreased in both PC-3 and HeLa cells, which meant that the PARP protein was cleaved after p53 expression. Meanwhile, PEN-mediated p53 transfection could reduce the expression of PARP to the utmost extent, consistent with the cell apoptosis results obtained by flow cytometry. These results confirmed that the cells treated with PEN/p53 nanocomplex could efficiently trigger the cleavage of PARP and
Fig. 7. Relative activity of caspase-3 (A), caspase-8 (B) and caspase-9 (C) in PC-3 and HeLa cells treated with p53 transfection. The data were expressed as mean value ± SD of three experiments.
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Fig. 8. Mitochondrial membrane potential analysis of PC-3 and HeLa cells after the p53 transfection for 36 h.
further activate the apoptosis pathway through the expression of exogenous p53 protein. Procaspase-3, the caspase-3’s precursor, with a molecular weight of 35 kDa, has been widely known to play a key role in the caspase family-related apoptotic cascade [27]. The activation of procaspase-3 to caspase-3 will generate the active executive caspase, which subsequently catalyzes the hydrolysis of many protein substrates. As shown in Fig. 6, the expression levels of procaspase-3 were obviously reduced after p53 transfection using Lipofectamine2000 , PEI25K or PEN in both PC3 and HeLa cells. Meanwhile, in PC-3 and HeLa cells treated with PEN/p53 complex, procaspase-3 expression was much lower than for Lipofectamine2000 or PEI25K, similar to the PARP results. The results were probably caused by the higher transfection efficiency of PEN and the further improved apoptosis-inducing ability after PEN-mediated p53 transfection. Furthermore, no matter whether in p53null or p53wt cells, expression of exogenous p53 would activate the intrinsic apoptosis pathway, as deduced from the hallmarks of apoptosis, such as activation of caspase-3 and PARP cleavage. To obtain a deeper insight into the mechanism of p53 transfection-induced cell apoptosis, the expression level of procaspase-8 and procaspase-9, and the caspase-3, caspase-8 and caspase-9 activities were measured after p53 transfection. As shown in Fig. 6, after p53 transfection, the expression level of procaspase-9 was obviously decreased in HeLa and PC-3 cells, indicating the activation of caspase-9. Meanwhile, the caspase-3 and caspase-9 activities were dramatically improved after exogenous p53 delivery into cells, and there were no significant differences between the Lipofectamine2000 /p53, PEI25K/p53 and PEN/p53 groups (Fig. 7). However, compared with the control, the expression level of procaspase-8 showed no obvious alternation after p53 transfection, and there were also no improvements in caspase-8 activity, illustrating no activation of caspase-8. Caspase-8 possesses a death receptor domain and acts as an initiator caspase in the TNF/FasL apoptosis pathway, which is referred as the extrinsic apoptosis pathway. Meanwhile, caspase-9 plays a proteolytic role, targeting procaspase-3 in the mitochondrial apoptosis pathway (intrinsic apoptosis pathway). Our data demonstrated that p53 transfection using the above-described carriers could activate the mitochondria-mediated pathway and had no effects on the death receptor-mediated pathway. As caspase-9 activation was associated with the mitochondriamediated pathway, the mitochondrial membrane potential was monitored using the JC-1 probe after p53 transfection. As shown
in Fig. 8, obvious red fluorescence (JC-1 aggregates) was observed in HeLa and PC-3 cells with no p53 treatment. After p53 transfection using Lipofectamine2000 , PEI25K or PEN as a carrier, changes in cell staining were detected: red fluorescence was decreased, and green fluorescence (JC-1 monomers) was obviously increased. In its normal state, the JC-1 probe would be located in the mitochondrial matrix in the form of aggregates (red), whereas due to a decrease in the mitochondrial membrane potential during the apoptotic process, it would converted to a monomeric state (green). Thus, in the present research, the three carriers could successfully mediate p53 transfection, triggering the mitochondrial apoptosis pathway, with evidence of activation of caspase-9 and a reduced mitochondrial membrane potential. Further in vivo studies for evaluating the therapeutic effect and mechanism of PEN-mediated p53 transfection are underway in our laboratory.
4. Conclusion The derivative PEN could successfully achieve the delivery of the tumor suppressor p53 into tumor cells. Through the PEN-mediated p53 transfection, cell apoptosis and cell cycle arrest could be efficiently triggered, accounting for a high anti-proliferative effect, and p53null cells exhibited a more sensitive response to exogenous p53 gene expression than p53wt cells did. Moreover, a detailed mechanistic analysis revealed that the cell apoptosis was mainly caused by activation of the mitochondria-mediated pathway after p53 transfection. In sum, this research provides a facile and effective tool for the construction and evaluation of gene carriers, and PEN-mediated p53 gene delivery might have the potential for clinical application in non-viral vector-mediated cancer gene therapy.
Acknowledgments The research was supported by Natural Science Foundation of China (No. 21204025, 81373344 and 81473142), the Ministry of Science and Technology of China (International Cooperation and Communication Program 2011DFR51090), the grants from Science & Technology Department of Jilin Province (No. 20130522005JH and 20140101140JC), and the Fundamental Research Funds for the Central Universities (JCKY-QKJC30).
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Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.colsurfb. 2015.03.032. References [1] C. Whibley, P.D. Pharoah, M. Hollstein, Nat. Rev. Cancer 9 (2009) 95. [2] L.J. Hofseth, S.P. Hussain, C.C. Harris, Trends Pharmacol. Sci. 25 (2004) 177. [3] M. Olivier, R. Eeles, M. Hollstein, M.A. Khan, C.C. Harris, P. Hainaut, Hum. Mutat. 19 (2002) 607. [4] T. Soussi, K.G. Wiman, Cancer Cell 12 (2007) 303. [5] T. Ramqvist, K.P. Magnusson, Y. Wang, L. Szekely, G. Klein, K.G. Wilman, Oncogene 8 (1993) 1495. [6] S.K. Misra, S. Naz, P. Kondaiah, S. Bhattacharya, Biomaterials 35 (2014) 1334. [7] L. Xu, K.F. Pirollo, E.H. Chang, J. Controlled Release 74 (2001) 115. [8] X. Shi, C. Li, S. Gao, L. Zhang, H. Han, J. Zhang, W. Shi, Q. Li, Colloids Surf. B: Biointerfaces 122 (2014) 498. [9] Y. He, Y. Nie, X. Li, H. Song, Z. Gu, Biomaterials 35 (2014) 1657. [10] A. El-Aneed, J. Controlled Release 94 (2014) 1. [11] T.G. Park, J.H. Jeong, S.W. Kim, Adv. Drug Deliv. Rev. 58 (2006) 467.
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H. Tian, J. Chen, X. Chen, Small 24 (2013) 2034. W.T. Godbey, K.K. Wu, A.G. Mikos, Proc. Natl. Acad. Sci. U. S. A. 96 (1996) 5177. M. Neu, D. Fischer, T. Kissel, J. Gene Med. 7 (2005) 992. H. Tian, L. Lin, J. Chen, X. Chen, T.G. Park, A. Maruyama, J. Controlled Release 155 (2011) 47. A.E. Nel, L. Madler, D. Velegol, T. Xia, E.M. Hoek, P. Somasundaran, F. Klaessig, V. Castranova, M. Thompson, Nat. Mater. 8 (2009) 543. Z. ur Rehman, D. Hoekstra, I.S. Zuhorn, ACS Nano 7 (2013) 3767. D. Fischer, Y. Li, B. Ahlemeyer, J. Krieglstein, T. Kissel, Biomaterials 24 (2003) 1121. Y. Zhao, R. Yang, D. Liu, M. Sun, L. Zhou, Z. Wang, Y. Wan, J. Biomed. Mater. Res. A 100 (2012) 134. A. Swami, A. Aggarwal, S. Pathak, P. Kumar, Y. Singh, K.C. Gupta, Int. J. Pharm. 335 (2007) 180. J. Xia, L. Chen, J. Chen, H. Tian, F. Li, X. Zhu, G. Li, X. Chen, Macromol. Biosci. 11 (2011) 211. H. Jia, H. Luo, H. Cheng, J. Yang, C. Li, C. Liu, J. Feng, X. Zhang, R. Zhuo, J. Mater. Chem. 22 (2012) 24092. J. Sun, F. Zeng, H. Jian, S. Wu, Polym. Chem. 4 (2013) 5810. H. Tian, F. Li, J. Chen, Y. Huang, X. Chen, Macromol. Biosci. 12 (2012) 1680. J. Xu, F. Zeng, H. Wu, C. Hu, C. Yu, S. Wu, Small 10 (2014) 3750. J. Morales, L. Li, F.J. Fattah, Y. Dong, E.A. Bey, M. Patel, J. Gao, D.A. Boothman, Crit. Rev. Eukaryot. Gene Expr. 24 (2014) 15. T.J. Yang, A. Haimovitz-Friedman, M. Verheij, Exp. Oncol. 34 (2012) 269.
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N-Isopropylacrylamide-modified polyethylenimine-mediated p53 gene delivery to prevent the proliferation of cancer cells Jianxu Zhang 1 , Di Wu 1 , Zhen Xing, Shaojun Liang, Haobo Han, Hui Shi, Yan Zhang, Yan Yang ∗ , Quanshun Li ∗ Key Laboratory for Molecular Enzymology and Engineering of Ministry of Education, School of Life Sciences, Jilin University, Changchun 130012, China
a r t i c l e
i n f o
Article history: Received 16 December 2014 Received in revised form 16 February 2015 Accepted 10 March 2015 Available online 18 March 2015 Keywords: N-Isopropylacrylamide Polyethylenimine p53 gene Gene therapy Cell apoptosis Cell cycle arrest
a b s t r a c t In this paper, N-isopropylacrylamide-modified polyethylenimine (PEN) was constructed through Michael addition and employed as a carrier to achieve the p53 gene delivery, using HeLa (p53wt) and PC-3 cells (p53null) as models. After PEN-mediated p53 transfection, expression level of p53 in HeLa and PC3 cells was up-regulated at both mRNA and protein levels. Due to the exogenous p53 expression, the inhibition of cell proliferation was observed through MTT analysis, attributing to the activation of apoptosis and cell cycle arrest. Using flow cytometric analysis, early apoptotic ratios of 54.95% and 27.06% after PENmediated p53 transfection were detected in PC-3 and HeLa cells, respectively, indicating that PC-3 cells were more sensitive to the exogenous p53 transfection than HeLa cells. Meanwhile, G1 phase arrest was detected in PC-3 cells while a unique G2 phase arrest was identified in HeLa cells after p53 transfection. Through Western blotting, activity analysis of caspase-3, caspase-8 and caspase-9 and mitochondrial membrane potential measurement, the apoptosis induced by PEN-mediated p53 transfection was conducted in a mitochondria-dependent apoptosis pathway. These results demonstrated that PEN could successfully mediate the p53 gene delivery and up-regulate the cellular p53 expression level, triggering a significant p53-dependent anti-proliferative effect on tumor cells. © 2015 Elsevier B.V. All rights reserved.
1. Introduction The p53 gene has been widely studied and is considered to be an effective tumor suppressor that plays key roles in apoptosis induction and cell cycle regulation [1]. The processes are mainly dependent on the DNA repair proteins and on pro-apoptotic proteins in the mitochondrial pathway induced by the p53 protein [2]. Nearly 50% of human tumors have been identified to possess mutations or deletions in the p53 gene [3,4], and thus, it is regarded as an important target in anticancer gene therapy. Previous reports have shown that transfection of exogenous p53 gene into p53null and p53mut cell lines could induce cell apoptosis and cell cycle arrest [5,6] and even improve the efficiency of conventional chemo/radiotherapy [7,8]. Meanwhile, enhancing the activity of wild-type p53 in p53wt cells can also trigger a variety of anti-proliferative effects (e.g., activation of apoptotic cell death) [9]. Thus, inducing the expression of wild-type p53 through gene transfer techniques or carriers could be an effective strategy for
∗ Corresponding authors. Tel.: +86 431 85155381; fax: +86 431 85155200. E-mail addresses: [email protected] (Y. Yang), [email protected] (Q. Li). 1 These authors contributed equally to the work. http://dx.doi.org/10.1016/j.colsurfb.2015.03.032 0927-7765/© 2015 Elsevier B.V. All rights reserved.
cancer treatment. However, constructing safe and efficient gene delivery carriers is still a key issue to be addressed in p53-based gene therapy. Non-viral carriers, and especially cationic polymers, have been widely used as gene delivery systems due to their low immunogenicity, high gene-loading capacity, flexible designability, potential safety and low production cost [10–12]. Among these carriers, branched polyethylenimine (PEI) with a weight-average molecular weight of 25 kDa (PEI25K) has exhibited superior gene transfection efficiency both in vitro and in vivo, because its high positive charge and proton-buffering capacity can condense and protect nucleic acids in serum and facilitate their internalization into cells and release from endosomes [13–17]. However, high positive charge density can lead to membrane-disruptive properties and nonspecific interaction with negatively charged proteins in the serum and thus cause severe problems [18,19], e.g., high cytotoxicity and hemolysis, and serum instability of the nanoparticles. To overcome these disadvantages, chemical modification of PEI25K to improve its transfection efficiency and/or to decrease its cytotoxicity has been actively investigated, especially grafting of hydrophobic groups [20–23]. These PEI derivatives exhibited amphiphilic characteristics, similar to the phospholipid layers in the cell membrane, leading to enhanced biocompatibility
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and transfection efficiency. Meanwhile, the grafted hydrophobic moieties were favorable for reducing the cationic density, which would not only reduce the cytotoxicity but also facilitate gene dissociation from polyplexes in the cytosol to further improve the transfection efficiency. Recently, Tian et al. [24] constructed a PEI derivative (namely PEN) with a higher transfection efficiency and lower cytotoxicity than PEI25K through modification of PEI25K with N-isopropylacrylamide via Michael addition. Compared with previous modification strategies, this approach did not reduce the content of protonated amines, and would not influence the formation of stable nanocomplexes between the carrier and pDNA through electrostatic interaction. The improved hydrophobicity would be favorable for the complexes’ interaction with cell membrane and gene dissociation in the cytosol, leading to enhanced endocytosis and transfection. Notably, this derivative showed an improved ability of nuclear uptake, despite no conjugation with targeting ligands or nuclear localization signals. Thus, the derivative PEN is of great potential in the delivery of plasmid harboring a tumor suppressor gene to cell nuclei to achieve gene expression. In the present research, the derivative PEN was employed as a carrier to achieve p53 gene delivery using the human cervical carcinoma cell line HeLa (p53wt) and the prostate tumor cell line PC-3 (p53null) as models. The performance of the gene transfection was evaluated at the mRNA and protein expression levels, and the inhibition of cancer cell proliferation after p53 gene transfection was investigated. Finally, the detailed mechanism of the therapeutic effect induced by cell apoptosis and cell cycle arrest was systematically elucidated. 2. Materials and methods
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36 h, 20 L of MTT solution (5 mg/mL in PBS) was added to each well, and the plates were incubated for an additional 4 h. The MTT solution was then removed, and 150 L of DMSO was added to dissolve the formazan crystals. The plates were incubated for an additional 10 min, after which the absorbance at 492 nm was recorded using a GF-M3000 microplate reader (Shandong, China). The cell viability (%) was calculated as Asample /Acontrol , where Asample and Acontrol were the absorbance values of the treated and untreated cells, respectively. Additionally, after treatment with polymer/p53 nanocomplex (2:1, wt/wt) with a plasmid mass of 2.5 g for 36 h, the cells in a 6-well plate were harvested, washed with ice-cold PBS and fixed with 70% ice-cold ethanol at 4 ◦ C for 5 min. The fixed cells were then washed with PBS, and stained with DAPI solution (1 g/mL) for 10 min at room temperature. The cellular morphology was observed through an Olympus IX71 fluorescence microscope (Japan).
2.3. Induction of cell apoptosis by p53 transfection HeLa and PC-3 cells were seeded at a density of 5 × 105 cells per well in 6-well plates and incubated for 24 h before transfection. As described above, the p53 transfection was performed using different polymer/p53 samples (2:1, wt/wt) with a plasmid mass of 2.5 g. After 36 h, the cells were harvested, washed with cold PBS twice, and re-suspended in binding buffer according to the manufacturer’s protocols. The cells were then incubated with annexin-V-FITC and PI, mixed and incubated at room temperature for 10 min in the dark. Apoptosis was determined by analyzing 15,000 ungated cells using FACSCalibur (BD Biosciences, Mountain View, USA) and CellQuest software (Becton-Dickinson, USA).
2.1. Materials 2.4. Cell cycle analysis after p53 transfection The plasmid p3XFLAG-CMV-p53 encoding wild-type p53 protein was kindly provided by Dr. Yuhang Zhang (Institute of Biochemistry and Cell Biology, CAS, China), amplified in Escherichia coli DH5␣ and purified using Plasmid Maxi kit (Axygen). Branched PEI25K, N-isopropylacrylamide and heparin were purchased from Aldrich and used as received. 3-(4,5-Dimethylthiazol-2yl)-2,5-diphenyltetrazolium bromide (MTT) and 4,6-diamidino-2phenylindile (DAPI) were purchased from Amersco (Solon, USA). Dulbecco’s modified Eagle’s medium (DMEM) and fetal bovine serum (FBS) were purchased from Gibco (Grand Island, USA). Lipofectamine2000 was purchased from Invitrogen, and polyvinylidene fluoride (PVDF) membrane was obtained from Millipore. Annexin-V-FITC/PI apoptosis detection kit, cell cycle detection kit, and caspase-3, 8 and 9 activity assay kits were purchased from Bestbio (Beijing, China). The mitochondrial membrane potential assay kit with JC-1 and BCA protein assay kit were obtained from Beyotime (Jiangsu, China). DNase I, PrimeScript TMRT Master Mix, RNAIso Plus and ExTaq reverse transcriptase were obtained from TaKaRa (Dalian, China). All the other chemicals were of the highest reagent grade commercially available and used as received. 2.2. Cell proliferation assay HeLa (p53wt) and PC-3 (p53null) cells were propagated to confluence in DMEM supplemented with 100 IU/mL penicillin, 100 mg/mL streptomycin and 10% FBS and were maintained at 37 ◦ C in a humidified atmosphere of 5% CO2 . The inhibition of cell proliferation induced by p53 gene transfection was evaluated by MTT assay. The cells were seeded onto 96-well plates at a density of 8.0 × 103 cells per well, pre-incubated in FBS-free DMEM for 24 h, and further incubated with polymer/p53 nanocomplex (2:1, wt/wt) with a plasmid mass of 0.1 g. After
For cell cycle analysis, HeLa and PC-3 cells were seeded at a density of 5 × 105 cells per well in 6-well plates and incubated for 24 h and then subjected to p53 transfection using different polymer/p53 samples (2:1, wt/wt) with a plasmid mass of 2.5 g. After 36 h, the cells were harvested, fixed in ice-cold 70% ethanol and then stored at 4 ◦ C overnight. Prior to analysis, the cells were washed twice with PBS, suspended in 0.5 mL of cold PI solution containing 10 L of RNase A (25 g/mL) and 10 L of PI (50 g/mL), and incubated at 37 ◦ C for an additional 30 min in the dark. The cell cycle was determined by analyzing 15,000 ungated cells using FACSCalibur (BD Biosciences, Mountain View, USA) and CellQuest software (Becton-Dickinson, USA).
2.5. Evaluation of p53 gene transfection at mRNA level The p53 transfection at the mRNA level was evaluated using the reverse transcription PCR (RT-PCR) technique, with -actin as a reference housekeeping gene. Total RNA was extracted from cells using RNAiso Plus, as recommended by the manufacturer. For reverse transcription, first-strand cDNA was synthesized from 1 g of total RNA, and the obtained cDNA was used as a template for classical PCR amplification, as follows: 1 cycle at 94 ◦ C for 2 min, followed by 30 cycles at 94 ◦ C for 30 s, 55 ◦ C for 30 s and 72 ◦ C for 1 min. The PCR products were detected by 2% agarose gel electrophoresis. The primers were as follows: p53 forward: 5 -GGCTCTGACTGTACCACCATCCA-3 ; reverse: 5 -GGCACAAACACGCACCTCAAAG-3 ; -actin forward: 5 -TCTGGCACCACACCTTCTACAATG-3 ; reverse: 5 -GGATAGCACAGCCTGGATAGCAA-3 .
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2.6. Western blotting analysis The p53 transfection at the protein level and the expression of other proteins associated with apoptosis were evaluated by Western blotting analysis, and the protein expression was normalized against -actin. After p53 transfection (polymer/p53 of 2:1, wt/wt) for 36 h, HeLa and PC-3 cells were harvested, washed with ice-cold PBS twice and lysed with RIPA lysis buffer on ice for 2 h. The lysates were centrifuged at 12,000 rpm for 10 min. The supernatants were collected, and the concentrations of protein were measured by BCA protein assay kit. Equal amounts of protein were subjected to electrophoresis by SDS-PAGE and transferred to PVDF membranes by electroblotting. The membranes were blocked in PBS solution containing 10% non-fat milk and 0.1% Tween-20 for 2 h and subsequently incubated with the desired antibodies at 4 ◦ C overnight. The membranes were then incubated with appropriate HRP-labeled secondary antibody for 1 h, and specific proteins were detected by enhanced chemical luminescence (Amersham, UK). 2.7. Caspase-3, 8 and 9 activity assay The caspase-3, 8 and 9 activities were determined through corresponding activity assay kits according to the manufacturer’s protocols. Briefly, HeLa and PC-3 cells after p53 transfection (polymer/p53 of 2:1, wt/wt) for 36 h were harvested, washed twice with PBS, and lysed in 100 mL lysis buffer supplied in the kit. The resulting suspension was centrifuged at 10,000 × g for 10 min (4 ◦ C), and 10 mL supernatant was used for detecting the caspase-3, 8 and 9 activities. The activities were monitored by measuring the changes of absorbance at 405 nm using a GF-M3000 microplate reader (Shandong, China). 2.8. Mitochondrial membrane potential measurement Mitochondrial membrane potential changes were detected using the fluorescent probe JC-1 according to a previous report [25]. Briefly, HeLa and PC-3 cells were seeded at a density of 5 × 105 cells per well in 6-well plates and incubated for 24 h before transfection. After p53 transfection (polymer/p53 of 2:1, wt/wt) for 36 h, the cells were rinsed with PBS, harvested with trypsin-EDTA solution and collected by centrifugation at 2000 × g for 2 min. The cell pellets were then stained with fluorescent probe JC-1 provided by the mitochondrial membrane potential assay kit, washed twice with PBS and examined through an Olympus IX71 fluorescence microscope (Japan). 3. Results and discussion 3.1. Synthesis and characterization of PEN The derivative PEN was synthesized through the modification of PEI25K with N-isopropylacrylamide via Michael addition according to the previous report [24]. Typical 1 H NMR spectra of PEI25K and PEN are shown in Fig. S1, and the number of grafted N-isopropylacrylamide units could be determined based on the integral ratio of the peak at 4.05 ppm to the peak groups of PEI25K (2.50–2.70 ppm). Based on a previous report on optimization of the grafting ratio [24], PEN175 was selected and employed in the present research. The binding affinity of PEN to plasmid pEGFP-N3 was examined by agarose gel retardation assay (Fig. S2), and complete pDNA retardation by PEN could be induced at a critical mass ratio of 0.4, with a similar binding ability as PEI25K (critical mass ratio of 0.3). Additionally, PEN could efficiently protect the plasmid from degradation of DNase I, indicating the formation of stable PEN/pDNA nanocomplex in the presence of DNase I (Fig. S3). The particle size and zeta potential of PEI25K/pDNA and PEN/pDNA at
Fig. 1. The cell viability assay of PC-3 (A) and HeLa cells (B) after the p53 transfection mediated by different carriers. The data were expressed as mean value ± SD of three experiments.
different mass ratios were then determined by Malvern Nano ZS90 Zetasizer. As shown in Fig. S4, the particle size values of nanocomplexes were in the range of 70–175 nm in the mass ratios studied, and the zeta potential exhibited an increasing tendency with the increasing ratio of carrier. For PEN/pDNA nanocomplex, the particle size and zeta potential were measured to be 108 nm and +15.3 mV at a mass ratio of 2.0, and thus it will be suitable for further transfection investigations especially for potential in vivo application. To study the interaction between the carriers and proteins, BSA was used as a model protein to simulate the carriers’ electrostatic adsorption of serum proteins. As shown in Fig. S5, the adsorption ability was slightly decreased after the modification, as the modification of N-isopropylacrylamide did not introduce the hydrophilic segments to resist the adsorption of negatively charged proteins. The hemoglobin release from erythrocytes after treatment with different concentration of carriers was then monitored to further investigate the nonspecific interactions of polymers with blood components (Fig. S6). Compared with PEI25K, PEN exhibited relatively lower hemolysis and less negative effects on the erythrocyte membranes, which meant an improved hemocompatibility.
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Fig. 2. Classical RT-PCR analysis of p53 expression in PC-3 and HeLa cells after the p53 transfection mediated by different carriers. Lane 1: control; lane 2: Lipofectamine2000 /p53 transfection; lane 3: PEI25K/p53 transfection; lane 4: PEN/p53 transfection.
Fig. 3. Western blotting analysis of p53 expression in PC-3 and HeLa cells after the p53 transfection mediated by different carriers. Lane 1: control; lane 2: Lipofectamine2000 /p53 transfection; lane 3: PEI25K/p53 transfection; lane 4: PEN/p53 transfection.
Fig. 4. Induction of apoptosis in PC-3 cells treated with p53 transfection determined by FACS: (a) control; (b) transfected with p3XFLAG-CMV-p53 plasmid only; (c) treated with PEN; (d) Lipofectamine2000 /p53 transfection; (e) PEI25K/p53 transfection; (f) PEN/p53 transfection.
The in vitro cytotoxicity of PEN was assessed in HeLa cells by MTT assay (Fig. S7), and the cell viabilities at various PEN concentrations were higher than those for PEI25K as anticipated, due to the shielding of positive charges after modification. Remarkably, the cell viability was nearly 70% when PEN was used at a concentration of 20 g/mL, whereas the value was found to be only 30% for PEI25K at the same concentration. Meanwhile, the transfection
of non-therapeutic plasmid pEGFP-N3 had almost no influences on the viability of cancer cells. Finally, the in vitro transfection efficiency of PEN was assessed in HeLa cells in the presence or absence of 10% FBS, using the plasmid pEGFP-N3 as a model. As shown in Fig. S8, the transfection efficiency exhibited an obviously improved tendency with an increasing mass ratio of PEN/pEGFPN3. At a mass ratio of 2.0, PEN exhibited slightly higher transfection
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Fig. 5. Induction of cell cycle arrest in PC-3 cells treated with p53 transfection determined by FACS. (A) Histograms of cell cycle distribution of control (a), Lipofectamine2000 /p53 transfection (b), PEI25K/p53 transfection (c) and PEN/p53 transfection (d) and (B) relative distribution of cell population in the cell cycle phases.
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Fig. 6. Western blotting analysis for the expression level of procaspase-3, procaspase-8, procaspase-9 and PARP. Lane 1: control; lane 2: Lipofectamine2000 /p53 transfection; lane 3: PEI25K/p53 transfection; lane 4: PEN/p53 transfection.
efficiency than Lipofectamine2000 did, and thus, a mass ratio of 2.0 was employed for further p53 gene delivery. In addition, in the presence of 10% FBS, the derivative PEN still possessed high transfection efficiency. In sum, these characteristics made the derivative PEN a potentially efficient carrier for p53 gene delivery. 3.2. Inhibition of cell proliferation induced by PEN-mediated p53 transfection The inhibition of cell proliferation after p53 transfection was detected by MTT assay (Fig. 1). In the present study, all of the carriers showed few effects on the cell viabilities of both PC-3 and HeLa cells, with values of more than 80%. In PC-3 cells, the commercial transfection agent Lipofectamine2000 exhibited relatively higher cytotoxicity, with a cell viability of 83% after 36 h of treatment. Compared with proliferation with the carriers alone, cell proliferation was greatly inhibited in the Lipofectamine2000 /p53, PEI25K/p53 and PEN/p53 groups. The inhibition of growth in these groups was maintained at a similar level in PC-3 cells, although PEN-mediated p53 transfection could inhibit proliferation more obviously than the other transfections could in HeLa cells. Moreover, among PC-3 cells, all of the p53 transfection groups exhibited stronger inhibitory effects than the corresponding groups of HeLa cells did. The phenomenon was probably caused by the fact that HeLa cells possess the wild-type p53 gene, so exogenous p53 expression will lead to less perturbation of the protein’s intrinsic signaling pathway; in contrast, PC-3 is a p53-deficient cell line, so exogenous p53 expression produces a significant inhibitory effect on the cells’ proliferation. The morphological analysis using DAPI staining revealed that compared with the control, the presence of nuclei with chromatin condensation and the formation of apoptotic bodies in cells were clearly observed in the PEI25K/p53 and PEN/p53 groups, and these phenomena were much more obvious in the PEN/p53 group (Fig. S9). However, in the Lipofectamine2000 /p53 group, many cell fragments were detected, indicating that the commercial transfection agent Lipofectamine2000 was of higher cytotoxicity and not suitable for gene delivery, especially at higher concentrations. To elucidate whether the inhibition of cell proliferation was induced by p53 transfection, the expression level of the p53 gene was evaluated at the mRNA and protein levels. As shown in Fig. 2,
no expression of p53 mRNA was detected in PC-3 cells, which was consistent with the cells’ p53 gene expression status, and p53 mRNA expression (549 bp) was markedly observed in cells transfected with p53 using Lipofectamine2000 , PEI25K or PEN. In HeLa cells, relatively lower expression of p53 mRNA was detected, and the intensity of the p53 bands was dramatically enhanced after p53 transfection. In addition, there were no significant differences in p53 expression at the mRNA level in Lipofectamine2000 /p53, PEI25K/p53 and PEN/p53 groups. In the protein-level analysis, the tendency was identical to that in the classical RT-PCR analysis, as shown in Fig. 3. In PC-3 cells, the control appeared not to express the p53 protein, and obvious p53 expression was observed in all of the p53 transfection groups. Similarly, after p53 transfection, HeLa cells showed considerably enhanced expression of the p53 protein compared with the control. The Western blotting analysis also validated that the expression level of p53 showed no remarkable differences between Lipofectamine2000 , PEI25K and PEN use, which indicated that PEN still possessed a stable and efficient transfection capability after modification. In this case, due to the lower cytotoxicity of PEN, it possessed superior potential to be used as a biocompatible gene carrier to attain the efficient delivery of exogenous p53 gene. 3.3. Induction of apoptosis and cell cycle arrest by PEN-mediated p53 transfection After the confirmation of successful p53 delivery and expression in HeLa and PC-3 cells, the anti-proliferation routes, including apoptosis induction and cell cycle arrest, were analyzed. Apoptosis induction was studied by flow cytometry using annexin V-FITC labeling for the detection of phosphatidylserine externalization, occurring as an early step during apoptosis. The proportions of early apoptotic cells (annexin V-FITC positive and PI negative) were determined. As shown in Fig. 4, compared with the control (0.27%), Lipofectamine2000 /p53, PEI25K/p53 and PEN/p53 transfection could efficiently induce cell apoptosis, with early apoptosis ratios of 50.88%, 50.81% and 54.95%, respectively. In contrast, the plasmid and the carrier PEN could only produce slight cell apoptosis, with early apoptosis ratios lower than 10%. A similar tendency was observed in HeLa cells, with PEN-mediated p53 transfection resulted in the highest apoptotic ratio (27.06%), as shown in
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Fig. S10. These results demonstrated that p53 transfection could induce early apoptosis, which is a key route for anti-proliferative effects. Although all of these carriers could achieve successful p53 transfection and further trigger cell apoptosis, PEN-mediated p53 transfection exhibited remarkably higher efficiency in inducing cell apoptosis than Lipofectamine2000 /p53 and PEI25K/p53 did in both PC-3 and HeLa cells. In addition, PC-3 cells were more sensitive to p53 transfection than HeLa cells were, which was consistent with MTT analysis and could be attributed to the difference in the p53 gene status of these two tumor cell lines. Additionally, cell cycle analysis via flow cytometry was performed to further elucidate whether the anti-proliferative effect after p53 transfection was associated with cell cycle arrest. As shown in Fig. 5, after transfection of the exogenous p53 gene using PEI25K or PEN, the cell cycle of PC-3 revealed a significant increase in cells in the G1 phase, and PEN-mediated p53 transfection could lead to obvious G1 phase arrest (66.89%), with much higher frequency than in the PEI25K/p53 group (60.13%). The results were consistent with the fact that p53 is generally known to arrest cell growth by holding the cell cycle at the G1/S phase [9]. Compared with the control, increased sub-G1 population values were observed in the Lipofectamine2000 /p53, PEI25K/p53 and PEN/p53 groups, indicating that p53 transfection induced significant cell cycle perturbation prior to apoptosis. Interestingly, in HeLa cells, p53 transfection yielded a different phenomenon in PC-3 cells (Fig. S11). Although a minor increase in cells in the G1 phase was caused in the PEI25K/p53 and PEN/p53 groups, a significant increase in cells in the G2 phase was detected indicating that p53 transfection mediated by PEI25K or PEN would lead to G2 phase arrest. As HeLa cells express the wild-type p53 gene, exogenous p53 expression did not yield obvious perturbation in the cell cycle at an early stage. The cells continuously divided until the G2 phase, which was then interrupted by induction of exogenous p53 protein. For Lipofectamine2000 /p53 transfection group, the increased proportion of cells in the S phase was probably caused by its intrinsic cytotoxicity. However, the precise mechanism of unique cell cycle arrest in p53wt cell lines is still yet to be elucidated. Besides the anti-proliferative effects mediated by p53 transfection, the wound healing assay was performed to check whether the p53 transfection could inhibit the cell migration. Compared with the control, obvious inhibition in cell migration was observed in PC-3 and HeLa cells treated by PEN or PEN/p53, indicating the inhibition of cell migration could be induced by both the carrier and p53 transfection (Figs. S12 and S13). Similar to cell apoptosis, PC-3 cells exhibited a higher sensitive response to exogenous p53 transfection than HeLa cells did. Thus, the PEN/p53 delivery system could be potentially employed to prevent or treat the metastasis-related death in clinic, and the detailed mechanism for p53 transfectionmediated anti-migration is yet to be elucidated in future. 3.4. Molecular mechanism analysis of inhibition of cell proliferation After p53 transfection, the expression level of proteins associated with cell apoptosis was detected. In cell apoptosis, an important feature is the cleavage of the DNA repair enzyme poly(ADP-ribose) polymerase (PARP), which will result in an enhancement in the level of cleaved PARP [26]. As shown in Fig. 6, after p53 transfection using Lipofectamine2000 , PEI25K or PEN, the expression levels of PARP (116 kDa) were obviously decreased in both PC-3 and HeLa cells, which meant that the PARP protein was cleaved after p53 expression. Meanwhile, PEN-mediated p53 transfection could reduce the expression of PARP to the utmost extent, consistent with the cell apoptosis results obtained by flow cytometry. These results confirmed that the cells treated with PEN/p53 nanocomplex could efficiently trigger the cleavage of PARP and
Fig. 7. Relative activity of caspase-3 (A), caspase-8 (B) and caspase-9 (C) in PC-3 and HeLa cells treated with p53 transfection. The data were expressed as mean value ± SD of three experiments.
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Fig. 8. Mitochondrial membrane potential analysis of PC-3 and HeLa cells after the p53 transfection for 36 h.
further activate the apoptosis pathway through the expression of exogenous p53 protein. Procaspase-3, the caspase-3’s precursor, with a molecular weight of 35 kDa, has been widely known to play a key role in the caspase family-related apoptotic cascade [27]. The activation of procaspase-3 to caspase-3 will generate the active executive caspase, which subsequently catalyzes the hydrolysis of many protein substrates. As shown in Fig. 6, the expression levels of procaspase-3 were obviously reduced after p53 transfection using Lipofectamine2000 , PEI25K or PEN in both PC3 and HeLa cells. Meanwhile, in PC-3 and HeLa cells treated with PEN/p53 complex, procaspase-3 expression was much lower than for Lipofectamine2000 or PEI25K, similar to the PARP results. The results were probably caused by the higher transfection efficiency of PEN and the further improved apoptosis-inducing ability after PEN-mediated p53 transfection. Furthermore, no matter whether in p53null or p53wt cells, expression of exogenous p53 would activate the intrinsic apoptosis pathway, as deduced from the hallmarks of apoptosis, such as activation of caspase-3 and PARP cleavage. To obtain a deeper insight into the mechanism of p53 transfection-induced cell apoptosis, the expression level of procaspase-8 and procaspase-9, and the caspase-3, caspase-8 and caspase-9 activities were measured after p53 transfection. As shown in Fig. 6, after p53 transfection, the expression level of procaspase-9 was obviously decreased in HeLa and PC-3 cells, indicating the activation of caspase-9. Meanwhile, the caspase-3 and caspase-9 activities were dramatically improved after exogenous p53 delivery into cells, and there were no significant differences between the Lipofectamine2000 /p53, PEI25K/p53 and PEN/p53 groups (Fig. 7). However, compared with the control, the expression level of procaspase-8 showed no obvious alternation after p53 transfection, and there were also no improvements in caspase-8 activity, illustrating no activation of caspase-8. Caspase-8 possesses a death receptor domain and acts as an initiator caspase in the TNF/FasL apoptosis pathway, which is referred as the extrinsic apoptosis pathway. Meanwhile, caspase-9 plays a proteolytic role, targeting procaspase-3 in the mitochondrial apoptosis pathway (intrinsic apoptosis pathway). Our data demonstrated that p53 transfection using the above-described carriers could activate the mitochondria-mediated pathway and had no effects on the death receptor-mediated pathway. As caspase-9 activation was associated with the mitochondriamediated pathway, the mitochondrial membrane potential was monitored using the JC-1 probe after p53 transfection. As shown
in Fig. 8, obvious red fluorescence (JC-1 aggregates) was observed in HeLa and PC-3 cells with no p53 treatment. After p53 transfection using Lipofectamine2000 , PEI25K or PEN as a carrier, changes in cell staining were detected: red fluorescence was decreased, and green fluorescence (JC-1 monomers) was obviously increased. In its normal state, the JC-1 probe would be located in the mitochondrial matrix in the form of aggregates (red), whereas due to a decrease in the mitochondrial membrane potential during the apoptotic process, it would converted to a monomeric state (green). Thus, in the present research, the three carriers could successfully mediate p53 transfection, triggering the mitochondrial apoptosis pathway, with evidence of activation of caspase-9 and a reduced mitochondrial membrane potential. Further in vivo studies for evaluating the therapeutic effect and mechanism of PEN-mediated p53 transfection are underway in our laboratory.
4. Conclusion The derivative PEN could successfully achieve the delivery of the tumor suppressor p53 into tumor cells. Through the PEN-mediated p53 transfection, cell apoptosis and cell cycle arrest could be efficiently triggered, accounting for a high anti-proliferative effect, and p53null cells exhibited a more sensitive response to exogenous p53 gene expression than p53wt cells did. Moreover, a detailed mechanistic analysis revealed that the cell apoptosis was mainly caused by activation of the mitochondria-mediated pathway after p53 transfection. In sum, this research provides a facile and effective tool for the construction and evaluation of gene carriers, and PEN-mediated p53 gene delivery might have the potential for clinical application in non-viral vector-mediated cancer gene therapy.
Acknowledgments The research was supported by Natural Science Foundation of China (No. 21204025, 81373344 and 81473142), the Ministry of Science and Technology of China (International Cooperation and Communication Program 2011DFR51090), the grants from Science & Technology Department of Jilin Province (No. 20130522005JH and 20140101140JC), and the Fundamental Research Funds for the Central Universities (JCKY-QKJC30).
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