Acylation of the antimicrobial peptide CAMEL for cancer gene therapy (2024)

Peptides synthesis

All peptides were synthesized using FMOC SPPS strategy. All the peptides were chemically synthesized manually using Fmoc chemistry on Rink amide MBHA resin. Briefly, the amino acid (3 equiv.) together with N-[(lH-benzotriazol-l-yl)(dimethylammo)methylene]-Nmethylmethanaminium hexafluorophosphate N-oxide (HBTU, 3 equiv.), 1-Hydroxybenzotriazole (HOBt, 3 equiv.) and N,N-Diisopropylethylamine (DIEA, 6 equiv.) in dimethylformamide (DMF) were coupled for 60 min. Fatty acids were coupled as amino acids. The Fmoc protecting group was removed by treatment with 20% piperidine in DMF. Following synthesis, the resin was washed with several portions of DMF, dichloromethane, and methanol before it was dried in a vacuum for at least 3 h. The final peptides were cleaved from the resin by treatment with a solution of trichloroacetic acid (TFA)/triisopropylsilane (TIS)/water (95:2.5:2.5, v/v/v) for 3 h at room temperature. TFA was removed by evaporation and the product precipitated in cold diethyl ether. Fractions were pooled and lyophilized. Acyl-peptides were synthesized by coupling the corresponding fatty acids to the N-terminus of peptide resins. After cleavage from the resin, the desired peptides were purified by reversed-phase high-performance liquid chromatography (RP-HPLC) on a C18 column. Purity analysis was checked by analytical RP-HPLC. The synthetic peptides were characterized by electrospray ionization-mass spectrometry (ESI-MS). The purity of all peptides used for experiments was ≥95%.

Cell culture and amplification of plasmid DNA

COS-7, U87, U251, MCF-7, MB-MDA-231, Hela, Hepg2 and B16 cell lines were cultured in DMEM supplemented with 10% fetal bovine serum (FBS) in a 5% CO₂ humidified atmosphere at 37 °C. All cell lines were obtained from the Chinese Academy of Sciences. pGL3 plasmid containing luciferase gene and pcDNA3.1 plasmid containing wild p53 gene were transformed in Escherichia coli DH5α and were amplified in LB medium at 37 °C overnight at 180 rpm. The plasmids were purified by an EndoFree Plasmid kit (TIANGEN, Beijing, China). Then the purified plasmids were dissolved in distilled water and stored at −20 °C.

Complexes formation

All complexes were formed by mixing peptides and plasmids (0.5 μg) in 50 μL of water at various N/P ratios (ratio of positive charges of the peptide to negative charges of the plasmid) and were incubated for 30 min at 37 °C to form stable nanoparticles. Then, these complexes were diluted to a final volume of 500 μL and used immediately.

In vitro transfections

Cells were seeded at 1 × 10⁵ cells/well in a 24-well plate 24 h before treatment. After washing with PBS, cells were treated with peptide/pGL3 plasmid complexes in 500 μL of DMEM containing FBS free or FBS at various concentrations for 4 h. Thereafter, the medium was replaced with 1 mL of DMEM containing 10% FBS. After incubation for 20 h, luciferase activity was measured using Promega’s luciferase detection kit. Data were normalized to protein content measured using a BCA protein assay kit (Pierce). Lipofectamine 2000 (LF2000, Invitrogen) served as a positive control. To evaluate the effect of chloroquine (CQ) on the transfection efficiency of C18-CAMEL, COS-7 cells were treated with peptides/pGL3 plasmid complexes and CQ (final concentration 100 μmol/L) for 4 h. After 20 h of incubation, luciferase activity was determined according to the above methods. Three independent experiments were performed.

Cellular uptake assay

To quantify the cellular uptake of peptides/pGL3 plasmid complexes, COS-7 cells were cultured in a 24-well plate 24 h before treatment. After washing with PBS, cells were incubated with peptides/Cy5-labeled pGL3 plasmid complexes at an N/P ratio of 2. Labeling of the pGL3 plasmid with the fluorescent probe Cy5 was performed using a Label IT Tracker kit (Mirus), as described by the manufacturer. After 4 h of incubation, the cells were washed with PBS and then incubated with 0.02% trypsin for 10 min. The cells were harvested and centrifuged at 1500 rpm for 5 min. Thereafter, the cell pellets were resuspended and detected with a BD FACS Caliber Flow Cytometer. To explore the cellular uptake pathways of C18-CAMEL/pGL3 plasmid complexes, COS-7 cells were preincubated with an endocytosis inhibitor chlorpromazine (10 μg/mL), amiloride (50 μM), or methyl-β-cyclodextrin (5 mM) for 30 min, and then were incubated with peptide/Cy5-labeled pGL3 plasmid complexes at an N/P ratio of 2. After 4 h of incubation, the cellular uptake of complexes was detected using the above method. Three independent experiments were performed.

Gel retardation assay

To explore the binding ability of CAMEL or C18-CAMEL with pDNA, peptides, and pGL3 plasmids were mixed at various ratios and at 37 °C for 30 min to form complexes. Thereafter, samples were electrophoresed through the 0.8% (W/V) agarose gel and imaged by staining the gel with EtBr.

DNA condensation assay

The DNA condensation ability of peptides was evaluated using the ethidium bromide (EtBr) exclusion assay. pGL3 plasmids (0.5 μg) were mixed with peptides at various N/P ratios in 50 μL of milli-Q water. After incubation for 1 h, 135 μL of water was added to each sample and transferred into a 96-well black plate. Thereafter, 15 μL of EtBr solution was added to give a final EtBr concentration of 400 nmol/L. After 10 min of incubation, the 96-well plate was shaken orbitally for 30 s and the fluorescence intensity measured. Three independent experiments were performed.

Transmission electron microscopy

TEM was used to observe the morphology of C18-CAMEL/pGL3 plasmid complexes at an N/P ratio of 2. C18-CAMEL/pGL3 plasmid complexes were prepared as described above. The TEM samples were prepared by dropping 10 μL of C18-CAMEL/pGL3 plasmid complex solution onto a copper grid and then staining by 0.2% (W/V) phosphotungstic acid solution before measurement.

Particle size and ζ-potential measurements

C18-CAMEL/pGL3 plasmid complexes were prepared as described above. These complexes were diluted in phosphate-buffered saline (PBS) to 1 mL volume, and then, the size and ζ-potential were measured by Nano-ZS ZEN3600 (Malvern Instruments, Malvern, UK). Three independent experiments were performed.

Antiproliferative assay

The antiproliferative effect of C18-CAMEL/p53 plasmid complexes was determined by the MTT assay. Cells were seeded in 96-well plates at a density of 5 × 10³ cells/well. After being washed, cells were treated with C18-CAMEL alone, p53 plasmid alone, and C18-CAMEL/p53 plasmid complexes in 100 μL of DMEM at various N/P ratios for 4 h. Thereafter, 100 μL of DMEM containing 10% FBS was added, and the cytotoxicity was determined by the MTT assay after 96 h. Three independent experiments were performed.

To study the antiproliferative effect of C18-CAMEL/p53 plasmid complexes in combination with MDM2 inhibitor nutlin-3a against different cell lines, cells were treated with C18-CAMEL/p53 plasmid complexes at an N/P ratio of 2, nutlin-3a at different concentrations, C18-CAMEL, a combination of C18-CAMEL and nutlin-3a, or combination of C18-CAMEL/p53 plasmid complexes and nutlin-3a in 100 μL of DMEM for 4 h. Thereafter, 100 μL of DMEM containing 10% FBS was added, and the cytotoxicity was determined by the MTT assay after 96 h. Three independent experiments were performed.

P53 expression assay

For mRNA level analysis, after transfection with C18-CAMEL/p53 plasmid complexes for 4 h, MCF-7 cells were collected and total RNA was extracted using the TRIzol reagent. Approximately, 1 μg of total RNA from each sample was converted to complementary cDNA using a commercially available RT-PCR kit. The obtained cDNA was then employed as a template for classical PCR amplification as follows. The PCR products were detected by 2% agarose gel electrophoresis. The primers used were as follows: p53 sense 50-CCTCAGCATCTTATCCGAGTGG-30 and antisense 50-TGGATGGTGGTACAGTCAGAGC-30. Three independent experiments were performed.

For p53 protein expression level analysis, western blot analysis was performed. Briefly, MCF-7 cells were transfected with peptide/p53 plasmid complex for 48 h. Thereafter, cells were harvested and lysed, and protein concentrations were determined by using a BCA protein assay kit (Pierce). The total amount of 40 μg of protein from each sample was loaded and separated on a 10% SDS − PAGE gel. After electrophoresis, the samples were transferred onto a PVDF membrane. The membranes were probed with the primary antibody specific for p53 followed by incubation with the HRP-conjugated secondary antibody. The signal was detected by an enhanced chemiluminescence detection system.

Statistical analysis

Experiments were performed three times and the data were expressed as means ± SEM. Statistical analysis was performed using Student’s t-tests. p < .05 was considered to be indicative of statistical significance.

Transfection efficiency of peptides

Delivery of therapeutic nucleic acids with poor bioavailability into cells is a prerequisite for cancer gene treatment. Recently, CPP-based vectors have attracted considerable interest as non-viral vectors due to their efficiency (Lehto etal., Citation2016; Sun etal., Citation2017). Given that AMPs share common features with CPPs in various aspects, such as physicochemical characteristics and strong membrane association, AMPs may provide us with inspiration to design more efficient gene vectors (Henriques etal., Citation2006; Splith & Neundorf, Citation2011). CAMEL is a hybrid antimicrobial peptide with membrane-lysis and cell-penetrating activity (Smolarczyk etal., Citation2010). It was reported that the attachment of fatty acids with different chain lengths can influence the transfection efficiency of acyl-CPPs (Katayama etal., Citation2011; Lehto etal., Citation2017; Morais etal., Citation2018). Therefore, a series of vectors were synthesized by attaching fatty acids with different chain lengths to the N-terminus of CAMEL in the present study ().

To identify satisfactory non-viral vectors for cancer gene therapy, we first evaluated the transfection efficiency of CAMEL and acyl-CAMEL in COS-7 cells using pGL3 plasmid, which contains a reporter gene encoding luciferase. As shown in , the luciferase expression in the COS-7 cells transfected with the CAMEL/plasmid complexes, the C4-CAMEL/plasmid complexes, and the C8-CAMEL/plasmid complexes displayed a slight increase over that of the naked plasmids at various N/P ratios. When the chain length exceeded 12 carbons, the transfection efficiency of C12, C16, and C18-CAMEL showed a strong increase. Among these vectors, C18-CAMEL, with the highest hydrophobicity, displayed the strongest transfection efficiency with Lipofectamine 2000 (LF2000). Our results are consistent with a previous study, where the transfection of acyl-peptides started to pronouncedly increase from a chain length of 12 carbons (Lehto etal., Citation2017). Importantly, the transfection efficiency of C18-CAMEL was higher than that of stearyl-rMel, which is constructed by stearylation of the retro isomer of antimicrobial peptide melittin and was reported to display high transfection efficiency in our previous study (Zhang etal., Citation2013). In addition, we unexpectedly found that stearyl-rMel with a reverse sequence exhibited approximately 10-fold transfection efficiency compared with stearyl-Mel. Therefore, we also constructed vectors by attaching C12, C16, and C18 to the N-terminus of the retro isomer of CAMEL (). Unfortunately, the transfection efficiency of C12-, C16- and C18-rCAMEL was substantially lower than that of C18-CAMEL (). This result demonstrates that this strategy is not suitable for all peptides to enhance their transfection efficiency.

To further address the impact of fatty acids with different chain lengths on the transfection efficiency of acyl-CAMEL, we used FACS to evaluate the cellular uptake efficiency of the complexes formed by acyl-CAMEL and Cy5-labeled pGL3 plasmids at an N/P ratio of 2. As shown in , the fluorescence intensity of the internalized complexes started to increase from an acyl chain length of 12 carbons. This result demonstrated that the vectors based on CAMEL with longer carbon chain lengths displayed higher plasmid delivery efficiency, strongly supporting the result derived from the transfection efficiency assay.

Taken together, the above results indicated that C18-CAMEL displayed the highest transfection efficiency among these vectors. Thus, in the following studies, we mainly evaluated the application potential of C18-CAMEL for cancer gene therapy.

Characterization of the peptide/plasmid complexes

A non-covalent strategy has proven to be a simple, cost-efficient, and effective methodology for CPP-based vectors to deliver nucleic acids into cells (Deshayes etal., Citation2008). This strategy predominantly relies on electrostatic- and hydrophobic interactions, which can facilitate cationic CPPs to condense anionic nucleic acids into more stable nanoparticles (Deshayes etal., Citation2008; Lehto etal., Citation2011). Attachment of fatty acids is an effective approach for many CPPs to efficiently condense nucleic acids (Lehto etal., Citation2011; Nakase etal., Citation2012; Lehto etal., Citation2016). Many factors, such as nucleic acid condensation, size, zeta potential, and shape, were reported to influence the transfection efficiency of nanoparticles formed by CPPs and nucleic acids (Wang etal., Citation2011; Sharma etal., Citation2013; Dutta etal., Citation2015; Lehto etal., Citation2017; Suchaoin etal., Citation2017). Therefore, we explored these physicochemical characteristics of C18-CAMEL/pGL3 plasmid complexes.

To evaluate the extent to which peptides are capable of binding and condensing pDNA, we performed a gel retardation assay and ethidium bromide (EtBr) exclusion assay. As shown in , the results derived from the gel retardation assay indicated that CAMEL completely retarded pDNA migration at an N/P ratio of 3, while C18-CAMEL exhibited the same pDNA binding capacity at an N/P ratio of 2. This result demonstrated that C18-CAMEL exhibits a stronger pDNA binding capacity than CAMEL. In addition, at N/P ratios of 3 and 4, the brightness of the pDNA bands in the sample wells containing C18-CAMEL/pDNA complexes was substantially lower than that in the sample wells containing CAMEL/plasmid complexes. These results implied that C18-CAMEL and pDNA could form stable complexes that can prevent EtBr interaction with pDNA (McCarthy etal., Citation2014). The results derived from the EtBr exclusion assay also showed that unmodified CAMEL condensed pDNA to a lower extent than C18-CAMEL (). This result coincided with that of the gel retardation assay, demonstrating that N-terminal stearylation can efficiently facilitate the pDNA binding and condensing capacity of CAMEL.

The size and zeta potentials of CPPs/pDNA complexes are critical features for efficient transfection. It was reported that the size of the complexes should not exceed 300 nm for efficient internalization (Hoyer & Neundorf, Citation2012). In this study, our results showed that the size of the C18-CAMEL/pDNA complexes at various N/P ratios was in the range of 190–260 nm (). Furthermore, TEM images revealed that C18-CAMEL/pDNA complexes at an N/P ratio of 2 displayed a spherical shape with smooth edges (), confirming that C18-CAMEL can indeed compact pDNA into regular nanoparticles. The zeta potential of the C18-CAMEL/pDNA complexes showed a negative value (−20.6 mV) at an N/P ratio of 1, while it increased to positive values ranging from 11.8 to 18.5 mV at an N/P ratio of 2 and greater (). The increased positive zeta potential values of the complexes mean that more cationic C18-CAMEL is engaged in pDNA binding, resulting in forming more stable nanoparticles. On the other hand, the increased zeta potential values can facilitate the binding of complexes to negatively charged cell membranes and subsequent translocation. (Hoyer & Neundorf, Citation2012; Raucher & Ryu, Citation2015).

Endocytic uptake pathway and endosomal escape of the C18-CAMEL/plasmid complexes

Although the cellular uptake mechanism of CPPs remains heavily controversial, there is a general consensus that CPP/nucleic acid complexes enter cells primarily by endocytosis (Margus etal., Citation2012; Arukuusk etal., Citation2013; Boisguerin etal., Citation2015; Lehto etal., Citation2016). Endocytosis may occur via different types of pathways, such as clathrin-mediated endocytosis, caveolin-mediated endocytosis, and micropinocytosis (Hoyer & Neundorf, Citation2012; Nakase etal., Citation2012; Lehto etal., Citation2016). To further elucidate the endocytic uptake pathways of the C18-CAMEL/pDNA complexes, cells were pretreated with pharmacological endocytosis inhibitors, including chlorpromazine (CPZ) to inhibit clathrin-mediated endocytosis, methyl-β-cyclodextrin (MβCD) to inhibit caveolin-mediated endocytosis, and amiloride to inhibit micropinocytosis (Xiang etal., Citation2012; Zhang etal., Citation2013). The results derived from the FACS assay showed that both CPZ and MβCD could reduce the uptake of complexes by 49.4 and 84.5%, respectively (). In contrast, amiloride displayed no notable effect on the uptake of complexes. This result demonstrated that the C18-CAMEL/pDNA complexes enter cells by clathrin- and caveolin-mediated endocytosis rather than macropinocytosis. Compared with that of CPZ, the strong inhibitory effect of MβCD indicated that caveolin-mediated endocytosis may be the predominant uptake pathway for the C18-CAMEL/pDNA complexes. Conclusively, our results demonstrated that the C18-CAMEL/pDNA complexes enter cells through endocytosis, and correspondingly, endosomal escape should be considered for efficient transfection.

Efficient endosomal escape is necessary for vector/nucleic acid complexes after internalization through endocytosis. For endosomal escape, attachment of membrane-lytic peptides has proven to be an effective strategy for non-viral vectors (Ferrer-Miralles etal., Citation2008; Varkouhi etal., Citation2011; Hou etal., Citation2015; Komin etal., Citation2017; Sun etal., Citation2017). Antimicrobial peptides have been used to enhance the endosomal escape of non-viral gene vectors due to their special membrane-lytic activity (Ferrer-Miralles etal., Citation2008; Hou etal., Citation2015). In our previous study, the gene vectors based on the antimicrobial peptide melittin displayed significant endosome-lytic activity (Zhang etal., Citation2013). Chloroquine (CQ), a well-known endosome-lytic agent, is commonly used to explore whether vector/nucleic acid complexes are trapped in endosomes (Erbacher etal., Citation1996). In the present study, the coaddition of CQ could only slightly elevate the transfection efficiency of the C18-CAMEL/pDNA complexes at a range of 1- to 2-fold at various N/P ratios (). However, the transfection efficiency of C18-NTAT, a control peptide with no membrane-lytic activity (data not shown), substantially increased by 7- to 15-fold at various N/P ratios in the presence of CQ. Taken together, the above results demonstrated that the excellent endosomal escape ability makes C18-CAMEL an ideal gene vector.

Transfection efficiency of C18-CAMEL in serum and different cancer cell lines

The serum has been described as an important limitation for the transfection efficiency of cationic gene vectors in various ways (Dash etal., Citation1999; Hoyer & Neundorf, Citation2012). For example, negatively charged serum proteins can induce aggregation or dissociation of the complexes after nonspecific binding, which substantially hinders cellular uptake and subsequent transfection of complexes (Pack etal., Citation2005; Hoyer & Neundorf, Citation2012; Chen J etal., Citation2019). Therefore, the transfection efficiency of the C18-CAMEL/plasmid complexes at an N/P ratio of 2 in the presence of serum at a range from 5 to 40% was assessed according to a previously reported method (Lehto etal., Citation2011). As shown in , the transfection efficiency of C18-CAMEL displayed a slight decrease in the presence of serum. This maybe because C18-CAMEL and pDNA can form stable complexes that are not susceptible to serum (Nguyen etal., Citation2000; Chen J etal., Citation2019).

Cell type is considered as an important factor that influences the transfection efficiency of gene delivery vectors. To validate the effectiveness of C18-CAMEL as a non-viral vector for cancer gene therapy, we performed transfection experiments in different cancer cell lines treated with C18-CAMEL/pGL3 plasmid complexes at an N/P ratio of 2. As expected, showed that C18-CAMEL exhibited a high transfection efficiency in the tested cancer cell lines, especially breast cancer cell lines (MDA-MB-231 and MCF-7) and glioma cell lines (U87 and U251). Conclusively, C18-CAMEL can transfect several cancer cell lines with satisfactory efficiency.

Antiproliferative effect of the C18-CAMEL/p53 plasmid complexes

The tumor suppressor p53 is an important transcription factor that can trigger cell cycle arrest, cell senescence, and apoptosis. p53 plays a crucial role in preventing tumorigenesis and treating cancer (Cheok etal., Citation2011; Duffy etal., Citation2014). However, mutations in the p53 gene are one of the most common events and occur in approximately 50% of human cancers (Cheok etal., Citation2011). In most cancers with no gene mutation, wild-type p53 is always inactivated by several different mechanisms (Wade etal., Citation2013). Due to the near-universal loss of p53 function in cancers, it can be assumed that restoring functional p53 will suppress tumor growth. Delivery of the wild-type p53 gene into cancer cells has proven to be an effective approach for cancer treatment (Cheok etal., Citation2011; Senzer etal., Citation2013; Duffy etal., Citation2014). In order to evaluate the potential of C18-CAMEL in cancer gene therapy, we assessed the anticancer activity of the C18-CAMEL/p53 plasmid complexes against two glioma cell lines (U87 cell line containing the wild-type p53 gene; U251 cell line containing a mutant p53 gene) and two breast cancer cell lines (MCF-7 cell line containing the wild-type p53 gene; MDA-MB-231 cell line containing a mutant p53 gene). Our results showed that the C18-CAMEL/p53 plasmid complexes exhibited substantially increased anticancer activity against the mutant p53-expressing U251 cells compared with the cells with the p53 plasmids and C18-CAMEL alone (). However, the C18-CAMEL/p53 plasmid complexes displayed slightly increased cytotoxicity against the wild-type p53-expressing U87 cells (). This result is in accordance with previous studies showing that mutant p53-expressing glioma cell lines are much more susceptible to p53-based gene treatment than wild-type p53-expressing glioma cell lines (Gomez-Manzano etal., Citation1996; Shono etal., Citation2002). In contrast, C18-CAMEL/p53 plasmid complexes displayed high antiproliferative activity against both MDA-MB-231 cells and MCF-7 cells (). This result demonstrated that the anticancer activity of the C18-CAMEL/p53 plasmid complexes against breast cancer cells does not depend on the p53 status of the cells.

The expression of p53 is a crucial step for p53-based gene therapy. Therefore, to verify whether cell death was attributed to p53 plasmid transfection, we evaluated the p53 mRNA and protein levels in the MCF-7 cells transfected with the C18-CAMEL/p53 plasmid complexes. As shown in , the p53 mRNA levels of MCF-7 cells transfected with C18-CAMEL/p53 plasmid complexes showed a substantial increase compared with those of the control cells. The MCF-7 cells transfected with naked p53 plasmids did not show obvious changes in the p53 mRNA levels. Subsequently, the results derived from the western blotting assay further confirmed that only the cells transfected with the C18-CAMEL/p53 plasmid complexes displayed a strong increase in the p53 protein expression levels (). These results fully confirmed that C18-CAMEL can deliver p53 plasmids into cancer cells, resulting in cell death induced by the expressed p53 protein.

Because of the pathological complexity of cancer, combined therapy has numerous benefits for cancer therapy. Cancer treatment involving a combination of nucleic acids and drugs has been proven a highly effective strategy (Li etal., Citation2013; Teo etal., Citation2016). Murine double minute 2 (MDM2), an E3 ubiquitin ligase that is highly expressed in many cancers, can inactivate wild-type p53 by mediating the nuclear export and degradation of this protein (Wade etal., Citation2013; Lemos etal., Citation2016). When the expression levels of p53 increase, the MDM2 gene will be transcriptionally upregulated accordingly by the negative p53-MDM2 feedback loop, resulting in a decrease in p53 levels (Wade etal., Citation2013; Lemos etal., Citation2016). Disruption of the p53-MDM2 feedback loop is a promising approach for restoring p53 activity. Nutlin-3a is a potent and selective MDM2 antagonist that is used for cancer therapy (Lemos etal., Citation2016). In addition, it was reported that high expression of MDM2 limited the anticancer effect of exogenous p53 (van Beusechem etal., Citation2005). Therefore, in this study, nutlin-3a was used to treat cancer with the C18-CAMEL/p53 plasmid complexes at an N/P ratio of 2. We assumed that nutlin-3a could protect both endogenous and exogenous p53 from being degraded, resulting in improved therapeutic efficiency. As shown in , nutlin-3a at various concentrations exhibited no obvious anticancer activity against the mutant p53-expressing U251 cells and MDA-MB-231 cells. In addition, nutlin-3a did not significantly increase the anticancer activity of the C18-CAMEL/p53 plasmid complexes against the mutant p53-expressing cells, suggesting that a synergistic antiproliferative effect did not occur. As shown in , nutlin-3a also did not show synergistic anticancer activity against the wild-type p53-expressing U87 cells with the C18-CAMEL/p53 plasmid complexes despite its high anticancer activity against U87 cells. This result may be related to the low anticancer activity of the C18-CAMEL/p53 plasmid complexes against U87 cells (). Gratifyingly, nutlin-3a, and the C18-CAMEL/p53 plasmid complexes displayed significantly synergistic anticancer activity against the wild-type p53-expressing MCF-7 cells (). The increased antiproliferative effect of the combined treatment was dependent on the expressed exogenous p53 and MDM2 inactivation by nutlin-3a. Our results confirmed that the combination of an MDM2 inhibitor with p53 gene therapy is an efficient approach for inhibiting the proliferation of cancer cells. However, this combined therapy is not a universal approach for treating all cancer cell lines, and screening cell lines sensitive to this combined therapy is necessary.

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