p53 DNA Nanocomplex as

Jul 31, 2014 - Adjuvant Therapy for Drug-Resistant Breast Cancer ... of apoptotic AVPI peptide and p53 DNA as apoptosis-induction adjuvant therapy for...
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Cell-Penetrating Apoptotic Peptide/p53 DNA Nanocomplex as Adjuvant Therapy for Drug-Resistant Breast Cancer Huiyuan Wang,† Huixin Wang,† Jianming Liang,†,‡ Yifan Jiang,† Qianqian Guo,† Huige Peng,† Qin Xu,‡ and Yongzhuo Huang*,† †

Shanghai Institute of Materia Medica, Chinese Academy of Sciences, 501 Hai-ke Road, Shanghai 201203, China Tropical Medicine Institute, Guangzhou University of TCM, 12 Jichang Road, Guangzhou 510450, China

‡

ABSTRACT: Drug resistance becomes a formidable challenge against effective cancer therapy. Defective apoptosis in cancer cells is a key factor responsible for chemoresistance or radioresistance. Promoting apoptosis is an important method to sensitize the resistant cells, thereby achieving successful treatment for MDR cancer. We present a strategy of codelivery of apoptotic AVPI peptide and p53 DNA as apoptosis-induction adjuvant therapy for combating the resistant breast cancer. AVPI tetrapeptide is poorly cell-permeable, thereby with very limited value for therapeutic use. Cell-penetrating chimeric AVPI derivative was developed by modification with an octa-arginine sequence (R8). The AVPIR8 is able to not only efficiently penetrate into tumor cells but also work as a vector for gene delivery by forming nanocomplexes based on its cationic R8 moiety. The combination of AVPIR8/p53 DNA was selected for targeting apoptotic pathways, thereby sensitizing the cancer cells to chemotherapeutics. The anti-MDR effect was demonstrated both in vitro and in vivo. The synergistic use of AVPIR8/p53 significantly increased the sensitivity of the resistant tumor cells to the cytotoxic agent doxorubicin by inducing apoptosis, as demonstrated in the cellular studies. Importantly, the treatment improvement was also observed in the animal studies with resistant breast tumor model. Coadministration of AVPIR8/p53 enabled a full arrest of tumor growth combined with a reduced DOX dose, yielding a productive and safe cancer treatment. KEYWORDS: cell-penetrating peptide, drug resistance, apoptosis, apoptotic peptide, p53, breast cancer, AVPI

1. INTRODUCTION Chemoresistance is a major barrier against effective treatment outcomes. This is particularly seen in epithelial tumors, exemplified by breast cancer.1 There are around 60% of patients with early breast cancer receiving chemotherapy, but only a minority could benefit from it.2 Responses to the firstline chemotherapy in metastatic breast cancer usually rate 30− 70%, but not durable, only with a time to progression of 6−10 months.3 Due the occurrence of resistance, treatments fail in most cases, and the 5-year survival rate of patients with metastatic breast cancer is merely 27%.4 Cancer cells become insensitive to chemotherapy and radiotherapy via the pathway of evasion of apoptosis.5 Apoptosis is a “suicide” program that is essential for maintaining a homeostatic balance between cell proliferation and death, and the normal life cycle as well. Defective apoptosis-related resistance of cell death has been considered the one among six hallmarks of cancer.6 Deficient apoptosis signaling is responsible for tumorigenesis, tumor maintenance, and drug resistance.1 Targeting the apoptosis pathway has become an emerging means for anti-MDR therapy.7 Cancer cells can activate multiple pathways to escape apoptosis and become resistant to cell death, but most commonly, dysfunction of TP53 tumor suppressor is the primary mechanism.6 Development of p53-based gene therapy has attracted great clinical interest.8 P53 regulates apoptosis in © XXXX American Chemical Society

both transcription-dependent and -independent pathways by activating the expression of proapoptotic proteins (e.g., PUMA, Bax, and BID), and blocking the antiapoptotic effect of Bcl-2/ Bcl-XL at the mitochondria.9 Transfection of the wild-type p53 (wtp53) for reprograming apoptosis has achieved success in reversal of MDR.10,11 Another common mechanism for evasion of apoptosis is accounted for the overexpression of inhibitors of apoptosis proteins (IAPs),12 which bind and inhibit both the initiator apoptotic caspase-9 and effector caspase-3 and -7. The second mitochondrial-derived activator of caspase/direct inhibitor of apoptosis protein-binding protein with low isoelectric point (pI) (Smac/DIABLO) is a mitochondria-derived proapoptotic protein that neutralizes and antagonizes IAPs, thus activating caspases and apoptosis. Upregulation of Smac/DIABLO can sensitize tumor cells to apoptotic death, and peptides derived from N-terminal Smac/DIABLO have been also demonstrated the mimic Smac/DIABLO functions to increase the apoptotic Special Issue: Recent Molecular Pharmaceutical Development in China Received: February 4, 2014 Revised: June 23, 2014 Accepted: July 31, 2014

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effect of chemo drugs.13 Among them, the Smac N-terminal tetrapeptide (AVPI) is one of the most widely investigated apoptotic peptides, with a high binding affinity to the X-linked IAPs.14 However, AVPI is poorly cell-permeable, thus with limited therapeutic values. We previously reported that the combination of AVPI/p53 with doxorubicin (DOX) could effectively arrest melanoma and cervical tumor growth.15 Considering the formidable challenge of MDR in cancer therapy and its close association with apoptosis, we thereby further investigated the feasibility of antiMDR through dual-activation of Smac/DIABLO- and p53mediated apoptosis pathways, and the potential application of AVPI/p53 adjuvant therapy in drug-resistant breast cancer. Cell-penetrating peptide (CPP)-based technique was used for codelivery of AVPI/p53 DNA, by which the C-terminal AVPI was modified by octa-arginine (R8) that could not only increase the cell-permeability of AVPI but also work as a drug carrier for p53 DNA (Scheme 1). Although the R8 fragment bears less

has been reported that the p53 can increase mitochondrial release of Smac and thus induce apoptosis.19,20 On the other hand, apoptosis induced by Ad-p53 was significantly inhibited by downregulation of Smac expression by using antisense Smac.21 These results suggested that p53 induced apoptosis may be associated with promotion of release of Smac into cytosol and Smac is required in p53-induced apoptosis. Because both Smac and p53 have been used as targets for enhancing apoptosis in cancer cells, and more importantly, they are related to each other, the combination strategy would be potential for anti-MDR. With the aim to develop a new activated apoptotic regulation for overcoming MDR tumor, we developed a codelivery system of AVPIR8/p53 to increase the expression of wild type p53 and the concentration of Smac sequence (AVPI) concurrently.

2. MATERIALS AND METHODS 2.1. Materials. N-Hydroxybenzotriazole (HOBt), 2-chlorotrityl chloride resin (100−200 mesh, loading capacity: 1.32 mmol/g), o-benzotriazol-N,N,N′,N′-tetramethyluronium hexafluorophosphate (HBTU), N-fluorenyl-9-methoxycarbonyl (FMOC) protected L-amino acids (FMOC-Arg(pbf)-OH, FMOC-Ala-OH, FMOC-Val-OH, FMOC-Pro-OH, FMOCIle-OH) were purchased from GL Biochem Ltd. Diisopropylethylamine (DiEA), piperidine, trifluoroacetic acid (TFA), methylacrylic acid polyethylene glycol single armor ether ester, N,N-dimethylformamide (DMF), and dichloromethane (DCM) were purchased from Shanghai Reagent Chemical Co. (Shanghai, China). Triisopropylsilane (TIS) was obtained from Sigma-Aldrich (St. Louis, MO, USA). DOX was purchased from Melone Pharmaceutical Co., Ltd. (Dalian, China). All other reagents were of analytical grade. QIAfilter plasmid purification Giga Kit (5) was purchased from Qiagen (Hilden, Germany). GelRed was purchased from Biotium (Hayward, USA). Fetal bovine serum (FBS), RPMI 1640 medium, penicillin−streptomycin, 0.25% trypsin−EDTA solution, dimethyl sulfoxide (DMSO), and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) were obtained from Invitrogen (Carlsbad, CA, USA). Micro BCA protein assay kit and caspase-3 activity assay kit were acquired from Beyotime Institute of Biotechnology (Haimen, China). Caspase-3 antibody and p53 antibody were purchased from Cell Signal Technology (USA), and P-glycoprotein antibody was obtained from Abcam (Cambridge, U.K.). 2.2. Cell Culture and Animals. Human breast cancer cell line MCF-7 and human umbilical vein endothelial cell line (HUVEC) were purchased from the Cell Bank of Chinese Academy of Sciences (Shanghai, China). Doxorubicin-resistant human breast cancer cells (MCF-7/ADR) were kindly provided by Prof. Yaping Li (Shanghai Institute of Materia Medica, Chinese Academy of Sciences). Cells were cultured in Gibco RPMI 1640 medium containing 10% FBS and 1% antibiotics (penicillin−streptomycin) at 37 °C in humidified atmosphere with 5% CO2. Healthy female BALB/c-nu nude mice (4−5 weeks old, 18− 22 g) were housed under specific pathogen-free conditions. Animals possessed continuous access to sterilized food pellets and distilled water, under a 12 h light/dark cycle. The animals were in quarantine for a week before treatment. All experimental procedures were approved by the Institutional Animal Care and Use Committee. 2.3. Solid Phase Synthesis of the Cell-Penetrating Smac Peptide (AVPIR8). The cell-penetrating Smac peptide

Scheme 1. Codelivery of Cell-Penetrating AVPI-R8/p53 DNA Nanocomplex as Adjuvant Therapy for Overcoming Drug Resistance

charge density compared to some cationic polymers (e.g., PEI), it is an efficient gene carrier due to the arginine’s natural affinity to nucleic acids. The binding between arginine-peptide/DNA involves multiple mechanisms, including electrostatic force, hydrogen bonding, and hydrophobic interaction, because arginine can form bidentate hydrogen bonds, but not solely based on charge interaction as PEI/DNA.16 Moreover, the additional advantages of arginine-rich peptides over low molecular weight PEI are the potent cell/tissue-penetrating ability and low toxicity, as well as the recently reported nuclearlocalization ability.17 Antiapoptosis is a major mechanism responsible for drug resistance.18 P53 is a key regulator for apoptosis, and AVPI is the proapoptotic Smac peptide. Both play an important role in apoptosis. The synergistic mechanism between the p53 and Smac signaling pathways has not been clearly understood yet. It B

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containing the first four N-terminal amino acids of Smac (AVPI) and the octa-arginine (R8) was synthesized by using the standard solid phase method based on classical Fmoc (1-(9Hfluoren-9-yl)methoxycarbonyl)/tert-butyl chemistry. Peptide identity was verified by liquid chromatography−mass spectrometry. 2.4. Preparation of AVPIR8/pDNA Nanocomplex. The nanocomplex was prepared by mixing a certain volume of AVPIR8 solution with 1 μg of p53 DNA (200 ng/μL in 40 mM Tris-HCl buffer solution). Subsequently, saline was added into the mixture to a total volume of 100 μL and vortexed for 5 s. The mixture was incubated at 37 °C for 30 min to allow formation of the nanocomplex. 2.5. Agarose Gel Electrophoresis. The stability of AVPIR8/pDNA nanocomplex at various w/w ratios ranging from 5 to 50 was measured by agarose gel electrophoresis. In brief, p53 was mixed with AVPIR8 at varying w/w ratios. Saline was then added to a total volume of 8 μL, and the mixture was incubated at 37 °C for 30 min. The resultant nanocomplex was loaded on the 0.8% (w/v) agarose gel containing 0.1 ‰ GelRed with tris-acetate (TAE) running buffer at 80 V for 60 min. Naked DNA was used as control. A Vilber Lourmat UVtransiluminator was used for the visualization of DNA. 2.6. Particle Size and Zeta Potential Measurement. The particle size and zeta potential of AVPIR8/p53 nanocomplex were measured by Zeta Sizer N particle analyzer (Malvern, U.K.) at 25 °C. The AVPIR8/p53 nanocomplexes at w/w ratios from 5 to 50 were prepared as described above, and diluted with deionized water to 1 mL volume for the sample before determination. The morphology examination of the AVPIR8/p53 nanocomplex was performed using transmission electron microscopy (TEM) operating at an acceleration voltage of 100 kV, after negative staining with sodium phosphotungstate solution (0.2 w/v %). 2.7. In Vitro Transfection. The optimal ratio for transfection was determined by luciferase expression experiments, and pGL-3 plasmid was used as a reporter gene. The cells were seeded in the 24-well plate and cultured in 1640 medium containing 10% FBS at 37 °C. After incubation for 24 h, the medium was replaced by fresh 1640 with 10% FBS containing the nanocomplex at a dose of 1 μg of pGL-3/well, followed by 4 h incubation. The cells were then cultured with fresh complete 1640 medium for 2 d to allow gene expression. To assess the luciferase expression of pGL-3, the medium was removed and cells were thoroughly washed with PBS and then lysed using 200 μL of lysis buffer. The luciferase activity was determined with a chemiluminometer (Lumat LB9507, EG&G Berthold, Germany) and normalized to the amount of total protein in the sample. Luciferase activity was designated as RLU/mg proteins. The p53 protein expression after AVPIR8/p53 transfection was also measured by Western blot. The treated MCF-7/ADR cells were harvested and washed with PBS. The cellular extracts were prepared by homogenization in 500 μL of lysis buffer, and incubation on ice for 30 min followed by centrifugation at 12,000 rpm for 10 min twice. Protein concentration of the extracts was determined using the BCA protein assay kit. Cellular proteins were then processed by SDS−PAGE and transferred to nitrocellulose membrane using the standard Western blotting technique. The membranes were incubated with anti-p53 and anti-actin antibodies, which was subsequently detected by secondary antibodies.

2.8. Cytotoxicity Assay in MCF-7/ADR Cells. Growth inhibition mediated by AVPIR8/p53 combination in MCF-7/ ADR cells was measured by a standard MTT assay. Cells were treated with DOX alone, DOX and AVPIR8, and the AVPIR8/ p53 plus DOX for 48 h. Specifically, in the (AVPIR8/p53 + DOX) group, AVPIR8/p53 complexes at optimal ratio (w/w = 30) were added into each well with peptide concentration of 1 mg/mL and incubated for 4 h. The medium was subsequently replaced with 1 mL of fresh complete medium containing 0.1 to 10 μg/mL DOX. Cytotoxicity studies were also conducted in HUVEC and MCF-7 cells with DOX concentrations ranging from 0.2 to 2 μg/mL and 0.3 mg/mL of peptide. AVPIR8/p53 complexes at optimal ratio (w/w = 30) were added into each well with peptide concentration of 0.3 mg/mL for 4 h; after further culture for 44 h, the medium was replaced with fresh culture medium containing MTT solution (0.5 mg/mL) for an additional incubation of 4 h at 37 °C. The medium was finally removed, and 200 μL of DMSO was added to dissolve the formazan crystals. The absorption was measured at 570 nm using a microplate reader (Thermo, USA). The cell viability was calculated according to the equation below. cell viability (%) = (ODsample − ODDMSO) /(ODcontrol − ODDMSO) × 100

2.9. Cell Apoptosis Analysis. Flow cytometry was used to determine the activation of apoptosis on drug-resistant cells induced by AVPIR8/p53. Apoptosis level was quantified by Annexin V-FITC/propidium iodide (PI) assay. In brief, MCF7/ADR cells were incubated with the nanocomplex. Cells were then harvested, washed with PBS, and stained with Annexin VFITC/PI according to the manufacturer’s instructions. Fluorescence was measured using a flow cytometer (BD FACSCalibur, USA). Analysis was performed with data management using FlowJo (TreeStar Inc., Oregon, USA). Cells treated with DOX alone were also measured as a control group. The caspase-3 is one of the effectors of caspases that lead to apoptosis. The levels of procaspase-3 after AVPIR8/p53 transfection were also measured by Western blot analysis with anti-aspase-3 antibody. Caspase-3 activity was determined with a caspase-3 assay kit. Briefly, MCF-7/ADR cells were treated with complexes and DOX for 24 h and then collected. The cells were washed twice with PBS, and the lysates were centrifuged at 12,000 rpm for 15 min at 4 °C. The caspase activity in the supernatant was measured by adding the caspase-3 substrate to each sample, with incubation at 37 °C for 12 h; the absorbance was recorded at 405 nm.22 2.10. In Vivo Therapeutic Study Using Drug-Resistant Breast Tumor Model. To establish the drug-resistant breast tumor model, nude female BALB/c mice (4 weeks old) were xenografted with 1 × 107 MCF-7/ADR cells/mouse by a 25gauge needle subcutaneously in the back. The tumor growth was monitored every 3 days using calipers, and tumor volume was calculated using the following formula.

V = W 2 × L/2 where W and L respectively are the shortest and longest diameters. The P-gp expression levels in xenografted tumors and resistance cells were also examined. Formaldehyde-fixed tumor C

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Figure 1. Characterization of the AVPIR8/pDNA nanocomplex. (A) Gel retardation assay with varying w/w ratios. (B) Measurement of particle size and zeta potential using dynamic light scattering (DLS). (C) TEM image.

particle size and morphology of complexes were also determined using TEM; Figure 1C showed that the diameter was less than 50 nm. Average particle size was small enough to achieve longevity during systemic circulation in vivo. However, the determination by DLS showed larger size compared to the TEM results. TEM provides information about the size and shape of dried nanoparticles, and the swelled layer will collapse under the high vacuum conditions used for TEM measurements. Therefore, a discrepancy often exists between TEM and DLS measurements that is attributed to factors associated with the high vacuum conditions of TEM and the hydrodynamic and electrokinetic effects operative in DLS measurements.24 By contrast, the zeta potential of complexes showed an increase trend with the elevating w/w ratio due to the surplus of cationic AVPIR8. The zeta potential turned positive when the w/w ratio was higher than 20. Positive charge of vectors is crucial for the complexes to enter cells due to the negatively charged cell membrane. The trends were similar to the change in particle sizes. The AVPIR8/p53 nanocomplexes were monodisperse and positively charged. They worked as a codelivery system for simultaneously delivering cell-penetrating apoptotic peptide AVPIR8 and p53. The in vitro gene transfection with varying ratios of AVPIR8/ pDNA was carried out in the presence of 10% serum to mimic the physiological conditions. The luciferase expression efficiency was assessed in MCF-7 and MCF-7/ADR cells using pGL-3 plasmid as a reporter gene. The optimal ratio of AVPIR8/pDNA determined by transfection studies was 30 (Figure 2A), because AVPIR8/pGL-3 showed the highest luciferase expression at a w/w ratio of 30 in the parallel experiments in the two cell lines. So the ratio was selected for the following studies. We also evaluated the transfection effect of AVPIR8 carrier mediated p53 gene delivery in the MCF-7/ADR cells, of which the wt-p53 expression was deficient. The p53 transfection efficiency was observed by Western blot analysis. The MCF-7/ ADR cells showed increased levels of p53 protein in accordance with the increased w/w ratios of AVPIR8/p53 complexes (Figure 2B), demonstrating the feasibility of p53-based gene therapy using the A VPIR8/p53 system. Drug resistance is the major barrier against effective cancer treatment, accounting for chemotherapy failure in over 90% of patients with metastatic cancer.23 Upregulation of apoptotic activity in resistant cancer cells can restore their sensitivity to chemo drugs.25 In our studies, drug-resistant MCF-7/ADR cells were pretreated with AVPIR8/p53 DNA for reinstallation of the “suicide” program, and then exposed to DOX to test the

tissues, MCF-7/ADR cells, and MCF-7 cells were used for Western blot analysis. P-gp detection was performed with antip53 antibody (Abcam, U.K.). When tumors reached an average volume of 100−200 mm3, the treatment regimens were carried out. The tumor-bearing mice were randomized into 4 groups (6 mice per group). Group (1) was peritumorally injected with PBS (control); group (2) with DOX (2.5 mg/kg/d); group (3) with DOX (1 mg/kg/d); group (4) with a mixture of DOX (1 mg/kg/d) and AVPIR8/p53 (1.2 mg of AVPIR8 plus 40 μg of p53 DNA per mouse). After 21 days, mice were killed, and their tumors and major organs including livers, lungs, spleens, kidneys, and hearts were immediately harvested and weighed to calculate the organ coefficient and process histological examination. The organ coefficient was calculated using the following formula: organ coefficient (%) = (weight of the organ/body weight) × 100%

2.11. Statistical Analysis. Data represent the mean and standard deviation in each experiment, all of which were repeated at least three times. The differences between the mean values were analyzed by Student’s test. The results were considered statistically significant at p < 0.05.

3. RESULTS AND DISCUSSION Oligoarginine is a class of CPP that is commonly investigated for use in gene delivery.23 The polycationic nature renders it strongly electrostatically binding with nucleic acid drugs, functioning as not only a condenser but also an enhanced cell-penetrating vector. Based on this feature, a codelivery strategy of AVPIR8/pDNA was developed. The binding capability of the cell-penetrating AVPIR8 to condense DNA was studied by gel retardation study (Figure 1A). The formation of the compact complexes was confirmed by resistance to electrophoresis, in which the electrophoretic mobility of DNA was completely retarded when the w/w ratio was 30 and above. The formation of a small nanoscale and compact complex (carrier/gene) is favorable in gene delivery. In this study, the particle sizes and zeta potentials of AVPIR8/pDNA complexes at w/w ratios ranging from 5 to 50 were measured. As shown in Figure 1B, a typical trend was seen that, with the increase of w/ w ratio, the particle size of peptide/p53 complexes was decreased gradually. Notably, a significant decline of size occurred when the w/w ratio was raised from 20 to 25, corresponding to the results of agarose gel electrophoresis. The D

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However, in nontumoral HUVEC cell line, the adjuvant AVPIR8/p53 therapy did not show obvious enhancement of cytotoxicity (Figure 3C). It indicated that the tumor cells were specifically sensitive to the p53 and AVPIR8 compared to the nontumoral cells, and this selective action demonstrated the safety advantage of reduced adverse effects of the system for breast cancer treatment.26 The apoptotic efficiency was also measured by using Annexin V-FITC/PI staining for fluorescence activated cell sorting (FACS). The FACS data (Figure 4A,B) showed an increased trend in apoptotic efficacy in the groups treated with the proapoptotic agents. Chemotherapeutic stimulus (4 mg/L of DOX alone) only induced a very minor apoptosis in MCF-7/ ADR cells, and the late apoptosis was measured to be merely 7.4%. The late apoptosis rate of DOX was increased to 24.2% in the presence of AVPIR8, which could activate the intrinsic pathway via a mechanism of lifting the IAPs’ inhibition on caspases. Moreover, dual action of AVPIR8/p53 further increased the late apoptosis rate up to 44.3%. The p53 transfection enhances the sensitivity to cytotoxic DOX through upregulation of wtp53 protein.11 However, it was reported that introduction of wtp53 alone was not sufficient to substantially sensitize the resistant cells to a chemo drug.27 Given the high genome instability of cancer cells, multiple action patterns (e.g., AVPI- and p53-mediated pathways) would help secure the reprogramming of apoptosis. In addition, procaspase-3 is highly expressed in many cancer cells, and its activated conversion typically induced apoptosis.28 Its high expression was also found in the tested MCF-7/ADR cells. After treatment with apoptotic stimuli, decreased concentration of procaspase-3 was observed (Figure 4C), and DOX combined with proapoptotic regulators of p53 and/or AVPIR8 showed a markedly lower level of procaspase-3 than single use of DOX. The downregulation of procaspase-3 was associated with upregulation of the corresponding cleaved caspase-3. Moreover, there was a dramatic increase in caspase-3 activity after 24 h of treatment in MCF-7/ADR cells (Figure 4D). For instance, the caspase-3 activity was 3.3-fold higher in tumor cells treated for 24 h with AVPIR8/p53/DOX, as compared with the control (DOX alone, 2 mg/L), and 3.1-fold higher compared to the high-dose control (DOX alone, 4 mg/ L). Taken together, these results clearly showed that the AVPIR8 apoptotic peptide and p53 gene can sensitize the resistant MCF-7/ADR cells, and the AVPIR8/p53 complexes promoted caspase-3-dependent apoptosis for overcoming MDR.

Figure 2. (A) In vitro gene transfection mediated by AVPIR8 in MCF7 and MCF-7/ADR cells. (B) Expression of p53 after transfection with AVPIR8/p53 DNA.

restored sensitivity. As shown in Figure 3A, the MCF-7/ADR cells displayed strong resistance to DOX, and still retained approximately 80% viability even at a high dose of 10 mg/L. By contrast, the AVPIR8 apoptotic peptide sensitized the resistant MCF-7/ADR cells, and dramatically increased DOX-directed cytotoxic effect. For instance, at a DOX dose of 2 mg/L, antitumor activity with combination of AVPIR8 and DOX showed a remarkable 17-fold enhancement compared to single use of DOX (34.7% vs 2.1% of inhibition rate). Furthermore, a remarkable synergetic effect was found in the use of AVPIR8/ p53 nanocomplex, which gave a 36-fold increase (76.5% vs 2.1% of inhibition rate), compared to the single use of DOX. In the high dose group (10 mg/L DOX), the inhibition rates of the single DOX and the DOX + AVPIR8 and DOX + AVPIR8/ p53 were 21.6%, 81.6%, and 95.1%, respectively. These results clearly demonstrated the antidrug resistance effect by activation of apoptosis using the AVPIR8/p53 nanocomplex. It should be noted that the AVPIR8/p53 also improved the antitumor effect in the nonresistant (or sensitive) MCF-7 cells, though the enhancement was not as significant as that in the MCDF-7/ ADR cells (Figure 3B).

Figure 3. Cytotoxicity and antitumor efficacy determined by MTT assay in (A) MCF-7/ADR cells, (B) MCF-7 cells, and (C) HUVEC cells. E

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Figure 4. Apoptosis-induced effect in MCF-7/ADR cells determined by flow cytometry. (A) The induced apoptosis determined by FACS (represented by early apoptosis and late apoptosis rates). (B) Cells that were negative for both Annexin V-FITC and PI staining were classified as alive, while cells that stained positive for Annexin V-FITC and negative for PI were classified as early apoptotic, while cells that stained positive for Annexin V-FITC and PI were classified as late apoptotic. (C) Procaspase-3 expression after treatment. (D) Assay of caspase-3 activity.

Based on the in vitro results, the synergetic AVPIR8/p53 combination displayed the best antitumor effect and, therefore, was examined further in vivo. The animal studies showed that the AVPIR8/p53 adjuvant therapy combined with DOX (1 mg/ kg/d) yielded a full arrest of the resistant breast tumor growth (Figure 5D). At the end point, the tumor was well restrained within the starting sizes, measured to be 183 mm3, compared to 440 mm3 in the high-DOX group (2.5 mg/kg/d) and 866 mm3 in the low-DOX group (1 mg/kg/d). The dual proapoptotic agents of AVPIR8/p53 successfully sensitized the resistant cells, and the tumor growth was almost completely arrested with exposure to 1 mg/kg/d DOX that otherwise was an ineffective dose to the resistant tumor. Notably, the use of relatively low chemotherapeutic dose in resistant breast cancer therapy would be beneficial for improving not only the treatment outcomes but also the life quality of the patients. Our results demonstrated that the apoptosis-targeting adjuvant therapy exhibited remarkable efficacy on anti-MDR tumor. The cocktail (or combination) chemotherapy containing a few drugs with different action patterns is often used in clinical practice. Biologics are an emerging class of drugs that has been booming for research and development for cancer therapy. In the presented work, we combined peptide, DNA, and chemical drugs as a cocktail, in which, assisted by AVPIR8/p53 adjuvant therapy, DOX yielded effective treatment outcomes. The apoptotic modulators of Smac peptide and p53 involve highly

The anti-MDR efficacy of the AVPIR8/p53 adjuvant therapy was further examined by using a resistant breast tumor mouse model that harbored MCF-7/ADR tumor xenograft. Pglycoprotein (P-gp) is one of the major mechanisms responsive for MDR. With Western blot assay, we confirmed the overexpression of P-gp in both MCF-7/ADR cells and the xenografted tumors (Figure 5A), but no expression was observed in normal MCF-7 cells. Two doses were set up in single DOX-treated mice in order to better monitor the dose−response in resistant breast tumor. Low dose of DOX (1 mg/kg/d) yielded only minor therapeutic effects, and although there was a 2.5-fold increase in dose, mice treated with 2.5 mg/kg/d of DOX produced unsatisfying arrest of tumor growth, because of the resistance to chemotherapy (Figure 5B). Therefore, further increase of the dose could do little help for improving the treatment in the tumors with low response to chemotherapeutics. Moreover, the high doseassociated side toxicity would outweigh the treatment benefits, as evidenced by four deaths out of six mice receiving 2.5 mg/ kg/d of DOX before reaching the end point (Figure 5C). A similar observation was made in our previous study, in which the increased DOX dose effectively inhibited cervical tumor growth, but nevertheless gave rise to an unacceptably high mortality rate.15 Indeed, such a dilemma between risks and benefits is one of the biggest challenges in chemotherapy practice. F

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Figure 5. In vivo anti-MDR efficacy in nude mice harboring resistant breast tumor. (A) Expression of P-gp in MCF-7/ADR tumors, MCF-7 cells, and MCF-7/ADR cells. (B) Tumor volume changes in the treatment regimen (n = 6). (C) The tumor weight at the end point and the calculated inhibition rate of tumor growth. (D) The survival rate in the treatment regimen. (E) The representative tumor tissues after treatment.

contrary, the animals receiving 2.5 mg/kg/d of DOX suffered apparent loss of body weight during the indicated treatment course. Moreover, the organ coefficient of spleen only in the high-dose DOX group displayed significant difference compared to the PBS-treated control group (Figure 6B). Histological examination showed that the high-dose DOX induced severe damages, e.g., hepatic fatty change, reduced area of spleen white pulp, and renal tubule necrosis (Figure 6C), but no obvious toxicity was found in lung and heart. Of note, the results in this animal model were different from the clinical observation that DOX is known for its cardiac toxicity, possibly on account of the variation between different species. Importantly, the AVPIR8/p53 cocktail did not cause obvious changes in the major organs (spleen, kidney, heart, lung, liver), further demonstrating the reduced toxicity and better tolerance of the apoptosis-induced adjuvant therapy.

complicated processes, but often not limited only to apoptosis pathway. For example, it was reported that the wtp53 expression in breast cancer cells resulted in downregulation of P-gp and the consequent decrease in drug efflux.11 Therefore, the treatment by the synergistic effect of AVPIR8/ p53 may benefit from a complex network of regulation. It should be mentioned that the focus of our studies was on exploration of the feasibility of this strategy for anti-MDR application; we thus used peritumoral administration for local delivery of the therapeutics so as to closely monitor the drug exposure−responses. Further investigation should be conducted for exploring the in vivo targeted delivery by modification of a targeting ligand. Moreover, the positive transfection results suggested the stability of the nanocomplexes in serum, but circulation stability should be further investigated for systemic application. The cytotoxic agent DOX is usually associated with severe adverse effects, resulting in an unsustainable therapy regimen and offset of treatment benefits. The side toxicity was initially evaluated by monitoring the body weight change. No significant difference in body weight changes was observed between the control and the group given the combination, suggesting the safety of AVPIR8/p53 adjuvant therapy (Figure 6A). On the

4. CONCLUSION In order to overcome drug-resistant breast cancer, a strategy based on codelivery of cell-penetrating peptide/p53 DNA nanocomplex was developed as adjuvant therapy by targeting the apoptosis pathways. Modified with an octa-arginine G

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sequence, the thus-formed cell-penetrating AVPIR8 is featured by its great enhancement of intracellular delivery, as well as its ability to function as gene carrier. The dual biological functions render codelivery of apoptotic peptide AVPI and p53 DNA for inducing apoptosis via multiple mechanisms. The cocktail containing AVPIR8/p53 and DOX demonstrated the capacity of overcoming drug resistance, yielding a productive treatment outcome against resistant breast tumor with significantly reduced adverse toxicity. The strategy offers a potential method for anti-MDR cancer therapy.

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AUTHOR INFORMATION

Corresponding Author

*Tel: +86-21-2023-1000 ext 1401. E-mail: [email protected]. cn. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by the following grants: National Basic Research Program of China (973 Program 2013CB932503, 2014CB931900) and NSFC, China (81172996, 81373357). This work was also supported in part by the Chinese Postdoctoral Science Foundation (2012M510097, 2013T60478).

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