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Arginine modified PAMAM dendrimer for interferon beta gene delivery to malignant glioma
International Journal of Pharmaceutics 445 (2013) 79–87
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International Journal of Pharmaceutics journal homepage: www.elsevier.com/locate/ijpharm
Pharmaceutical nanotechnology
Arginine modified PAMAM dendrimer for interferon beta gene delivery to malignant glioma Cheng Zhe Bai a,1 , Sunghyun Choi a,1 , Kihoon Nam b , Songhie An a , Jong-Sang Park a,∗ a b
Department of Chemistry, Seoul National University, Seoul, Republic of Korea Department of Pharmaceutics and Pharmaceutical Chemistry, Center for Controlled Chemical Delivery – CCCD, University of Utah, Salt Lake City, USA
a r t i c l e
i n f o
Article history: Received 12 September 2012 Received in revised form 9 December 2012 Accepted 24 January 2013 Available online 4 February 2013 Keywords: Malignant glioma Apoptosis Gene delivery PAMAM-R Interferon beta
a b s t r a c t A xenograft brain tumor model was established by the subcutaneous injection of U87MG cells into nude mice to investigate the efficacy of a non-viral vector, arginine-modified polyamidoamine dendrimer (PAMAM-R), in delivering a therapeutic gene, human interferon beta (IFN-). We used 4 ,6-diamidino2-phenylindole staining, the terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL) assay, and the caspase-3 activity assay to determine the induction of apoptosis upon transfection with the PAMAM-R/IFN- gene polyplex in vitro. The polyplex was injected into xenograft brain tumors. Mice treated with PAMAM-R/pORF-IFN- exhibited a significantly smaller tumor size than control mice and PAMAM-R/pORF treated mice. Hematoxylin/eosin staining and immunohistochemistry with the endothelial growth factor receptor antibody also revealed inhibition of tumor growth. Furthermore, reverse transcription polymerase chain reaction and the TUNEL assay also verified the expression of IFN- and induction of apoptosis in vivo. These results indicate that the PAMAM-R/pORF-IFN- polyplex is an effective therapeutic candidate for glioblastoma multiforme due to its selective induction of apoptosis in tumor cells. © 2013 Elsevier B.V. All rights reserved.
1. Introduction Glioblastoma multiforme (GBM) is the most malignant astrocytoma (World Health Organization grade IV), and the most aggressive among all primary brain tumors, with overall median survival of <1 year following diagnosis (Sathornsumetee and Rich, 2006). Despite advances in conventional therapy including surgery, radiotherapy, and chemotherapy alone or in combination, the diffuse and highly infiltrative nature of GBM allows it to expand from the tumor site into the surrounding brain. Complete surgical removal of the tumor is nearly impossible and inevitably, recurrent tumors appear at the site of the initial lesion (Lawler et al., 2006; Maguire et al., 2008). Therefore, gene therapy has been investigated as a method to target GBM tumor cells. A number of gene therapy strategies for malignant glioma, such as the direct killing of tumor cells using “suicide” genes, restoration of a defective tumor suppressor gene, drug resistance genes and immune-gene therapy have been developed for GBM (Klatzmann et al., 1998; Lang et al., 2003; Pulkkanen and Yla-Herttuala, 2005). Among these
∗ Corresponding author at: Department of Chemistry, Seoul National University, 599 Gwanak-ro, Gwanak-gu, Seoul 151-747, Republic of Korea. Tel.: +82 2 871 5355; fax: +82 2 877 5110. E-mail addresses: [email protected], [email protected] (J.-S. Park). 1 These authors contributed equally to this study. 0378-5173/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.ijpharm.2013.01.057
approaches, transfection of immune gene has been considered as therapeutically promising tools for GBM. Interferon (IFN)- as a immune gene is known to exert anti-tumor activity via to the induction of apoptosis in IFN- protein-resistant tumor cells, including melanoma, renal cell carcinoma, and glioma cells; Apoptosis induction was closely correlated with the level of intracellular IFN- mRNA, a prolonged phosphorylation time of signal transducer and activator of transcription (STAT)-1, a molecule related to the IFN- signal transduction pathway, and the level of activated DNase ␥ (Yoshida et al., 2004). Although the associated mechanisms are not yet clearly understood, much evidence indicates that IFN- expression correlated with tumor cell suppression (Bulbul et al., 1986; Juang et al., 2004; Qin et al., 1998; Wang et al., 2010). Thus, delivery and expression of IFN- could be an effective approach for GBM. To study brain tumor, allogenic graft model, xenograft model and genetically engineered mouse (GEM) model are used (Chen et al., 2011). Among them, one of the most widely used models is the human tumor xenograft due to variety of advantages such as using actual human tumor tissue that featuring complexity of genetic and epigenetic abnormalities, suitable for multiple therapeutic applications from a single tumor biopsy and timesaving until obtaining the results (Fomchenko and Holland, 2006; Richmond and Su, 2008). Although xenograft model cannot study stage of tumor progression and produce specific genetic abnormalities, there are several important successes in clinical trials that
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used xenograft model as a preclinical trial such as bortezomib curing multiple myeloma, herceptin that enhance anti-tumor activity of paclitaxel and doxorubixin in human breast cancer (Baselga et al., 1998; LeBlanc et al., 2002). Therefore xenograft model could be a useful candidate for understanding cancer treatment. In previous study, we reported that PAMAM-R, which consists of cationic arginine residues conjugated to the periphery of the polyamidoamine (PAMAM) dendrimer G4, exhibits greatly enhanced transfection efficiency and lower cytotoxicity than do other commonly used non-viral vectors (Choi et al., 2004). Furthermore, PAMAM-R was shown to increase the gene expression level not only in primary cortical cultures but also in glial cells (Kim et al., 2006). In present study, we focused to investigate the expression of IFN- delivered by PAMAM-R prevent tumor suppression both in vitro and in mouse brain tumor xenograft model. 2. Materials and methods 2.1. Materials PAMAM dendrimer (ethylene diamine core, G4), N,Ndiisopropylethylamine (DIPEA), piperidine, triisopropylsilane, trifluoroacetic acid, N,N-dimethylformamide (DMF), 3-[4,5dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT), Trizma base, paraformaldehyde, Tween 20, ethylenediaminetetraacetic acid (EDTA), Harris’s hematoxylin solution, and eosin were purchased from Sigma–Aldrich (St. Louis, MO). N-hydroxybenzotriazole (HOBt) and 2-(1H-benzotriazole-1-yl)1,1,3,3,-tetramethyluronium hexafluorophosphate (HBTU) were purchased from Anaspec, Inc. (San Jose, CA). Fmoc-L-Arg(pbf)-OH was purchased from Novabiochem (San Diego, CA). Dulbecco’s phosphate-buffered saline (DPBS), Dulbecco’s modified Eagle’s medium (DMEM), and fetal bovine serum (FBS) were purchased from GIBCO (Gaithersburg, MD). The VeriKineTM Human IFN- enzyme-linked immunosorbent assay (ELISA) Kit was purchased from PBL, Inc. (Piscataway, NJ). The peroxidase-blocking solution, REALTM EnVisionTM Detection System, was purchased from Dako (Glostrup, Denmark). Epidermal growth factor receptor (EGFR) antibody was purchased from Abcam (Cambridge, UK). 2.2. Cell lines U87MG (human glioblastoma cells), Neuro2A (mouse neuroblastoma cells), NIH3T3 (mouse embryo fibroblast cells), and HT22 (mouse hippocampal cells) were maintained in DMEM medium supplemented with 10% FBS at 37 ◦ C in an atmosphere of 5% CO2 . 2.3. Preparation of plasmid The human IFN- plasmid, pORF-IFN- was purchased from Invivogen (San Diego, CA). The pORF plasmid was cloned using a synthesized primer, which eliminates the part of IFN- sequence from pORF-IFN- vector. The sequence of the forward primer was 5 -ATCGTCGACTACTAACCTTCTTCTCTTTCCTACAGC-3 , containing the SalI recognition sequence, and that of the reverse primer was 5 -CTCTAATTCGTCGACGTCCGATCGTCT-3 , containing the PstI recognition sequence and an additional NheI site. The IFN- gene sequence and the pORF-IFN- plasmid were digested separately with the restriction enzymes SalI and NheI, and then ligated together to obtain the pORF plasmid. 2.4. Anti-tumor activity of human interferon beta with PAMAM-R U87MG cells were seeded in 24-well plates at a density of 1 × 104 cells in DMEM medium containing 10% FBS. After 1 day, the cells
were treated for 48 h at 37 ◦ C with either a polyplex solution containing 1 g pORF-IFN- plasmid DNA at a weight ratio of 4, or a control polyplex solution containing 1 g pORF plasmid DNA at the same weight ratio. Following this, 100 L of MTT (2 mg/mL in PBS) solution was added to each well. After incubating at 37 ◦ C for 4 h, the medium was removed from each well and 150 L of DMSO was added to dissolve the insoluble formazan crystals formed by proliferating cells. The absorbance in each well was measured at 570 nm using a microplate reader (Molecular Devices Co., Menlo Park, CA). These experiments were performed in Neuro2A cells, NIH3T3 cells, and HT22 cells using the same method as described above. 2.5. Quantification of human IFN-ˇ protein level by ELISA U87MG cells were seeded in a 24-well plate at a density of 1 × 104 cells in DMEM containing 10% FBS. After 1 day, cells were transfected with either the PAMAM-R/pORF-IFN- polyplex or the PAMAM-R/pORF polyplex as described above for 48 h. The medium was collected from each well and centrifuged to remove cell debris. The samples, along with IFN- protein standards, were assayed using the human VeriKineTM human IFN- ELISA kit. Briefly, sample diluent was added to each well, and then the standards, blanks, and samples were added to individual wells and incubated for 1 h. After the IFN- antibody was added and incubated for 1 h, a horseradish peroxidase (HRP) solution was added to each well and incubated for 1 h. Before adding the stop solution, tetramethyl-benzidine (TMB) substrates were added to each well and incubated for 15 min in the dark. Finally, the absorbance at 450 nm was measured using a microplate reader within 5 min after the addition of the stop solution. The final results were reported in terms of pg/mg protein. 2.6. Nuclear morphological change U87MG cells were seeded in LabTek chambers at a cell density of 3 × 104 in a DMEM medium containing 10% FBS. After 1 day, cells were transfected with the PAMAM-R/pORF-IFN- or PAMAMR/pORF polyplex as described above for 48 h. The medium was removed and the cells were rinsed twice with PBS to discard residual medium. The cells were then fixed in 4% paraformaldehyde for 10 min and washed twice with PBS. Thirty minutes after mounting with mounting solution containing 4 ,6-diamidino-2-phenylindole (DAPI), the cells were observed using fluorescence microscopy. 2.7. Terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL) assay in vitro U87MG cells were plated in LabTek chambers at a cell density of 3 × 104 in DMEM medium containing 10% FBS. After 1 day, the cells were transfected with either PAMAM-R/pORF-IFN or PAMAM-R/pORF polyplexes as described above for 48 h. The medium was removed and the cells were rinsed with PBS twice to eliminate residual medium. The cells were then processed using the DeadEndTM Colorimetric TUNEL System. Briefly, the cells were fixed with 4% paraformaldehyde for 25 min at room temperature and permeabilized in 0.2% Triton X-100 solution, and then equilibrated with equilibration buffer at room temperature for 10 min. The cells were incubated with a reaction mix containing equilibration buffer, biotinylated nucleotide mix, and the terminal deoxynucleotidyl transferase (TdT) enzyme at 37 ◦ C for 60 min inside a humidified chamber to allow the end-labeling reaction to occur. After immersing in 2× SSC to stop the reaction, endogenous peroxidase was blocked by treatment with 0.3% hydrogen peroxide for 10 min at room temperature. Subsequently, a streptavidin HRP solution was added for 30 min at room temperature. A solution of 3,3 diaminobenzidine (DAB) was then added to the cells and developed for 5 min. Cells were then rinsed several times in deionized water
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and mounted in aqueous medium. Stained cells were observed under a light microscope. 2.8. Caspase-3 activity assay U87MG cells (3 × 106 ) were seeded in DMEM medium containing 10% FBS. After 1 day, cells were incubated with the PAMAM-R/pORF-IFN- or PAMAM-R/pORF polyplexes for 48 h. The cells were then collected and assayed using the caspase-3 assay kit. Briefly, the collected cells were incubated in 1× lysis buffer for 20 min. Lysed cells were centrifuged at 16,000 × g for 15 min at 4 ◦ C, and the supernatants were transferred to fresh tubes. 5 L of cell lysate was added to each well of a 96-well plate along with 85 L of 1× assay buffer and 10 L of the caspase-3 substrate Ac-DEVDpNA. The samples were incubated in a humidified chamber at 37 ◦ C for 24 h. Following this, the absorbance at 405 nm was measured using a microplate reader and recorded as a percentage relative to untreated cells. 2.9. Mouse xenograft brain tumor model Four- to six-week-old BALB/C nude mice were kept at 25 ◦ C in a specific pathogen-free environment, in positive pressure rooms with filtered and humidified air. The mice were maintained in the pathogen-free Biogen animal facility for 1 week prior to each experiment. After the mice were anesthetized using Zoletil and Rompun, a total of 1 × 106 cells, harvested with trypsin/EDTA solution in a 50 L volume, were injected subcutaneously into the left flanks of nude mice. The tumor volumes were measured by the formula (w2 × l)/2, where w is the smallest and l is the largest diameter of the tumor (Engebraaten et al., 2002). When the tumor size reached 20 mm3 , the mice were divided into 3 groups. The control group was injected with 50 L of PBS, the empty vector group with 50 L of polyplex solution containing 5 g of pORF plasmid DNA at a weight ratio of 4, and the gene therapy group with the same volume of polyplex solution containing 5 g pORF-IFN- plasmid DNA at the same weight ratio. All 3 groups received a single injection directly into the center of the tumor. Tumor sizes were measured every other day. After 3 weeks, the animals were sacrificed and tumor tissues were excised for immunohistochemistry. 2.10. Tissue preparation and morphologic immunohistochemistry The excised tissues were fixed in 4% paraformaldehyde solution and embedded in paraffin. Serial sections of 5 m thickness were cut from the tissue blocks and floated onto glass slides. All sections were deparaffinized in xylene and rehydrated in a graded series of solutions from ethanol to deionized water for staining. Hematoxylin and eosin (H&E) staining: sections were stained in Harris’s hematoxylin solution for 8 min, differentiated in 1% acid alcohol for 30 s, and subjected to bluing in 0.2% ammonia water for 45 s to remove unreacted hematoxylin. After rinsing in 95% alcohol, the slides were counterstained in eosin-phloxine B solution for 45 s. After being dehydrated in a graded series of ethanol and xylene solutions, the sections were treated with mounting solution for observation under the scan scope. Immunostaining for EGFR: following antigen retrieval overnight at 60 ◦ C in 10 mM Tris base solution (pH 9.0) containing 1 mM EDTA and 0.05% (v/v) Tween 20, the slides were washed in Tris-buffered saline (TBS) containing 0.05% (v/v) Tween 20. Endogenous peroxidase was blocked by incubating with peroxidase-blocking solution for 30 min. The sections were treated with 10% normal goat serum for 1 h to further reduce nonspecific background staining. The slides were then incubated in a 200:1 dilution of EGFR antibody overnight at 4 ◦ C. The sections were then washed with TBS, and treated with goat anti-rabbit immunoglobulin (IgG) for 1 h and DAB+ chromogen
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with substrate buffer for 2 min (Dako REALTM EnVisionTM Detection System). After washing with TBS and tap water, the sections were incubated in hematoxylin for counterstaining. The slides were differentiated in 1% acid alcohol and subjected to bluing in 0.2% ammonia water. After dehydration, the sections were treated with mounting solution and observed in the scan scope. 2.11. Reverse transcription polymerase chain reaction (RT-PCR) analysis To detect the expression of IFN- in tissue, animals treated with the PAMAM-R/pORF-IFN- polyplex, the PAMAM-R/pORF polyplex and the control, animals were sacrificed and the tumor and skin at the injection site were collected. Tissues were homogenized and total RNA was prepared using the Trizol® Reagent according to the manufacturer’s instructions (Invitrogen, Carlsbad, CA). Reverse transcription followed by PCR was done using the OneStep RT-PCR Kit (QIAGEN, Valencia, CA) with manually designed PCR primers: 5 -GGCCATGACCAACAAGTGTCTCCTCC-3 (forward), 5 -CTTACAGGTTACCTCCGAAACTGAG-3 (reverse) for human IFN-, and 5 -CGCTGGGTCAGAAGGACTCCTATG-3 (forward), 5 -CGGAGAAGAGCTATGAGCTGCCTG-3 (reverse) for the housekeeping gene -actin. 2.12. TUNEL assay in vivo All the sections were deparaffinized in xylene and rehydrated in a graded series of solutions from ethanol to deionized water. Then the specimens were processed using the DeadEndTM Fluorometric TUNEL System. Briefly, the sections were washed in 0.85% NaCl for 5 min at room temperature and fixed in 4% paraformaldehyde for 15 min at room temperature. After permeabilizing in proteinase K for 10 min, the sections were fixed in 4% paraformaldehyde and exposed to the TdT reaction mix containing equilibration buffer, biotinylated nucleotide mix, and the TdT enzyme at 37 ◦ C for 60 min inside a humidified chamber to allow the end-labeling reaction to occur. After immersing in 2× SSC to stop the reaction, the slides were rinsed in PBS solution and mounted in aqueous medium containing DAPI. Finally, the sections were observed in a fluorescence microscope. 2.13. Statistical analysis All data are expressed as mean ± SD. One-way analysis of variance (ANOVA) was used to determine statistically significant differences between groups. Dunnett’s test (SPSS v.12.0, Chicago, IL) was used to correct for multiple comparisons when statistical significances were identified in the ANOVA. 3. Results 3.1. Construction of the pORF plasmid To confirm the specific effect of IFN- expressed by PAMAMR/pORF-IFN-, the empty pORF vector was constructed as a control plasmid DNA for PAMAM-R/pORF. pORF-IFN- contains restriction enzyme sites for SalI, PstI, and NheI. To eliminate the IFN- gene from pORF-IFN-, the portion of the sequence between the SalI and PstI sites was first obtained; additionally, the NheI site was added to the PstI sequence in order to generate a sequence containing SalI and NheI recognition sequences. Since both the pORF-IFN- plasmid and the generated sequence contained the SalI and NheI sites, the empty pORF vector could be constructed by ligating the 2 sequences together after treating with the same
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Fig. 1. Anti-tumor activity of human interferon beta expressed by the transfection of PAMAM-R/pORF-IFN- polyplex into U87MG (A), Neuro2A (B), HT22 (C), and NIH-3T3 (D) cells. Relative cell viability (RCV) is represented as relative absorbance (%) to that of cells alone. Data represent the mean ± SD of 3 independent experiments.
restriction enzymes. This constructed pORF plasmid was used in further experiments. 3.2. PAMAM-R/pORF-IFN-ˇ inhibit growth of selected tumor cells in vitro The antitumor activity of PAMAM-R with pORF-IFN- was assayed using MTT in order to investigate the potency of PAMAM-R as a non-viral gene delivery carrier to deliver the human IFN- gene. Normal cells such as NIH-3T3 and HT22 cells, and cancer cells such as Neuro2A and U87MG cells, were selected for the experiment to illustrate cell diversity. As shown in Fig. 1, treatment with the PAMAM-R/pORF-IFN- polyplex resulted in approximately 46% cell viability in Neuro2A cells and 27% in U87MG cells, whereas treatment with the PAMAM-R/pORF polyplex exhibited viability similar to that seen in the control group. On the other hand, PAMAMR/pORF-IFN--treated HT22 and NIH3T3 cells displayed almost the same cell viability as seen in control and PAMAM-R/pORF-treated cells. 3.3. Apoptosis induction in vitro and in vivo To confirm whether tumor cell death and prevention of tumor growth were caused by the induction of apoptosis by transfected IFN-, we performed several experiments to investigate apoptosis in vivo and in vitro. For the detection of IFN- expression in vitro as well as the expression of the GFP and luciferase proteins, an ELISA detection kit was used. Samples were taken 2 days after transfection, at the same time point as in the anti-tumor activity test. The PAMAM-R/pORFIFN- polyplex-treated sample contained 3372 pg/mg protein of
IFN- although PAMAM-R/pORF polyplex treated sample contained 142 pg/mg protein of IFN-. Cell death can be classified as necrosis and apoptosis. Necrosis is evidenced by irregular clumping of chromatin, disruption of the nucleus, and swelling of the cell and organelles, whereas apoptosis is related to regular defined condensation of chromatin, nuclear fragmentation, cytoplasmic condensation, and preserved cytoplasmic organelles (Vinatier et al., 1996). Staining of the nucleus with DAPI is a widely used method to assess apoptosis (Benachour and Seralini, 2009; Edwards et al., 2008; Kim et al., 2004). Nuclear morphological changes in U87MG cells were observed by DAPI staining following transfection with the PAMAM-R/pORF-IFN- polyplex. The PAMAM-R/pORF polyplex was used as a control. As shown in Fig. 2(B and C), cells treated with the PAMAM-R/pORF-IFN- polyplex formed crescents around the periphery of the nucleus and had a condensed nucleus compared to cells transfected with PAMAM-R/pORF. The presence of these apoptotic features was supported by the TUNEL assay, which indicates DNA fragmentation. The assay labels 3 -OH DNA ends using the recombinant terminal deoxynucleotidyl transferase (rTdT) enzyme. The cells were then stained with DAB to measure the level of apoptosis after transfection. Neither control cells nor cells transfected with PAMAM-R/pORF showed any apoptotic bodies after 48 h. However, PAMAM-R/pORF-IFN--transfected cells showed dark brown spots, indicating apoptosis, at 48 h after transfection (Fig. 2 (D–F)). We evaluated caspase-3 activity, another key effecter in apoptosis. As shown in Fig. 2G, PAMAM-R/pORF-IFN-treated U87MG cells displayed 3-fold higher caspase-3 activity than PAMAM-R/pORF treated cells. For the in vivo detection of IFN- expression as well as detected by ELISA in vitro, total mRNA was isolated from tissue and analyzed by RT-PCR. As shown in Fig. 3, the housekeeping gene -actin was
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Fig. 2. Apoptosis determined by DAPI, TUNEL and caspase-3activity assay. DAPI: (A) Control, (B) PAMAM-R/pORF, and (C) PAMAM-R/pORF-IFN-. White arrows indicate shrinkage and smaller nuclei. Blue circles represent cell nuclei stained by DAPI (20× magnification); TUNEL: (D) Control, (E) PAMAM-R/pORF, (F) PAMAM-R/pORF-IFN-. The brown dots represent DNA fragments stained by DAB (10× magnification); caspase-3 activity (G): data represent the mean ± SD of 3 independent experiments. ***P < 0.001 compared with control and pORF polyplex-treated cells.
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counterstained with DAPI to highlight the nucleus. As shown in Fig. 4, control and PAMAM-R/pORF-treated cells exhibited few green spots, which represent the apoptotic bodies. The PAMAMR/pORF-IFN--treated group displayed a larger number of green spots, indicating apoptosis. 3.4. U87MG cell glioblastoma in nude mice
Fig. 3. RT-PCR analysis of in vivo samples.
positive in all 3 groups. In contrast, IFN- was only expressed in the therapeutic gene-treated group. Both the control and PAMAMR/p-ORF-treated groups were negative for IFN-. It means that IFN- expression was also achieved by the transfection of PAMAMR/pORF-IFN-. The TUNEL assay revealed the green fluorescence of fluorescein-12-dUTP, which was directly incorporated to the 3 -OH DNA ends of apoptotic cells using rTdT; the cells were
To evaluate the antitumor effect of the PAMAM-R/pORF-IFN- polyplex in vivo, we established a xenograft subcutaneous brain tumor model in the flanks of nude mice using human glioblastoma U87MG cells. Mice were treated with saline (control), the PAMAMR/pORF polyplex, or the PAMAM-R/pORF-IFN- polyplex. As shown in Fig. 5, average tumor sizes were 19.2 mm3 in the control group, 28.7 mm3 in the PAMAM-R/pORF group and 41.0 mm3 in the PAMAM-R/pORF-IFN- group at the time of sample injection. There was no significant difference in tumor size among the 3 groups until 10 days after treatment. Subsequently, tumor sizes gradually increased to 77.0 mm3 (control), and 90.7 mm3 (PAMAM-R/pORF)
Fig. 4. TUNEL assay of in vivo samples of (A and D) control, (B and E) PAMAM-R/pORF-treated, and (C and F) PAMAM-R/pORF-IFN--treated cells. Apoptotic body staining with green fluorescence (A–C) and counterstaining with DAPI (D, E, F) (40× magnification).
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Fig. 5. Anti-tumor activity of human interferon beta in vivo. (A) Morphology of the xenograft brain tumor. (B) Time course of tumor size in control (䊉), PAMAM-R/pORFtreated () and PAMAM-R/pORF-IFN- treated () cells. All presented data were expressed as the mean ± S. E. ***P < 0.001 compared with control and pORF polyplex-treated cells.
at 16 days, whereas tumors treated with PAMAM-R/pORF-IFN- did not change significantly in size. By the 21st day, the end point of the experiment, the tumor size in PAMAM-R/pORF treated animals reached a significantly greater size of 358.1 mm3 , similar to that in control mice (373.1 mm3 ). In contrast, tumor size in PAMAMR/pORF-IFN--treated animals was 26.7 mm3 , indicating that the tumors barely grew in the treated mice.
tumor between the epidermis and substructure, but this was not seen in the PAMAM-R/pORF-IFN--treated group (Fig. 6E). EGFR staining also supported this observation. The results shown in panels B and D of Fig. 6 are EGFR positive but those in panel F are negative. We observed a small lump in the PAMAM-R/pORF-IFN-treated group before sacrifice; however, signs of a tumor could be observed in H&E staining and EGFR immunostaining (panels E and F, Fig. 6), which showed that the layers were tightly bound.
3.5. H&E staining and immunohistochemistry 4. Discussion Sections were stained with H&E (hematoxylin stains the cell nucleus blue, whereas eosin stains the cell cytoplasm and adjacent tissue) to observe the overall tissue structure, and also immunostained using an antibody against EGFR to determine the cellular phenotype of the subcutaneous injection site and the adjacent mouse skin, because the EGFR is expressed at a high level in a variety of solid tumors commonly (Ciardiello and Tortora, 2001, 2003; Mendelsohn, 2001). Fig. 6 shows representative sections from control, PAMAM-R/pORF-treated and PAMAM-R/pORF-IFN-treated mice at 21 days after sample injection. Control (Fig. 6A) and PAMAM-R/pORF-treated (Fig. 6 C) groups showed an inserted
In the present study, we evaluated a gene therapy method using the non-viral gene delivery carrier PAMAM-R with human IFN- for the treatment of brain tumors. A significant decrease in tumor size was found in mice treated with PAMAM-R/pORF-IFN-, compared to that seen in control animals or PAMAM-R/pORF treated animals. In general, brain cancer is one of the most devastating and difficult forms of cancer to treat. Although enormous have been made in the treatment of malignant gliomas, no curative treatment or long-term control has been achieved. Despite advances in surgical and imaging techniques, multiple problems remain including
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Fig. 6. Histological images of xenograft brain tumors on day 21. Xenograft control mice (A, B), PAMAM-R/pORF polyplex-treated mice (C, D) and PAMAM-R/pORF-IFN- polyplex-treated mice. A, C, E: H&E staining; B, D, F: immunohistochemistry of EGFR.
dismal prognosis, extensive infiltration of tumor cells, and invasion into normal brain parenchyma or other sites. Further, because of the location of these tumors in the brain, the impermeability of the blood-brain barrier is also a difficulty. Furthermore, the total removal of the tumor from the brain by surgery is almost impossible with chemotherapy and radiotherapy (Fomchenko and Holland, 2006; Lam and Breakefield, 2001). In addition, there may be resistance to adjuvant therapies used to treat brain tumors. Therefore, gene therapy is considered a feasible strategy for malignant gliomas. For the effective expression of the therapeutic gene targeted to malignant gliomas, the use of a gene delivery carrier with no adverse effects such as immunogenicity and cytotoxicity is important. PAMAM-R is an excellent gene delivery carrier due to its enhanced gene expression and low cytotoxicity in various cell lines including HepG2 and HEK293, Neuro 2A, and primary rat vascular smooth muscle cells (Choi et al., 2004). Additionally, PAMAM-R exhibits transfection levels of 35–40% in primary cortical culture, significantly higher than those of commercially available reagents such as Lipofectamine and branched polyethylenimine, and efficient transfection was also observed in neurons and all tested CNS glial cells such as astrocytes, microglia, and oligodendrocytes (Kim et al., 2006). This is presumably due to the multiple arginine residues grafted on the surface of the PAMAM dendrimer. Arginine, a basic amino acid, is found abundantly in protein transduction domains and in membrane translocational signals, such as the TAT sequence of HIV, which are able to efficiently translocate biologically active materials (Schwarze et al., 1999; Tung and Weissleder, 2003). This characteristic indicates that PAMAM-R could be a suitable gene delivery carrier for achieving an efficient transfection rate for targeting brain tumors in vivo. The activation of caspase-3, a member of the cysteine-aspartic acid protease family that plays a central role in the execution phase of apoptosis, was achieved by PAMAM-R-mediated hIFN- gene transfer in tumor cells. Our study showed that PAMAMR/pORF-IFN--treated cells had 3-fold higher activity than the control samples. Caspase-3 activity was probably initiated by IFN- through IFN type I receptors, because the IFN- that was detected by ELISA in the IFN- gene-treated group was expressed endogenously by the transfection of PAMAM-R/pORF-IFN-; endogenous IFN- served as an additional source of IFN-. STAT-1, an IFN type I
receptor, has been reported to have a pro-apoptotic effect through transcription-dependent activation of caspases and transcriptionindependent interaction with p53, the tumor necrosis factor receptor type 1-associated DEATH domain protein, and nuclear factor Bp65 (Kim and Lee, 2007). Activation of effector caspases was associated with the expression of inflammatory caspase-11, which converted pro-caspase-3 to active caspase-3 (Kang et al., 2000; Martinon and Tschopp, 2007). Caspase-3 is also associated with the activation of caspaseactivated DNase (CAD), which can cleave nuclear DNA into nucleosomal units. CAD is activated with the help of the inhibitor of CAD (ICAD) that serves as specific chaperon for CAD. Activation of caspase-3 allows CAD to dissociate from the CAD:ICAD complex, allowing CAD to cleave chromosomal DNA (Nagata, 2000). The TUNEL assay in vitro showed the presence of more condensed and dark brown spots in the therapeutic gene-treated group, indicating that DNA fragmentation occurred as a result of IFN--induced apoptosis. Interestingly, slightly dark brown spots were observed in pORF-treated samples, which were lacking in the control sample. This indicates that the PAMAM-R/DNA complex is slightly toxic. This may give rise to safety concerns associated with the use of this polyplex; however, the therapeutic gene-treated samples did not show any side effects, despite the TUNEL assay in vivo showing a pattern similar to that seen in the in vitro assay. Based in this, we conclude that the polyplex exhibited sufficiently low toxicity during gene delivery. In contrast to other published reports that have investigated the therapeutic effect in terms of reduction of tumor size, we have used only a single injection into the tumor region to evaluate the distinct therapeutic efficacy of IFN- therapy. Fig. 5 shows that tumor size degradation continued for almost 3 weeks after a single administration of the therapeutic gene. Additionally H&E and EGFR staining also indicate the tumor regression inside of the tissue by the expression of IFN-. This demonstrates that the PAMAM-R/pORF-IFN- complex clearly results in a high level of tumor regression. However, this effect does not last permanently, as evidenced by a slight increase in tumor size 18 days after injection due to the decreasing expression of IFN-. After the administration of the therapeutic gene, cancer cell death is determined by 2 factors: IFN- activity and tumor growth. In other words, when IFN- expression is faster than tumor growth, the tumor size is likely to decrease and vice
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versa. On the other hand, IFN- is still present and active but the tumor cells adapt to its presence by altering gene expression and signaling pathways so that IFN- resistance was appeared. Therefore repeated injection of IFN- or the other therapeutic genes may be necessary for the treatment of brain tumors. In summary, PAMAM-R can deliver the human IFN- gene to U87MG cells transplanted into mice, and can reduce tumor size effectively. The apoptotic effect of the PAMAM-R/IFN- polyplex was observed specifically in tumor cells. Therefore, we conclude that PAMAM-R/IFN- is a potential candidate for the gene therapy of malignant gliomas. However, additional research is needed to investigate the apoptotic efficacy of the PAMAM-R/IFN- polyplex in models of spontaneous tumor formation in genetically engineered mice, and to elucidate the mechanism of IFN- action. Acknowledgements This study was supported by a grant from the Gene Therapy Project of the Ministry of Education, Science and Technology in the Republic of Korea (200110018684), the Basic Science Research Program through a National Research Foundation of Korea (NRF) funded by the Korean government (2001K000818). References Baselga, J., Norton, L., Albanell, J., Kim, Y.M., Mendelsohn, J., 1998. Recombinant humanized anti-HER2 antibody (Herceptin) enhances the antitumor activity of paclitaxel and doxorubicin against HER2/neu overexpressing human breast cancer xenografts. Cancer Res. 58, 2825–2831. Benachour, N., Seralini, G.E., 2009. Glyphosate formulations induce apoptosis and necrosis in human umbilical, embryonic, and placental cells. Chem. Res. Toxicol. 22, 97–105. Bulbul, M.A., Huben, R.P., Murphy, G.P., 1986. Interferon-beta treatment of metastatic prostate cancer. J. Surg. Oncol. 33, 231–233. Chen, H., Dong, J., Huang, Q., 2011. Xenograft model of human brain tumor, brain-tumors. In: Dr. Abujamara, A.L. (Ed.), Current and Emerging Therapeutics Strategies. InTech, ISBN 978-953-307-588-4, http://www.intechopen.com/ books/brain-tumors-current-and-emerging-therapeuticstrategies/xenograftmodel-of-human-brain-tumor Choi, J.S., Nam, K., Park, J.Y., Kim, J.B., Lee, J.K., Park, J.S., 2004. Enhanced transfection efficiency of PAMAM dendrimer by surface modification with l-arginine. J. Control. Release 99, 445–456. Ciardiello, F., Tortora, G., 2001. A novel approach in the treatment of cancer: targeting the epidermal growth factor receptor. Clin. Cancer Res. 7, 2958–2970. Ciardiello, F., Tortora, G., 2003. Epidermal growth factor receptor (EGFR) as a target in cancer therapy: understanding the role of receptor expression and other molecular determinants that could influence the response to anti-EGFR drugs. Eur. J. Cancer 39, 1348–1354. Edwards, L.A., Woo, J., Huxham, L.A., Verreault, M., Dragowska, W.H., Chiu, G., Rajput, A., Kyle, A.H., Kalra, J., Yapp, D., Yan, H., Minchinton, A.I., Huntsman, D., Daynard, T., Waterhouse, D.N., Thiessen, B., Dedhar, S., Bally, M.B., 2008. Suppression of VEGF secretion and changes in glioblastoma multiforme microenvironment by inhibition of integrin-linked kinase (ILK). Mol Cancer Ther 7, 59–70. Engebraaten, O., Hjortland, G.O., Juell, S., Hirschberg, H., Fodstad, O., 2002. Intratumoral immunotoxin treatment of human malignant brain tumors in immunodeficient animals. Int. J. Cancer 97, 846–852. Fomchenko, E.I., Holland, E.C., 2006. Mouse models of brain tumors and their applications in preclinical trials. Clin. Cancer Res. 12, 5288–5297.
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Contents lists available at SciVerse ScienceDirect
International Journal of Pharmaceutics journal homepage: www.elsevier.com/locate/ijpharm
Pharmaceutical nanotechnology
Arginine modified PAMAM dendrimer for interferon beta gene delivery to malignant glioma Cheng Zhe Bai a,1 , Sunghyun Choi a,1 , Kihoon Nam b , Songhie An a , Jong-Sang Park a,∗ a b
Department of Chemistry, Seoul National University, Seoul, Republic of Korea Department of Pharmaceutics and Pharmaceutical Chemistry, Center for Controlled Chemical Delivery – CCCD, University of Utah, Salt Lake City, USA
a r t i c l e
i n f o
Article history: Received 12 September 2012 Received in revised form 9 December 2012 Accepted 24 January 2013 Available online 4 February 2013 Keywords: Malignant glioma Apoptosis Gene delivery PAMAM-R Interferon beta
a b s t r a c t A xenograft brain tumor model was established by the subcutaneous injection of U87MG cells into nude mice to investigate the efficacy of a non-viral vector, arginine-modified polyamidoamine dendrimer (PAMAM-R), in delivering a therapeutic gene, human interferon beta (IFN-). We used 4 ,6-diamidino2-phenylindole staining, the terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL) assay, and the caspase-3 activity assay to determine the induction of apoptosis upon transfection with the PAMAM-R/IFN- gene polyplex in vitro. The polyplex was injected into xenograft brain tumors. Mice treated with PAMAM-R/pORF-IFN- exhibited a significantly smaller tumor size than control mice and PAMAM-R/pORF treated mice. Hematoxylin/eosin staining and immunohistochemistry with the endothelial growth factor receptor antibody also revealed inhibition of tumor growth. Furthermore, reverse transcription polymerase chain reaction and the TUNEL assay also verified the expression of IFN- and induction of apoptosis in vivo. These results indicate that the PAMAM-R/pORF-IFN- polyplex is an effective therapeutic candidate for glioblastoma multiforme due to its selective induction of apoptosis in tumor cells. © 2013 Elsevier B.V. All rights reserved.
1. Introduction Glioblastoma multiforme (GBM) is the most malignant astrocytoma (World Health Organization grade IV), and the most aggressive among all primary brain tumors, with overall median survival of <1 year following diagnosis (Sathornsumetee and Rich, 2006). Despite advances in conventional therapy including surgery, radiotherapy, and chemotherapy alone or in combination, the diffuse and highly infiltrative nature of GBM allows it to expand from the tumor site into the surrounding brain. Complete surgical removal of the tumor is nearly impossible and inevitably, recurrent tumors appear at the site of the initial lesion (Lawler et al., 2006; Maguire et al., 2008). Therefore, gene therapy has been investigated as a method to target GBM tumor cells. A number of gene therapy strategies for malignant glioma, such as the direct killing of tumor cells using “suicide” genes, restoration of a defective tumor suppressor gene, drug resistance genes and immune-gene therapy have been developed for GBM (Klatzmann et al., 1998; Lang et al., 2003; Pulkkanen and Yla-Herttuala, 2005). Among these
∗ Corresponding author at: Department of Chemistry, Seoul National University, 599 Gwanak-ro, Gwanak-gu, Seoul 151-747, Republic of Korea. Tel.: +82 2 871 5355; fax: +82 2 877 5110. E-mail addresses: [email protected], [email protected] (J.-S. Park). 1 These authors contributed equally to this study. 0378-5173/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.ijpharm.2013.01.057
approaches, transfection of immune gene has been considered as therapeutically promising tools for GBM. Interferon (IFN)- as a immune gene is known to exert anti-tumor activity via to the induction of apoptosis in IFN- protein-resistant tumor cells, including melanoma, renal cell carcinoma, and glioma cells; Apoptosis induction was closely correlated with the level of intracellular IFN- mRNA, a prolonged phosphorylation time of signal transducer and activator of transcription (STAT)-1, a molecule related to the IFN- signal transduction pathway, and the level of activated DNase ␥ (Yoshida et al., 2004). Although the associated mechanisms are not yet clearly understood, much evidence indicates that IFN- expression correlated with tumor cell suppression (Bulbul et al., 1986; Juang et al., 2004; Qin et al., 1998; Wang et al., 2010). Thus, delivery and expression of IFN- could be an effective approach for GBM. To study brain tumor, allogenic graft model, xenograft model and genetically engineered mouse (GEM) model are used (Chen et al., 2011). Among them, one of the most widely used models is the human tumor xenograft due to variety of advantages such as using actual human tumor tissue that featuring complexity of genetic and epigenetic abnormalities, suitable for multiple therapeutic applications from a single tumor biopsy and timesaving until obtaining the results (Fomchenko and Holland, 2006; Richmond and Su, 2008). Although xenograft model cannot study stage of tumor progression and produce specific genetic abnormalities, there are several important successes in clinical trials that
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used xenograft model as a preclinical trial such as bortezomib curing multiple myeloma, herceptin that enhance anti-tumor activity of paclitaxel and doxorubixin in human breast cancer (Baselga et al., 1998; LeBlanc et al., 2002). Therefore xenograft model could be a useful candidate for understanding cancer treatment. In previous study, we reported that PAMAM-R, which consists of cationic arginine residues conjugated to the periphery of the polyamidoamine (PAMAM) dendrimer G4, exhibits greatly enhanced transfection efficiency and lower cytotoxicity than do other commonly used non-viral vectors (Choi et al., 2004). Furthermore, PAMAM-R was shown to increase the gene expression level not only in primary cortical cultures but also in glial cells (Kim et al., 2006). In present study, we focused to investigate the expression of IFN- delivered by PAMAM-R prevent tumor suppression both in vitro and in mouse brain tumor xenograft model. 2. Materials and methods 2.1. Materials PAMAM dendrimer (ethylene diamine core, G4), N,Ndiisopropylethylamine (DIPEA), piperidine, triisopropylsilane, trifluoroacetic acid, N,N-dimethylformamide (DMF), 3-[4,5dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT), Trizma base, paraformaldehyde, Tween 20, ethylenediaminetetraacetic acid (EDTA), Harris’s hematoxylin solution, and eosin were purchased from Sigma–Aldrich (St. Louis, MO). N-hydroxybenzotriazole (HOBt) and 2-(1H-benzotriazole-1-yl)1,1,3,3,-tetramethyluronium hexafluorophosphate (HBTU) were purchased from Anaspec, Inc. (San Jose, CA). Fmoc-L-Arg(pbf)-OH was purchased from Novabiochem (San Diego, CA). Dulbecco’s phosphate-buffered saline (DPBS), Dulbecco’s modified Eagle’s medium (DMEM), and fetal bovine serum (FBS) were purchased from GIBCO (Gaithersburg, MD). The VeriKineTM Human IFN- enzyme-linked immunosorbent assay (ELISA) Kit was purchased from PBL, Inc. (Piscataway, NJ). The peroxidase-blocking solution, REALTM EnVisionTM Detection System, was purchased from Dako (Glostrup, Denmark). Epidermal growth factor receptor (EGFR) antibody was purchased from Abcam (Cambridge, UK). 2.2. Cell lines U87MG (human glioblastoma cells), Neuro2A (mouse neuroblastoma cells), NIH3T3 (mouse embryo fibroblast cells), and HT22 (mouse hippocampal cells) were maintained in DMEM medium supplemented with 10% FBS at 37 ◦ C in an atmosphere of 5% CO2 . 2.3. Preparation of plasmid The human IFN- plasmid, pORF-IFN- was purchased from Invivogen (San Diego, CA). The pORF plasmid was cloned using a synthesized primer, which eliminates the part of IFN- sequence from pORF-IFN- vector. The sequence of the forward primer was 5 -ATCGTCGACTACTAACCTTCTTCTCTTTCCTACAGC-3 , containing the SalI recognition sequence, and that of the reverse primer was 5 -CTCTAATTCGTCGACGTCCGATCGTCT-3 , containing the PstI recognition sequence and an additional NheI site. The IFN- gene sequence and the pORF-IFN- plasmid were digested separately with the restriction enzymes SalI and NheI, and then ligated together to obtain the pORF plasmid. 2.4. Anti-tumor activity of human interferon beta with PAMAM-R U87MG cells were seeded in 24-well plates at a density of 1 × 104 cells in DMEM medium containing 10% FBS. After 1 day, the cells
were treated for 48 h at 37 ◦ C with either a polyplex solution containing 1 g pORF-IFN- plasmid DNA at a weight ratio of 4, or a control polyplex solution containing 1 g pORF plasmid DNA at the same weight ratio. Following this, 100 L of MTT (2 mg/mL in PBS) solution was added to each well. After incubating at 37 ◦ C for 4 h, the medium was removed from each well and 150 L of DMSO was added to dissolve the insoluble formazan crystals formed by proliferating cells. The absorbance in each well was measured at 570 nm using a microplate reader (Molecular Devices Co., Menlo Park, CA). These experiments were performed in Neuro2A cells, NIH3T3 cells, and HT22 cells using the same method as described above. 2.5. Quantification of human IFN-ˇ protein level by ELISA U87MG cells were seeded in a 24-well plate at a density of 1 × 104 cells in DMEM containing 10% FBS. After 1 day, cells were transfected with either the PAMAM-R/pORF-IFN- polyplex or the PAMAM-R/pORF polyplex as described above for 48 h. The medium was collected from each well and centrifuged to remove cell debris. The samples, along with IFN- protein standards, were assayed using the human VeriKineTM human IFN- ELISA kit. Briefly, sample diluent was added to each well, and then the standards, blanks, and samples were added to individual wells and incubated for 1 h. After the IFN- antibody was added and incubated for 1 h, a horseradish peroxidase (HRP) solution was added to each well and incubated for 1 h. Before adding the stop solution, tetramethyl-benzidine (TMB) substrates were added to each well and incubated for 15 min in the dark. Finally, the absorbance at 450 nm was measured using a microplate reader within 5 min after the addition of the stop solution. The final results were reported in terms of pg/mg protein. 2.6. Nuclear morphological change U87MG cells were seeded in LabTek chambers at a cell density of 3 × 104 in a DMEM medium containing 10% FBS. After 1 day, cells were transfected with the PAMAM-R/pORF-IFN- or PAMAMR/pORF polyplex as described above for 48 h. The medium was removed and the cells were rinsed twice with PBS to discard residual medium. The cells were then fixed in 4% paraformaldehyde for 10 min and washed twice with PBS. Thirty minutes after mounting with mounting solution containing 4 ,6-diamidino-2-phenylindole (DAPI), the cells were observed using fluorescence microscopy. 2.7. Terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL) assay in vitro U87MG cells were plated in LabTek chambers at a cell density of 3 × 104 in DMEM medium containing 10% FBS. After 1 day, the cells were transfected with either PAMAM-R/pORF-IFN or PAMAM-R/pORF polyplexes as described above for 48 h. The medium was removed and the cells were rinsed with PBS twice to eliminate residual medium. The cells were then processed using the DeadEndTM Colorimetric TUNEL System. Briefly, the cells were fixed with 4% paraformaldehyde for 25 min at room temperature and permeabilized in 0.2% Triton X-100 solution, and then equilibrated with equilibration buffer at room temperature for 10 min. The cells were incubated with a reaction mix containing equilibration buffer, biotinylated nucleotide mix, and the terminal deoxynucleotidyl transferase (TdT) enzyme at 37 ◦ C for 60 min inside a humidified chamber to allow the end-labeling reaction to occur. After immersing in 2× SSC to stop the reaction, endogenous peroxidase was blocked by treatment with 0.3% hydrogen peroxide for 10 min at room temperature. Subsequently, a streptavidin HRP solution was added for 30 min at room temperature. A solution of 3,3 diaminobenzidine (DAB) was then added to the cells and developed for 5 min. Cells were then rinsed several times in deionized water
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and mounted in aqueous medium. Stained cells were observed under a light microscope. 2.8. Caspase-3 activity assay U87MG cells (3 × 106 ) were seeded in DMEM medium containing 10% FBS. After 1 day, cells were incubated with the PAMAM-R/pORF-IFN- or PAMAM-R/pORF polyplexes for 48 h. The cells were then collected and assayed using the caspase-3 assay kit. Briefly, the collected cells were incubated in 1× lysis buffer for 20 min. Lysed cells were centrifuged at 16,000 × g for 15 min at 4 ◦ C, and the supernatants were transferred to fresh tubes. 5 L of cell lysate was added to each well of a 96-well plate along with 85 L of 1× assay buffer and 10 L of the caspase-3 substrate Ac-DEVDpNA. The samples were incubated in a humidified chamber at 37 ◦ C for 24 h. Following this, the absorbance at 405 nm was measured using a microplate reader and recorded as a percentage relative to untreated cells. 2.9. Mouse xenograft brain tumor model Four- to six-week-old BALB/C nude mice were kept at 25 ◦ C in a specific pathogen-free environment, in positive pressure rooms with filtered and humidified air. The mice were maintained in the pathogen-free Biogen animal facility for 1 week prior to each experiment. After the mice were anesthetized using Zoletil and Rompun, a total of 1 × 106 cells, harvested with trypsin/EDTA solution in a 50 L volume, were injected subcutaneously into the left flanks of nude mice. The tumor volumes were measured by the formula (w2 × l)/2, where w is the smallest and l is the largest diameter of the tumor (Engebraaten et al., 2002). When the tumor size reached 20 mm3 , the mice were divided into 3 groups. The control group was injected with 50 L of PBS, the empty vector group with 50 L of polyplex solution containing 5 g of pORF plasmid DNA at a weight ratio of 4, and the gene therapy group with the same volume of polyplex solution containing 5 g pORF-IFN- plasmid DNA at the same weight ratio. All 3 groups received a single injection directly into the center of the tumor. Tumor sizes were measured every other day. After 3 weeks, the animals were sacrificed and tumor tissues were excised for immunohistochemistry. 2.10. Tissue preparation and morphologic immunohistochemistry The excised tissues were fixed in 4% paraformaldehyde solution and embedded in paraffin. Serial sections of 5 m thickness were cut from the tissue blocks and floated onto glass slides. All sections were deparaffinized in xylene and rehydrated in a graded series of solutions from ethanol to deionized water for staining. Hematoxylin and eosin (H&E) staining: sections were stained in Harris’s hematoxylin solution for 8 min, differentiated in 1% acid alcohol for 30 s, and subjected to bluing in 0.2% ammonia water for 45 s to remove unreacted hematoxylin. After rinsing in 95% alcohol, the slides were counterstained in eosin-phloxine B solution for 45 s. After being dehydrated in a graded series of ethanol and xylene solutions, the sections were treated with mounting solution for observation under the scan scope. Immunostaining for EGFR: following antigen retrieval overnight at 60 ◦ C in 10 mM Tris base solution (pH 9.0) containing 1 mM EDTA and 0.05% (v/v) Tween 20, the slides were washed in Tris-buffered saline (TBS) containing 0.05% (v/v) Tween 20. Endogenous peroxidase was blocked by incubating with peroxidase-blocking solution for 30 min. The sections were treated with 10% normal goat serum for 1 h to further reduce nonspecific background staining. The slides were then incubated in a 200:1 dilution of EGFR antibody overnight at 4 ◦ C. The sections were then washed with TBS, and treated with goat anti-rabbit immunoglobulin (IgG) for 1 h and DAB+ chromogen
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with substrate buffer for 2 min (Dako REALTM EnVisionTM Detection System). After washing with TBS and tap water, the sections were incubated in hematoxylin for counterstaining. The slides were differentiated in 1% acid alcohol and subjected to bluing in 0.2% ammonia water. After dehydration, the sections were treated with mounting solution and observed in the scan scope. 2.11. Reverse transcription polymerase chain reaction (RT-PCR) analysis To detect the expression of IFN- in tissue, animals treated with the PAMAM-R/pORF-IFN- polyplex, the PAMAM-R/pORF polyplex and the control, animals were sacrificed and the tumor and skin at the injection site were collected. Tissues were homogenized and total RNA was prepared using the Trizol® Reagent according to the manufacturer’s instructions (Invitrogen, Carlsbad, CA). Reverse transcription followed by PCR was done using the OneStep RT-PCR Kit (QIAGEN, Valencia, CA) with manually designed PCR primers: 5 -GGCCATGACCAACAAGTGTCTCCTCC-3 (forward), 5 -CTTACAGGTTACCTCCGAAACTGAG-3 (reverse) for human IFN-, and 5 -CGCTGGGTCAGAAGGACTCCTATG-3 (forward), 5 -CGGAGAAGAGCTATGAGCTGCCTG-3 (reverse) for the housekeeping gene -actin. 2.12. TUNEL assay in vivo All the sections were deparaffinized in xylene and rehydrated in a graded series of solutions from ethanol to deionized water. Then the specimens were processed using the DeadEndTM Fluorometric TUNEL System. Briefly, the sections were washed in 0.85% NaCl for 5 min at room temperature and fixed in 4% paraformaldehyde for 15 min at room temperature. After permeabilizing in proteinase K for 10 min, the sections were fixed in 4% paraformaldehyde and exposed to the TdT reaction mix containing equilibration buffer, biotinylated nucleotide mix, and the TdT enzyme at 37 ◦ C for 60 min inside a humidified chamber to allow the end-labeling reaction to occur. After immersing in 2× SSC to stop the reaction, the slides were rinsed in PBS solution and mounted in aqueous medium containing DAPI. Finally, the sections were observed in a fluorescence microscope. 2.13. Statistical analysis All data are expressed as mean ± SD. One-way analysis of variance (ANOVA) was used to determine statistically significant differences between groups. Dunnett’s test (SPSS v.12.0, Chicago, IL) was used to correct for multiple comparisons when statistical significances were identified in the ANOVA. 3. Results 3.1. Construction of the pORF plasmid To confirm the specific effect of IFN- expressed by PAMAMR/pORF-IFN-, the empty pORF vector was constructed as a control plasmid DNA for PAMAM-R/pORF. pORF-IFN- contains restriction enzyme sites for SalI, PstI, and NheI. To eliminate the IFN- gene from pORF-IFN-, the portion of the sequence between the SalI and PstI sites was first obtained; additionally, the NheI site was added to the PstI sequence in order to generate a sequence containing SalI and NheI recognition sequences. Since both the pORF-IFN- plasmid and the generated sequence contained the SalI and NheI sites, the empty pORF vector could be constructed by ligating the 2 sequences together after treating with the same
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Fig. 1. Anti-tumor activity of human interferon beta expressed by the transfection of PAMAM-R/pORF-IFN- polyplex into U87MG (A), Neuro2A (B), HT22 (C), and NIH-3T3 (D) cells. Relative cell viability (RCV) is represented as relative absorbance (%) to that of cells alone. Data represent the mean ± SD of 3 independent experiments.
restriction enzymes. This constructed pORF plasmid was used in further experiments. 3.2. PAMAM-R/pORF-IFN-ˇ inhibit growth of selected tumor cells in vitro The antitumor activity of PAMAM-R with pORF-IFN- was assayed using MTT in order to investigate the potency of PAMAM-R as a non-viral gene delivery carrier to deliver the human IFN- gene. Normal cells such as NIH-3T3 and HT22 cells, and cancer cells such as Neuro2A and U87MG cells, were selected for the experiment to illustrate cell diversity. As shown in Fig. 1, treatment with the PAMAM-R/pORF-IFN- polyplex resulted in approximately 46% cell viability in Neuro2A cells and 27% in U87MG cells, whereas treatment with the PAMAM-R/pORF polyplex exhibited viability similar to that seen in the control group. On the other hand, PAMAMR/pORF-IFN--treated HT22 and NIH3T3 cells displayed almost the same cell viability as seen in control and PAMAM-R/pORF-treated cells. 3.3. Apoptosis induction in vitro and in vivo To confirm whether tumor cell death and prevention of tumor growth were caused by the induction of apoptosis by transfected IFN-, we performed several experiments to investigate apoptosis in vivo and in vitro. For the detection of IFN- expression in vitro as well as the expression of the GFP and luciferase proteins, an ELISA detection kit was used. Samples were taken 2 days after transfection, at the same time point as in the anti-tumor activity test. The PAMAM-R/pORFIFN- polyplex-treated sample contained 3372 pg/mg protein of
IFN- although PAMAM-R/pORF polyplex treated sample contained 142 pg/mg protein of IFN-. Cell death can be classified as necrosis and apoptosis. Necrosis is evidenced by irregular clumping of chromatin, disruption of the nucleus, and swelling of the cell and organelles, whereas apoptosis is related to regular defined condensation of chromatin, nuclear fragmentation, cytoplasmic condensation, and preserved cytoplasmic organelles (Vinatier et al., 1996). Staining of the nucleus with DAPI is a widely used method to assess apoptosis (Benachour and Seralini, 2009; Edwards et al., 2008; Kim et al., 2004). Nuclear morphological changes in U87MG cells were observed by DAPI staining following transfection with the PAMAM-R/pORF-IFN- polyplex. The PAMAM-R/pORF polyplex was used as a control. As shown in Fig. 2(B and C), cells treated with the PAMAM-R/pORF-IFN- polyplex formed crescents around the periphery of the nucleus and had a condensed nucleus compared to cells transfected with PAMAM-R/pORF. The presence of these apoptotic features was supported by the TUNEL assay, which indicates DNA fragmentation. The assay labels 3 -OH DNA ends using the recombinant terminal deoxynucleotidyl transferase (rTdT) enzyme. The cells were then stained with DAB to measure the level of apoptosis after transfection. Neither control cells nor cells transfected with PAMAM-R/pORF showed any apoptotic bodies after 48 h. However, PAMAM-R/pORF-IFN--transfected cells showed dark brown spots, indicating apoptosis, at 48 h after transfection (Fig. 2 (D–F)). We evaluated caspase-3 activity, another key effecter in apoptosis. As shown in Fig. 2G, PAMAM-R/pORF-IFN-treated U87MG cells displayed 3-fold higher caspase-3 activity than PAMAM-R/pORF treated cells. For the in vivo detection of IFN- expression as well as detected by ELISA in vitro, total mRNA was isolated from tissue and analyzed by RT-PCR. As shown in Fig. 3, the housekeeping gene -actin was
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Fig. 2. Apoptosis determined by DAPI, TUNEL and caspase-3activity assay. DAPI: (A) Control, (B) PAMAM-R/pORF, and (C) PAMAM-R/pORF-IFN-. White arrows indicate shrinkage and smaller nuclei. Blue circles represent cell nuclei stained by DAPI (20× magnification); TUNEL: (D) Control, (E) PAMAM-R/pORF, (F) PAMAM-R/pORF-IFN-. The brown dots represent DNA fragments stained by DAB (10× magnification); caspase-3 activity (G): data represent the mean ± SD of 3 independent experiments. ***P < 0.001 compared with control and pORF polyplex-treated cells.
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counterstained with DAPI to highlight the nucleus. As shown in Fig. 4, control and PAMAM-R/pORF-treated cells exhibited few green spots, which represent the apoptotic bodies. The PAMAMR/pORF-IFN--treated group displayed a larger number of green spots, indicating apoptosis. 3.4. U87MG cell glioblastoma in nude mice
Fig. 3. RT-PCR analysis of in vivo samples.
positive in all 3 groups. In contrast, IFN- was only expressed in the therapeutic gene-treated group. Both the control and PAMAMR/p-ORF-treated groups were negative for IFN-. It means that IFN- expression was also achieved by the transfection of PAMAMR/pORF-IFN-. The TUNEL assay revealed the green fluorescence of fluorescein-12-dUTP, which was directly incorporated to the 3 -OH DNA ends of apoptotic cells using rTdT; the cells were
To evaluate the antitumor effect of the PAMAM-R/pORF-IFN- polyplex in vivo, we established a xenograft subcutaneous brain tumor model in the flanks of nude mice using human glioblastoma U87MG cells. Mice were treated with saline (control), the PAMAMR/pORF polyplex, or the PAMAM-R/pORF-IFN- polyplex. As shown in Fig. 5, average tumor sizes were 19.2 mm3 in the control group, 28.7 mm3 in the PAMAM-R/pORF group and 41.0 mm3 in the PAMAM-R/pORF-IFN- group at the time of sample injection. There was no significant difference in tumor size among the 3 groups until 10 days after treatment. Subsequently, tumor sizes gradually increased to 77.0 mm3 (control), and 90.7 mm3 (PAMAM-R/pORF)
Fig. 4. TUNEL assay of in vivo samples of (A and D) control, (B and E) PAMAM-R/pORF-treated, and (C and F) PAMAM-R/pORF-IFN--treated cells. Apoptotic body staining with green fluorescence (A–C) and counterstaining with DAPI (D, E, F) (40× magnification).
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Fig. 5. Anti-tumor activity of human interferon beta in vivo. (A) Morphology of the xenograft brain tumor. (B) Time course of tumor size in control (䊉), PAMAM-R/pORFtreated () and PAMAM-R/pORF-IFN- treated () cells. All presented data were expressed as the mean ± S. E. ***P < 0.001 compared with control and pORF polyplex-treated cells.
at 16 days, whereas tumors treated with PAMAM-R/pORF-IFN- did not change significantly in size. By the 21st day, the end point of the experiment, the tumor size in PAMAM-R/pORF treated animals reached a significantly greater size of 358.1 mm3 , similar to that in control mice (373.1 mm3 ). In contrast, tumor size in PAMAMR/pORF-IFN--treated animals was 26.7 mm3 , indicating that the tumors barely grew in the treated mice.
tumor between the epidermis and substructure, but this was not seen in the PAMAM-R/pORF-IFN--treated group (Fig. 6E). EGFR staining also supported this observation. The results shown in panels B and D of Fig. 6 are EGFR positive but those in panel F are negative. We observed a small lump in the PAMAM-R/pORF-IFN-treated group before sacrifice; however, signs of a tumor could be observed in H&E staining and EGFR immunostaining (panels E and F, Fig. 6), which showed that the layers were tightly bound.
3.5. H&E staining and immunohistochemistry 4. Discussion Sections were stained with H&E (hematoxylin stains the cell nucleus blue, whereas eosin stains the cell cytoplasm and adjacent tissue) to observe the overall tissue structure, and also immunostained using an antibody against EGFR to determine the cellular phenotype of the subcutaneous injection site and the adjacent mouse skin, because the EGFR is expressed at a high level in a variety of solid tumors commonly (Ciardiello and Tortora, 2001, 2003; Mendelsohn, 2001). Fig. 6 shows representative sections from control, PAMAM-R/pORF-treated and PAMAM-R/pORF-IFN-treated mice at 21 days after sample injection. Control (Fig. 6A) and PAMAM-R/pORF-treated (Fig. 6 C) groups showed an inserted
In the present study, we evaluated a gene therapy method using the non-viral gene delivery carrier PAMAM-R with human IFN- for the treatment of brain tumors. A significant decrease in tumor size was found in mice treated with PAMAM-R/pORF-IFN-, compared to that seen in control animals or PAMAM-R/pORF treated animals. In general, brain cancer is one of the most devastating and difficult forms of cancer to treat. Although enormous have been made in the treatment of malignant gliomas, no curative treatment or long-term control has been achieved. Despite advances in surgical and imaging techniques, multiple problems remain including
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Fig. 6. Histological images of xenograft brain tumors on day 21. Xenograft control mice (A, B), PAMAM-R/pORF polyplex-treated mice (C, D) and PAMAM-R/pORF-IFN- polyplex-treated mice. A, C, E: H&E staining; B, D, F: immunohistochemistry of EGFR.
dismal prognosis, extensive infiltration of tumor cells, and invasion into normal brain parenchyma or other sites. Further, because of the location of these tumors in the brain, the impermeability of the blood-brain barrier is also a difficulty. Furthermore, the total removal of the tumor from the brain by surgery is almost impossible with chemotherapy and radiotherapy (Fomchenko and Holland, 2006; Lam and Breakefield, 2001). In addition, there may be resistance to adjuvant therapies used to treat brain tumors. Therefore, gene therapy is considered a feasible strategy for malignant gliomas. For the effective expression of the therapeutic gene targeted to malignant gliomas, the use of a gene delivery carrier with no adverse effects such as immunogenicity and cytotoxicity is important. PAMAM-R is an excellent gene delivery carrier due to its enhanced gene expression and low cytotoxicity in various cell lines including HepG2 and HEK293, Neuro 2A, and primary rat vascular smooth muscle cells (Choi et al., 2004). Additionally, PAMAM-R exhibits transfection levels of 35–40% in primary cortical culture, significantly higher than those of commercially available reagents such as Lipofectamine and branched polyethylenimine, and efficient transfection was also observed in neurons and all tested CNS glial cells such as astrocytes, microglia, and oligodendrocytes (Kim et al., 2006). This is presumably due to the multiple arginine residues grafted on the surface of the PAMAM dendrimer. Arginine, a basic amino acid, is found abundantly in protein transduction domains and in membrane translocational signals, such as the TAT sequence of HIV, which are able to efficiently translocate biologically active materials (Schwarze et al., 1999; Tung and Weissleder, 2003). This characteristic indicates that PAMAM-R could be a suitable gene delivery carrier for achieving an efficient transfection rate for targeting brain tumors in vivo. The activation of caspase-3, a member of the cysteine-aspartic acid protease family that plays a central role in the execution phase of apoptosis, was achieved by PAMAM-R-mediated hIFN- gene transfer in tumor cells. Our study showed that PAMAMR/pORF-IFN--treated cells had 3-fold higher activity than the control samples. Caspase-3 activity was probably initiated by IFN- through IFN type I receptors, because the IFN- that was detected by ELISA in the IFN- gene-treated group was expressed endogenously by the transfection of PAMAM-R/pORF-IFN-; endogenous IFN- served as an additional source of IFN-. STAT-1, an IFN type I
receptor, has been reported to have a pro-apoptotic effect through transcription-dependent activation of caspases and transcriptionindependent interaction with p53, the tumor necrosis factor receptor type 1-associated DEATH domain protein, and nuclear factor Bp65 (Kim and Lee, 2007). Activation of effector caspases was associated with the expression of inflammatory caspase-11, which converted pro-caspase-3 to active caspase-3 (Kang et al., 2000; Martinon and Tschopp, 2007). Caspase-3 is also associated with the activation of caspaseactivated DNase (CAD), which can cleave nuclear DNA into nucleosomal units. CAD is activated with the help of the inhibitor of CAD (ICAD) that serves as specific chaperon for CAD. Activation of caspase-3 allows CAD to dissociate from the CAD:ICAD complex, allowing CAD to cleave chromosomal DNA (Nagata, 2000). The TUNEL assay in vitro showed the presence of more condensed and dark brown spots in the therapeutic gene-treated group, indicating that DNA fragmentation occurred as a result of IFN--induced apoptosis. Interestingly, slightly dark brown spots were observed in pORF-treated samples, which were lacking in the control sample. This indicates that the PAMAM-R/DNA complex is slightly toxic. This may give rise to safety concerns associated with the use of this polyplex; however, the therapeutic gene-treated samples did not show any side effects, despite the TUNEL assay in vivo showing a pattern similar to that seen in the in vitro assay. Based in this, we conclude that the polyplex exhibited sufficiently low toxicity during gene delivery. In contrast to other published reports that have investigated the therapeutic effect in terms of reduction of tumor size, we have used only a single injection into the tumor region to evaluate the distinct therapeutic efficacy of IFN- therapy. Fig. 5 shows that tumor size degradation continued for almost 3 weeks after a single administration of the therapeutic gene. Additionally H&E and EGFR staining also indicate the tumor regression inside of the tissue by the expression of IFN-. This demonstrates that the PAMAM-R/pORF-IFN- complex clearly results in a high level of tumor regression. However, this effect does not last permanently, as evidenced by a slight increase in tumor size 18 days after injection due to the decreasing expression of IFN-. After the administration of the therapeutic gene, cancer cell death is determined by 2 factors: IFN- activity and tumor growth. In other words, when IFN- expression is faster than tumor growth, the tumor size is likely to decrease and vice
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versa. On the other hand, IFN- is still present and active but the tumor cells adapt to its presence by altering gene expression and signaling pathways so that IFN- resistance was appeared. Therefore repeated injection of IFN- or the other therapeutic genes may be necessary for the treatment of brain tumors. In summary, PAMAM-R can deliver the human IFN- gene to U87MG cells transplanted into mice, and can reduce tumor size effectively. The apoptotic effect of the PAMAM-R/IFN- polyplex was observed specifically in tumor cells. Therefore, we conclude that PAMAM-R/IFN- is a potential candidate for the gene therapy of malignant gliomas. However, additional research is needed to investigate the apoptotic efficacy of the PAMAM-R/IFN- polyplex in models of spontaneous tumor formation in genetically engineered mice, and to elucidate the mechanism of IFN- action. Acknowledgements This study was supported by a grant from the Gene Therapy Project of the Ministry of Education, Science and Technology in the Republic of Korea (200110018684), the Basic Science Research Program through a National Research Foundation of Korea (NRF) funded by the Korean government (2001K000818). References Baselga, J., Norton, L., Albanell, J., Kim, Y.M., Mendelsohn, J., 1998. Recombinant humanized anti-HER2 antibody (Herceptin) enhances the antitumor activity of paclitaxel and doxorubicin against HER2/neu overexpressing human breast cancer xenografts. Cancer Res. 58, 2825–2831. Benachour, N., Seralini, G.E., 2009. Glyphosate formulations induce apoptosis and necrosis in human umbilical, embryonic, and placental cells. Chem. Res. Toxicol. 22, 97–105. Bulbul, M.A., Huben, R.P., Murphy, G.P., 1986. Interferon-beta treatment of metastatic prostate cancer. J. Surg. Oncol. 33, 231–233. Chen, H., Dong, J., Huang, Q., 2011. Xenograft model of human brain tumor, brain-tumors. In: Dr. Abujamara, A.L. (Ed.), Current and Emerging Therapeutics Strategies. InTech, ISBN 978-953-307-588-4, http://www.intechopen.com/ books/brain-tumors-current-and-emerging-therapeuticstrategies/xenograftmodel-of-human-brain-tumor Choi, J.S., Nam, K., Park, J.Y., Kim, J.B., Lee, J.K., Park, J.S., 2004. Enhanced transfection efficiency of PAMAM dendrimer by surface modification with l-arginine. J. Control. Release 99, 445–456. Ciardiello, F., Tortora, G., 2001. A novel approach in the treatment of cancer: targeting the epidermal growth factor receptor. Clin. Cancer Res. 7, 2958–2970. Ciardiello, F., Tortora, G., 2003. Epidermal growth factor receptor (EGFR) as a target in cancer therapy: understanding the role of receptor expression and other molecular determinants that could influence the response to anti-EGFR drugs. Eur. J. Cancer 39, 1348–1354. Edwards, L.A., Woo, J., Huxham, L.A., Verreault, M., Dragowska, W.H., Chiu, G., Rajput, A., Kyle, A.H., Kalra, J., Yapp, D., Yan, H., Minchinton, A.I., Huntsman, D., Daynard, T., Waterhouse, D.N., Thiessen, B., Dedhar, S., Bally, M.B., 2008. Suppression of VEGF secretion and changes in glioblastoma multiforme microenvironment by inhibition of integrin-linked kinase (ILK). Mol Cancer Ther 7, 59–70. Engebraaten, O., Hjortland, G.O., Juell, S., Hirschberg, H., Fodstad, O., 2002. Intratumoral immunotoxin treatment of human malignant brain tumors in immunodeficient animals. Int. J. Cancer 97, 846–852. Fomchenko, E.I., Holland, E.C., 2006. Mouse models of brain tumors and their applications in preclinical trials. Clin. Cancer Res. 12, 5288–5297.
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