Delivery of Hypoxia and Glioma Dual-Specific Suicide Gene Using

Jan 27, 2014 - Gene therapy has been considered a promising approach for glioblastoma therapy. To avoid side effects and increase the specificity of g...
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Delivery of Hypoxia and Glioma Dual-Specific Suicide Gene Using Dexamethasone Conjugated Polyethylenimine for GlioblastomaSpecific Gene Therapy Hyun Ah Kim,†,‡ Jin Hyeong Park,† Na Yi,† and Minhyung Lee*,†,‡ †

Department of Bioengineering, College of Engineering, Hanyang University, Seoul 133-791, Republic of Korea Institute for Bioengineering and Biopharmaceutical Research, Hanyang University, Seoul 133-791, Republic of Korea

‡

ABSTRACT: Gene therapy has been considered a promising approach for glioblastoma therapy. To avoid side effects and increase the specificity of gene expression, gene expression should be tightly regulated. In this study, glioma and hypoxia dual-specific plasmids (pEpo-NI2-SV-Luc and pEpo-NI2-SVHSVtk) were developed by combining the erythropoietin (Epo) enhancer and nestin intron 2 (NI2). In the in vitro studies, pEpo-NI2-SV-Luc showed higher gene expression under hypoxia than normoxia in a glioblastoma-specific manner. The MTT and caspase assays demonstrated that pEpo-NI2-SV-HSVtk specifically induced caspase activity and cell death in hypoxic glioblastoma cells. For in vivo evaluation, subcutaneous and intracranial glioblastoma models were established. Dexamethasone-conjugated-polyethylenimine (PEI-Dexa) was used as a gene carrier, since PEI-Dexa efficiently delivers plasmid to glioblastoma cells and also has an antitumor effect due to the effect of dexamethasone. In the in vivo study in the subcutaneous and intracranial glioblastoma models, the tumor size was reduced more effectively in the pEpo-NI2-SV-HSVtk group than in the control and pSV-HSVtk groups. In addition, higher levels of HSVtk gene expression and TUNEL-positive cells were observed in the pEpo-NI2-SV-HSVtk group compared with the control and pSV-HSVtk groups, suggesting that pEpo-NI2-SV-HSVtk increased the therapeutic efficacy in hypoxic glioblastoma. Therefore, pEpo-NI2-SV-HSVtk/PEI-Dexa complex may be useful for glioblastoma-specific gene therapy. KEYWORDS: gene regulation, gene therapy, glioblastoma, hypoxia, suicide gene

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has a “bystander effect” that is mediated by gap junctions. Through gap junctions, the transfer of toxic metabolites from HSVtk-expressing cancer cells are transferred to neighboring cancer cells.10 Currently, HSVtk/GCV gene therapy approach is being used in several clinical trials.9,11−13 Although anticancer effects were observed in some patients, there were some problems such as the recurrence of cancers from the residual cancer cells and nonspecific toxicity in normal tissues. Therefore, further improvements are required to address these problems.12,13 The targeting of therapeutic gene expression is important for safe and efficient cancer gene therapy. To overcome the nonspecific toxicity of therapeutic genes, transcription regulation has been investigated using tissue-specific promoters.14 For this purpose, several promoters have been examined for brain tumor-specific expression. For example, the glial fibrillary acidic protein (GFAP) promoter is useful for glioma-specific expression.15,16 Nestin, an intermediate filament protein, is also expressed specifically in glioma cells. Furthermore, the quantity

INTRODUCTION Glioblastoma multiforme (glioblastoma) is an aggressively malignant primary brain tumor and is considered to be one of the most malignant human cancers. Glioblastoma usually causes death within one to two years after conventional therapies such as surgery, radiation, and chemotherapy. After conventional therapies, the remaining tumor cells grow, and glioblastoma recurs, and thus, the patients have a short median survival of two to three months.1−3 Despite technological advances in surgical resection, radiation, and chemotherapy, glioblastoma still has a very poor prognosis.4 Therefore, novel therapies that have long-lasting anticancer effects with minimal side effects are needed. In this context, gene therapy is an emerging strategy to overcome the limitations of current therapeutic tools for glioblastoma.5−7 Gene expression has a longer half-life than conventional therapy. Glioblastoma is usually localized in the central nervous system with limited metastasis and is thus a suitable target for gene therapy using local gene therapy. Herpes simplex virus thymidine kinase (HSVtk) is a suicide gene that has been investigated for glioblastoma gene therapy.8,9 In HSVtk/ganciclovir (GCV) gene therapy, the HSV-TK protein converts nontoxic GCV into a toxic phosphorylated form that inhibits DNA synthesis, resulting in the death of dividing cells. Moreover, the HSVtk/GCV strategy © 2014 American Chemical Society

Received: Revised: Accepted: Published: 938

October 11, 2013 December 30, 2013 January 27, 2014 January 27, 2014 dx.doi.org/10.1021/mp4006003 | Mol. Pharmaceutics 2014, 11, 938−950

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primer, 5′-CCG CTCGAG TGC AGG ATC ACT CTT TAT3′. For cloning convenience, the XhoI and HindIII sites were introduced to the forward and backward primers of the nestin promoter, and the MluI and XhoI sites were introduced to the forward and backward primers of NI2, respectively (the enzyme sites are underlined). Then, pSV-Luc was digested with XhoI and HindIII to remove the SV40 promoter. The amplified nestin promoter was digested with XhoI and HindIII and purified by 1% agarose gel electrophoresis and elution. The purified nestin promoter was inserted at the XhoI and HindIII sites of pSV-Luc, which produced pNestin-Luc. The amplified NI2 was digested with MluI and XhoI and inserted at the MluI and XhoI sites of pNestin-Luc, producing pNI2-Nestin-Luc. In addition, pNI2-SV-Luc was constructed by the insertion of the MluI- and XhoI-digested NI2 upstream of the SV40 promoter of pSV-Luc. The procedure for constructing pEpo-SV-Luc was as described previously.34 To construct pEpo-NI2-SV-Luc, the Epo enhancer was amplified by PCR using pEpo-SV-Luc as a template. The sequences of the primers were as follows: forward primer (or backward primer) 5′-GGGGTACCGCCCTACGTGCTGTCTCA-3′. For cloning convenience, the KpnI site was introduced to the primer. The amplified Epo enhancer was digested with KpnI and inserted upstream of the NI2 of pNI2-SV-Luc, producing pEpo-NI2-SV-Luc. The resulting vectors, pNestin-Luc, pNI2-Nestin-Luc, and pNI2-SV-Luc, were confirmed by restriction enzyme study and direct sequencing. Finally, pNSE-Luc was purchased from Addgene (Cambridge, MA, USA). An expression vector containing the herpes simplex virus thymidine kinase gene, pORF-HSVtk, was purchased from InvivoGen (San Diego, CA, USA) and used as a PCR template. The sequences of the primers for the HSVtk gene were as follows: forward primer, 5′-CCCAAGCTTATGGCTTCGTACCCCTGC-3′; backward primer 5′-GCTCTAGATCAGTTAGCCTCCCCCAT-3′. The HindIII and XbaI sites were introduced to the forward and reverse primers, respectively (the enzyme sites are underlined). The amplified HSVtk gene was digested with HindIII and XbaI and purified by agarose gel electrophoresis and elution. Next, pSV-HSVtk and pEpo-NI2-SV-HSVtk were constructed by the insertion of the HSV-TK cDNA at the position of the luciferase gene of pSV-Luc and pEpo-NI2-SV-Luc. The resulting vectors, pSVHSVtk and pEpo-NI2-SV-HSVtk, were confirmed by restriction enzyme study and direct sequencing. Gel Retardation Assay. The complexes were prepared at various weight ratios by mixing a fixed amount of pEpo-NI2SV-HSVtk (0.5 μg) with increasing amounts of PEI-Dexa in 5% glucose. After 30 min incubation, the mixtures were electrophoresed through a 1% agarose gel for 40 min. Measurement of Zeta Potential and Complex Size. PEI-Dexa and pEpo-NI2-SV-HSVtk were mixed for complex formation at various weight ratios. The amount of pEpo-NI2SV-HSVtk was fixed at 2 μg. After 30 min incubation, the complex size and zeta potential were measured by the Zetasizer Nano ZS system (Malvern Instruments, UK). The size and zeta potential were presented as the average values from triplicated experiments. Cell Culture and Transfection. The C6 rat glioblastoma, U87 and A172 human glioblastoma, and human embryonic kidney (HEK) 293 cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS) at 37 °C in a 5% CO2 incubator. For the transfection assays, the cells were seeded at a density of 5.0

of nestin increases as the grade of glioma becomes more malignant toward glioblastoma.17 In contrast, nestin protein was not found in normal brain tissue.18 The glioma-specific expression of the nestin gene is controlled by the regulatory promoter and enhancer of its second intron, NI2.19−21 Therefore, the nestin promoter and enhancer may be suitable for the gene therapy of glioblastoma. One of the important characteristics of solid tumors is hypoxia. Most solid tumors, including glioblastoma, have intratumoral hypoxic areas. These hypoxic regions are associated with resistance to conventional anticancer treatments.22,23 Therefore, hypoxia is another important target for tumor targeting strategies. Hypoxia-specific promoters have been evaluated for tumor hypoxia-specific gene therapies. These promoters include the glyceraldehyde 3-phosphate dehydrogenase (GAPDH), vascular endothelial growth factor (VEGF), and phosphoglycerate kinase-1 (PGK-1) promoters.17,24−27 In vitro and in vivo evaluations have shown that these promoters are useful for tumor hypoxia-specific gene therapy. The combination of gene regulation systems for glioma and hypoxia may be useful to specifically target gene expression for glioblastoma. Dexamethasone (Dexa) is a potent glucocorticoid that is widely used as a therapeutic for inflammatory disease and in brain cancer for the treatment of edema.28 Several studies have shown that Dexa treatment decreases the permeability of the blood−brain barrier.29,30 Due to its frequent use in brain cancer treatment, many researchers have investigated the effects of Dexa on the proliferation of tumor cells.30,31 Some studies have reported that Dexa inhibited the growth of glioma cells.31−33 In our previous report, Dexa conjugated low-molecular-weight polyethylenimine (2 kDa, PEI2k), and PEI-Dexa was synthesized. PEI-Dexa was evaluated as an efficient drug and gene delivery carrier in acute lung injury and ischemic stroke animal models. PEI-Dexa had a high transfection efficiency similar to that of high-molecular-weight polyethylenimine (25 kDa, PEI25k). In addition, the cytotoxicity of PEI-Dexa to normal cells was lower than that of PEI25k, suggesting that PEI-Dexa may be a useful carrier of the glioblastoma-specific HSVtk plasmid. In this study, the glioma and hypoxia dual-specific HSVtk gene expression system, pEpo-NI2-SV-HSVtk, was developed for glioblastoma-specific suicide gene expression. In addition, the dual-specific HSVtk plasmid was delivered into the glioblastoma using PEI-Dexa as a gene carrier. In vitro and in vivo study results indicated that a gene expression plasmid dually specific for glioma and hypoxia might be useful for glioblastoma-targeting gene therapy.

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MATERIALS AND METHODS Plasmid Construction. pSV-Luc was purchased from Promega (Madison, WI, USA). Cloning of the nestin promoter and NI2 was performed by PCR. Chromosomal DNA was extracted from mouse brain tissue by a chromosomal DNA isolation kit (Qiagen, Valencia, CA, USA). The concentration of DNA was measured by the absorbance at 260/280 nm. The fragments coding the nestin promoter and the NI2 were amplified by PCR using Tfl DNA polymerase (Promega). The sequences of the primers were as follows: nestin promoter, forward primer, 5′-CCG CTCGAG GGG CCC AGT TCT GTG CA-3′, backward primer, 5′-CCC AAGCTT GTG GAG CAC TAG AGA AGG-3′; NI2, forward primer, 5′-CG ACGCGT GAA TTC CCA CTT CCC CT-3′, backward 939

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× 104 cells/well in 12-well plates (Falcon, Becton Dickenson, Franklin Lakes, NJ, USA). PEI-Dexa and various pDNAs were prepared at an 8:1 weight ratio based on previous reports.35,36 The PEI2k/pDNA complexes and the PEI25k/pDNA complexes were prepared at a 40:1 N/P ratio and a 5:1 N/P ratio, respectively, based on a previous report.37 The lipofectamine/ pDNA complexes were prepared at a 2.5:1 weight ratio according to the manufacturer’s instructions. The same amount of dexamethasone as used in the conjugation procedure of PEIDexa was simply mixed with empty plasmid for the simple mixture of dexamethasone and pEmpty. The amount of pDNA was fixed at 1 μg/well. Before transfection, the cells were washed twice with serum-free DMEM, and then 1 mL of fresh serum-free medium was added. Then, the carrier/pDNA complexes were added to each well, and the cells were incubated for 4 h. After the incubation, the transfection mixtures were removed, and 1 mL of fresh DMEM, containing 10% FBS with or without GCV (10 μg/mL), was added. The cells were incubated for an additional 20 h under normoxic or hypoxic conditions (normoxia, 20% oxygen; hypoxia, 1% oxygen). In Vitro Luciferase Assay. After the incubation, the cells were washed twice with phosphate-buffered saline (PBS), and 120 μL of reporter lysis buffer (Promega) was added to each well. After 15 min of incubation at room temperature (RT), the cells were harvested and transferred to a microcentrifuge tube. After 15 s of vortexing, the cells were centrifuged at 13 000 rpm for 5 min. The supernatants were transferred to fresh tubes. The protein concentrations of the extracts were measured by a bicinchoninic protein assay (Pierce, Rockford, IL, USA). Luciferase activity was measured in terms of relative light units (RLU) using a 96-well plate luminometer (Berthold Detection System, GmbH, Pforzheim, Germany). The luciferase activity was monitored and integrated over a period of 20 s. The final values of the luciferases were reported in terms of RLU/mg total protein. MTT Assay. To evaluate cytotoxicity or proliferation, the C6, A172, U87, and HEK293 cells were seeded at a density of 1.0 × 104 cells/well in 96-well plates and incubated for 24 h before transfection. The cells were then transfected with the carrier/pDNA complexes. The amount of pDNA was fixed at 0.5 μg/well, and the complexes were prepared as described above. The cells were incubated for 4 h at 37 °C, and then the transfection mixtures were removed. A portion of 200 μL of fresh DMEM containing 10% (v/v) FBS and GCV (10 μg/mL) were added to each well. The cells were incubated for an additional 24 h at 37 °C. After incubation, 20 μL of 5 mg/mL MTT solution in 1× PBS were added to each well. Cells were incubated for an additional 4 h at 37 °C. The MTT-containing medium was removed, and 200 μL of dimethyl sulfoxide were added to dissolve the formazan crystals formed by the living cells. Absorbance was measured at 570 nm using a microplate reader. Cell viability (%) was calculated according to the following equation:

performed as described above, and the cells were incubated under normoxic and hypoxic conditions. After 24 h of incubation, the cells were harvested to fresh tubes and washed twice with cold PBS. The apoptosis level was determined by annexin V staining according to the instructions of the manufacturer (BD Pharmingen, San Jose, CA, USA). Flow cytometry was performed using BD FACSCalibur (BD Biosciences, San Jose, CA, USA). Caspase 3/7 Assay. To determine the apoptosis level in the HSVtk gene-transfected C6 cells, the enzymatic activity of caspase 3 was measured with a caspase 3/7 assay kit (Promega). For the transfection, the C6 cells were seeded at a density of 1.0 × 104 cells/well in 96-well plates. Transfection was performed as described in the MTT assay section. After 24 h of incubation, the caspase 3/7 assay was performed according to the manufacturer’s instructions. The caspase 3/7 activity was measured in RLUs using a 96-well plate luminometer (Berthold Detection System, GmbH, Pforzheim, Germany). In Vivo Evaluation in a Subcutaneous Glioblastoma Model. All in vivo experimental protocols were approved by the Animal Research Committee of Hanyang University College of Medicine. Under general anesthesia, subcutaneous tumors were established in five-week-old Balb/cSlc nude mice by a single inoculation of 1 × 105 C6 cells into the dorsal flank of each mouse. After the tumor diameters reached 4−6 mm, the mice were randomly allocated into groups (cancer control, saline, PEI25k/pEmpty, PEI2k/pEmpty, PEI-Dexa/pEmpty, PEI-Dexa/pSV-HSVtk, PEI-Dexa/pEpo-NI2-SV-HSVtk, and Dexa-only groups) and treated accordingly with the carriers/ pDNA complexes. Each group consisted of 12 mice. pSI was used as an empty plasmid (pEmpty). The complexes were prepared at the optimized ratios in 50 μL of saline. The amount of pDNA was fixed at 20 μg/mouse. Dexa-only was prepared with the same amount of Dexa used in the conjugation procedure for PEI-Dexa. The complex injection was performed a total of three times with one injection every three days. After the first complex injection, GCV (25 mg/kg) was injected intraperitoneally every 24 h for 16 days. Tumor volumes were measured at the first, second, and third injection points and every week after injection up to three weeks. Tumor volumes were calculated using the formula L × W2 × 0.5, where L and W represent the largest and smallest diameters, respectively. The mice were sacrificed three weeks after the final injection. In Vivo Experiments in the Intracranial Tumor Rat Model. Intracranial tumor rat models were developed in sevenweek-old male Sprague−Dawley rats. Intracerebral tumors were generated by stereotactic injections. The rats were anesthetized, and each skull was exposed to bore a 2 mm hole. The monitoring point was 2 mm lateral to the bregma and carefully drilled using a gentle saline (0.89% NaCl) drip to avoid a disrupted dura (coordinates to bregma: anteroposterior, 0 mm; lateral, 2.0 mm; ventral, 4.0 mm) using a 26-gauge Hamilton microsyringe (80330; Hamilton, Reno, NV, USA). After each hole was made, 1 × 105/10 μL C6 cells were injected into the cerebral cortex. The injections were performed by first rapidly injecting 1 μL of cells and then slowly injecting 9 μL of cells (at 0.9 μL/min). One week after tumor implantation, the rats were randomly allocated into groups (cancer control, saline, PEI25k/ pEmpty, PEI2k/pEmpty, PEI-Dexa/pEmpty, PEI-Dexa/pSVHSVtk, PEI-Dexa/pEpo-NI2-SV-HSVtk, and Dexa-only groups). Each group consisted of eight rats. pSI was used as an empty plasmid. The complexes were prepared at the optimized weight ratios and injected at the same location as the

cell viability(%) = [OD570(sample)/OD570(control)] × 100

where OD570 is the optical density at 570 nm. Annexin V Assay. C6 cells were seeded at a density of 1 × 105 cells/well in six-well plates (Falcon, Becton Dickinson) 24 h before transfection. The carrier/pDNA complexes were prepared at optimized weight ratios. Transfection was 940

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Figure 1. Construction of glioblastoma-specific gene expression plasmids. The diagram shows the structures of the luciferase reporter constructs with the SV promoter, the nestin promoter, the NI2-SV promoter, the NI2-nestin promoter, and the NSE promoter.

Figure 2. Glioma-specific gene expression. To evaluate promoter activities in glioblastoma cells, pSV-Luc, pNestin-Luc, pNI2-SV-Luc, pNI2-NestinLuc, and pNSE-Luc were transfected into (A) C6 cells or (B) U87 cells. (C) To evaluate the cell specificity of the NI2-SV promoter, pNI2-SV-Luc was transfected into C6 or N2A cells. The cells were incubated for 24 h and assessed for luciferase activity. Luciferase activity (RLU/mg protein) was expressed by relative luciferase activity (% of pSV-Luc). The data are expressed as the mean value (±standard deviation) of triplicate experiments. *P < 0.002 compared with pNestin-Luc, **P < 0.003 compared with pNI2-Nestin-Luc, ***P < 0.006 compared with pNI2-SV-Luc of N2A neuroblastoma.

tumor implantation. The pDNA amount was fixed at 1 μg/ mouse. A total volume of 10 μL of carrier/pDNA complexes was stereotaxically injected into the site of the tumor cell

implantation. In total, 30 mg/kg GCV were administered intraperitoneally every 24 h for one week. Two weeks after tumor implantation, the rats were sacrificed by perfusion, and 941

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Figure 3. Structures of pSV-Luc, pEpo-NI2-SV-Luc, pSV-HSVtk, and pEpo-NI2-SV-HSVtk. (A) The diagram shows the structure of the luciferase reporter or HSV-TK gene constructs containing the Epo enhancer and NI2-SV promoter. E indicates the Epo enhancer. (B) Restriction enzyme study was performed to confirm the construction of the plasmids. 1: the HSVtk cDNA of pSV-HSVtk, 2: the HSVtk cDNA of pEpo-NI2-SV-HSVtk, 3: the Epo-NI2-SV promoter of pEpo-NI2-SV-HSVtk. 1kb M indicates the size markers.

the brains were harvested and fixed with 4% paraformaldyhyde solution. Nissl Staining. The fixed brain samples were embedded in paraffin and cut into sections (5 μm thick), and Nissl staining was performed to determine the histopathological features of the brains.38 After deparaffinization, brain section slides were incubated in cresyl violet (0.1%; Sigma, St. Louis, MO, USA). The sections were passed through a destaining solution (70% ethanol, 10% acetic acid), dehydrated (100% ethanol and xylene), and coverslipped with mounting solution. The brain total size and cancer volume were measured using Image J 1.42 software (National Institutes of Health, Bethesda, MD, USA). Immunohistochemistry. Three days after the last injection into the brain, the animals were sacrificed with perfusion. Brains were inflated and fixed with 4% paraformaldehyde solution. The samples were embedded in paraffin and cut into 5-μmthick sections. After deparaffinization of the brain section slides, the endogenous peroxidase activity was blocked in 0.5% hydrogen peroxide diluted in methanol for 10 min at RT. The slides were then rehydrated with graded alcohols and Trisbuffered saline (TBS). Antigen retrieval was performed in a sodium citrate buffer (2.94 mg/mL, pH 6.0) for 30 min at 95− 100 °C. After being cooled on ice for 20 min, the slides were rinsed in TBS and incubated in a 10% normal horse serum dilution with PBS for 30 min at RT. All antibodies were diluted in TBS with 1% bovine serum albumin. HSVtk antibody (rabbit monoclonal, Abcam, Cambridge, MA, USA) was diluted to 1:200 and incubated overnight at 4 °C. After being rinsed in TBS, the slides were incubated with secondary antibody (horseradish peroxidase-conjugated antirabbit, 1:500, Abcam) for 30 min at RT. Signal detection was performed with the 3,3′diaminobenzidine substrate (Pierce). The slides were counterstained in Mayer’s hematoxylin for 30 s, dehydrated, and mounted for microscopic examination.

described in the Materials and Methods and confirmed by restriction enzyme study and direct sequencing. In vitro transfection assays were performed to evaluate the glioma-specific promoter activities of pNestin-Luc, pNI2Nestin-Luc, pNI2-SV-Luc, and pNSE-Luc. The pDNAs were transfected into C6 rat glioblastoma cells and U87 human glioblastoma cells using PEI25k as a gene carrier. Next, pSVLuc was transfected into the cells as a control pDNA. Twentyfour hours after the transfection, the cells were harvested, and luciferase assays were performed with the cell extracts. The pNI2-SV-Luc group showed significantly higher gene expression than did the pSV-Luc, pNestin-Luc, pNI2-NestinLuc, or pNSE-Luc group in the C6 and U87 cells (Figure 2A and B). Glioma-specific gene expression was confirmed by transfection into glioblastoma cells (C6) and neuroblastoma cells (N2A) (Figure 2C). Since the N2A cells originated from neurons, the luciferase expression by pNI2-SV-Luc was much lower in the N2A cells than in the C6 cells, showing the gliomaspecific activity of NI2. These results suggest that the NI2 and SV promoter had the highest glioma specificity among the tested promoters. Therefore, the NI2 and SV promoter was used for further modification for hypoxia-specific gene expression in the following experiments. Construction and in Vitro Transfection Assays of Glioma and Hypoxia Dual-Specific pDNA. One of the characteristics of solid tumors is that the tumors have hypoxic regions in their cores, due to the low supply of blood. Therefore, tumor hypoxia may be an excellent target for gene expression. To increase gene expression under tumor hypoxia, pEpo-NI2-SV-Luc was constructed by the insertion of the Epo enhancer upstream of the NI2 of pNI2-SV-Luc (Figure 3A). It was previously shown that the Epo enhancer contains a hypoxia response element (HRE) and is bound to hypoxia-inducible factor 1 (HIF-1).34,39 The insertion of the Epo enhancer can increase the expression of the downstream gene under hypoxia.34 Therefore, the Epo enhancer in combination with the NI2 and SV promoter may have dual specificity for hypoxia and glioma in gene expression. The constructed therapeutic vectors were digested by HindIII and XbaI restriction enzymes for 4 h and, then, analyzed by 1% agarose gel electrophoresis (Figure 3B). The results confirmed the constuctions of the plasmids.

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RESULTS Construction of Glioma-Specific Plasmid and in Vitro Transfection Assay. As glioma-specific gene expression plasmids incorporating luciferase (Luc), pNestin-Luc, pNI2SV-Luc, pNI2-Nestin-Luc, and pNSE-Luc were constructed with the various glioma-specific promoters (Figure 1). The construction of plasmid DNAs (pDNAs) was performed as 942

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Figure 4. Gene expression by pEpo-NI2-SV-Luc under normoxia or hypoxia. First, pSV-Luc, pNI2-SV-Luc, and pEpo-NI2-SV-Luc were transfected into (A) C6 or (B) U87 cells. The cells were incubated for 24 h under normoxic or hypoxic conditions and assessed for luciferase activity. The data were expressed as the mean value (±standard deviation) of triplicate experiments. *P < 0.05 compared with pSV-Luc (under normoxia and hypoxia), pNI2-SV-Luc (under normoxia and hypoxia), and pNI2-SV-Luc (under hypoxia).

Figure 5. Apoptosis induced by hypoxia/glioma dual-specific HSVtk gene expression plasmid. (A) The cell viability was evaluated by MTT assay. (B) Apoptosis level was measured by caspase 3/7 assay. First, pSV-HSVtk and pEpo-NI2-SV-HSVtk were transfected into C6 cells. The cells were incubated under normoxia or hypoxia for 24 h with or without GCV. Caspase 3/7 activity was assessed. The data are expressed as the mean value (±standard deviation) of quadruplicate experiments. *P < 0.0001 compared with control (under normoxia and hypoxia), pSV-HSVtk (under normoxia and hypoxia), and pEpo-NI2-SV-HSVtk (under normoxia).

SV-Luc, confirming that the Epo enhancer was effective in hypoxia-inducible gene expression (Figure 4). Hypoxia and glioma dual-specific HSVtk expression pDNA was constructed with the Epo enhancer and the NI2-SV promoter, and pSV-HSVtk was constructed as a control pDNA. The construction of pDNAs was confirmed by restriction enzyme study and direct sequencing. To evaluate the hypoxiaspecific gene expression and induction of cell death, pEpo-NI2SV-HSVtk was transfected into C6 cells, and pSV-HSVtk was transfected as a negative control. Then, the cells were incubated with GCV under hypoxia or normoxia. The level of cell death was measured by an MTT assay. The results showed that pEpoNI2-SV-HSVtk induced cell death more efficiently in hypoxic cells than in normoxic cells (Figure 5A). GCV treatment

To evaluate the hypoxia specificity of pEpo-NI2-SV-Luc, pEpo-NI2-SV-Luc was transfected into C6 and U87 cells, while pSV-Luc and pNI2-SV-Luc were transfected as negative controls. After transfection, the cells were incubated under normoxia or hypoxia for 24 h. In both C6 and U87 glioblastoma cells, pEpo-NI2-SV-Luc showed significantly higher luciferase gene expression under hypoxic conditions compared with the expression under normoxic conditions (Figure 4). Likewise, pNI2-SV-Luc showed increased gene expression under hypoxia compared with normoxia. This may be due to the stimulation protein 1 (Sp1) binding sites in the SV promoter, since Sp1 is one of the transcription factors for hypoxia-inducible gene expression.40,41 However, the induction-fold of the pNI2-SV-Luc was less than that of pEpo-NI2943

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Figure 6. Physical characterization of PEI-Dexa/pDNA complex. (A) Gel retardation assay. The PEI-Dexa/pDNA complexes were analyzed by 1% agarose gel electrophoresis. (B) Particle size and (C) zeta potential. The particle sizes and zeta potentials of the PEI-Dexa/pDNA complexes were measured at various weight ratios. Data are expressed as mean values (±standard deviation) of triplicate experiments.

Figure 7. Viability of glioblastoma cells after delivery of the PEI-Dexa/pEmpty complexes. The carrier/pEmpty complexes were transfected into the (A) C6, (B) A172, (C) U87, and (D) HEK293 cells under optimal conditions. After 24 h, the cell viability was measured by MTT assay. The data are expressed as the mean value (±standard deviation) of triplicate experiments. *P < 0.05 compared with PEI25k and lipofectamine. **P < 0.05 compared with PEI25k. ***P < 0.05 compared with PEI25k and lipofectamine.

without the transfection of pDNA did not change the viability of cells (Figure 5A). The incorporation of GCV phosphate into the replicated DNA induced instability of the DNA and apoptosis of the cells. Therefore, the apoptosis level of the

transfected C6 cells in the presence of GCV was measured by caspase assay (Figure 5B). The caspase 3/7 assay showed that pEpo-NI2-SV-HSVtk and GCV treatment increased the caspase level under hypoxia, suggesting that apoptosis was induced by 944

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dexamethasone did not induce cell death in HEK293 cells as much as in C6 cells. The results suggest that dexamethasone may induce cell death significantly in the C6 tumor cells, but not in the HEK293 nontumor cells. In addition, the results showed that PEI-Dexa had a higher antitumor effect than dexamethasone only. The transfection efficiency of PEI-Dexa into glioblastoma cells was evaluated by a luciferase assay. The PEI-Dexa/pSVLuc complexes were prepared and transfected into C6, U87, and A172 cells. PEI2k, PEI25k, and lipofectamine were used as control carriers. In all of these cell lines, the transfection assays showed that PEI-Dexa had a higher transfection efficiency than PEI2k (Figure 9). Although the transfection efficiency of PEIDexa was lower than that of PEI25k in the glioblastoma cells, it was similar to that of lipofectamine in the tested cells (Figure 9). This higher transfection efficiency of PEI-Dexa than PEI2k may be due to two factors, considering that the PEI-Dexa was synthesized with PEI2k. First, PEI-Dexa forms micelle structures in aqueous solution and may behave like a higher molecular weight PEI. Second, the binding of PEI-Dexa to the glucocorticoid receptor may facilitate the nuclear translocation of the PEI-Dexa/pDNA complex.42,43 The glucocorticoid receptor is a nuclear receptor, which is localized without its ligand. Upon its binding to its ligand, the glucocorticoid receptor is transported into the nucleus. During this process, the receptor may facilitate the translocation of the PEI-Dexa/ pDNA complex into the nucleus. Therefore, the conjugation of dexamethasone may increase transfection efficiency of PEI2k and PEI-Dexa has a high transfection efficiency comparable PEI25k. Therefore, PEI-Dexa may be useful for gene delivery into glioblastoma cells with high transfection efficiency and an anticancer effect. In Vivo Characterization of PEI-Dexa as a Gene Carrier for Glioblastoma Treatment. For our in vivo studies, a subcutaneous glioblastoma model and intracranial glioblastoma model were established in five-week-old Balb/cSlc nude mice. The subcutaneous glioblastoma model was established by the subcutaneous inoculation of C6 cells into the dorsal flanks of the mice. The PEI-Dexa/pEmpty complexes were prepared and intratumorally injected into the subcutaneous tumors a total of three times with one injection every three days. The PEI2k/ pEmpty complex and PEI25k/pEmpty complex injection groups were used as carrier controls. The saline-only and Dexa-only injection groups were also prepared as negative control groups. The tumor sizes of the groups were followed for up to three weeks after the third injection of the complexes. The results showed that Dexa inhibited tumor growth more efficiently compared with the control, saline, PEI2k/pEmpty, and PEI25k/pEmpty injection groups (Figure 10). Furthermore, the PEI-Dexa/pEmpty group reduced tumor growth more efficiently than the Dexa-only injection group (Figure 10). The suppression in tumor growth was improved by the PEI-Dexa/pEmpty complex compared with the Dexa-only group. This may be due to the more efficient delivery of Dexa. Our previous report showed that the PEI-Dexa/pEmpty complex formed nanoparticles and that the nanoparticles of the PEI-Dexa/pEmpty complex were efficiently taken up by cells.42 However, PEI-Dexa without pDNA did not form nanoparticles, and the cellular entry efficiency of PEI-Dexa was lower than that of the PEI-Dexa/pDNA nanoparticles.42 Therefore, nanoparticle formation may be an important step for the enhanced delivery of Dexa. Furthermore, PEI-Dexa is watersoluble and does not require a toxic organic solvent for

the increased expression of HSV-TK in the hypoxic cells (Figure 5B). Taken together, these results suggest that the Epo enhancer−NI2-SV promoter specifically induced HSVtk gene expression and apoptosis in hypoxic glioma cells. In Vitro Characterization of PEI-Dexa as a Carrier for Hypoxia/Glioma Dual-Specific HSVtk Expression pDNA. In our previous study, PEI-Dexa was demonstrated to be an efficient gene carrier to the brain and lungs.42,43 Dexa has been widely used for the treatment of brain edema in brain cancer. Furthermore, it has been suggested that Dexa suppressed the growth of glioma cells.31−33 Therefore, it is likely that PEI-Dexa may induce the cell death of glioblastoma cells. If this is the case, PEI-Dexa may have dual effects as a gene carrier and Dexa carrier. To confirm the complex formation, physical characterizations of the PEI-Dexa/pDNA complex were performed. A fixed amount of pDNA (0.5 μg/μL) was mixed with increasing amounts of PEI-Dexa for the gel retardation assay. In Figure 6A, the DNA band was completely retarded above a 3:1 (PEIDexa:pEpo-NI2-SV-HSVtk) weight ratio. The complex had a 110−120 nm mean diameter and positive surface charge with an average of 35−40 mV (Figure 6B and C). The results suggested that PEI-Dexa formed stable complexes with pEpoNI2-SV-HSVtk. To evaluate the anticancer effect of PEI-Dexa in glioblastoma cells, the PEI-Dexa/pEmpty complexes were transfected into C6, A172 (human glioblastoma), U87, and HEK293 (human embryonic kidney) cells. After 24 h of incubation, the cell viability was evaluated by an MTT assay. The assay indicated that the transfection of the PEI-Dexa/pEmpty complex reduced the viability of the glioblastoma cells (Figure 7A, B, and C). However, the viability of the HEK293 cells was not decreased by the transfection of the PEI-Dexa/pEmpty complex (Figure 7D). These results suggest that the Dexa in PEI-Dexa may have an anticancer effect in glioblastoma cells. PEI25k and lipofectamine had high toxicities in all of the transfected cells (Figure 7), suggesting that PEI25k and lipofectamine are toxic to all types of cells. The antitumor effect of the PEI-Dexa/pEmpty complex was compared with that of the simple mixture of dexamethasone and pEmpty. In Figure 8, PEI-Dexa induced higher cell death than dexamethasone in the C6 cells. However, PEI-Dexa and

Figure 8. Antitumor effect comparison of PEI-Dexa and dexamethasone only. The PEI-Dexa/pEmpty complex or the simple mixture of dexamethasone and pEmpty was transfected into the C6 and HEK293 cells. After 24 h, the cell viability was measured by MTT assay. The data are expressed as the mean value (±standard deviation) of quadruplicate experiments. *P < 0.0002 compared with dexamethasone of C6. 945

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Figure 9. Transfection efficiency of PEI-Dexa into glioblastoma cells. PEI2k, PEI25k, lipofectamine, and PEI-Dexa/pSV-Luc complexes were transfected into (A) C6, (B) U87, and (C) A172 cells. The carrier/pSV-Luc complexes were prepared under optimal conditions. After 24 h, gene expression activity was measured by luciferase assay. The data are expressed as the mean value (±standard deviation) of quadruplicate experiments. *P < 0.02 compared with PEI2k. **P < 0.0007 compared with PEI2k. ***P < 0.0006 compared with PEI2k.

The anticancer effect of PEI-Dexa was also measured in an intracranial glioblastoma model. The intracranial glioblastoma model was established by the stereotaxic injection of C6 cells into the cerebral cortexes of seven-week-old male Sprague− Dawley rats. One week after the implantation of the tumor cells, the PEI-Dexa/pEmpty complexes were injected at the position of the tumor cell implantation. PEI2k/pEmpty, PEI25k/pEmpty, Dexa, and saline were injected as controls. The brains were harvested, and Nissl staining was performed using cresyl violet to measure the sizes of the tumors. The Nissl staining results showed that the tumor size was decreased by Dexa (Figure 11). The Dexa-only and PEI-Dexa/pEmpty groups showed smaller tumor sizes than the PEI2k/pEmpty complex or PEI25k/pEmpty complex groups (Figure 11). In Vivo Therapeutic Effects of the Delivery of pEpoNI2-SV-TK Using PEI-Dexa. To evaluate the hypoxia/glioma dual-specific HSVtk pDNA in the glioblastoma animal models, pSV-HSVtk and pEpo-NI2-SV-HSVtk were delivered into the tumors using PEI-Dexa as a gene carrier. For the subcutaneous glioblastoma model, the PEI-Dexa/pSV-HSVtk and PEI-Dexa/ pEpo-NI2-SV-HSVtk complexes were intratumorally injected into the subcutaneous tumors a total of three times with one injection every three days. After the delivery of the pDNAs, GCV was supplied by intraperitoneal injection every 24 h for 15 days. The PEI-Dexa/pSV-HSVtk complex group was used as a control. The saline-only and Dexa-only injection groups were also prepared as negative controls. The tumor sizes of the groups were followed for up to three weeks after the third

Figure 10. Anticancer effect of PEI-Dexa in a subcutaneous glioblastoma model. The complexes were directly injected into the tumors as described in the Materials and Methods. The amount of pDNA was fixed at 20 μg for each carrier. The tumor volume was calculated using the formula L × W2 × 0.5, where L and W represent the largest and smallest diameters, respectively. Each group consisted of 12 mice (n = 12). The data are expressed as the mean value (±standard deviation). *P < 0.05 compared with saline and PEI25k/ pEmpty.

solubilization. Therefore, these factors suggest that PEI-Dexa may be an efficient Dexa carrier as well as a pDNA carrier. 946

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Figure 11. Anticancer effect of PEI-Dexa in intracranial glioblastoma model. (A) Tumor size and (B) Nissl staining. The PEI-Dexa/pDNA complexes were injected into the intracranial glioblastoma model with stereotaxic equipment. One week after the injection, brain section and Nissl staining were performed. Each group consisted of eight rats (n = 8). The brain total size and tumor volume were measured using Image J 1.42 software (National Institutes of Health).*P < 0.02 compared with control, saline, PEI25k, and PEI2k.

Figure 12. Anticancer effect of pEpo-NI2-SV-HSVtk in the subcutaneous glioblastoma model. (A) Tumor size and (B) the images of the tumors at two weeks after the injection. The complexes were directly injected into the tumor. The amount of pDNA was fixed at 20 μg. After the first complex injection, GCV (25 mg/kg) was injected intraperitoneally every day for 15 days. The tumor volume was calculated using the formula L × W2 × 0.5, where L and W represent the largest and smallest diameters, respectively. Each group consisted of 12 mice (n = 12). *P < 0.05 compared with all other groups.

injection of the complexes. The PEI-Dexa/pEpo-NI2-SVHSVtk complex injection group showed more effective suppression of tumor growth compared with the PEI-Dexa/ pSV-HSVtk and PEI-Dexa/pEmpty groups (Figure 12). This result suggests that pEpo-NI2-SV-HSVtk expressed a higher level of HSV-HSVtk in the hypoxic glioma cells and more efficiently induced apoptosis of the cells than pSV-HSVtk. To confirm the antitumor efficiency of pEpo-NI2-SV-HSVtk, an intracranial glioblastoma model was also established. One week after tumor implantation, the PEI-Dexa/pEpo-NI2-SVHSVtk complex was injected at the site of the tumor implantation. GCV was injected intraperitoneally every 24 h. One week after pDNA injection, the brains were harvested and stained with cresyl violet to measure the tumor sizes. The results showed that the pEpo-NI2-SV-HSVtk group had significantly smaller tumors than any other injection group (Figure 13A and B). Although the pSV-HSVtk group showed the decreased tumor size, pEpo-NI2-SV-HSVtk showed greater decreases in the tumor size in the brain.

After three days of the last injection, the HSVtk gene expression level was evaluated by immunohistochemistry with HSVtk antibody. As shown in Figure 13C, higher HSVtk gene expression was observed in the pEpo-NI2-SV-HSVtk injection group than in the pSV-HSVtk injection group. In addition, the pEpo-NI2-SV-HSVtk group showed the highest apoptosis level in the TUNEL assay (Figure 13D). This result suggests that pEpo-NI2-SV-HSVtk induces HSVtk gene expression and apoptosis in ischemic glioblastoma cells.

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DISCUSSION Glioblastoma is the most common malignant primary brain tumor. Since glioma cells grow invasively in intracranial central nervous system tissues, the complete elimination of tumors by surgical resection is difficult to achieve. In addition, supplemental treatments, including irradiation and anticancer chemotherapy, have many side effects. To overcome the limitations of current treatments, gene therapy has emerged as a 947

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Figure 13. Suppression of tumor growth by pEpo-NI2-SV-HSVtk in the intracranial model. The PEI-Dexa/pDNA complexes were injected into the glioblastoma rat model. GCV (25 mg/kg) was injected intraperitoneally every day for one week. One week after the injection, the brain section and Nissl staining were performed. Each group consisted of eight rats (n = 8). The total brain size and tumor size were measured using Image J 1.42 software (National Institutes of Health). (A) Tumor size; *P < 0. as compared with the control, saline, and pSV-HSVtk group; (B) images of tumors after Nissl staining; (C) immunostaining of HSVtk; (D) TUNEL assay.

new treatment strategy for glioblastoma.5−7 Several studies have reported successful antitumor effects of gene therapy. For example, the intratumoral injection of a retroviral vector expressing HSVtk/interleukin 2 activated a systemic cytokine cascade, with tumor responses in 50% of cases.44 Gene therapy has been improved by employing tissue-specific promoters. For example, the human telomerase reverse transcriptase and GFAP promoters were used for glioblastoma gene therapies.45,46 The tissue-specific promoters reduced the side effects of gene therapy and increased its specificity. In this study, a hypoxia/glioma dual-specific gene expression system was constructed for stronger glioblastoma-specific gene therapy compared with the single-specific expression systems. The system was composed of two regulatory elements. NI2 increases gene expression in glioblastoma. However, nestin has been also reported to be induced in neural stem cells. Therefore, a single tissue-specific expression system with the nestin intron may induce gene expression in neural stem cells and cause side effects. This suggests that NI2 alone is not sufficient for tumor specificity. We combined the Epo enhancer with the NI2, which increased gene expression in the ischemic region. This combination has increased specificity of the HSVtk expression in the ischemic glioblastoma tissues, reducing nonspecific expression in other tissues. The in vivo therapeutic effect of pEpo-NI2-SV-HSVtk was evaluated in the subcutaneous glioblastoma and intracranial glioblastoma models. The results showed that pEpo-NI2-SVHSVtk had a significantly greater antitumor effect compared with the control groups (Figures 12 and 13). The in vivo results showed that pEpo-NI2-SV-HSVtk efficiently inhibited the

tumor growth of the glioblastomas. This suggests that the dual-specific promoter increases the efficiency of gene therapy in ischemic glioblastomas by increasing gene expression. The Epo enhancer has been studied for the gene therapies of various ischemic diseases. These diseases include ischemic heart disease, spinal cord injury, and islet transplantations. The Epo enhancers contain HREs, which are the targets of HIF-1 binding. The Epo enhancer can be combined with various promoters. For the application of the Epo enhancer to ischemic diseases, it was combined with the SV40 promoter. Due to the characteristics of the enhancer, the Epo enhancer can induce the closest promoter under hypoxia. Therefore, it can be combined with various tissue-specific promoters. Dual-specific promoters with the Epo enhancer can be constructed for various ischemic diseases. The HREs from the various hypoxiainducible promoters were combined with the tissue-specific promoters. For example, the HREs were combined with the estrogen response elements for breast cancer-specific gene expression. In a previous report, PEI-Dexa was evaluated as a gene carrier for acute lung injury.42,43 In addition, PEI-Dexa delivered Dexa more efficiently than Dexa alone. PEI-Dexa with pDNA forms nanoparticles. The nanoparticles, with a size around 100 nm, fit the clathrin-coated pits and are easily taken up by cells via endocytosis. PEI-Dexa did not form nanoparticles without pDNA.42 The complex formation of PEI-Dexa with pDNA may be an important step for efficient Dexa delivery. However, there are some drawbacks in using PEI-Dexa in gene delivery. The ratio between pDNA and PEI-Dexa was fixed at a 1:8 weight ratio to obtain the highest transfection rate. This means that, if 948

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the dose of pDNA is fixed, the dose of Dexa is fixed. Therefore, in this system, the doses for both Dexa and pDNA may not be optimized at the same time. Second, the inhibition effects of Dexa on tumor growth have not been uniformly observed in various types of cancers.47 Some types of cancers are protected against cell apoptosis by Dexa.48,49 Therefore, the application of Dexa should be carefully controlled. In the case of glioblastoma, Dexa has been clinically used as a drug in the case of edema. This usage of Dexa is based on the fact that Dexa reduces vascular permeability. Therefore, the application of Dexa for glioblastoma is acceptable and may be beneficial in causing a reduction of tumor growth.31−33 In summary, the HSVtk gene expression vector pEpo-NI2SV-HSVtk, which is dually specific for hypoxia and glioma, induced gene expression and apoptosis in ischemic glioblastoma tissue. In combination with PEI-Dexa as a gene carrier, pEpo-NI2-SV-HSVtk achieved greater therapeutic effects than pSV-HSVtk. Therefore, pEpo-NI2-SV-HSVtk with PEI-Dexa as a carrier may be useful for glioblastoma-specific gene therapy.

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AUTHOR INFORMATION

Corresponding Author

*Department of Bioengineering, College of Engineering, Hanyang University, 17 Haengdang-dong, Seongdong-gu, Seoul 133-791, Republic of Korea. Phone: +82-2-2220-0484. Fax: +82-2-2220-1998. E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was financially supported by the grants from the National Research Foundation of Korea, funded by the Ministry of Science, ICT and Future Planning (2013K000257 and 2013059236).

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