Ultrasound in Med. & Biol., Vol. 40, No. 11, pp. 2662–2670, 2014 Copyright Ó 2014 World Federation for Ultrasound in Medicine & Biology Printed in the USA. All rights reserved 0301-5629/$ - see front matter
http://dx.doi.org/10.1016/j.ultrasmedbio.2014.05.012
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Original Contribution TRANSFECTION OF wtp53 AND Rb94 GENES INTO RETINOBLASTOMAS OF NUDE MICE BY ULTRASOUND-TARGETED MICROBUBBLE DESTRUCTION RUIQI GAO,*y XIYUAN ZHOU,* YINGXUE YANG,* and ZHIGANG WANGzx * Department of Ophthalmology, Second Affiliated Hospital of Chongqing Medical University, Chongqing, China; y Department of Ophthalmology, Dujiangyan Medical Center, Sichuan, China; z Institute of Ultrasonic Imaging, Second Affiliated Hospital of Chongqing Medical University, Chongqing, China; and x Chongqing Key Laboratory of Ophthalmology, Chongqing, China (Received 12 November 2013; revised 9 May 2014; in final form 19 May 2014)
Abstract—Using ultrasound-targeted microbubble destruction (UTMD), we transfected both wild-type p53 (wtp53) and Rb94 genes into retinoblastomas (RBs) of nude mice to investigate the inhibitory role of these two genes in RB development. The 40 tumor-bearing mice, which had been established by sub-retinal injection of an HXO-Rb44 cell suspension, were randomly divided into five groups: blank control group (C); blank plasmid group (M); wtp53 plasmid group (p53); Rb94 plasmid group (Rb94); wtp53 1 Rb94 plasmid group (p53 1 Rb94). For preparation of the DNA-loaded microbubbles, a pre-determined amount of blank plasmid, pVIVO1-p53, pVIVO1-Rb94 or pVIVO1-p53-Rb94 was added and mixed with the microbubbles. Then, these DNA-loaded microbubbles were respectively transfected into the animal model by UTMD. Vascular endothelial growth factor level and microvessel density of the tumor were determined by immunohistochemical staining. Apoptosis of tissues was detected by terminal deoxynucleotidyl transferase dUTP nick end labeling staining. Expression of wtp53 and Rb94 at both the gene and protein levels was detected by RT-PCR (reverse transcription polymerase chain reaction) and Western blot, respectively. Transfection of both genes had greater inhibitory effects on RB development and resulted in lower levels of vascular endothelial growth factor, lower microvessel density and more obvious apoptosis than single-gene transfection (p , 0.05). The results indicate that the transfection of both genes into the RB by UTMD more strongly inhibited RB growth than transfection of a single gene. (E-mail: [email protected]) Ó 2014 World Federation for Ultrasound in Medicine & Biology. Key Words: Ultrasound microbubbles, wtp53 gene, Rb94 gene, Retinoblastoma.
and survival rate by using systemic chemotherapy combined with serial aggressive local therapy (Ferris and Chew 1996; Li et al. 2011). Thus, it is necessary to explore new therapeutic means that can improve the clinical outcomes. Recently, attention has been focused on gene therapy in RB (Zhang et al. 2006). It is now generally recognized that the relationship between the mutation in the RB gene and loss of function in tumor suppressor genes determines the development of RB. Rb94, which lacks the N-terminal 112 amino acid residues of the full-length protein, has a strong tumordepressing effect in head and neck and bladder cancers (Friend et al. 1986; Li et al. 2002; Zhang et al. 2003). The p53 gene has a very close relationship to human malignant neoplasms (Tomkova et al. 2008). The normal p53 gene is also known as wild-type p53 (wtp53), and its expression product plays roles in growth, stress, differentiation, senescence and apoptosis. Mutated p53 (mtp53) is widespread in various human malignancies, including most solid RBs (Ghule et al. 2006; Nork et al. 1997).
INTRODUCTION Retinoblastoma (RB) is the most common malignant intraocular tumor and occurs mainly in children (Huang and Zhang 2008). The worldwide incidence of RB is one case per 15,000–20,000 live births, which amounts to more than 1100 new cases in China every year (Kivela 2009; Luo and Deng 2013). In China, traditional treatment for RB consists of ophthalmectomy, supplemented by radiotherapy and chemotherapy (Li et al. 2011). However, most children still suffer from seriously damaged vision and a decreased quality of life. Therefore, the treatment for RB has gradually developed to improve quality of life
Address correspondence to: Xiyuan Zhou, Department of Ophthalmology, Second Affiliated Hospital of Chongqing Medical University, 74 Linjiang Road, Chongqing 400010, China. E-mail: [email protected] Conflicts of Interest: The authors have indicated that they have no conflicts of interest regarding the content of this article. 2662
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Rather than inhibiting tumor development, mtp53 can promote the occurrence of tumors (Desilet et al. 2010). However, the occurrence and growth of a tumor are the result of mutant gene accumulation and interactions in a complicated gene network. Multiple genes and factors with inhibitory roles in the development of RB have been reported, such as wtp53 and RB (Lai et al. 2012; Sherr and McCormick 2002). It remains unclear whether the combined synergy of wtp53 and RB genes can enhance the inhibition of RB growth. Therefore, we chose the two most prominent tumor suppressor genes, Rb94 and p53, to investigate the inhibitory effect of this combination of genes on RB proliferation. Ultrasound-targeted microbubble destruction (UTMD) is a new research hotspot in gene therapy and drug delivery (Chen et al. 2013a, 2013b; Yang et al. 2013). UTMD can improve the transfection efficiency of another gene vector to enhance inhibition of tumor growth in renal cell carcinoma (Li et al. 2014) and can achieve release of targeted drugs and genes in targeted cells or tumors (Chen et al. 2013a, 2013b; Lin et al. 2013; Ling et al. 2013). In our previous studies, we reported that wtp53 could be effectively transfected into RB cells and RB xenograft tumor tissues with the use of UTMD (Luo et al. 2010) and that the Rb94 plasmid could be transfected into RB cells by UTMD (Zheng et al. 2012). In addition, microbubble-assisted p53, RB and p130 gene transfer in combination with radiation therapy has been reported in prostate cancer (Nande et al. 2013). Moreover, we have found few prior reports on the effects of the combination of p53 and Rb94 on inhibition of RB growth. Thus, we transfected both the wtp53 and Rb94 genes into RBs of nude mice by UTMD and investigated the joint inhibitory role of these two genes in RB development in a mouse model. Vascular endothelial growth factor (VEGF) is one of the principal factors that can induce tumor neovascularization, thus promoting vascular endothelial cell proliferation (Kaio et al. 2003). In addition, microvessel density (MVD), which is the microvascular count of a specific tumor area, is one of the most common indices in tumor angiogenesis research. Angiogenesis is a fundamental event in the process of tumor growth, development and metastasis (Hicklin and Ellis 2005; Xin et al. 2012). Therefore, in the present study, we investigated the inhibitory effects of gene transfection on tumor growth as assessed with these indicators. METHODS Cell culture The human RB tumor cell line HXO-Rb44 (Xu and Zhu 1994), obtained from Hunan Medical University, China, was propagated in RPMI-1640 medium (Invitro-
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gen, Carlsbad, CA, USA) supplemented with 10% fetal bovine serum (FBS), 100 mg/mL penicillin (Sigma, St. Louis, MO, USA) and 100 mg/mL streptomycin (Sigma) at 37 C in 5% CO2. Cell suspensions were prepared after cells were centrifuged at 100g for 5 min, rinsed twice with Hanks’ saline solution and suspended in RPMI-1640 medium without FBS at a concentration of (5.5–6.5) 3 106 cells/mL. Animals The experiment was approved by the local institutional care and animal research committee. Animals were handled in strict accordance with the National Institutes of Health’s Guide for the Care and Use of Laboratory Animals, as well as the Association for Research in Vision and Ophthalmology Statement for the Use of Animals in Ophthalmic and Vision Research. Fifty nude mice (athymic, BALB/c nu/nu) (4 wk old, 15–16 g) originally purchased from the Experimental Animal Center of the University of Chongqing Medical University were used. All mice were housed in specific pathogen-free (SPF) animal care facilities and maintained with free access to food and water. Xenograft tumors in vivo Surgeries were performed in strict accordance with sterile surgical procedures. The ultraclean operative table was exposed to ultraviolet light for 45 min. Nude mice were administered Mydrin-p eyedrops (Santen Pharmaceutical Co., Ltd., Japan) before intraperitoneal anesthesia with 50 mg/kg sodium pentobarbital. Sodium hyaluronate was used to coat the eyes of mice to simulate a supplementary lens. Sub-retinal injections under a dissecting microscope were performed according to previous publications with modifications (Lei et al. 2009; Pang et al. 2006). An aperture within the pupil area was carefully introduced through the superior corneal opening, lens suspensory ligament and sub-retinal space with a 30-gauge needle mounted on a 10-mL syringe. Careful attention was paid to avoid lens damage. The NanoFil sub-microliter injection system (World Precision Instruments, Sarasota, FL, USA) was used to inject 1 mL of the HXO-Rb44 cell suspension at a concentration of (5.5–6.5) 3 106 cells/mL. After subretinal injection, lincomycin eyedrops and antibiotic ophthalmic ointment were topically applied to the operated eye, respectively. Growth of the RB tumor in the mice was observed every 3 d after pentobarbital anesthesia. Anterior and posterior segments of the operated eye were detected using a slit lamp and a direct ophthalmoscope. Throughout the experiment, there was no significant weight loss in the nude mice. Preparation of microbubbles Microbubbles were provided by the Ultrasonographic Image Research Institute of Chongqing Medical
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University, China. They were prepared and characterized according to the published procedure (Ren et al. 2008), with minor modifications: Five milligrams of distearoyl phosphatidylcholine (DSPC, Sigma), 2 mg of dipalmitoyl phosphatidylethanolamine (DPPE, Sigma) and 10% glucose were mixed in a final volume of 0.5 mL of phosphate-buffered saline (PBS) in 1.5-mL vials. The vials were incubated at 37 C for 30 min. Each vial was filled with perfluoropropane gas (C3F8), mechanically shaken for 60 s in a dental amalgamator (Medical Apparatus and Instrument, YJT, Shanghai, China) and kept still for 5 min. This solution was washed with PBS three times and sterilized with 60Co irradiation before use (Zheng et al. 2012). The density of the microbubbles was 1.8 3 109/mL, with a diameter of 3–5 mm. Plasmid construction and in vivo transfections The recombinant plasmids pVIVO1-p53, pVIVO1Rb94 and pVIVO1-p53-Rb94 were constructed at plasmid concentrations of 1 mg/mL. For preparation of the DNA-loaded microbubbles, a pre-determined amount of blank plasmid, plasmid pVIVO1-p53, pVIVO1-Rb94 or pVIVO1-p53-Rb94 was added and gently mixed with the microbubbles. A total of 1 mL of plasmid was gently blended with 1 mL of microbubble suspension, and this 2-mL mixture was incubated for 20 min at 4 C to increase adhesion. The 40 tumor-bearing mice were randomly divided into five groups each containing eight mice: blank control group (C); blank plasmid group (M); wtp53 plasmid group (p53); Rb94 plasmid group (Rb94); wtp53 1 Rb94 plasmid group (p53 1 Rb94). Tail vein injections (1-mL syringe) of 0.1 mL of microbubble suspension containing the respective plasmid were performed manually on the latter four groups. Eyeballs from each group were then immediately exposed to ultrasound. Ultrasonic gene transfection (UGT1025) was carried out by the Ultrasonographic Image Research Institute of Chongqing Medical University, China. The ultrasound probe was placed directly on the eyeball of the nude mouse. Ultrasound (300 kHz, 0.5 W/cm2) was applied, and the working time was controlled at 20% of the 60-s period (4 s irradiation 1 24 s rest 1 4 s irradiation 1 24 s rest 1 4 s irradiation). The tumor tissue was removed after 7 d (Luo et al. 2010). Some tumor tissues were fixed in a 4% paraformaldehyde solution, and other tissues were used for extraction of total RNA and total protein. RT-PCR A 50-mg sample of tumor tissue pestle homogenate was taken from each group. Total RNA was isolated by phenol–chloroform extraction and ethanol precipitation using the Trizol RNA extraction reagent (Life Technol-
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ogy, Gaithersburg, MD, USA). RNA integrity was determined on 2% agarose gels containing ethidium bromide. Polymerase chain reaction was performed with a twostep reverse transcription polymerase chain reaction (RT-PCR) kit (AMV, Takara, Tokyo, Japan) using a PCR system (MyCycler, Bio-Rad), according to a protocol provided by the manufacturer. The primer sequences for wtp53 were 50 -GAGAATCTCCGCAAGAAAGG-30 (sense) and 50 -GCAAGCAAGGGTTCAAAGAC-30 (anti-sense). The primer sequences for Rb94 were 50 (sense) ATGTCGTTCACTTTTACTGAGCTACA-30 and 50 -TCATTTCTCTTCCTTGTTTGAGGTA-30 (antisense). Duplicate PCRs were run using the following amplification protocol: initial denaturation at 94 C for 5 min; 35 cycles of 94 C for 30 s, 58 C for 30 s and 72 C for 2 min; 72 C for 10 min. PCR products were electrophoresed on 2% agarose gels containing ethidium bromide. The mean fluorescence intensity was calculated and statistically analyzed using a gel scanning imaging system (Bio-Rad, Hercules, CA, USA). The bands were normalized with b-actin. Western blot analysis Proteins were extracted using a protein extraction reagent (Pierce, Rockford, IL, USA), according to the protocol provided by the manufacturer. The total protein concentration was determined with the Bradford protein assay. Protein samples were mixed in Laemmli loading buffer and boiled for 5 min. Tissue lysates containing equal amounts of protein were electrophoresed using a 4% stacking gel and a 10% separating gel. The electrophoresed proteins were transferred to a polyvinylidene fluoride membrane (Millipore, Bedford, MA, USA) using a Trans-Blot SD (Pierce). The membranes were washed and blocked in TRIS-buffered saline (1 3 TBS) with 5% non-fat dried milk. Samples were incubated for 1 h at room temperature with mouse anti-human wtp53 antibody (ABCAM, London, UK), mouse anti-human Rb94 antibody (ABCAM) and rabbit-anti-human VEGF antibody (GeneTex, Irvine, CA, USA) at a concentration of 1:1000 in TBS. After incubation for 2 h with the appropriate species-specific horseradish peroxidase-conjugated secondary antibody (ABCAM) at a 1:2000 dilution, immunoreactive bands were visualized with a chemiluminescent substrate (Santa Cruz Biotechnology, Santa Cruz, CA, USA) according to the manufacturer’s instructions. Protein bands were normalized with b-actin, and all blots were quantified with the software Quantity One (Bio-Rad). Histopathology and immunohistochemistry analysis Tumor tissue was embedded in paraffin, and five serial 4-mm-thick paraffin sections from each sample were selected for immunohistochemistry detection using SP-9001 immunohistochemistry assay kits (Beijing
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Fig. 1. Mouse model. (a) Fifteen days after injection: The right eyeball is larger than the left. (b) Twenty-one days after injection: The left eyeball is larger than the right. Tumors induced ‘‘leukocoria,’’ a white pupillary reflex found in the retrolental area. (c) Twenty-eight days after injection: Neoplasms of nude mice have grown out of the eyeballs.
Zhongshan Golden Bridge, Beijing, China). Samples were immunolabeled with rabbit anti-human CD34, a vascular endothelium marker (1:100) (GeneTex), at 37 C for 2 h and then incubated with goat anti-rabbit IgG-HRP secondary antibodies. The specimens were treated with 3,30 -diaminobenzidine, and photographs were taken of the light microscopy images. MVD was calculated by counting the brown-stained CD34positive cells in the vascular endothelial cell capsule per unit area. Tumor stage positively correlates with MVD (Weidner 1995). The cells were counted according to a previously reported method (R€ ossler et al. 2004; Weidner 1995), with minor modifications: The areas with the richest vascularity at 1003 magnification were chosen. Each brown-stained cell or cell cluster was counted as a CD34-positive vessel, which was counted with a square field of 0.25 mm2 at 2003 magnification. The average density of five randomly selected fields of vision was determined to be the MVD.
Detection of tumor tissue apoptosis with the TUNEL assay The TUNEL (terminal deoxynucleotidyl transferase dUTP nick end labeling) Apoptosis Detection Kit (Roche) was used to observe the brown-stained RB nucleus of apoptotic cells. Three high-magnification fields of vision were randomly selected for each slice to count 200 cells, and the apoptotic index (AI) was calculated. AI (%) 5 apoptotic cells/total cells 3 100%.
Fig. 2. Histopathologic characterization of tumor tissues. (a) Tumor cells are rounded or oval with little cytoplasm and karyokinesis (3200). (b) The nuclei are stained dark blue and large, and the cytoplasm is stained pale red; heteromorphism is obvious in the tumor cells (3400).
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Fig. 3. Confirmation of p53 mRNA expression in the different groups by reverse transcription polymerase chain reaction. M 5 DNA marker, 1 5 blank control group, 2 5 blank plasmid group, 3 5 p53 group, 4 5 Rb94 group, 5 5 p53 1 Rb94 group.
Statistical analysis Statistical analyses were performed using SPSS 17.0 software (IBM, Armonk, NY, USA). All data were presented as means 6 standard deviations (SD) and were subjected to analysis of variance (ANOVA). A p-value ,0.05 was considered to indicate a statistically significant difference. RESULTS RB model construction and identification In the present study, 40 nude mice successfully grew tumors and became the animal model of RB (Fig. 1). Manifestations of RB varied with the development of the disease. In the early stage of RB, some large tumors induced leukocoria, and this white pupillary reflex could be found in the retrolental area. In the later stage of RB, the tumors occupied the entire eye, destroyed the normal structure and grew outside of the eye. Tumors that did not exactly grow out of the eye were about 4–5 mm in diameter, and tumors that grew out of the eye were about 7–9 mm in diameter. One nude mouse that had a grown tumor was from each group selected for histopathologic detection to confirm the presence of RB tissue. Hematoxylin–eosin staining revealed that the nucleolus of RB cells became larger and bluer, the cytoplasm became
Fig. 4. Confirmation of Rb94 mRNA expression in the different groups by reverse transcription polymerase chain reaction. M 5 DNA marker, 1 5 blank control group, 2 5 blank plasmid group, 3 5 p53 group, 4 5 Rb94 group, 5 5 p53 1 Rb94 group.
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Fig. 5. Western blot analysis using antibodies against wtp53 and Rb94 in the different groups. ß-Actin was used as an internal reference. 1 5 blank control group, 2 5 blank plasmid group, 3 5 p53 group, 4 5 Rb94 group, 5 5 p53 1 Rb94 group.
smaller and less pink and cell growth was mass-like. The histologic features of the tumor were observed using an optical microscope (Fig. 2). Confirmation of transfected wtp53 and Rb94 recombinant plasmids To verify the validity of transfection, we detected wtp53 and Rb94 expression at the mRNA and protein levels in each group using RT-PCR and Western blot, respectively. RT-PCR results indicated the relative expression of p53 in each group: 0.177 6 0.014 (C), 0.168 6 0.012 (M), 0.650 6 0.070 (p53), 0.158 6 0.017 (Rb94), 0.450 6 0.020 (p53 1 Rb94). p53 mRNA expression was significantly greater in the p53 and p53 1 Rb94 groups than in the other three groups (all p-values,0.05) (Fig. 3). Similarly, expression of the
Fig. 6. (a) Western blot detection of vascular endothelial growth factor (VEGF) protein expression in the different groups. 1 5 blank control group, 2 5 blank plasmid group, 3 5 p53 group, 4 5 Rb94 group, 5 5 p53 1 Rb94 group. (b) Optical density ratio of VEGF/GAPDH in the different groups.
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Fig. 7. Immunohistochemical staining to determine average microvessel density (MVD) in the different groups (3 200). (a) Blank control group. (b) Blank plasmid group. Retinoblastoma cells reacted positively, staining brown in vascular endothelial cells. (c) p53 group. (d) Rb94. Arrows indicate positive reactions (brown staining) in vascular endothelial cells. (e) p53 1 Rb94. Arrows indicate rare positive reactions (brown staining) in VECs. (f) MVD in the different groups.
Rb94 gene was greater in the Rb94 and p53 1 Rb94 groups; the relative levels of expression of Rb94 in each group were 0.00 (C), 0.00 (M), 0.00 (p53), 0.480 6 0.030 (Rb94) and 0.350 6 0.030 (p53 1 Rb94) (Fig. 4). Consistent with the RT-PCR results, Western blot analysis revealed that the wtp53, Rb94 and p53 1 Rb94 groups had an obvious protein band, indicating the imported gene expression at the protein level (Fig. 5). VEGF levels, MVD and tumor cell apoptosis By Western blot analysis, the VEGF protein levels, expressed as the optical density ratio of VEGF/GAPDH (glyceraldehyde-phosphate dehydrogenase), were 0.825 6 0.031 (C), 0.803 6 0.028 (M), 0.761 6 0.026 (p53), 0.776 6 0.021 (Rb94) and 0.568 6 0.020 (p53 1 Rb94). There was significantly less VEGF expression in the p53 1 Rb94 group than in the other four groups (p , 0.05) (Fig. 6).
Microvessel density was calculated by counting the anti-CD34 monoclonal antibody-marked vascular endothelial cells, which were located in the cytomembrane of vascular endothelial cells and contained brownishyellow granules. The MVDs were 14.32 6 2.21 (C), 13.21 6 1.21 (M), 8.95 6 1.01 (p53), 9.34 6 1.12 (Rb94) and 4.21 6 1.01 (p53 1 Rb94). The MVD in the p53 1 Rb94 group was significantly lower than those of the other four groups (p , 0.05) (Fig. 7). Terminal deoxynucleotidyl transferase dUTP nick end labeling staining results indicating that the tumor tissue of the p53 1 Rb94 group had more apoptotic cells with brown-stained nuclei, whereas the p53 and Rb94 groups had only a few apoptotic cells. In addition, the AI of the p53 1 Rb94 group was significantly higher than those of the other four groups (C: 0.46 6 0.05; M: 0.48 6 0.06; wtp53: 5.05 6 0.80; Rb94: 6.43 6 1.02; p53 1 Rb94: 20.35 6 2.14) (p , 0.01) (Fig. 8).
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Fig. 8. Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining for cell apoptosis of tumor tissue in the different groups (3200). (a) Blank control group. (b) Blank plasmid group. Retinoblastoma cells exhibit no obvious positive reaction. (c) p53. (d) Rb94. Arrows indicate rare positive reactions (brown staining) in the nucleus. (e) p53 1 Rb94. Retinoblastoma cells react positively (brown staining) in the nucleus. (f) Apoptosis of tumor tissue in the different groups.
DISCUSSION In the present study, we used a new type of gene carrier, ultrasound microbubble contrast agent, to carry therapeutic genes into the target tissue. It has been reported that this carrier can be injected intravenously and delivered to tissues by the pulmonary circulation. After that, the microbubbles are destroyed by ultrasound irradiation, and the genes enter the target tissues to achieve their therapeutic purpose (Bekeredjian et al. 2003; Feril and Kondo 2004). In previous studies, we tested UTMD parameters to ensure the safety of the animals’ retina and found that the targeted gene could be delivered by UTMD to the retina and inhibit the development of choroidal neovascularization (Deng et al. 2006; Xu et al. 2007; Zhou et al. 2009). At present, some studies have reported the injection of microbubbles containing wtp53
genes into nude mice through tail veins; after ultrasonic irradiation of the RB for a period, the therapeutic genes can be directionally transfected (Luo et al. 2010). The transfection efficiency of two-gene injection was lower than that of single-gene injection, which is one of the inadequacies of this study. We hypothesize that the possible reason may be that under the conditions of the same carrier, the molecular weight of the two genes is greater than that of the single gene, which largely affected transfection efficiency. Determination of the exact reason requires further investigation. Currently, the utilization of microbubbles as a vector in RB treatment under lowintensity ultrasound is still in the preliminary testing stage. Here, we used ultrasonic microbubbles to transfect the plasmids of wpt53 with Rb94 into the tissues of mouse RB to observe the effects of double-gene therapy.
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In the early stage of tumorigenesis, the genes were imported, the eyeballs were removed after 7 d and the tumor was stripped. PT-PCR and Western blot analyses revealed that wtp53 and Rb94 genes were transfected successfully into the tumor tissues. Thus, our results indicate that ultrasonic microbubbles can successfully transfect single genes and combined genes into tumor tissues at both the mRNA and protein levels, although many have reported that it is very difficult to detect the expression of wtp53 and Rb94 proteins in tumor tissues because of the short half-life and low wtp53 protein expression, as well as scarce Rb94 protein products in most RBs (Desilet et al. 2010; Hernando et al. 2004). It was found that VEGF expression was significantly inhibited, the mean MVD was lower and the AI was clearly greater in the group injected with two genes than in the other four groups. On the basis of this experiment, we speculate that the combination of wtp53 and Rb94 genes may increase their anti-cancer versatility and induce the senility and apoptosis of tumor cells. Two crucial issues remain to be resolved. First, more effective transfection of the combined gene delivery by ultrasound with microbubbles is necessary. Second, interactions between the wpt53 and Rb94 genes and how their combination can provide positive anti-tumor effects need to be clarified. Acknowledgments—This project was supported by the Program of the National Natural Science Foundation of China (30872826). This work was also supported by grants from the Institute of Ultrasonic Imaging in the Second Affiliated Hospital and the Chongqing Key Laboratory of Ophthalmology.
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Xin GH, Zhao XH, Liu D, Gong Q, Hou L, Li JY, Pan BR, Li X, Cheng YJ. Effect of VEGF-targeted antisense gene therapy on retinoblastoma cell line SO-RB50 in vitro and in vivo. Int J Ophthalmol 2012;5:440–447. Xu HP, Zhu HC. Characteristics of an established retinoblastoma cell line HXO-Rb44. Chinese Ophthalmol Res 1994;12:183–186. Xu Y, Zhou XY, Wang ZG, Li XS. Experimental study on transferring EGFP gene into the retina of rat mediated by microbubbles. Chin J Med Imaging Technol 2007;2:188–190. Zhang QX, Wang ZG, Ran HT, Fu XP, Li XD, Zheng YY, Peng ML, Chen M, Schutt CE. Enhanced gene delivery into skeletal muscles with ultrasound and microbubble techniques. Acad Radiol 2006; 13:363–367.
Volume 40, Number 11, 2014 Zhang X, Multani AS, Zhou JH, Shay JW, McConkey D, Dong L, Kim CS, Rosser CJ, Pathak S, Benedict WF. Adenoviral-mediated retinoblastoma 94 produces rapid telomere erosion, chromosomal crisis, and caspase-dependent apoptosis in bladder cancer and immortalized human urothelial cells but not in normal urothelial cells. Cancer Res 2003;63:760–765. Zheng MM, Zhou XY, Wang LP, Wang ZG. Experimental research RB94 gene transfection into retinoblastoma cells using ultrasoundtargeted microbubble destruction. Ultrasound Med Biol 2012;38: 1058–1066. Zhou XY, Liao Q, Pu YM, Tang YQ, Gong X, Li J, Xu Y, Wang ZG. Ultrasound-mediated microbubble delivery of pigment epitheliumderived factor gene into retina inhibits choroidal neovascularization. Chin Med J (Engl) 2009;122:2711–2717.
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Original Contribution TRANSFECTION OF wtp53 AND Rb94 GENES INTO RETINOBLASTOMAS OF NUDE MICE BY ULTRASOUND-TARGETED MICROBUBBLE DESTRUCTION RUIQI GAO,*y XIYUAN ZHOU,* YINGXUE YANG,* and ZHIGANG WANGzx * Department of Ophthalmology, Second Affiliated Hospital of Chongqing Medical University, Chongqing, China; y Department of Ophthalmology, Dujiangyan Medical Center, Sichuan, China; z Institute of Ultrasonic Imaging, Second Affiliated Hospital of Chongqing Medical University, Chongqing, China; and x Chongqing Key Laboratory of Ophthalmology, Chongqing, China (Received 12 November 2013; revised 9 May 2014; in final form 19 May 2014)
Abstract—Using ultrasound-targeted microbubble destruction (UTMD), we transfected both wild-type p53 (wtp53) and Rb94 genes into retinoblastomas (RBs) of nude mice to investigate the inhibitory role of these two genes in RB development. The 40 tumor-bearing mice, which had been established by sub-retinal injection of an HXO-Rb44 cell suspension, were randomly divided into five groups: blank control group (C); blank plasmid group (M); wtp53 plasmid group (p53); Rb94 plasmid group (Rb94); wtp53 1 Rb94 plasmid group (p53 1 Rb94). For preparation of the DNA-loaded microbubbles, a pre-determined amount of blank plasmid, pVIVO1-p53, pVIVO1-Rb94 or pVIVO1-p53-Rb94 was added and mixed with the microbubbles. Then, these DNA-loaded microbubbles were respectively transfected into the animal model by UTMD. Vascular endothelial growth factor level and microvessel density of the tumor were determined by immunohistochemical staining. Apoptosis of tissues was detected by terminal deoxynucleotidyl transferase dUTP nick end labeling staining. Expression of wtp53 and Rb94 at both the gene and protein levels was detected by RT-PCR (reverse transcription polymerase chain reaction) and Western blot, respectively. Transfection of both genes had greater inhibitory effects on RB development and resulted in lower levels of vascular endothelial growth factor, lower microvessel density and more obvious apoptosis than single-gene transfection (p , 0.05). The results indicate that the transfection of both genes into the RB by UTMD more strongly inhibited RB growth than transfection of a single gene. (E-mail: [email protected]) Ó 2014 World Federation for Ultrasound in Medicine & Biology. Key Words: Ultrasound microbubbles, wtp53 gene, Rb94 gene, Retinoblastoma.
and survival rate by using systemic chemotherapy combined with serial aggressive local therapy (Ferris and Chew 1996; Li et al. 2011). Thus, it is necessary to explore new therapeutic means that can improve the clinical outcomes. Recently, attention has been focused on gene therapy in RB (Zhang et al. 2006). It is now generally recognized that the relationship between the mutation in the RB gene and loss of function in tumor suppressor genes determines the development of RB. Rb94, which lacks the N-terminal 112 amino acid residues of the full-length protein, has a strong tumordepressing effect in head and neck and bladder cancers (Friend et al. 1986; Li et al. 2002; Zhang et al. 2003). The p53 gene has a very close relationship to human malignant neoplasms (Tomkova et al. 2008). The normal p53 gene is also known as wild-type p53 (wtp53), and its expression product plays roles in growth, stress, differentiation, senescence and apoptosis. Mutated p53 (mtp53) is widespread in various human malignancies, including most solid RBs (Ghule et al. 2006; Nork et al. 1997).
INTRODUCTION Retinoblastoma (RB) is the most common malignant intraocular tumor and occurs mainly in children (Huang and Zhang 2008). The worldwide incidence of RB is one case per 15,000–20,000 live births, which amounts to more than 1100 new cases in China every year (Kivela 2009; Luo and Deng 2013). In China, traditional treatment for RB consists of ophthalmectomy, supplemented by radiotherapy and chemotherapy (Li et al. 2011). However, most children still suffer from seriously damaged vision and a decreased quality of life. Therefore, the treatment for RB has gradually developed to improve quality of life
Address correspondence to: Xiyuan Zhou, Department of Ophthalmology, Second Affiliated Hospital of Chongqing Medical University, 74 Linjiang Road, Chongqing 400010, China. E-mail: [email protected] Conflicts of Interest: The authors have indicated that they have no conflicts of interest regarding the content of this article. 2662
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Rather than inhibiting tumor development, mtp53 can promote the occurrence of tumors (Desilet et al. 2010). However, the occurrence and growth of a tumor are the result of mutant gene accumulation and interactions in a complicated gene network. Multiple genes and factors with inhibitory roles in the development of RB have been reported, such as wtp53 and RB (Lai et al. 2012; Sherr and McCormick 2002). It remains unclear whether the combined synergy of wtp53 and RB genes can enhance the inhibition of RB growth. Therefore, we chose the two most prominent tumor suppressor genes, Rb94 and p53, to investigate the inhibitory effect of this combination of genes on RB proliferation. Ultrasound-targeted microbubble destruction (UTMD) is a new research hotspot in gene therapy and drug delivery (Chen et al. 2013a, 2013b; Yang et al. 2013). UTMD can improve the transfection efficiency of another gene vector to enhance inhibition of tumor growth in renal cell carcinoma (Li et al. 2014) and can achieve release of targeted drugs and genes in targeted cells or tumors (Chen et al. 2013a, 2013b; Lin et al. 2013; Ling et al. 2013). In our previous studies, we reported that wtp53 could be effectively transfected into RB cells and RB xenograft tumor tissues with the use of UTMD (Luo et al. 2010) and that the Rb94 plasmid could be transfected into RB cells by UTMD (Zheng et al. 2012). In addition, microbubble-assisted p53, RB and p130 gene transfer in combination with radiation therapy has been reported in prostate cancer (Nande et al. 2013). Moreover, we have found few prior reports on the effects of the combination of p53 and Rb94 on inhibition of RB growth. Thus, we transfected both the wtp53 and Rb94 genes into RBs of nude mice by UTMD and investigated the joint inhibitory role of these two genes in RB development in a mouse model. Vascular endothelial growth factor (VEGF) is one of the principal factors that can induce tumor neovascularization, thus promoting vascular endothelial cell proliferation (Kaio et al. 2003). In addition, microvessel density (MVD), which is the microvascular count of a specific tumor area, is one of the most common indices in tumor angiogenesis research. Angiogenesis is a fundamental event in the process of tumor growth, development and metastasis (Hicklin and Ellis 2005; Xin et al. 2012). Therefore, in the present study, we investigated the inhibitory effects of gene transfection on tumor growth as assessed with these indicators. METHODS Cell culture The human RB tumor cell line HXO-Rb44 (Xu and Zhu 1994), obtained from Hunan Medical University, China, was propagated in RPMI-1640 medium (Invitro-
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gen, Carlsbad, CA, USA) supplemented with 10% fetal bovine serum (FBS), 100 mg/mL penicillin (Sigma, St. Louis, MO, USA) and 100 mg/mL streptomycin (Sigma) at 37 C in 5% CO2. Cell suspensions were prepared after cells were centrifuged at 100g for 5 min, rinsed twice with Hanks’ saline solution and suspended in RPMI-1640 medium without FBS at a concentration of (5.5–6.5) 3 106 cells/mL. Animals The experiment was approved by the local institutional care and animal research committee. Animals were handled in strict accordance with the National Institutes of Health’s Guide for the Care and Use of Laboratory Animals, as well as the Association for Research in Vision and Ophthalmology Statement for the Use of Animals in Ophthalmic and Vision Research. Fifty nude mice (athymic, BALB/c nu/nu) (4 wk old, 15–16 g) originally purchased from the Experimental Animal Center of the University of Chongqing Medical University were used. All mice were housed in specific pathogen-free (SPF) animal care facilities and maintained with free access to food and water. Xenograft tumors in vivo Surgeries were performed in strict accordance with sterile surgical procedures. The ultraclean operative table was exposed to ultraviolet light for 45 min. Nude mice were administered Mydrin-p eyedrops (Santen Pharmaceutical Co., Ltd., Japan) before intraperitoneal anesthesia with 50 mg/kg sodium pentobarbital. Sodium hyaluronate was used to coat the eyes of mice to simulate a supplementary lens. Sub-retinal injections under a dissecting microscope were performed according to previous publications with modifications (Lei et al. 2009; Pang et al. 2006). An aperture within the pupil area was carefully introduced through the superior corneal opening, lens suspensory ligament and sub-retinal space with a 30-gauge needle mounted on a 10-mL syringe. Careful attention was paid to avoid lens damage. The NanoFil sub-microliter injection system (World Precision Instruments, Sarasota, FL, USA) was used to inject 1 mL of the HXO-Rb44 cell suspension at a concentration of (5.5–6.5) 3 106 cells/mL. After subretinal injection, lincomycin eyedrops and antibiotic ophthalmic ointment were topically applied to the operated eye, respectively. Growth of the RB tumor in the mice was observed every 3 d after pentobarbital anesthesia. Anterior and posterior segments of the operated eye were detected using a slit lamp and a direct ophthalmoscope. Throughout the experiment, there was no significant weight loss in the nude mice. Preparation of microbubbles Microbubbles were provided by the Ultrasonographic Image Research Institute of Chongqing Medical
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University, China. They were prepared and characterized according to the published procedure (Ren et al. 2008), with minor modifications: Five milligrams of distearoyl phosphatidylcholine (DSPC, Sigma), 2 mg of dipalmitoyl phosphatidylethanolamine (DPPE, Sigma) and 10% glucose were mixed in a final volume of 0.5 mL of phosphate-buffered saline (PBS) in 1.5-mL vials. The vials were incubated at 37 C for 30 min. Each vial was filled with perfluoropropane gas (C3F8), mechanically shaken for 60 s in a dental amalgamator (Medical Apparatus and Instrument, YJT, Shanghai, China) and kept still for 5 min. This solution was washed with PBS three times and sterilized with 60Co irradiation before use (Zheng et al. 2012). The density of the microbubbles was 1.8 3 109/mL, with a diameter of 3–5 mm. Plasmid construction and in vivo transfections The recombinant plasmids pVIVO1-p53, pVIVO1Rb94 and pVIVO1-p53-Rb94 were constructed at plasmid concentrations of 1 mg/mL. For preparation of the DNA-loaded microbubbles, a pre-determined amount of blank plasmid, plasmid pVIVO1-p53, pVIVO1-Rb94 or pVIVO1-p53-Rb94 was added and gently mixed with the microbubbles. A total of 1 mL of plasmid was gently blended with 1 mL of microbubble suspension, and this 2-mL mixture was incubated for 20 min at 4 C to increase adhesion. The 40 tumor-bearing mice were randomly divided into five groups each containing eight mice: blank control group (C); blank plasmid group (M); wtp53 plasmid group (p53); Rb94 plasmid group (Rb94); wtp53 1 Rb94 plasmid group (p53 1 Rb94). Tail vein injections (1-mL syringe) of 0.1 mL of microbubble suspension containing the respective plasmid were performed manually on the latter four groups. Eyeballs from each group were then immediately exposed to ultrasound. Ultrasonic gene transfection (UGT1025) was carried out by the Ultrasonographic Image Research Institute of Chongqing Medical University, China. The ultrasound probe was placed directly on the eyeball of the nude mouse. Ultrasound (300 kHz, 0.5 W/cm2) was applied, and the working time was controlled at 20% of the 60-s period (4 s irradiation 1 24 s rest 1 4 s irradiation 1 24 s rest 1 4 s irradiation). The tumor tissue was removed after 7 d (Luo et al. 2010). Some tumor tissues were fixed in a 4% paraformaldehyde solution, and other tissues were used for extraction of total RNA and total protein. RT-PCR A 50-mg sample of tumor tissue pestle homogenate was taken from each group. Total RNA was isolated by phenol–chloroform extraction and ethanol precipitation using the Trizol RNA extraction reagent (Life Technol-
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ogy, Gaithersburg, MD, USA). RNA integrity was determined on 2% agarose gels containing ethidium bromide. Polymerase chain reaction was performed with a twostep reverse transcription polymerase chain reaction (RT-PCR) kit (AMV, Takara, Tokyo, Japan) using a PCR system (MyCycler, Bio-Rad), according to a protocol provided by the manufacturer. The primer sequences for wtp53 were 50 -GAGAATCTCCGCAAGAAAGG-30 (sense) and 50 -GCAAGCAAGGGTTCAAAGAC-30 (anti-sense). The primer sequences for Rb94 were 50 (sense) ATGTCGTTCACTTTTACTGAGCTACA-30 and 50 -TCATTTCTCTTCCTTGTTTGAGGTA-30 (antisense). Duplicate PCRs were run using the following amplification protocol: initial denaturation at 94 C for 5 min; 35 cycles of 94 C for 30 s, 58 C for 30 s and 72 C for 2 min; 72 C for 10 min. PCR products were electrophoresed on 2% agarose gels containing ethidium bromide. The mean fluorescence intensity was calculated and statistically analyzed using a gel scanning imaging system (Bio-Rad, Hercules, CA, USA). The bands were normalized with b-actin. Western blot analysis Proteins were extracted using a protein extraction reagent (Pierce, Rockford, IL, USA), according to the protocol provided by the manufacturer. The total protein concentration was determined with the Bradford protein assay. Protein samples were mixed in Laemmli loading buffer and boiled for 5 min. Tissue lysates containing equal amounts of protein were electrophoresed using a 4% stacking gel and a 10% separating gel. The electrophoresed proteins were transferred to a polyvinylidene fluoride membrane (Millipore, Bedford, MA, USA) using a Trans-Blot SD (Pierce). The membranes were washed and blocked in TRIS-buffered saline (1 3 TBS) with 5% non-fat dried milk. Samples were incubated for 1 h at room temperature with mouse anti-human wtp53 antibody (ABCAM, London, UK), mouse anti-human Rb94 antibody (ABCAM) and rabbit-anti-human VEGF antibody (GeneTex, Irvine, CA, USA) at a concentration of 1:1000 in TBS. After incubation for 2 h with the appropriate species-specific horseradish peroxidase-conjugated secondary antibody (ABCAM) at a 1:2000 dilution, immunoreactive bands were visualized with a chemiluminescent substrate (Santa Cruz Biotechnology, Santa Cruz, CA, USA) according to the manufacturer’s instructions. Protein bands were normalized with b-actin, and all blots were quantified with the software Quantity One (Bio-Rad). Histopathology and immunohistochemistry analysis Tumor tissue was embedded in paraffin, and five serial 4-mm-thick paraffin sections from each sample were selected for immunohistochemistry detection using SP-9001 immunohistochemistry assay kits (Beijing
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Fig. 1. Mouse model. (a) Fifteen days after injection: The right eyeball is larger than the left. (b) Twenty-one days after injection: The left eyeball is larger than the right. Tumors induced ‘‘leukocoria,’’ a white pupillary reflex found in the retrolental area. (c) Twenty-eight days after injection: Neoplasms of nude mice have grown out of the eyeballs.
Zhongshan Golden Bridge, Beijing, China). Samples were immunolabeled with rabbit anti-human CD34, a vascular endothelium marker (1:100) (GeneTex), at 37 C for 2 h and then incubated with goat anti-rabbit IgG-HRP secondary antibodies. The specimens were treated with 3,30 -diaminobenzidine, and photographs were taken of the light microscopy images. MVD was calculated by counting the brown-stained CD34positive cells in the vascular endothelial cell capsule per unit area. Tumor stage positively correlates with MVD (Weidner 1995). The cells were counted according to a previously reported method (R€ ossler et al. 2004; Weidner 1995), with minor modifications: The areas with the richest vascularity at 1003 magnification were chosen. Each brown-stained cell or cell cluster was counted as a CD34-positive vessel, which was counted with a square field of 0.25 mm2 at 2003 magnification. The average density of five randomly selected fields of vision was determined to be the MVD.
Detection of tumor tissue apoptosis with the TUNEL assay The TUNEL (terminal deoxynucleotidyl transferase dUTP nick end labeling) Apoptosis Detection Kit (Roche) was used to observe the brown-stained RB nucleus of apoptotic cells. Three high-magnification fields of vision were randomly selected for each slice to count 200 cells, and the apoptotic index (AI) was calculated. AI (%) 5 apoptotic cells/total cells 3 100%.
Fig. 2. Histopathologic characterization of tumor tissues. (a) Tumor cells are rounded or oval with little cytoplasm and karyokinesis (3200). (b) The nuclei are stained dark blue and large, and the cytoplasm is stained pale red; heteromorphism is obvious in the tumor cells (3400).
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Fig. 3. Confirmation of p53 mRNA expression in the different groups by reverse transcription polymerase chain reaction. M 5 DNA marker, 1 5 blank control group, 2 5 blank plasmid group, 3 5 p53 group, 4 5 Rb94 group, 5 5 p53 1 Rb94 group.
Statistical analysis Statistical analyses were performed using SPSS 17.0 software (IBM, Armonk, NY, USA). All data were presented as means 6 standard deviations (SD) and were subjected to analysis of variance (ANOVA). A p-value ,0.05 was considered to indicate a statistically significant difference. RESULTS RB model construction and identification In the present study, 40 nude mice successfully grew tumors and became the animal model of RB (Fig. 1). Manifestations of RB varied with the development of the disease. In the early stage of RB, some large tumors induced leukocoria, and this white pupillary reflex could be found in the retrolental area. In the later stage of RB, the tumors occupied the entire eye, destroyed the normal structure and grew outside of the eye. Tumors that did not exactly grow out of the eye were about 4–5 mm in diameter, and tumors that grew out of the eye were about 7–9 mm in diameter. One nude mouse that had a grown tumor was from each group selected for histopathologic detection to confirm the presence of RB tissue. Hematoxylin–eosin staining revealed that the nucleolus of RB cells became larger and bluer, the cytoplasm became
Fig. 4. Confirmation of Rb94 mRNA expression in the different groups by reverse transcription polymerase chain reaction. M 5 DNA marker, 1 5 blank control group, 2 5 blank plasmid group, 3 5 p53 group, 4 5 Rb94 group, 5 5 p53 1 Rb94 group.
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Fig. 5. Western blot analysis using antibodies against wtp53 and Rb94 in the different groups. ß-Actin was used as an internal reference. 1 5 blank control group, 2 5 blank plasmid group, 3 5 p53 group, 4 5 Rb94 group, 5 5 p53 1 Rb94 group.
smaller and less pink and cell growth was mass-like. The histologic features of the tumor were observed using an optical microscope (Fig. 2). Confirmation of transfected wtp53 and Rb94 recombinant plasmids To verify the validity of transfection, we detected wtp53 and Rb94 expression at the mRNA and protein levels in each group using RT-PCR and Western blot, respectively. RT-PCR results indicated the relative expression of p53 in each group: 0.177 6 0.014 (C), 0.168 6 0.012 (M), 0.650 6 0.070 (p53), 0.158 6 0.017 (Rb94), 0.450 6 0.020 (p53 1 Rb94). p53 mRNA expression was significantly greater in the p53 and p53 1 Rb94 groups than in the other three groups (all p-values,0.05) (Fig. 3). Similarly, expression of the
Fig. 6. (a) Western blot detection of vascular endothelial growth factor (VEGF) protein expression in the different groups. 1 5 blank control group, 2 5 blank plasmid group, 3 5 p53 group, 4 5 Rb94 group, 5 5 p53 1 Rb94 group. (b) Optical density ratio of VEGF/GAPDH in the different groups.
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Fig. 7. Immunohistochemical staining to determine average microvessel density (MVD) in the different groups (3 200). (a) Blank control group. (b) Blank plasmid group. Retinoblastoma cells reacted positively, staining brown in vascular endothelial cells. (c) p53 group. (d) Rb94. Arrows indicate positive reactions (brown staining) in vascular endothelial cells. (e) p53 1 Rb94. Arrows indicate rare positive reactions (brown staining) in VECs. (f) MVD in the different groups.
Rb94 gene was greater in the Rb94 and p53 1 Rb94 groups; the relative levels of expression of Rb94 in each group were 0.00 (C), 0.00 (M), 0.00 (p53), 0.480 6 0.030 (Rb94) and 0.350 6 0.030 (p53 1 Rb94) (Fig. 4). Consistent with the RT-PCR results, Western blot analysis revealed that the wtp53, Rb94 and p53 1 Rb94 groups had an obvious protein band, indicating the imported gene expression at the protein level (Fig. 5). VEGF levels, MVD and tumor cell apoptosis By Western blot analysis, the VEGF protein levels, expressed as the optical density ratio of VEGF/GAPDH (glyceraldehyde-phosphate dehydrogenase), were 0.825 6 0.031 (C), 0.803 6 0.028 (M), 0.761 6 0.026 (p53), 0.776 6 0.021 (Rb94) and 0.568 6 0.020 (p53 1 Rb94). There was significantly less VEGF expression in the p53 1 Rb94 group than in the other four groups (p , 0.05) (Fig. 6).
Microvessel density was calculated by counting the anti-CD34 monoclonal antibody-marked vascular endothelial cells, which were located in the cytomembrane of vascular endothelial cells and contained brownishyellow granules. The MVDs were 14.32 6 2.21 (C), 13.21 6 1.21 (M), 8.95 6 1.01 (p53), 9.34 6 1.12 (Rb94) and 4.21 6 1.01 (p53 1 Rb94). The MVD in the p53 1 Rb94 group was significantly lower than those of the other four groups (p , 0.05) (Fig. 7). Terminal deoxynucleotidyl transferase dUTP nick end labeling staining results indicating that the tumor tissue of the p53 1 Rb94 group had more apoptotic cells with brown-stained nuclei, whereas the p53 and Rb94 groups had only a few apoptotic cells. In addition, the AI of the p53 1 Rb94 group was significantly higher than those of the other four groups (C: 0.46 6 0.05; M: 0.48 6 0.06; wtp53: 5.05 6 0.80; Rb94: 6.43 6 1.02; p53 1 Rb94: 20.35 6 2.14) (p , 0.01) (Fig. 8).
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Fig. 8. Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining for cell apoptosis of tumor tissue in the different groups (3200). (a) Blank control group. (b) Blank plasmid group. Retinoblastoma cells exhibit no obvious positive reaction. (c) p53. (d) Rb94. Arrows indicate rare positive reactions (brown staining) in the nucleus. (e) p53 1 Rb94. Retinoblastoma cells react positively (brown staining) in the nucleus. (f) Apoptosis of tumor tissue in the different groups.
DISCUSSION In the present study, we used a new type of gene carrier, ultrasound microbubble contrast agent, to carry therapeutic genes into the target tissue. It has been reported that this carrier can be injected intravenously and delivered to tissues by the pulmonary circulation. After that, the microbubbles are destroyed by ultrasound irradiation, and the genes enter the target tissues to achieve their therapeutic purpose (Bekeredjian et al. 2003; Feril and Kondo 2004). In previous studies, we tested UTMD parameters to ensure the safety of the animals’ retina and found that the targeted gene could be delivered by UTMD to the retina and inhibit the development of choroidal neovascularization (Deng et al. 2006; Xu et al. 2007; Zhou et al. 2009). At present, some studies have reported the injection of microbubbles containing wtp53
genes into nude mice through tail veins; after ultrasonic irradiation of the RB for a period, the therapeutic genes can be directionally transfected (Luo et al. 2010). The transfection efficiency of two-gene injection was lower than that of single-gene injection, which is one of the inadequacies of this study. We hypothesize that the possible reason may be that under the conditions of the same carrier, the molecular weight of the two genes is greater than that of the single gene, which largely affected transfection efficiency. Determination of the exact reason requires further investigation. Currently, the utilization of microbubbles as a vector in RB treatment under lowintensity ultrasound is still in the preliminary testing stage. Here, we used ultrasonic microbubbles to transfect the plasmids of wpt53 with Rb94 into the tissues of mouse RB to observe the effects of double-gene therapy.
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In the early stage of tumorigenesis, the genes were imported, the eyeballs were removed after 7 d and the tumor was stripped. PT-PCR and Western blot analyses revealed that wtp53 and Rb94 genes were transfected successfully into the tumor tissues. Thus, our results indicate that ultrasonic microbubbles can successfully transfect single genes and combined genes into tumor tissues at both the mRNA and protein levels, although many have reported that it is very difficult to detect the expression of wtp53 and Rb94 proteins in tumor tissues because of the short half-life and low wtp53 protein expression, as well as scarce Rb94 protein products in most RBs (Desilet et al. 2010; Hernando et al. 2004). It was found that VEGF expression was significantly inhibited, the mean MVD was lower and the AI was clearly greater in the group injected with two genes than in the other four groups. On the basis of this experiment, we speculate that the combination of wtp53 and Rb94 genes may increase their anti-cancer versatility and induce the senility and apoptosis of tumor cells. Two crucial issues remain to be resolved. First, more effective transfection of the combined gene delivery by ultrasound with microbubbles is necessary. Second, interactions between the wpt53 and Rb94 genes and how their combination can provide positive anti-tumor effects need to be clarified. Acknowledgments—This project was supported by the Program of the National Natural Science Foundation of China (30872826). This work was also supported by grants from the Institute of Ultrasonic Imaging in the Second Affiliated Hospital and the Chongqing Key Laboratory of Ophthalmology.
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Volume 40, Number 11, 2014 Zhang X, Multani AS, Zhou JH, Shay JW, McConkey D, Dong L, Kim CS, Rosser CJ, Pathak S, Benedict WF. Adenoviral-mediated retinoblastoma 94 produces rapid telomere erosion, chromosomal crisis, and caspase-dependent apoptosis in bladder cancer and immortalized human urothelial cells but not in normal urothelial cells. Cancer Res 2003;63:760–765. Zheng MM, Zhou XY, Wang LP, Wang ZG. Experimental research RB94 gene transfection into retinoblastoma cells using ultrasoundtargeted microbubble destruction. Ultrasound Med Biol 2012;38: 1058–1066. Zhou XY, Liao Q, Pu YM, Tang YQ, Gong X, Li J, Xu Y, Wang ZG. Ultrasound-mediated microbubble delivery of pigment epitheliumderived factor gene into retina inhibits choroidal neovascularization. Chin Med J (Engl) 2009;122:2711–2717.
