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Experimental Research of RB94 Gene Transfection Into Retinoblastoma Cells Using Ultrasound-Targeted Microbubble Destruction
Ultrasound in Med. & Biol., Vol. 38, No. 6, pp. 1058–1066, 2012 Copyright Ó 2012 World Federation for Ultrasound in Medicine & Biology Printed in the USA. All rights reserved 0301-5629/$ - see front matter
doi:10.1016/j.ultrasmedbio.2012.02.007
Original Contribution
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EXPERIMENTAL RESEARCH OF RB94 GENE TRANSFECTION INTO RETINOBLASTOMA CELLS USING ULTRASOUND-TARGETED MICROBUBBLE DESTRUCTION MIN-MING ZHENG,*z XI-YUAN ZHOU,*z LI-PING WANG,*z and ZHI-GANG WANGy * Department of Ophthalmology, The Second Affiliated Hospital of Chongqing Medical University, Chongqing, P. R. China; Institute of Ultrasonic Imaging, The Second Affiliated Hospital of Chongqing Medical University, Chongqing, P. R. China; and z Chongqing Key Laboratory of Ophthalmology, Chongqing, P. R. China
y
(Received 8 March 2011; revised 5 January 2012; in final form 8 February 2012)
Abstract—The purpose of this study was to explore the transfection of the recombinant expression plasmid pEGFP-C1/RB94 into human retinoblastoma cells (HXO-Rb44) using ultrasound-targeted microbubble destruction (UTMD). pEGFP-C1/RB94 was transfected into HXO-Rb44 in vitro by UTMD, with liposome as the positive control. After 24 to 72 h, the expression of the reporter gene enhanced green fluorescent protein (EGFP) was observed using fluorescent microscopy and flow cytometry. The cell viability of HXO-Rb44 was measured by a MTT assay. The mRNA and proteins of RB94, caspase-3 and Bax were analyzed by reverse transcription polymerase chain reaction (RT-PCR) and Western blot. Moreover, the apoptosis rate and cell cycle progression of the cells were detected by flow cytometry. This study demonstrated that UTMD can enhance the transfection efficiency of RB94, which has an obvious impact on the inhibition of the growth process of retinoblastoma cells, suggesting that the combination of UTMD and RB94 compounds might be a useful tool for use in the gene therapy of retinoblastoma. (E-mail: [email protected]) Ó 2012 World Federation for Ultrasound in Medicine & Biology. Key Words: Ultrasound, Microbubble, Retinoblastoma, Gene transfection, RB94.
genetic defects including both RB(1) and RB(–) tumors (Pirollo et al. 2008). Differences reported thus far include the observations that RB94 has a longer half-life than RB110, remains in its active hypophosphorylated form for an extended period of time and causes unusual nuclear morphologic changes (Zhou et al. 2009a). Thus, it appears that RB94 is a more potent tumor-suppressing agent than RB110. Albeit recent advances in enucleation and conservative treatments, there has been no improvement in the 5-year survival rate in children. Therefore, it is urgent to explore novel therapeutic strategies that can improve the clinical outcomes. Recently, gene therapy has been gaining necessity in RB therapy (Zhang et al. 2006). Nevertheless, efficient, systemic delivery of the gene encoding RB94 specifically to tumors, is thought to be an obstacle to clinical application as an anticancer therapeutic. Updated studies have shown that ultrasoundtargeted microbubble destruction (UTMD) is regarded as a noninvasive gene transfer technology that provides a new means of gene therapy for retina disease (Zhou et al. 2009b). Microbubbles currently used as ultrasound
INTRODUCTION Retinoblastoma (RB) is the most common malignant intraocular tumor in children representing 2.5% to 4% of all pediatric cancers. There are about 1000 new cases of RB annually in China, accounting for 20% of new cases worldwide (Chen 2007). RB gene, which is located on a region of the long (q) arm of chromosome 13 designated 13q14, is most closely related with the incidence of retinoblastoma. As a fact, mutations in the RB gene are responsible for most cases of retinoblastoma. RB94 is produced by the translation of the RB gene from the second in-frame AUG codon and lacks the 112 amino acids on the NH2 terminus present in RB110 (Xu et al. 1994). RB94 has markedly increased tumor suppressor potency compared with RB110 and is active against all tumor types examined to date, despite their specific
Address correspondence to: Xi-Yuan Zhou, Department of Ophthalmology, The Second Affiliated Hospital of Chongqing Medical University, 74# Linjiang Road, Chongqing 400010, P. R. China. E-mail: [email protected] 1058
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contrast agents can lower the energy threshold of ultrasound for cavitation, which transiently perforates the cell membrane or disrupts the capillary wall to allow delivery of bioactive agents into the cells or the interstitial space (Taniyama et al. 2002a). In this regard, ultrasound with microbubbles has been considered as a novel vehicle for gene therapy. In our previous studies, we have demonstrated that the efficiency of wild-type p53 (wtp53) plasmid transfection into Y79 cells and RB xenograft tumour tissues mediated by UTMD (Luo et al. 2010). In this context, we used RB94 transfecting into RB cells in the same way and examined the efficiency of RB94 on the inhibition of the growth process of retinoblastoma. RB94, compared with other tumor suppressor gene such as p53, is strongly associated with the incidence of retinoblastoma (Calo et al. 2010; Laurie et al. 2006). Therefore, RB94 is a better candidate gene for gene therapy of retinoblastoma than wtp53. In addition, we have found no prior reports studying the effects of using RB94 to inhibit the growth of retinoblastoma. Importantly, we first explored the transfection of RB94 into human retinoblastoma cells using UTMD. Hereby, we employed a eukaryotic coexpression vector (pEGFP-C1/RB94) coding with enhanced green fluorescent protein (EGFP) and RB94, which was efficiently carried by an ultrasound contrast agent that we developed. The UTMD was used for the treatment of retinoblastoma. The study provided reliable experimental evidence for studying and evaluating gene transfer mediated by UTMD, which could be a novel strategy for the gene therapy of RB. MATERIALS AND METHODS Cell culture Human retinoblastoma cells (HXO-Rb44) were obtained from Hunan Medical University in China and grown in RIPA 1640 medium (Gibco, San Jose, CA, USA) containing 10% fetal bovine serum (FBS), 100 mg/mL streptomycin (Sigma, St. Louis, MO, USA), 100 U/mL penicillin (Sigma). Microbubble preparation The microbubble was prepared and characterized as previously described (Ren et al. 2008), with minor modifications. Briefly, 5 mg 1,2-distearoyl-sn-glycerophosphatidylcholine (DSPC; Sigma), 2 mg 1,2-dipalmitoyl-sn-glycero-3-phosphor-ethanolamine (DPPE; Sigma) and 10% glucose were dissolved in phosphate-buffered saline (PBS) to a final volume of 0.5 mL in 1.5-mL vials. The vials were incubated at 37 C for 30 min. The headspace of each vial was filled with the perfluoropropane gas and then the vial was mechanically shaken for 60 s
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using a dental amalgamator (medical apparatus and instrument, YJT, Shanghai, China). This solution was then diluted with 0.5 mL PBS and sterilized by 60Co irradiation. The density of the microbubbles was 1.2 3 109/mL, with a diameter of 4–6 mm and a half-life of about 6 h. Cell transfection A eukaryotic coexpression vector (pEGFP-C1/ RB94) coded with EGFP and RB94 was constructed in our previous work (Zheng et al. 2010). The pEGFP-C1/ RB94 and pEGFP-C1 plasmids grown in Escherichia coli were purified and suspended in 2.5 mM Tris-HCL (pH 8.5) at a concentration of 1.0 mg/mL. For preparation of the DNA-loaded microbubbles, a quantified amount of plasmid pEGFP-C1/RB94 and pEGFP-C1 plasmids were respectively added and gently mixed with the microbubbles. We lightly blended 4.0 mg of plasmid (4.0 mL) with 200 mL of the microbubble suspension and gently incubated the mixture for a few minutes at 4 C to add in adhesion. The cells were transfected with pEGFP-C1 vector using LipofectamineÔ2000 (Invitrogen, Carlsbad, CA, USA), which has a better membrane affinity and DNA condensation and can enhance the in vitro transfection efficiency, according to the manufacturer’s protocol. We also lightly blended 4.0 mg of plasmid (4.0 mL) with 250 mL of RPMI-1640 medium or 12 mL of liposome plus 250 mL of RPMI-1640 medium and then mixed the two at room temperature for 20 min. The gene transfer machine (UGT 1025 type, Chongqing, China) was produced by Ultrasonographic Image Research Institute of Chongqing Medical University. The following parameters for the ultrasound treatment of HXO-Rb44 cells were used: continuous wave, 300 KHz, 0.5 W/cm2, 30 s and a 10% concentration of microbubbles (Luo and Zhou 2010). Cultured HXO-Rb44 cells were divided into six groups: (1) control (no treatment); (2) pEGFPC1/RB94 plasmid (RB94 plasmid) with ultrasound; (3) RB94 plasmid with microbubbles; (4) RB94 plasmid with microbubbles and ultrasound; (5) pEGFP-C1 plasmid (C1 plasmid) with microbubbles and ultrasound; and (6) RB94 plasmid with liposome. After incubating for 6 h at 37 C with 5% CO2, the compound solution was replaced with 500 mL of fresh media containing 10% fetal bovine serum and 1% penicillin/streptomycin. The cells were cultured for another 24 h and the reporter EGFP expression was analyzed. All transfection experiments were performed in triplicate. Cell transfection analysis Forty-eight hours after transfection, the EGFP expression of all groups in HXO-Rb44 was observed using fluorescent microscopy. The cells were washed three times with warm sterile D-Hamks and treated with 0.125% trypsin for 2 min at 37 C. Cells were
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collected by centrifugation at 1000 rpm and were resuspended in 500 mL PBS. Transfection efficiency was analyzed using flow cytometry (FACS-Calibur; Becton Dickinson, San Jose, CA, USA) with the excitation setting at 488 nm. At least 10,000 cells were acquired and the data were analyzed by the software CellQuest (BD Company, New York, NY, USA). MTT assay The influence of all treated factors in HXO-Rb44 on cell viability was assessed with the 3-(4,5-dimthylthiazol2-yl)-2,5 diphenyl-tetrazolium bromide (MTT) assay. HXO-Rb44 cells were seeded in 96-well plates at a density of 13104 cells per well. When the cells grew to about 60%–80% in confluence, the old medium was replaced with 200 mL of 1640 without FBS and penicillin/streptomycin. Then, every treated factor was added to each respective well and all samples were generated in triplicate. Cells were incubated at 37 C and inspected daily for 5 days. Cell growth was determined by adding 20 mL of MTT (5 mg/mL) to each well and incubating for an additional 4 h at 37 C. The supernatant was discarded and 150 mL of dimethyl sulfoxide were added. Absorbance was determined by spectrophotometry using a wavelength of 570 nm, with 630 nm as a reference. Western blotting Proteins were extracted using a protein extraction reagent (Pierce, Rockford, IL, USA), following the protocol provided by the manufacturer. The total protein concentration was determined with the Bradford protein assay (Bradford 1976). Protein samples were mixed in Laemmli loading buffer and boiled for 5 min. Equal protein levels of cell lysates were electrophoresed using a 4% stacking gel and a 10% separating gel. The electrophoresed proteins were transferred to a polyvinylidene fluoride membrane (PVDF; Millipore, Bedford, MA, USA) using a trans-blot SD semi-dry transfer cell (Pierce). Completion of protein transfer from the gels to the membranes was checked by staining the gels with Coomassie Blue R-250. Western blotting was performed as described by Towbin et al. (1979). The membranes were washed and blocked in TRISbuffered saline (13TBS) with 5.0% nonfat dried milk. Samples were incubated for 1 h at room temperature with primary caspase-3 antibody (ab2302; Abcam, London, UK), Bax antibody (ab10813; Abcam) and mouseantihuman RB (ab24; Abcam), which recognizes both full-length RB and NH2-terminal-truncated RB94 proteins, at a concentration of 1:1000 in TBS. After 2 h of incubation with the appropriate species-specific horseradish peroxidase-conjugated secondary antibody (ab8227; Abcam) at a 1:2000 dilution rate, immunoreactive bands were visualized with a chemiluminescent substrate (Santa Cruz Biotechnology, Santa Cruz, CA, USA) according to
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the manufacturer’s instructions. The protein bands were normalized with b-actin and all blots were quantified with the software Quantity One (Bio-Rad, Hercules, CA, USA). Reverse transcription-polymerase chain reaction (RT-PCR) The total RNA was extracted by phenol-chloroform extraction and ethanol precipitation using TRIZOL (Life Technology, Gaithersburg, MD, USA). The integrity of RNA was detected by 1.5% agarose gels containing ethidium bromide. The polymerase chain reaction was performed with a two-step RT-PCR kit (AMV; Takara, Tokyo, Japan) using a PCR system (MyCycler; BioRad), following a protocol provided by the manufacturer. Sequences of the primers for RB94 are: 50 -CCTCTCGTC AGGCTTGAGTTTG-30 (sense) and 50 -TTGAGCAACA TGGGAGGTGAG-30 (antisense). Sequences of the primers for caspase-3 are: 50 -TGAAGGCAAGGTGCTA AA-30 (sense) and 50 -CTGGCTCAAACCACATTCTC-30 (antisense). Sequences of primers for Bax are: 50 -ATG CGTCCACCAAGAAGCTGAG-30 (sense) and 50 -CCCC AGTTGAAGTTGCCATCAG-30 (antisense). The PCR was performed at 94 C for 2 min at first, 94 C for 30 s, 55 C for 30 s, 72 C for 45 s followed by 35 cycles, 72 C for 10 min at last. The PCR products were affirmed on 1.5% agarose gels containing ethidium bromide. The products were separated alongside a 100-bp DNA molecular weight ladder (Promega, Southampton, UK) for sizing. The bands were normalized with b-actin. Cell cycle analysis Both the control and treated cells were harvested with trypsin-EDTA and washed in 1 mL of cold PBS. Approximately 13106 cells from each treatment were resuspended in 0.5 mL of propidium iodide solution (50 mg/ mL propidium iodide, 100 mg/mL RNase A and 0.2% Triton X-100). Cells were then incubated at 4 C for 30 min in the dark. All measurements of cell cycle distribution were performed on a flow cytometer (FACS-Calibur; Becton Dickinson, CA, USA). Data from over 30,000 cells were collected and analyzed by the Multiple Software (Phoenix Flow Systems, San Diego, CA, USA). Detection of apoptosis rate HXO-Rb44 cells were collected by centrifugation (1000 r/min 3 5 min) and washed twice with incubation buffer (10 mmol/L HEPES/NaOH, 140 mmol/L NaCl, 5 mmol/L CaCl2). Approximately 5 3 106 cells were collected. We added 100 mL of binding buffer to suspend the cells and then added 2 mL of the FITCAnnexin V Apoptosis Detection Kit (eBioscience, San Diego, CA, USA) and 5 mL of propidium iodide. The reaction was left in the dark at room temperature for 10–15 min. The cells were collected again by
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centrifugation (500 r/min 3 5 min) and washed in incubation buffer. SA-FLOUS solution was applied for 20 min in the dark at 4 C. The apoptosis rate was detected by flow cytometry. Statistical analysis The statistical analyses were all performed by using SPSS 17.0 software (SPSS Inc., Chicago, IL, USA). All the data were presented as mean 6 SD and were analyzed by using ANOVA tests. Means with P , 0.05 were considered to be statistically different from one another. RESULTS Fluorescent microscopy At 24 h post transfection, EGFP expression of all groups in HXO-Rb44 was observed using fluorescent microscopy (Fig. 1). Figure 1 shows that only minute amounts of green fluorescence were observed in the blanked control group while the obvious green fluorescence was very prominent in the other groups. Green fluorescence was more intense for the RB94 plasmid with microbubbles and ultrasound, the C1 plasmid with microbubbles and ultrasound and the RB94 plasmid with liposome than in other groups. Transfection efficiency Quantitative analysis of gene expression was undertaken to determine the efficiency of gene transfection for all groups. Positive EGFP expression was detected using flow cytometry 24 h post transfection. Transfection efficiency was measured by the percentage of cells that expressed EGFP in the population (Fig. 2). Figure 2 shows that the transfection efficiencies of the control, RB94 plasmid1microbubbles, RB94 plasmid1ultrasound, RB94 plasmid1microbubbles1ultrasound, C1 plasmid1 microbubbles1ultrasound and RB94 plasmid1liposome groups were 0.48%, 6.51%, 7.07%, 23.59%, 22.09% and 26.04%, respectively. The transfection efficiencies of the last three groups were higher than those of the other groups. The difference between the three groups was not statistically significant. MTT assay The influence of all treated factors in HXO-Rb44 on cell viability was evaluated by the MTT assay (Fig. 3). Those cells treated with RB94 plasmid1microbubbles1 ultrasound and RB94 plasmid1liposome were seen to undergo an apoptotic-like cytological change, losing their anchorage dependence and assuming a small spherical shape. Cells were collected and counted from day 0 through day 5 and cell growth curves were generated. In multiple trials, cell growth curves were seen to flatten and rise slightly in RB94 plasmid1microbubbles1 ultrasound and RB94 plasmid1liposome groups
Fig. 1. Fluorescent microscopic images of pEGFP-C1 expression in HXO-Rb44(3200). Cells were transfected with all treated factors. Cells were cultured for 24 h and the reporter gene expression was observed using fluorescent microscopy. (a) and (b) Blanked control group. (c) and (d) HXO-Rb44 transfected with pEGFP-C1/RB94 using ultrasound. (e) and (f) HXO-Rb44 transfected with pEGFP-C1/RB94 using microbubble. (g) and (h) HXO-Rb44 transfected with pEGFP-C1 using ultrasoundtargeted microbubble destruction. (i) and (j) HXO-Rb44 transfected with pEGFP-C1/RB94 using ultrasound-targeted microbubble destruction. (k) and (l) HXO-Rb44 transfected with pEGFP-C1/RB94 using liposome. Bright field images (b),(d), (f), (h), (j) and (l); fluorescence images (a), (c), (e), (g), (i) and (k).
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Fig. 2. Transfection efficiency in HXO-Rb44. The percentage of EGFP-positive cells was determined using flow cytometry 24 h post transfection (b) along with a computer-generated bar graph analysis (a). Data are represented as mean 6 SD (n 5 3). C indicates the blanked control group; RB94 indicates the pEGFP-C1/RB94 plasmid; M indicates microbubbles; U indicates ultrasound; L indicates liposome; and C1 indicates the pEGFP-C1 plasmid. The transfection efficiencies of the last three groups were higher than those of the other groups. The difference between the three groups was not statistically significant.
compared with other groups where cellular division continued at a brisk pace.
microbubbles1ultrasound and RB94 plasmid1liposome groups were more prominent than those of the RB94 plasmid1microbubbles and RB94 plasmid1ultrasound groups.
Evaluation of RB94 expression after transfection
RT-PCR. Consistent with the results of the Western blot analysis, RT-PCR revealed that the expression of RB94 mRNA in the RB94 plasmid1microbubbles1 ultrasound and RB94 plasmid1liposome groups was more remarkable than those of the other groups (Fig. 5).
Western blotting. Western blot analysis of RB protein expression following RB94 gene transfer revealed a band reactive to the anti-RB antibody at 94 kDa (RB94) in the RB94 plasmid1microbubbles1ultrasound, RB94 plasmid1 liposome, RB94 plasmid1microbubbles and RB94 plasmid1ultrasound groups (Fig. 4). Furthermore, 94-kDa bands were not seen in the other two groups. However, the bands of the RB94 plasmid1
Cell cycle analysis. To investigate the mechanism behind the antitumor activity, cell cycle studies were performed to determine the fate of the cells after RB94 gene transfer. Flow cytometry was performed along with cell
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Fig. 3. RB94 gene transfer suppresses tumor cell growth. Growth curves for HXO-Rb44 cell after transfection with all treated factors. Mean cell numbers were assessed for 6 consecutive days. Transfection with pEGFP-C1/RB94 using ultrasound-targeted microbubble destruction and liposome caused a significant flattening of cell growth curves in HXORb44 when compared with other groups. C indicates the blanked control group; RB94 indicates the pEGFP-C1/RB94 plasmid; M indicates microbubbles; U indicates ultrasound; L indicates liposome; and C1 indicates the pEGFP-C1 plasmid.
cycle analysis (Fig. 6). In repeated experiments, flow cytometry showed that the RB94 plasmid1 microbubbles1 ultrasound and RB94 plasmid1liposome groups go through cell arrest in the G2-M phase of the cell cycle. Apoptosis studies. Having witnessed the cell cycle of HXO-Rb44 cells transfected with the RB94 gene, an examination of apoptosis-related indicators was performed. The Western blot analysis showed that the protein expressions of caspase-3 and Bax in the RB94 plasmid1microbubbles1ultrasound and RB94 plasmid1liposome groups were more conspicuous than those in other groups (Fig. 4). The results of RT-PCR were
Fig. 4. Western blot analysis for the presence of RB protein, caspase-3 protein and Bax protein from HXO-Rb44. Cells were transfected with all treated factors. The cells were harvested and the proteins were subjected to SDSpolyacrylamide gel electrophoresis and probed by Western blot analysis with antibodies to RB, caspase-3 and Bax. Cells transfected with pEGFP-C1/RB94 using ultrasound-targeted microbubble destruction and liposome showed more prominent bands than other groups. C indicates the blanked control group; RB94 indicates the pEGFP-C1/RB94 plasmid; M indicates microbubbles; U indicates ultrasound; L indicates liposome; and C1 indicates the pEGFP-C1 plasmid. b-actin was used as an inner reference.
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Fig. 5. RT-PCR analysis of the expression of RB94 mRNA, caspase-3 mRNA and Bax mRNA in HXO-Rb44. Cells were transfected with all treated factors. RNA was prepared and analyzed by RT-PCR. Cells transfected with pEGFP-C1/RB94 using ultrasound-targeted microbubble destruction and liposome showed more prominent bands than other groups. C indicates the blanked control group; RB94 indicates the pEGFP-C1/ RB94 plasmid; M indicates microbubbles; U indicates ultrasound; L indicates liposome; and C1 indicates the pEGFP-C1 plasmid. b-actin was used as an inner reference.
well-matched with those of the Western blot analysis. Caspase-3 and Bax mRNA expressions in the RB94 plasmid1microbubbles1ultrasound and RB94 plasmid1liposome groups were noticeable compared with those in the other groups (Fig. 5). The apoptotic indexes for the RB94 plasmid1microbubbles1ultrasound and RB94 plasmid1liposome groups were found to be 28.7% and 28.6%, respectively, which are significantly higher than those obtained for the other groups (Fig. 7). The difference between the two groups was not statistically significant. DISCUSSION This study demonstrates a new method for gene therapy. Ultrasound can create transient nonlethal perforations in cells and other membranes, which allow large molecules from the surrounding medium to enter the cell without remarkable damage so that the cell can reseal its membrane and survive (Unger et al. 2001; Nozaki et al. 2003). The effect is potentiated by the use of microbubbles, which act as cavitation nuclei. Microbubbles were applied in combination with the use of ultrasound and are particularly effective for gene delivery (Taniyama et al. 2002b). Based on the theory mentioned above, we prepared ultrasound microbubbles in which the gene was loaded efficiently. Then, the UTMD method was used to transfer the gene into the cells. Our previous work showed that the most amounts of cells were able to survive under the conditions of a 0.5 W/cm2 sound intensity, 30 s irradiation time and 10% microbubble concentration. Therefore, we chose these optimal conditions in the present study. Under fluorescent microscopy, the intensity of green fluorescence of the RB94 plasmid with microbubbles and
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Fig. 6. RB94 gene transfer induces G2-M cell cycle arrest. DNA content flow cytometric profiles after transfection with pEGFP-C1/RB94 using ultrasound-targeted microbubble destruction and liposome in HXO-Rb44 (a) along with a computer-generated bar graph analysis (b) and (c). (b) indicated the cell distribution of ultrasound- targeted microbubble transfection of the RB94 gene in RB cells. (c) indicated the cell cycle distribution of liposome-targeted transfection of the RB94 gene in RB cell. Transfection with pEGFP-C1/RB94 using ultrasound-targeted microbubble destruction and liposome induced G2-M cell cycle arrest was observed in both of them.
ultrasound, C1 plasmid with microbubbles and ultrasound and RB94 plasmid with liposome was more intense than those of the other groups. Flow cytometry showed that the transfection efficiency of RB94 plasmid with microbubbles and ultrasound group was 23.59%. Compared
with RB94 plasmid with liposome group, the difference between the two groups was not statistically significant. This revealed that we successfully transfected the RB94 gene into the target cells by UTMD method. The MTT values of RB94 plasmid with microbubbles and
Fig. 7. Flow cytometry was used to detect the apoptosis rate in the six groups. The apoptotic index for the RB94 plasmid1microbubbles1ultrasound and RB94 plasmid1 liposome groups was found to be 28.7% and 28.6%, respectively, whereas only a few apoptotic cells were detected in the controls. The difference between the two groups was not statistically significant. C the indicates blanked control group; RB94 indicates the pEGFP-C1/RB94 plasmid; M indicates microbubbles; U indicates ultrasound; L indicates liposome; and C1 indicates the pEGFP-C1 plasmid.
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ultrasound group and RB94 plasmid with liposome group were dramatically less than those in the other groups, indicating that the growth of the two groups of tumor cells had been restrained. Western blot analysis and RT-PCR were used to detect the RB94 gene expression. The results demonstrated that the RB94 protein was well expressed in RB94 plasmid with microbubbles and ultrasound group and RB94 plasmid with liposome group, whereas there was less expression, if at all, in other groups. This was confirmed at the mRNA level of RB94 by RT-PCR analysis as well. The results mentioned above suggest that, as a novel transfection medium, microbubbles can meet the need for efficient gene transfer. These conclusions were also confirmed by cell cycle analysis and apoptosis studies. It has been reported that Ad-RB94 gene therapy in pancreatic cancer cells result in antiproliferative effects, apoptosis induction and S-G2 cell cycle arrest in tumor cells (Roig et al. 2004). Another study confirmed that Ad-RB94 gene transfer induces G2-M cell cycle arrest in head and neck cancer cells (Li et al. 2002). The common factor of these findings was the cell cycle progression through the G1 phase and the shift of the cell cycle arrest to G2 after Ad-RB94 gene therapy (Araki et al. 2008). Accordingly, cancer cells transfected with the RB94 gene had a striking effect on cell cycle distribution, resulting in an increase in cells arrested in the S-G2 phase. In our experiments, flow cytometry showed that the RB94 plasmid1microbubbles1 ultrasound and RB94 plasmid1liposome groups of cells arrested in the G2-M phase of the cell cycle after 48 h. Apoptosis appears to be the main phenomenon resulting in significant cell death and cell growth inhibition (Zheng et al. 2010). Several factors contribute to apoptosis but the key elements are categorized into two main families of proteins: the caspase enzymes and the Bcl-2 family (Thornberry and Lazebnik 1998). The bcl-2 family is a set of cytoplasmic proteins that regulate apoptosis. The two main groups of this family, Bcl-2 and Bax proteins, are functionally opposed: Bcl-2 acts to inhibit apoptosis, whereas Bax counteracts this effect (Ashkenazi and Dixit 1998; Kroemer 1997). Caspases are crucial mediators of programmed cell death (apoptosis). Among them, caspase-3 is a frequently activated death protease, catalyzing the specific cleavage of many key cellular proteins (Porter and Janicke 1999; Kroemer 1997). Thus, we studied Bax and caspase-3 protein in HXORb44 cells. Detection of caspase-3 and Bax in cancer cells treated with RB94 would be indicative of ongoing apoptosis. We discovered that the expression of caspase-3 and Bax protein was evident in the RB94 plasmid1 microbubbles1ultrasound and RB94 plasmid1liposome groups while it showed less expression in the other groups. In the current study, we observed that the apoptosis rates in the RB94 plasmid1microbubbles1ultrasound and RB94
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plasmid1liposome groups were significantly higher compared with those of the other groups, indicating apoptotic-like cytological changes in the cells. Above all, cells transfected successfully with the RB94 gene induced apoptosis. This study approved for the first time that UTMD can build up the transfection efficiency of RB94, which has an obvious impact on the inhibition of the growth process of retinoblastoma cells, indicating that the combination of UTMD and RB94 could be a useful method for application in the gene therapy of retinoblastoma. Nevertheless, gene therapy for retinoblastoma using an ultrasound-mediated microbubble in the experimental study is still in its infancy and it is still far from ready to be used in clinical applications. There are still some unresolved issues. The key problem is that gene transfection efficiency is still low compared with the vital vectors, which cannot live up to the expectation for clinical gene therapy in retinoblastoma. Two crucial issues need to be addressed: (1) the further optimization of the ultrasound, microbubble and gene parameters and (2) an increase in the gene-loading efficiency for the microbubbles. With these issues resolved, the prospect of gene therapy for retinoblastoma using ultrasoundmediated microbubbles will have a broader application. Acknowledgments—This work was supported by grants from the Institute of Ultrasonic Imaging in the Second Affiliated Hospital and Chongqing Key Laboratory of Ophthalmology. This project was also supported by the Program of National Natural Science Foundation of China (30872826). Dr. Z. G. Wang is the Director of the Institute of Ultrasonic Imaging, whose help was instrumental in allowing us to complete this study.
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Nozaki T, Ogawa R Jr, Feril LB, Kagiya G, Fuse H, Kondo T. Enhancement of ultrasound-mediated gene transfection by membrane modification. J Gene Med 2003;5:1046–1055. Pirollo KF, Rait A, Zhou Q, Zhang XQ, Zhou J, Kim CS, Benedict WF, Chang EH. Tumor-targeting nanocomplex delivery of novel tumor suppressor RB94 chemosensitizes bladder carcinoma cells in vitro and in vivo. Clin Cancer Res 2008;14:2190–2198. Porter AG, Janicke RU. Emerging roles of caspase-3 in apoptosis. Cell Death Differ 1999;6:99–104. Ren JL, Wang ZG, Zhang Y, Zheng YY, Li XS, Zhang QX, Wang ZX, Xu CS. Transfection efficiency of TDL compound in HUVEC enhanced by ultrasound-targeted microbubble destruction. Ultrasound Med Biol 2008;34:1857–1867. Roig JM, Molina MA, Cascante A, Calbo J, Carbo N, Wirtz U, Sreedharan S, Fillat C, Mazo A. Adenovirus-mediated retinoblastoma 94 gene transfer induces human pancreatic tumor regression in a mouse xenograft model. Clin Cancer Res 2004;10:1454–1462. Taniyama Y, Tachibana K, Hiraoka K, Aoki M, Yamamoto S, Matsumoto K, Nakamura T, Ogihara T, Kaneda Y, Morishita R. Development of safe and efficient novel nonviral gene transfer using ultrasound: Enhancement of transfection efficiency of naked plasmid DNA in skeletal muscle. Gene Ther 2002a;9:372–380. Taniyama Y, Tachibana K, Hiraoka K, Namba T, Yamasaki K, Hashiya N, Aoki M, Ogihara T, Yasufumi K, Morishita R. Local delivery of plasmid DNA into rat carotid artery using ultrasound. Circulation 2002b;105:1233–1239.
Volume 38, Number 6, 2012 Thornberry NA, Lazebnik Y. Caspases: Enemies within. Science 1998; 281:1312–1316. Towbin H, Staehelin T, Gordon J. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc Natl Acad Sci USA 1979;76:4350–4354. Unger EC, Hersh E, Vannan M, McCreery T. Gene delivery using ultrasound contrast agents. Echocardiography 2001;18:355–361. Xu HJ, Xu K, Zhou Y, Li J, Benedict WF, Hu SX. Enhanced tumor cell growth suppression by an N-terminal truncated retinoblastoma protein. Proc Natl Acad Sci U S A 1994;91:9837–9841. Zhang Q, Wang Z, Ran H, Fu X, Li X, Zheng Y, Peng M, Chen M, Schutt CE. Enhanced gene delivery into skeletal muscles with ultrasound and microbubble techniques. Acad Radiol 2006;13: 363–367. Zheng Y, Zhou M, Ye A, Li Q, Bai Y, Zhang Q. The conformation change of Bcl-2 is involved in arsenic trioxide-induced apoptosis and inhibition of proliferation in SGC7901 human gastric cancer cells. World J Surg Oncol 2010;8:31. Zhou J, Zhang XQ, Ashoori F, McConkey DJ, Knowles MA, Dong L, Benedict WF. Early RB94-produced cytotoxicity in cancer cells is independent of caspase activation or 50 kb DNA fragmentation. Cancer Gene Ther 2009a;16:13–19. 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) 2009b;122:2711–2717.
doi:10.1016/j.ultrasmedbio.2012.02.007
Original Contribution
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EXPERIMENTAL RESEARCH OF RB94 GENE TRANSFECTION INTO RETINOBLASTOMA CELLS USING ULTRASOUND-TARGETED MICROBUBBLE DESTRUCTION MIN-MING ZHENG,*z XI-YUAN ZHOU,*z LI-PING WANG,*z and ZHI-GANG WANGy * Department of Ophthalmology, The Second Affiliated Hospital of Chongqing Medical University, Chongqing, P. R. China; Institute of Ultrasonic Imaging, The Second Affiliated Hospital of Chongqing Medical University, Chongqing, P. R. China; and z Chongqing Key Laboratory of Ophthalmology, Chongqing, P. R. China
y
(Received 8 March 2011; revised 5 January 2012; in final form 8 February 2012)
Abstract—The purpose of this study was to explore the transfection of the recombinant expression plasmid pEGFP-C1/RB94 into human retinoblastoma cells (HXO-Rb44) using ultrasound-targeted microbubble destruction (UTMD). pEGFP-C1/RB94 was transfected into HXO-Rb44 in vitro by UTMD, with liposome as the positive control. After 24 to 72 h, the expression of the reporter gene enhanced green fluorescent protein (EGFP) was observed using fluorescent microscopy and flow cytometry. The cell viability of HXO-Rb44 was measured by a MTT assay. The mRNA and proteins of RB94, caspase-3 and Bax were analyzed by reverse transcription polymerase chain reaction (RT-PCR) and Western blot. Moreover, the apoptosis rate and cell cycle progression of the cells were detected by flow cytometry. This study demonstrated that UTMD can enhance the transfection efficiency of RB94, which has an obvious impact on the inhibition of the growth process of retinoblastoma cells, suggesting that the combination of UTMD and RB94 compounds might be a useful tool for use in the gene therapy of retinoblastoma. (E-mail: [email protected]) Ó 2012 World Federation for Ultrasound in Medicine & Biology. Key Words: Ultrasound, Microbubble, Retinoblastoma, Gene transfection, RB94.
genetic defects including both RB(1) and RB(–) tumors (Pirollo et al. 2008). Differences reported thus far include the observations that RB94 has a longer half-life than RB110, remains in its active hypophosphorylated form for an extended period of time and causes unusual nuclear morphologic changes (Zhou et al. 2009a). Thus, it appears that RB94 is a more potent tumor-suppressing agent than RB110. Albeit recent advances in enucleation and conservative treatments, there has been no improvement in the 5-year survival rate in children. Therefore, it is urgent to explore novel therapeutic strategies that can improve the clinical outcomes. Recently, gene therapy has been gaining necessity in RB therapy (Zhang et al. 2006). Nevertheless, efficient, systemic delivery of the gene encoding RB94 specifically to tumors, is thought to be an obstacle to clinical application as an anticancer therapeutic. Updated studies have shown that ultrasoundtargeted microbubble destruction (UTMD) is regarded as a noninvasive gene transfer technology that provides a new means of gene therapy for retina disease (Zhou et al. 2009b). Microbubbles currently used as ultrasound
INTRODUCTION Retinoblastoma (RB) is the most common malignant intraocular tumor in children representing 2.5% to 4% of all pediatric cancers. There are about 1000 new cases of RB annually in China, accounting for 20% of new cases worldwide (Chen 2007). RB gene, which is located on a region of the long (q) arm of chromosome 13 designated 13q14, is most closely related with the incidence of retinoblastoma. As a fact, mutations in the RB gene are responsible for most cases of retinoblastoma. RB94 is produced by the translation of the RB gene from the second in-frame AUG codon and lacks the 112 amino acids on the NH2 terminus present in RB110 (Xu et al. 1994). RB94 has markedly increased tumor suppressor potency compared with RB110 and is active against all tumor types examined to date, despite their specific
Address correspondence to: Xi-Yuan Zhou, Department of Ophthalmology, The Second Affiliated Hospital of Chongqing Medical University, 74# Linjiang Road, Chongqing 400010, P. R. China. E-mail: [email protected] 1058
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contrast agents can lower the energy threshold of ultrasound for cavitation, which transiently perforates the cell membrane or disrupts the capillary wall to allow delivery of bioactive agents into the cells or the interstitial space (Taniyama et al. 2002a). In this regard, ultrasound with microbubbles has been considered as a novel vehicle for gene therapy. In our previous studies, we have demonstrated that the efficiency of wild-type p53 (wtp53) plasmid transfection into Y79 cells and RB xenograft tumour tissues mediated by UTMD (Luo et al. 2010). In this context, we used RB94 transfecting into RB cells in the same way and examined the efficiency of RB94 on the inhibition of the growth process of retinoblastoma. RB94, compared with other tumor suppressor gene such as p53, is strongly associated with the incidence of retinoblastoma (Calo et al. 2010; Laurie et al. 2006). Therefore, RB94 is a better candidate gene for gene therapy of retinoblastoma than wtp53. In addition, we have found no prior reports studying the effects of using RB94 to inhibit the growth of retinoblastoma. Importantly, we first explored the transfection of RB94 into human retinoblastoma cells using UTMD. Hereby, we employed a eukaryotic coexpression vector (pEGFP-C1/RB94) coding with enhanced green fluorescent protein (EGFP) and RB94, which was efficiently carried by an ultrasound contrast agent that we developed. The UTMD was used for the treatment of retinoblastoma. The study provided reliable experimental evidence for studying and evaluating gene transfer mediated by UTMD, which could be a novel strategy for the gene therapy of RB. MATERIALS AND METHODS Cell culture Human retinoblastoma cells (HXO-Rb44) were obtained from Hunan Medical University in China and grown in RIPA 1640 medium (Gibco, San Jose, CA, USA) containing 10% fetal bovine serum (FBS), 100 mg/mL streptomycin (Sigma, St. Louis, MO, USA), 100 U/mL penicillin (Sigma). Microbubble preparation The microbubble was prepared and characterized as previously described (Ren et al. 2008), with minor modifications. Briefly, 5 mg 1,2-distearoyl-sn-glycerophosphatidylcholine (DSPC; Sigma), 2 mg 1,2-dipalmitoyl-sn-glycero-3-phosphor-ethanolamine (DPPE; Sigma) and 10% glucose were dissolved in phosphate-buffered saline (PBS) to a final volume of 0.5 mL in 1.5-mL vials. The vials were incubated at 37 C for 30 min. The headspace of each vial was filled with the perfluoropropane gas and then the vial was mechanically shaken for 60 s
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using a dental amalgamator (medical apparatus and instrument, YJT, Shanghai, China). This solution was then diluted with 0.5 mL PBS and sterilized by 60Co irradiation. The density of the microbubbles was 1.2 3 109/mL, with a diameter of 4–6 mm and a half-life of about 6 h. Cell transfection A eukaryotic coexpression vector (pEGFP-C1/ RB94) coded with EGFP and RB94 was constructed in our previous work (Zheng et al. 2010). The pEGFP-C1/ RB94 and pEGFP-C1 plasmids grown in Escherichia coli were purified and suspended in 2.5 mM Tris-HCL (pH 8.5) at a concentration of 1.0 mg/mL. For preparation of the DNA-loaded microbubbles, a quantified amount of plasmid pEGFP-C1/RB94 and pEGFP-C1 plasmids were respectively added and gently mixed with the microbubbles. We lightly blended 4.0 mg of plasmid (4.0 mL) with 200 mL of the microbubble suspension and gently incubated the mixture for a few minutes at 4 C to add in adhesion. The cells were transfected with pEGFP-C1 vector using LipofectamineÔ2000 (Invitrogen, Carlsbad, CA, USA), which has a better membrane affinity and DNA condensation and can enhance the in vitro transfection efficiency, according to the manufacturer’s protocol. We also lightly blended 4.0 mg of plasmid (4.0 mL) with 250 mL of RPMI-1640 medium or 12 mL of liposome plus 250 mL of RPMI-1640 medium and then mixed the two at room temperature for 20 min. The gene transfer machine (UGT 1025 type, Chongqing, China) was produced by Ultrasonographic Image Research Institute of Chongqing Medical University. The following parameters for the ultrasound treatment of HXO-Rb44 cells were used: continuous wave, 300 KHz, 0.5 W/cm2, 30 s and a 10% concentration of microbubbles (Luo and Zhou 2010). Cultured HXO-Rb44 cells were divided into six groups: (1) control (no treatment); (2) pEGFPC1/RB94 plasmid (RB94 plasmid) with ultrasound; (3) RB94 plasmid with microbubbles; (4) RB94 plasmid with microbubbles and ultrasound; (5) pEGFP-C1 plasmid (C1 plasmid) with microbubbles and ultrasound; and (6) RB94 plasmid with liposome. After incubating for 6 h at 37 C with 5% CO2, the compound solution was replaced with 500 mL of fresh media containing 10% fetal bovine serum and 1% penicillin/streptomycin. The cells were cultured for another 24 h and the reporter EGFP expression was analyzed. All transfection experiments were performed in triplicate. Cell transfection analysis Forty-eight hours after transfection, the EGFP expression of all groups in HXO-Rb44 was observed using fluorescent microscopy. The cells were washed three times with warm sterile D-Hamks and treated with 0.125% trypsin for 2 min at 37 C. Cells were
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collected by centrifugation at 1000 rpm and were resuspended in 500 mL PBS. Transfection efficiency was analyzed using flow cytometry (FACS-Calibur; Becton Dickinson, San Jose, CA, USA) with the excitation setting at 488 nm. At least 10,000 cells were acquired and the data were analyzed by the software CellQuest (BD Company, New York, NY, USA). MTT assay The influence of all treated factors in HXO-Rb44 on cell viability was assessed with the 3-(4,5-dimthylthiazol2-yl)-2,5 diphenyl-tetrazolium bromide (MTT) assay. HXO-Rb44 cells were seeded in 96-well plates at a density of 13104 cells per well. When the cells grew to about 60%–80% in confluence, the old medium was replaced with 200 mL of 1640 without FBS and penicillin/streptomycin. Then, every treated factor was added to each respective well and all samples were generated in triplicate. Cells were incubated at 37 C and inspected daily for 5 days. Cell growth was determined by adding 20 mL of MTT (5 mg/mL) to each well and incubating for an additional 4 h at 37 C. The supernatant was discarded and 150 mL of dimethyl sulfoxide were added. Absorbance was determined by spectrophotometry using a wavelength of 570 nm, with 630 nm as a reference. Western blotting Proteins were extracted using a protein extraction reagent (Pierce, Rockford, IL, USA), following the protocol provided by the manufacturer. The total protein concentration was determined with the Bradford protein assay (Bradford 1976). Protein samples were mixed in Laemmli loading buffer and boiled for 5 min. Equal protein levels of cell lysates were electrophoresed using a 4% stacking gel and a 10% separating gel. The electrophoresed proteins were transferred to a polyvinylidene fluoride membrane (PVDF; Millipore, Bedford, MA, USA) using a trans-blot SD semi-dry transfer cell (Pierce). Completion of protein transfer from the gels to the membranes was checked by staining the gels with Coomassie Blue R-250. Western blotting was performed as described by Towbin et al. (1979). The membranes were washed and blocked in TRISbuffered saline (13TBS) with 5.0% nonfat dried milk. Samples were incubated for 1 h at room temperature with primary caspase-3 antibody (ab2302; Abcam, London, UK), Bax antibody (ab10813; Abcam) and mouseantihuman RB (ab24; Abcam), which recognizes both full-length RB and NH2-terminal-truncated RB94 proteins, at a concentration of 1:1000 in TBS. After 2 h of incubation with the appropriate species-specific horseradish peroxidase-conjugated secondary antibody (ab8227; Abcam) at a 1:2000 dilution rate, immunoreactive bands were visualized with a chemiluminescent substrate (Santa Cruz Biotechnology, Santa Cruz, CA, USA) according to
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the manufacturer’s instructions. The protein bands were normalized with b-actin and all blots were quantified with the software Quantity One (Bio-Rad, Hercules, CA, USA). Reverse transcription-polymerase chain reaction (RT-PCR) The total RNA was extracted by phenol-chloroform extraction and ethanol precipitation using TRIZOL (Life Technology, Gaithersburg, MD, USA). The integrity of RNA was detected by 1.5% agarose gels containing ethidium bromide. The polymerase chain reaction was performed with a two-step RT-PCR kit (AMV; Takara, Tokyo, Japan) using a PCR system (MyCycler; BioRad), following a protocol provided by the manufacturer. Sequences of the primers for RB94 are: 50 -CCTCTCGTC AGGCTTGAGTTTG-30 (sense) and 50 -TTGAGCAACA TGGGAGGTGAG-30 (antisense). Sequences of the primers for caspase-3 are: 50 -TGAAGGCAAGGTGCTA AA-30 (sense) and 50 -CTGGCTCAAACCACATTCTC-30 (antisense). Sequences of primers for Bax are: 50 -ATG CGTCCACCAAGAAGCTGAG-30 (sense) and 50 -CCCC AGTTGAAGTTGCCATCAG-30 (antisense). The PCR was performed at 94 C for 2 min at first, 94 C for 30 s, 55 C for 30 s, 72 C for 45 s followed by 35 cycles, 72 C for 10 min at last. The PCR products were affirmed on 1.5% agarose gels containing ethidium bromide. The products were separated alongside a 100-bp DNA molecular weight ladder (Promega, Southampton, UK) for sizing. The bands were normalized with b-actin. Cell cycle analysis Both the control and treated cells were harvested with trypsin-EDTA and washed in 1 mL of cold PBS. Approximately 13106 cells from each treatment were resuspended in 0.5 mL of propidium iodide solution (50 mg/ mL propidium iodide, 100 mg/mL RNase A and 0.2% Triton X-100). Cells were then incubated at 4 C for 30 min in the dark. All measurements of cell cycle distribution were performed on a flow cytometer (FACS-Calibur; Becton Dickinson, CA, USA). Data from over 30,000 cells were collected and analyzed by the Multiple Software (Phoenix Flow Systems, San Diego, CA, USA). Detection of apoptosis rate HXO-Rb44 cells were collected by centrifugation (1000 r/min 3 5 min) and washed twice with incubation buffer (10 mmol/L HEPES/NaOH, 140 mmol/L NaCl, 5 mmol/L CaCl2). Approximately 5 3 106 cells were collected. We added 100 mL of binding buffer to suspend the cells and then added 2 mL of the FITCAnnexin V Apoptosis Detection Kit (eBioscience, San Diego, CA, USA) and 5 mL of propidium iodide. The reaction was left in the dark at room temperature for 10–15 min. The cells were collected again by
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centrifugation (500 r/min 3 5 min) and washed in incubation buffer. SA-FLOUS solution was applied for 20 min in the dark at 4 C. The apoptosis rate was detected by flow cytometry. Statistical analysis The statistical analyses were all performed by using SPSS 17.0 software (SPSS Inc., Chicago, IL, USA). All the data were presented as mean 6 SD and were analyzed by using ANOVA tests. Means with P , 0.05 were considered to be statistically different from one another. RESULTS Fluorescent microscopy At 24 h post transfection, EGFP expression of all groups in HXO-Rb44 was observed using fluorescent microscopy (Fig. 1). Figure 1 shows that only minute amounts of green fluorescence were observed in the blanked control group while the obvious green fluorescence was very prominent in the other groups. Green fluorescence was more intense for the RB94 plasmid with microbubbles and ultrasound, the C1 plasmid with microbubbles and ultrasound and the RB94 plasmid with liposome than in other groups. Transfection efficiency Quantitative analysis of gene expression was undertaken to determine the efficiency of gene transfection for all groups. Positive EGFP expression was detected using flow cytometry 24 h post transfection. Transfection efficiency was measured by the percentage of cells that expressed EGFP in the population (Fig. 2). Figure 2 shows that the transfection efficiencies of the control, RB94 plasmid1microbubbles, RB94 plasmid1ultrasound, RB94 plasmid1microbubbles1ultrasound, C1 plasmid1 microbubbles1ultrasound and RB94 plasmid1liposome groups were 0.48%, 6.51%, 7.07%, 23.59%, 22.09% and 26.04%, respectively. The transfection efficiencies of the last three groups were higher than those of the other groups. The difference between the three groups was not statistically significant. MTT assay The influence of all treated factors in HXO-Rb44 on cell viability was evaluated by the MTT assay (Fig. 3). Those cells treated with RB94 plasmid1microbubbles1 ultrasound and RB94 plasmid1liposome were seen to undergo an apoptotic-like cytological change, losing their anchorage dependence and assuming a small spherical shape. Cells were collected and counted from day 0 through day 5 and cell growth curves were generated. In multiple trials, cell growth curves were seen to flatten and rise slightly in RB94 plasmid1microbubbles1 ultrasound and RB94 plasmid1liposome groups
Fig. 1. Fluorescent microscopic images of pEGFP-C1 expression in HXO-Rb44(3200). Cells were transfected with all treated factors. Cells were cultured for 24 h and the reporter gene expression was observed using fluorescent microscopy. (a) and (b) Blanked control group. (c) and (d) HXO-Rb44 transfected with pEGFP-C1/RB94 using ultrasound. (e) and (f) HXO-Rb44 transfected with pEGFP-C1/RB94 using microbubble. (g) and (h) HXO-Rb44 transfected with pEGFP-C1 using ultrasoundtargeted microbubble destruction. (i) and (j) HXO-Rb44 transfected with pEGFP-C1/RB94 using ultrasound-targeted microbubble destruction. (k) and (l) HXO-Rb44 transfected with pEGFP-C1/RB94 using liposome. Bright field images (b),(d), (f), (h), (j) and (l); fluorescence images (a), (c), (e), (g), (i) and (k).
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Fig. 2. Transfection efficiency in HXO-Rb44. The percentage of EGFP-positive cells was determined using flow cytometry 24 h post transfection (b) along with a computer-generated bar graph analysis (a). Data are represented as mean 6 SD (n 5 3). C indicates the blanked control group; RB94 indicates the pEGFP-C1/RB94 plasmid; M indicates microbubbles; U indicates ultrasound; L indicates liposome; and C1 indicates the pEGFP-C1 plasmid. The transfection efficiencies of the last three groups were higher than those of the other groups. The difference between the three groups was not statistically significant.
compared with other groups where cellular division continued at a brisk pace.
microbubbles1ultrasound and RB94 plasmid1liposome groups were more prominent than those of the RB94 plasmid1microbubbles and RB94 plasmid1ultrasound groups.
Evaluation of RB94 expression after transfection
RT-PCR. Consistent with the results of the Western blot analysis, RT-PCR revealed that the expression of RB94 mRNA in the RB94 plasmid1microbubbles1 ultrasound and RB94 plasmid1liposome groups was more remarkable than those of the other groups (Fig. 5).
Western blotting. Western blot analysis of RB protein expression following RB94 gene transfer revealed a band reactive to the anti-RB antibody at 94 kDa (RB94) in the RB94 plasmid1microbubbles1ultrasound, RB94 plasmid1 liposome, RB94 plasmid1microbubbles and RB94 plasmid1ultrasound groups (Fig. 4). Furthermore, 94-kDa bands were not seen in the other two groups. However, the bands of the RB94 plasmid1
Cell cycle analysis. To investigate the mechanism behind the antitumor activity, cell cycle studies were performed to determine the fate of the cells after RB94 gene transfer. Flow cytometry was performed along with cell
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Fig. 3. RB94 gene transfer suppresses tumor cell growth. Growth curves for HXO-Rb44 cell after transfection with all treated factors. Mean cell numbers were assessed for 6 consecutive days. Transfection with pEGFP-C1/RB94 using ultrasound-targeted microbubble destruction and liposome caused a significant flattening of cell growth curves in HXORb44 when compared with other groups. C indicates the blanked control group; RB94 indicates the pEGFP-C1/RB94 plasmid; M indicates microbubbles; U indicates ultrasound; L indicates liposome; and C1 indicates the pEGFP-C1 plasmid.
cycle analysis (Fig. 6). In repeated experiments, flow cytometry showed that the RB94 plasmid1 microbubbles1 ultrasound and RB94 plasmid1liposome groups go through cell arrest in the G2-M phase of the cell cycle. Apoptosis studies. Having witnessed the cell cycle of HXO-Rb44 cells transfected with the RB94 gene, an examination of apoptosis-related indicators was performed. The Western blot analysis showed that the protein expressions of caspase-3 and Bax in the RB94 plasmid1microbubbles1ultrasound and RB94 plasmid1liposome groups were more conspicuous than those in other groups (Fig. 4). The results of RT-PCR were
Fig. 4. Western blot analysis for the presence of RB protein, caspase-3 protein and Bax protein from HXO-Rb44. Cells were transfected with all treated factors. The cells were harvested and the proteins were subjected to SDSpolyacrylamide gel electrophoresis and probed by Western blot analysis with antibodies to RB, caspase-3 and Bax. Cells transfected with pEGFP-C1/RB94 using ultrasound-targeted microbubble destruction and liposome showed more prominent bands than other groups. C indicates the blanked control group; RB94 indicates the pEGFP-C1/RB94 plasmid; M indicates microbubbles; U indicates ultrasound; L indicates liposome; and C1 indicates the pEGFP-C1 plasmid. b-actin was used as an inner reference.
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Fig. 5. RT-PCR analysis of the expression of RB94 mRNA, caspase-3 mRNA and Bax mRNA in HXO-Rb44. Cells were transfected with all treated factors. RNA was prepared and analyzed by RT-PCR. Cells transfected with pEGFP-C1/RB94 using ultrasound-targeted microbubble destruction and liposome showed more prominent bands than other groups. C indicates the blanked control group; RB94 indicates the pEGFP-C1/ RB94 plasmid; M indicates microbubbles; U indicates ultrasound; L indicates liposome; and C1 indicates the pEGFP-C1 plasmid. b-actin was used as an inner reference.
well-matched with those of the Western blot analysis. Caspase-3 and Bax mRNA expressions in the RB94 plasmid1microbubbles1ultrasound and RB94 plasmid1liposome groups were noticeable compared with those in the other groups (Fig. 5). The apoptotic indexes for the RB94 plasmid1microbubbles1ultrasound and RB94 plasmid1liposome groups were found to be 28.7% and 28.6%, respectively, which are significantly higher than those obtained for the other groups (Fig. 7). The difference between the two groups was not statistically significant. DISCUSSION This study demonstrates a new method for gene therapy. Ultrasound can create transient nonlethal perforations in cells and other membranes, which allow large molecules from the surrounding medium to enter the cell without remarkable damage so that the cell can reseal its membrane and survive (Unger et al. 2001; Nozaki et al. 2003). The effect is potentiated by the use of microbubbles, which act as cavitation nuclei. Microbubbles were applied in combination with the use of ultrasound and are particularly effective for gene delivery (Taniyama et al. 2002b). Based on the theory mentioned above, we prepared ultrasound microbubbles in which the gene was loaded efficiently. Then, the UTMD method was used to transfer the gene into the cells. Our previous work showed that the most amounts of cells were able to survive under the conditions of a 0.5 W/cm2 sound intensity, 30 s irradiation time and 10% microbubble concentration. Therefore, we chose these optimal conditions in the present study. Under fluorescent microscopy, the intensity of green fluorescence of the RB94 plasmid with microbubbles and
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Fig. 6. RB94 gene transfer induces G2-M cell cycle arrest. DNA content flow cytometric profiles after transfection with pEGFP-C1/RB94 using ultrasound-targeted microbubble destruction and liposome in HXO-Rb44 (a) along with a computer-generated bar graph analysis (b) and (c). (b) indicated the cell distribution of ultrasound- targeted microbubble transfection of the RB94 gene in RB cells. (c) indicated the cell cycle distribution of liposome-targeted transfection of the RB94 gene in RB cell. Transfection with pEGFP-C1/RB94 using ultrasound-targeted microbubble destruction and liposome induced G2-M cell cycle arrest was observed in both of them.
ultrasound, C1 plasmid with microbubbles and ultrasound and RB94 plasmid with liposome was more intense than those of the other groups. Flow cytometry showed that the transfection efficiency of RB94 plasmid with microbubbles and ultrasound group was 23.59%. Compared
with RB94 plasmid with liposome group, the difference between the two groups was not statistically significant. This revealed that we successfully transfected the RB94 gene into the target cells by UTMD method. The MTT values of RB94 plasmid with microbubbles and
Fig. 7. Flow cytometry was used to detect the apoptosis rate in the six groups. The apoptotic index for the RB94 plasmid1microbubbles1ultrasound and RB94 plasmid1 liposome groups was found to be 28.7% and 28.6%, respectively, whereas only a few apoptotic cells were detected in the controls. The difference between the two groups was not statistically significant. C the indicates blanked control group; RB94 indicates the pEGFP-C1/RB94 plasmid; M indicates microbubbles; U indicates ultrasound; L indicates liposome; and C1 indicates the pEGFP-C1 plasmid.
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ultrasound group and RB94 plasmid with liposome group were dramatically less than those in the other groups, indicating that the growth of the two groups of tumor cells had been restrained. Western blot analysis and RT-PCR were used to detect the RB94 gene expression. The results demonstrated that the RB94 protein was well expressed in RB94 plasmid with microbubbles and ultrasound group and RB94 plasmid with liposome group, whereas there was less expression, if at all, in other groups. This was confirmed at the mRNA level of RB94 by RT-PCR analysis as well. The results mentioned above suggest that, as a novel transfection medium, microbubbles can meet the need for efficient gene transfer. These conclusions were also confirmed by cell cycle analysis and apoptosis studies. It has been reported that Ad-RB94 gene therapy in pancreatic cancer cells result in antiproliferative effects, apoptosis induction and S-G2 cell cycle arrest in tumor cells (Roig et al. 2004). Another study confirmed that Ad-RB94 gene transfer induces G2-M cell cycle arrest in head and neck cancer cells (Li et al. 2002). The common factor of these findings was the cell cycle progression through the G1 phase and the shift of the cell cycle arrest to G2 after Ad-RB94 gene therapy (Araki et al. 2008). Accordingly, cancer cells transfected with the RB94 gene had a striking effect on cell cycle distribution, resulting in an increase in cells arrested in the S-G2 phase. In our experiments, flow cytometry showed that the RB94 plasmid1microbubbles1 ultrasound and RB94 plasmid1liposome groups of cells arrested in the G2-M phase of the cell cycle after 48 h. Apoptosis appears to be the main phenomenon resulting in significant cell death and cell growth inhibition (Zheng et al. 2010). Several factors contribute to apoptosis but the key elements are categorized into two main families of proteins: the caspase enzymes and the Bcl-2 family (Thornberry and Lazebnik 1998). The bcl-2 family is a set of cytoplasmic proteins that regulate apoptosis. The two main groups of this family, Bcl-2 and Bax proteins, are functionally opposed: Bcl-2 acts to inhibit apoptosis, whereas Bax counteracts this effect (Ashkenazi and Dixit 1998; Kroemer 1997). Caspases are crucial mediators of programmed cell death (apoptosis). Among them, caspase-3 is a frequently activated death protease, catalyzing the specific cleavage of many key cellular proteins (Porter and Janicke 1999; Kroemer 1997). Thus, we studied Bax and caspase-3 protein in HXORb44 cells. Detection of caspase-3 and Bax in cancer cells treated with RB94 would be indicative of ongoing apoptosis. We discovered that the expression of caspase-3 and Bax protein was evident in the RB94 plasmid1 microbubbles1ultrasound and RB94 plasmid1liposome groups while it showed less expression in the other groups. In the current study, we observed that the apoptosis rates in the RB94 plasmid1microbubbles1ultrasound and RB94
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plasmid1liposome groups were significantly higher compared with those of the other groups, indicating apoptotic-like cytological changes in the cells. Above all, cells transfected successfully with the RB94 gene induced apoptosis. This study approved for the first time that UTMD can build up the transfection efficiency of RB94, which has an obvious impact on the inhibition of the growth process of retinoblastoma cells, indicating that the combination of UTMD and RB94 could be a useful method for application in the gene therapy of retinoblastoma. Nevertheless, gene therapy for retinoblastoma using an ultrasound-mediated microbubble in the experimental study is still in its infancy and it is still far from ready to be used in clinical applications. There are still some unresolved issues. The key problem is that gene transfection efficiency is still low compared with the vital vectors, which cannot live up to the expectation for clinical gene therapy in retinoblastoma. Two crucial issues need to be addressed: (1) the further optimization of the ultrasound, microbubble and gene parameters and (2) an increase in the gene-loading efficiency for the microbubbles. With these issues resolved, the prospect of gene therapy for retinoblastoma using ultrasoundmediated microbubbles will have a broader application. Acknowledgments—This work was supported by grants from the Institute of Ultrasonic Imaging in the Second Affiliated Hospital and Chongqing Key Laboratory of Ophthalmology. This project was also supported by the Program of National Natural Science Foundation of China (30872826). Dr. Z. G. Wang is the Director of the Institute of Ultrasonic Imaging, whose help was instrumental in allowing us to complete this study.
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