Six-week-old female severe combined immune deficient (SCID) mice (weighing 18–25 g) were purchased from the Guangdong Medical Laboratory Animal Center (Guangzhou, China) and housed under specific pathogen-free conditions (21±2°C and 12/12 h light/dark) with free access to food and water. The animal experimental procedures were reviewed and approved by the Institutional Animal Care and Use Committee of Nanjing Medical University Affiliated Suzhou Hospital.

The ovarian cancer cell line A2780 purchased from the American Type Culture Collection (ATCC; Manassas, VA, USA) was cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (FBS) (both from Gibco, Rockville, MD, USA), 100 U/ml penicillin and 0.1 mg/ml streptomycin (Sigma, St. Louis, MO, USA). Cells were routinely cultured in a humidified incubator containing 5% CO₂ at 37°C.

The CD133⁺ ovarian cancer stem cells were isolated by magnetic bead sorting as previously described (22). Briefly, cells were dissociated with 0.25% trypsin (Sigma) and suspended in ice-cold phosphate-buffered saline (PBS). Then, single cells were incubated with microbeads labeled with anti-CD133/1 monoclonal antibodies (Miltenyi Biotech, Auburn, CA, USA) for 20 min at 4°C in the dark. The samples were then washed with ice-cold PBS and sorted on a BD fluorescence-activated cell sorting (FACS)Aria system (BD Biosciences, San Jose, CA, USA). The CD133⁺ cells were cultured in serum-free medium containing 10 ng/ml human recombinant basic fibroblast growth factor and 20 ng/ml human recombinant epidermal growth factor (Invitrogen, Carlsbad, CA, USA).

The cDNA fragments of the CD133 promoter and Cre were amplified and inserted into adenovirus pShuttle plasmid (Stratagene, Santa Clara, CA, USA) to construct the Ad-CD133-Cre recombinant adenovirus. The cDNA fragments of LoxP-Neo-LoxP and tBid were inserted into pShuttle-CMV plasmid (Stratagene) to construct the Ad-CMV-LoxP-Neo-LoxP-tBid recombinant adenovirus. The recombinant pShuttle and pAdEasy-1 plasmids (Stratagene) were homologously recombined in BJ5183 bacteria. The recombined plasmids were linearized and transfected into 293T cells (ATCC) to produce the recombinant virus. After 14 days, the cells were harvested and lyzed by freeze-thawing. The supernatants were collected and concentrated by CsCl gradient centrifugation. The titers were determined using the 50% tissue culture infectious dose method (23). Cells were infected with Ad-CD133-Cre at a multiplicity of infection (MOI) of 20 and Ad-CMV-LoxP-Neo-LoxP-tBid at MOI 30.

Mitochondrial and cytosolic proteins were extracted using a mitochondria isolation kit (Pierce, Rockford, IL, USA). The proteins were separated by 12.5% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and then transferred onto a nitrocellulose membrane (Bio-Rad, Hercules, CA, USA). The membrane was then blocked in 2.5% non-fat milk at 37°C for 1 h. The membrane was blotted with primary antibodies at 4°C overnight. After washing three times with Tris-buffered saline (TBS)-Tween (TBST) and once with TBS, the membrane was incubated with horseradish peroxidase (HRP)-conjugated secondary antibody (1:2,000; Bioss Antibodies, Beijing, China) for 1 h at 37°C. After washing with TBST, the protein bands were visualized using a chemiluminescence detection procedure (Amersham Biosciences, Beijing, China). The band intensity was quantified using Image Pro Plus 6.0 software (Media Cybernetics, Inc., Rockville, MD, USA). The primary antibodies used in this experiment were as follows: anti-Bid, anti-Cre, anti-cytochrome c, anti-cleaved caspase-9 and anti-β-actin (Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA) and anti-cleaved caspase-3 and anti-COX IV (Abcam, Cambridge, MA, USA).

Cell growth and viability were detected by MTT assay. Briefly, cells were plated onto a 96-well plate at a density of 1×10⁴ cells/well and cultured overnight. Thereafter, the cells were infected with Ad-CD133-Cre or/and Ad-CMV-LoxP-Neo-LoxP-tBid and incubated for 48 h. The medium was replaced with fresh medium and 20 µl of MTT stock solution (5 mg/ml; Sigma) was added to each well. After culturing for 4 h, 200 µl of dimethyl sulfoxide was added to each well to dissolve the formazan crystals. Then, absorbance at a wavelength of 490 nm was detected using a microplate reader (BioTek Instruments, Winooski, VT, USA).

Cell apoptosis was assessed using the terminal deoxynucleotidyl transferase dUTP nick end-labeling (TUNEL) kit (Roche, Indianapolis, IN, USA). Briefly, the infected cells were fixed with 4% paraformaldehyde and permeabilized with 0.2% Triton X-100. Then, the cells were incubated with TUNEL reaction mixtures for 1 h at 37°C. Then, the cells were observed using a fluorescence microscope (Olympus, Tokyo, Japan), and apoptotic cells were counted in five random fields/slide.

Annexin V-fluorescein isothiocyanate (FITC) and propidium iodide (PI) stained cells were detected by flow cytometric analysis according to the manufacturer's instructions (Jiancheng Bioengineering Institute, Nanjing, China). Briefly, cells were dissociated with trypsin, washed with ice-cold PBS and resuspended in binding buffer at 1×10⁶ cells/ml. Then, Annexin V and PI stock solution were added and incubated at 4°C in the dark. Thereafter, the samples were detected by FACS and the data were analyzed by FlowJo software (BD Biosciences).

CD133⁺ ovarian cancer stem cells (1×10⁵ cells) were suspended in 200 µl of PBS and subcutaneously injected into the right flank of SCID mice (n=6 mice/group). Approximately 1×10¹⁰ plaque-forming units of recombinant adenovirus diluted in 50 µl of PBS were intratumorally injected every 3 days. Tumor volume (V) was measured using an external caliper and calculated according to the formula: V = length × width² × π/6. The mice were sacrificed by subcutaneous injection with sodium pentobarbital (100 mg/kg) 21 days after inoculation and the tumors were excised for western blot analysis and TUNEL assay.

Data are presented as mean ± standard deviation (SD). Statistical analyses were performed by one-way analysis of variance followed by Bonferroni post hoc test using SPSS version 11.5 (SPSS, Inc., Chicago, IL, USA). Differences were considered statistically significant at p<0.05.

To achieve conditional tBid overexpression, we introduced a Cre/LoxP-mediated expression system (Fig. 1A). Briefly, we cloned the CD133 promoter into an adenovirus and inserted the open reading frame of Cre downstream from the CD133 promoter. Therefore, Cre was expressed only when the CD133 promoter was activated. Meanwhile, we constructed another recombinant adenovirus containing a flanked LoxP cassette upstream of tBid. Thus, tBid was expressed only when the LoxP cassette was deleted. To test whether this Cre/LoxP system was successful, we infected these recombinant adenoviruses into CD133⁺ ovarian cancer stem cells. The results showed that Cre was expressed in the Ad-CD133-Cre-infected CD133⁺ ovarian cancer stem cells (Fig. 1B). The single infection of Ad-CMV-LoxP-Neo-LoxP-tBid did not result in tBid overexpression whereas co-infection of Ad-CD133-Cre with Ad-CMV-LoxP-Neo-LoxP-tBid into CD133⁺ ovarian cancer stem cells resulted in tBid overexpression (Fig. 2B). In contrast, in CD133⁻ cells, infection of Ad-CD133-Cre or/and Ad-CMV-LoxP-Neo-LoxP-tBid did not produce Cre or tBid (Fig. 1C). These results indicated that the Cre/LoxP-mediated tBid overexpression system was successfully established in the CD133⁺ ovarian cancer stem cells.

To determine the efficiency of Cre/LoxP system-mediated tBid overexpression in gene therapy against ovarian cancer, we assessed its biological effect on cell growth and apoptosis in CD133⁺ ovarian cancer stem cells in vitro. The results showed that the single infection of Ad-CD133-Cre or Ad-CMV-LoxP-Neo-LoxP-tBid had no obvious effect on cell growth or viability, as detected by MTT assay (Fig. 2A). However, the co-infection of Ad-CD133-Cre and Ad-CMV-LoxP-Neo-LoxP-tBid significantly inhibited the growth of CD133⁺ ovarian cancer stem cells (Fig. 2A). Furthermore, Annexin V-FITC/PI assay also showed a significantly high apoptosis rate in the CD133⁺ ovarian cancer stem cells co-infected with Ad-CD133-Cre and Ad-CMV-LoxP-Neo-LoxP-tBid (Fig. 2B and C). Similarly, TUNEL assay showed that the co-infection of Ad-CD133-Cre and Ad-CMV-LoxP-Neo-LoxP-tBid markedly increased cell apoptosis of the CD133⁺ ovarian cancer stem cells compared with single infection of Ad-CD133-Cre or Ad-CMV-LoxP-Neo-LoxP-tBid (Fig. 2D and E). Taken together, these results suggested that Cre/LoxP system-mediated tBid overexpression effectively promoted the apoptosis of CD133⁺ ovarian cancer stem cells.

To further verify the effect of Cre/LoxP system-mediated tBid overexpression on induction of cell apoptosis, we assessed its effect on the pro-apoptotic signaling pathway. The results showed that the co-infection of Ad-CD133-Cre and Ad-CMV-LoxP-Neo-LoxP-tBid significantly upregulated the release of cytochrome c from mitochondria to the cytosol compared with the other groups (Fig. 3A and B). Furthermore, the activated forms of caspase-9 and caspase-3 (Fig. 3C) were also significantly upregulated by co-infection of Ad-CD133-Cre and Ad-CMV-LoxP-Neo-LoxP-tBid in the CD133⁺ ovarian cancer stem cells. Taken together, these results indicated that the Cre/LoxP system-mediated tBid overexpression significantly activated the pro-apoptotic signaling pathway in the CD133⁺ ovarian cancer stem cells.

To further determine the efficiency of Cre/LoxP system-mediated tBid overexpression in gene therapy against ovarian cancer, we investigated the effect of Cre/LoxP system-mediated tBid overexpression on cisplatin-induced cytotoxicity. The results showed that cisplatin treatment inhibited the growth and viability of CD133⁺ ovarian cancer stem cells, and that the single infection of Ad-CD133-Cre or Ad-CMV-LoxP-Neo-LoxP-tBid had no obvious effect on cisplatin-induced cytotoxicity (Fig. 4A). However, co-infection with Ad-CD133-Cre and Ad-CMV-LoxP-Neo-LoxP-tBid significantly augmented the cytotoxic effect of cisplatin in the CD133⁺ ovarian cancer stem cells (Fig. 4A). Furthermore, cisplatin-induced cell apoptosis was also enhanced by co-infection with Ad-CD133-Cre and Ad-CMV-LoxP-Neo-LoxP-tBid in comparison with single infection of Ad-CD133-Cre or Ad-CMV-LoxP-Neo-LoxP-tBid (Fig. 4B-E). Taken together, these results suggested that Cre/LoxP system-mediated tBid overexpression increased the sensitivity of CD133⁺ ovarian cancer stem cells to cisplatin.

To further confirm the antitumor effect of Cre/LoxP system-mediated tBid overexpression against CD133⁺ ovarian cancer stem cells, we subcutaneously inoculated the tumor cells into SCID mice. The recombinant adenoviruses were intratumorally injected into the mice and the tumor volume was monitored. The results showed that the single infection of Ad-CD133-Cre or Ad-CMV-LoxP-Neo-LoxP-tBid had no obvious effect on tumor growth compared with the non-infected control group (Fig. 5A). As expected, the co-infection of Ad-CD133-Cre and Ad-CMV-LoxP-Neo-LoxP-tBid significantly suppressed tumor growth compared with the other groups (Fig. 5A). TUNEL assay showed that the apoptotic cells were not significantly altered in the Ad-CD133-Cre or Ad-CMV-LoxP-Neo-LoxP-tBid single-infected group, but markedly increased in the Ad-CD133-Cre and Ad-CMV-LoxP-Neo-LoxP-tBid co-infected group (Fig. 5B). By analysis of xenograft tumor tissues, we found that tBid was overexpressed in the Ad-CD133-Cre and Ad-CMV-LoxP-Neo-LoxP-tBid co-infected group (Fig. 5C and D). Moreover, caspase-3 activity was also markedly upregulated by co-infection with Ad-CD133-Cre and Ad-CMV-LoxP-Neo-LoxP-tBid (Fig. 5C and D). Taken together, these results revealed that Cre/LoxP system-mediated tBid overexpression triggered cell apoptosis and suppressed tumor growth in vivo.