Adenovirus-mediated truncated Bid overexpression induced by the Cre/LoxP system promotes the cell apoptosis of CD133+ ovarian cancer stem cells
- Authors:
- Published online on: November 21, 2016 https://doi.org/10.3892/or.2016.5263
- Pages: 155-162
Abstract
Introduction
Ovarian cancer is one of the most lethal gynecologic cancers with high morbidity in women worldwide (1). Surgical resection followed by chemotherapy has been considered the most effective treatment for ovarian cancer; however, most patients are diagnosed at advanced stages and undergo recurrence after treatment, which leads to a poor survival rate (2,3). To date, there is still lack of an effective therapeutic method for ovarian cancer. It is of great importance to develop promising strategies to improve the treatment of ovarian cancer.
Cancer stem cells, a small subset of cancer cells, are capable of self-renewal and multi-lineage differentiation into cells that can form tumors (4). These cells have been identified in various types of cancer and contribute to cancer progression, metastasis, chemoresistance and recurrence (5–8). Therefore, the elimination of cancer stem cells may be a potential and promising therapy for the treatment of cancer. Cancer stem cells express distinct cell surface markers such as CD24, CD44, CD119 and CD133 (6,9,10). Among these, CD133+ cancer stem cells have been widely studied in ovarian cancers as CD133+ cells isolated from ovarian cancer cells or primary ovarian tumor tissues show self-renewal, multi-lineage differentiation and tumor initiation capabilities (10–12). Given these findings, the elimination of CD133+ ovarian cancer stem cells could be a novel and effective therapeutic strategy for preventing ovarian cancer.
Truncated Bid (tBid), the C-terminal of full-length Bid (a BH3-only subgroup of the Bcl-2 family member) cleaved by caspase-8, with a molecular weight of 15 kDa, is a potent inducer of cell apoptosis (13,14). tBid translocates to mitochondria and activates the release of cytochrome c from mitochondria to the cytosol wherein cytochrome c activates caspase-9/−3/−7 to trigger cell apoptosis (14–16). Thus, tBid has been proposed as a promising molecular target for killing cancer cells (17,18). Recombinant adenoviruses have been suggested as an effective gene delivery tool for cancer treatment. However, adenovirus-mediated tBid expression initiated by the CMV promoter may kill packaging cells during the production of the recombinant adenovirus, making it difficult to obtain high titers of recombinant adenovirus. Therefore, a conditional expression system is needed.
Cre (‘Causes recombination’) is a site-specific recombinase that can mediate the specific recombinant process between two LoxP (locus of crossover P1) sequences (19,20). In the present study, we designed a recombinant adenovirus carrying tBid (with CMV promoter) and a null gene expression cassette (Neo) flanked by LoxP which was inserted upstream of tBid to block tBid expression (Ad-CMV-LoxP-Neo-LoxP-tBid). To conditionally initiate tBid expression, we introduced another recombinant adenovirus carrying Cre (without CMV promoter), with a CD133 promoter inserted upstream of Cre. Thus, Cre may only be expressed when the CD133 promoter was activated (Ad-CD133-Cre). We hypothesized that infection of Ad-CD133-Cre into CD133+ ovarian cancer stem cells may trigger the expression of Cre which may then mediate the LoxP recombinant and cut-off the null gene expression cassette between the two LoxP, ultimately leading to tBid overexpression. The activity of the CD133 promoter is weaker than that of the CMV promoter, but it has been reported that a very small amount of Cre is able to cut-off LoxP sequences effectively (19,21). Therefore, CD133 promoter-mediated Cre expression in CD133+ ovarian cancer stem cells is sufficient to trigger tBid overexpression. Together, this Cre/LoxP system mediated-tBid overexpression could specifically function in and kill CD133+ ovarian cancer stem cells, representing a potential and promising adjuvant therapy for the prevention of ovarian cancer metastasis and recurrence.
Materials and methods
Animals
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.
Ovarian cancer cell culture
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.
Generation of CD133+ ovarian cancer stem cells
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).
Construction, production and purification of recombinant adenovirus
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.
Western blot analysis
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 viability assay
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).
TUNEL assay
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.
Flow cytometric analysis
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).
In vivo xenograft experiments
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 analysis
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.
Results
Cre/LoxP system-mediated tBid overexpression in CD133+ ovarian cancer stem cells
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.
Cre/LoxP system-mediated tBid overexpression promotes cell apoptosis of 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.
Cre/LoxP system-mediated tBid overexpression activates the pro-apoptotic signaling pathway
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.
Cre/LoxP system-mediated tBid overexpression augments the cytotoxic effect of cisplatin in 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.
Cre/LoxP system-mediated tBid overexpression inhibits tumor growth in vivo
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.
Discussion
In the present study, we successfully established conditional truncated Bid (tBid) overexpression induced by the Cre/LoxP system. tBid overexpression mediated by the Cre/LoxP system was triggered in CD133+ ovarian cancer stem cells, significantly inducing cell apoptosis and inhibiting CD133+ ovarian cancer stem cell growth in vitro and in vivo. The present study suggests that adenovirus-mediated tBid overexpression induced by the Cre/LoxP system may represent a potential therapeutic tool for anti-ovarian cancer therapy.
CD133 has been suggested as a stem cell marker in many types of stem cells including hematopoietic, prostate epithelial and renal stem cells, and endothelial progenitor cells (24–27), implying that CD133 is associated with the original cell differentiation state. In malignant tumors, the promoter region of CD133 is in a hypomethylated state and CD133 is highly expressed in various types of cancers, including ovarian (6), breast (28), brain (5), prostate (29), colon (30) and pancreatic cancer (31). Increasing evidence suggests that CD133+ cells isolated from ovarian cancer cells or primary ovarian tumor tissues have self-renewal, multi-lineage differentiation and tumor initiation capabilities and are thus defined as ovarian cancer stem cells (10–12). A very small amount of CD133+ ovarian cancer stem cells can form tumors in SCID (12). Therefore, CD133 serves as a potential molecular target for eliminating CD133+ cancer stem cells. One approach for molecular-targeted therapy is monoclonal antibody therapy, for example using monoclonal antibodies targeting CD44 and CD123 in the removal of human acute myeloid leukemic stem cells (32,33). Smith et al reported that the CD133 single-chain antibody conjugated with cytotoxic drugs inhibited hepatocellular and gastric cancer cell growth (34). However, the effect of CD133 antibody targeted therapy is still limited due to the complex molecular structure of CD133 (35,36).
Utilizing specific transcriptional regulation is another strategy. The carcinoembryonic antigen (CEA) promoter-mediated Cre/LoxP system was found to induce the expression of the cytosine deaminase and specifically inhibit CEA-producing gastric cancer cells in vitro and in vivo (37). Similarly, in CD133+ cells, the CD133 promoter can be used to control selective gene expression. In the present study, we wanted to introduce tBid, a potent suicide gene, into CD133+ ovarian cancer stem cells in order to kill the cells. However, the recombinant adenovirus carrying tBid driven by the CMV potent promoter is difficult to obtain since the tBid expression may kill packaging cells during the production of the recombinant adenovirus. Therefore, a conditional expression system was needed. Miao et al constructed a recombinant adenovirus containing a tBid gene driven by an α-fetoprotein (AFP) promoter and tBid was overexpressed in AFP-producing hepatocellular carcinoma cells (38). Similarly, a recent study demonstrated that a selected fragment of the AFP promoter designated as EA4D exhibited the highest reporter activity and that the tBid driven by EA4D was highly and specifically expressed in AFP-producing hepatocellular carcinoma cells (17). In CD133+ cancer stem cells, we used the CD133 promoter to drive tBid expression. However, the expression level of tumor-specific promoters is generally low and the activity of the CD133 promoter is weaker than that of the CMV promoter (39). The tBid expression driven directly by the CD133 promoter is so low that it cannot effectively kill CD133+ ovarian cancer stem cells. Therefore, a way of augmenting the selective expression of the tBid gene was needed.
In the present study, we used the Cre/LoxP regulation system to selectively augment tBid expression. We constructed two recombinant adenoviruses, Ad-CMV-LoxP-Neo-LoxP-tBid and Ad-CD133-Cre. The tBid cannot be expressed in Ad-CMV-LoxP-Neo-LoxP-tBid, and thus we were able to obtain a high titer of recombinant adenovirus. With regards to Ad-CD133-Cre, the Cre is expressed in CD133+ cancer stem cells. Even though the CD133 promoter was a weaker promoter, a very small amount of Cre was able to cut-off LoxP sequences effectively (19,21). After LoxP was cut-off in Ad-CMV-LoxP-Neo-LoxP-tBid, the tBid was highly overexpressed as driven by the CMV promoter in CD133+ cancer stem cells. Therefore, a selective and augmented tBid expression system was established. Co-infection with Ad-CMV-LoxP-Neo-LoxP-tBid and Ad-CD133-Cre effectively inhibited cell growth and induced cell apoptosis in the CD133+ ovarian cancer stem cells. tBid is a potent apoptosis inducer and sensitizes apoptosis induced by chemotherapeutic drugs in cancer cells (40). The present study also revealed that Cre/LoxP system-mediated tBid overexpression augmented the cytotoxic effect of cisplatin in CD133+ ovarian cancer stem cells. The data indicate that Cre/LoxP system mediated-tBid overexpression represents a potential and promising adjuvant therapy to prevent ovarian cancer metastasis and recurrence.
In conclusion, the present study applied the Cre/loxP system to augment the selective expression of the tBid gene in CD133+ ovarian cancer stem cells. The Cre/LoxP system mediated-tBid overexpression significantly induced the apoptosis of CD133+ ovarian cancer stem cells in vitro and in vivo, representing a future clinical approach for preventing ovarian cancer metastasis and recurrence. Furthermore, the adenovirus-mediated tBid overexpression induced by the Cre/LoxP system could be applicable to other CD133+ cancer stem cells.
Acknowledgements
The present study was supported by grants from the Natural Science Foundation of Jiangsu Province (BK2012599), the Suzhou Municipal Science and Technology Development Project (SYSD2012088), and the Suzhou Key Medical Center (SZZX201506).
Glossary
Abbreviations
Abbreviations:
|
tBid |
truncated Bid |
|
Cre |
causes recombination |
|
LoxP |
locus of crossover P1 |
|
SCID |
severe combined immune-deficient |
|
AFP |
α-fetoprotein |
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