Background
Deregulated cyclin-dependent kinase (cdk) activity is a hallmark of human cancer. Ectopic expression of cdk inhibitors in tumor cell lines usually results in cell cycle arrest in G1 or G2 or both, and this has translated into therapeutic benefit in xenograft models with slowed tumor growth and improved host survival [
1]. Flavopiridol is a small molecule potent inhibitor of cdks, which is structurally related to a compound derived from the plant
Dysoxylumm binectariferum. It strongly inhibits cdk1, cdk2, cdk4, and cdk7 and exerts cytostatic or cytotoxic activities against various human cancer cell lines. It also inhibits various kinases such as protein kinase A and C and epidermal growth factor-receptor tyrosine kinase at micromolar concentrations. It also broadly suppressed the transcription of genes, including cyclin D1, and binds to DNA [
1]-[
4]. To date, the results from phase II clinical trials of flavopiridol as a single agent are unsatisfactory [
5]. Combination therapy of flavopiridol with other drugs have been attempted to improve the efficacy against various tumor types [
6],[
7].
Caspase-3 plays a major role in the transduction of the apoptotic signal and execution of apoptosis in mammalian cells. There has been interest in using the pro-apoptotic caspase-3 gene for therapy against cancer. Yamabe
et al. showed that caspase-3 transgene therapy in combination with an additional death-inducing therapeutic agent could be effective against various tumor types [
8]. Shinoura
et al. also showed that caspase-3 gene therapy required a pro-apoptotic signal to induce effective tumor cell killing [
9]. Our group constructed constitutively active recombinant caspase-3 precursors (rev-caspase-3) that were capable of auto-processing and inducing significant apoptosis
in vivo independent of the upstream caspases [
10]. We also generate an amplified hTERT promoter system for efficient transcriptional targeting of active caspase-3 by using a two-step transcription amplification (TSTA) system. Our data have demonstrated a potent, hTERT-restricted apoptosis which is induced by adenovirus-mediated active caspase-3 gene driven by hTERTp-TSTA system in human ovarian cancer cells.
We hypothesized that adenovirus mediated delivery of constitutively active recombinant caspase-3 precursors to tumor cells in combination with flavopiridol may exhibit enhanced cytotoxicities against tumor cells when compared with either agent used alone. In the current study, we investigated the effect of the combination of flavopiridol and recombinant caspase-3 on OVCAR3 cells in vitro and in mouse xenograft model. We observed a very potent induction of apoptosis by the sequential combination treatment of these agents both at low doses in the cancer cells.
Materials and methods
Cell culture and drug preparation
Human ovarian adenocarcinoma cell line OVCAR3(Nanjing Biotechnology Development Co., Ltd.) was maintained in RPMI1640 (Gibco, Grand Island, NY) supplemented with 10% heat-inactivated fetal bovine serum (FBS) at 37°C with 5% CO2 in air. Flavopiridol, which was kindly provided by Professor Geoffrey I. Shapiro at the US National Cancer Institute, was prepared in dimethyl sulfoxide (DMSO) at a working concentration of 25 mM and stored at - 20°C and was diluted in fresh medium before use.
Adenoviral vectors
AdHTVP2G5-rev-casp3, a recombinant adenoviral vector expressing rev-caspase-3 driven by the hTERTp-TSTA system, was constructed by our laboratory. Details of the recombinant vector are available upon request.
Cell viability assays
OVCAR3 cells were seeded at a density of 1.7 × 104 cells per well on 96-well plates and after an overnight incubation were treated with flavopiridol and/or AdHTVP2G5-rev-casp3 at the indicated doses. Cell viability was assessed using the Dojindo Cell Counting Kit-8 (Dojindo Laboratories, Gaithersburg, MD) according to the supplier’s recommendations. Absorbance was read at 450 nm and cell viability was expressed as the percentage of viable cells relative to untreated cells. All experiments were performed in triplicate and at least three times independently.
Cell-cycle analysis
Treated OVCAR3 cells were washed once with phosphate buffered saline (PBS), trypsinized, and washed again in PBS with 2% FBS and then fixed in ice-cold ethanol for at least 1 h at −20°C. Then the cells were stained with propidium iodide (30 μg/ml) and treated with RNase (0.6 mg/ml) in PBS plus 0.5% (v/v) Tween20 and 2% FBS. Stained cells were analyzed on a FACS Calibur flow cytometer (BD Bioscience) using the Cellquest software, and the ModFit program (Verity Software House Inc, Topsham, ME) was used to analyze the cell-cycle profiles.
Real time PCR
Total cellular RNA was isolated using the Trizol Reagent (Invitrogen, Carlsbad, CA). First-strand cDNA synthesis was carried out using reverse transcriptase (Invitrogen) by incubation at 25°C for 10 min, 37°C for 60 min and 95°C for 5 min. The sequences of primers used for real-time PCR were as follows: cyclin D: 5’-GAGGTCTGCGAGGAACAGAAGT-3’ (sense) and 5’-TTGAGCTTGTTCACCAGGAGC-3’ (anti-sense); active caspase-3: 5’-CCATGCTGAAACAGTATGCCG-3’ (sense) and 5’-TTCCAGAGTCCATTGATTCGCT-3’ (anti-sense); GAPDH: 5’-ACCACAGTCCATGCCATCAC-3’ (sense) and 5’-TCCACCACCCTGTTGCTGTA-3 (anti-sense). Amplification was performed in a 25-μL reaction containing 2 μL sample cDNA, 0.4 × Taq Man Universal PCR Master Mix (Applied Biosystems, USA), 120 nM each primer, and 1 nM DNA probe. The PCR was run at 94°C for 2 min followed by 40 cycles of 94°C for 5 s, 62°C for 10s and 60°C for 30s. Amplification was performed according to the manufacturer’s specifications.
Immunoblotting studies
Cell lysates were prepared as previously described. Immunoblotting procedure was carried out as depicted earlier [
11]. The following antibody was used for immunoblotting: anti-PARP antibody and anti-caspase-3 antibody (Cell Signaling Technology, Danvers, MA).
Xenograft studies
The study protocol was approved by the local institution review board at the authors’ affiliated institution. Female mice, 4 to 6 weeks old and weighing 16 to 20 g (Animal Research Institution of Chinese Academy of Medical Sciences, Beijing, China), were used for the experiment. All animals were cared for according to the Guidelines for the Care and Use of Laboratory Animals and the institutional guidelines of Chinese Academy of Medical Sciences. For the subcutaneous tumor model, 1 × 107 OVCAR3 cells in 0.2 ml PBS were inoculated subcutaneously into the dorsal flank of nude mice. When the tumor reached a volume of ~150 mm3, these mice received intratumoral injections of PBS, AdHTVP2G5-rev-casp3 (7 × 108 tissue culture infectious dose 50 (TCID50)/tumor), flavopiridol (10 mg/kg) and the sequential combination treatment in which flavopiridol (5 mg/kg) was injected 72 h after treatment with AdHTVP2G5-rev-casp3 (7 × 108 TCID50/tumor). Three intratumoral injections were given every 10 d and 5 mice from each group were followed up once per three days to measure tumor size by calipers. Tumor volumes were calculated using the formula a × b2 × 0.5, where a and b represent the larger and smaller diameters, respectively. Mice were sacrificed according to the institutional guidelines when the tumor reached 2000 mm3 in volume.
For the peritoneal tumor model, 1 × 10
7 OVCAR3 cells in 0.5 ml PBS were injected intraperitoneally into the nude mice. Subsequently, mice were weighed once every two days. Twenty-one days after inoculation, the mice received intra peritoneal injections of PBS, AdHTVP2G5-rev-casp3 (7 × 10
8 TCID
50/tumor), flavopiridol (10 mg/kg) and the sequential combination treatment in which flavopiridol (5 mg/kg) was injected 72 h after treatment with AdHTVP2G5-rev-casp3 (7 × 10
8 TCID
50/tumor). Three intraperitoneal injections were given every 10 days. Survival was defined as the study endpoint. Histopathological changes in the liver, spleen, intestine, lung, kidney, ovary, pancreas and heart were examined and serum contents of alanine transaminase (ALT) and aspartate transaminase (AST) were performed as described previously [
12]. Blood samples were collected via the tail vein on d 1 and 14 to monitor liver damage, and, specifically, the serum levels of AST and ALT in each group.
Statistical analysis
Statistical differences among the treatment groups were assessed by ANOVA using SPSS11.5 software program. A value P < 0.05 was considered significant. Additionally, the survival data was summarized and plotted using the Kaplan-Meier method, and survival curves were compared using the log-rank test.
Discussion
Cdk inhibitor flavopiridol has been demonstrated to exert potent antitumor activities in various preclinical tumor models [
12],[
13]. It is possible that cdk inhibition by flavopiridol is primarily cytostatic in the majority of solid tumors not typically predisposed to apoptotic responses. In the study, we also found that flavopiridol was only moderately cytotoxic against human ovarian carcinoma cells
in vitro but it demonstrated significantly improved survival of tumor-bearing mice compared with controls at 106 ± 11d post treatment. This survival advantage, however, disappeared at 134 ± 10 d post treatment, suggesting the modest activities of the agent. By contrast, the sequential combination treatment with AdHTVP2G5-rev-casp3 and flavopiridol not only showed synergistically enhanced cytotoxicities against human ovarian carcinoma cells
in vitro but also significantly prolonged the survival of tumor-bearing mice with a median survival of 283 ± 7 d.
In our study, significant synergism with flavopiridol was observed when cells were treated with AdHTVP2G5-rev-casp3 at low doses. There is a very strong increase in cell death and the presence of a pronounced sub-G1 fraction, when the cells were infected by AdHTVP2G5-rev-casp3 at MOI of 5 ~ 20, followed by flavopiridol for 48 hs at 300 nM. The sub-G1 fraction increased to 43.4 ~ 71.9% and the cell survival rates decreased to 39.6% ~ 5.8%, accompanied with a significant decrease in S-phase content in the sequential combination treatment, compared with either flavopiridol or AdHTVP2G5-rev-casp3 treatment at drug concentrations above that alone produce almost no cell death. The cell-killing synergism of them was the most potent when the cells were infected by AdHTVP2G5-rev-casp3 at MOI of 20. However, the synergism became weak with the MOIs increased to more than 40, at which dose AdHTVP2G5-rev-casp3 treatment alone began to induce significant cell apoptosis with no recruitment of cells to S-phase. No significant synergism was observed when cells were treated at MOI of 70 ~ 100.
Moreover, the extent of cell death by the sequential combination was time dependent. We observed the cell survival rates when cells were infected by AdHTVP2G5-rev-casp3(MOI = 20), and then 0 ~ 72 h later, followed by flavopiridol (300 nM) for 24 ~ 48 hs. The results showed that the cell-killing synergism of them was the most potent when flavopiridol was added into cells at 72 h after the viral infection and lasted for 48 hs.
Utilization of hTERT promoter that is predominantly active in tumor cells would be an effective system to restrict rev-caspase-3 expression [
14]-[
16]. However, in most cancer cells, the hTERT promoter activity is more than 10-fold lower than that of the CMV promoter, and is too weak to achieve sufficient transgene expression. It has been shown that transgene expression from a tumor-specific promoter can be augmented by using TSTA system [
17]-[
19]. In the present study, we constructed the TSTA system using the hTERT promoter to drive a chimeric transcription factor consisting of the powerful herpes simplex virus VP16 transcriptional activation domain fused to the DNA-binding domain of the yeast protein GAL4, which then binds to the GAL4-binding sites upstream of a minimal promoter to activate rev-caspase-3 gene expression. Our studies showed that the TSTA system elevated the activity of the hTERTp up to a 200-fold in human ovarian cancer cell lines (data available upon request). Our
in vitro and
in vivo data about AdHTVP2G5-rev-casp3 demonstrated a strong, hTERT-restricted antitumor activity with significant reduction in liver toxicity.
It has been shown that cell lines can be sensitized to flavopiridol after recruitment to the S-phase. Apart from rev-caspase-3, treatment with non-cytotoxic concentrations of chemotherapeutic agents, including gemcitabine, cisplatin, and topoisomerase I and II inhibitors, are capable of retarding S-phase progression, suggesting that a common mechanism may exist for enhancing cytotoxicities against tumor cells by flavopiridol [
20]-[
22]. Our flow cytometric data showed here that AdHTVP2G5-rev-casp3 caused a significant increase in the percentage of OVCAR3 cells in the S-phase
in vitro. We further showed that AdHTVP2G5-rev-casp3 sensitized OVCAR3 cells to flavopiridol with an apoptotic rate of 71.9% compared with no apparent apoptotic activities in OVCAR3 cells treated with either agent alone. We additionally found that the synergism between AdHTVP2G5-rev-casp3 and flavopiridol was sequence dependent and time dependent. It required that rev-caspase-3 precede flavopiridol by 72 hs. Exposure to flavopiridol preceding or concomitantly with rev-caspase-3 did not enhance cell death.
We have checked the levels of ALT and AST for their toxicity, and found that the acute toxicity by flavopiridol(10 mg/kg) alone was significantly increased after 1 days, much higher than that of the combination of flavopiridol (5 mg/kg) and AdHTVP2G5-rev-casp3 (2.5 × 108). 10 mg/kg is the maximum tolerated dose(MTD) of flavopiridol in vivo, and this dose of flavopiridol can induce high toxicity levels in acute phase. But the combination of half of the MTD of flavopiridol (5 mg/kg) and low dose of AdHTVP2G5-rev-casp3 (7 × 108 TCID50/mouse) induce much lower ALT and AST level than that in mice receiving 10 mg/kg flavopiridol alone (88.1 ± 7.8 vs. 289.3 ± 15.7, 91.5 ± 9.4 vs. 548.1 ± 42.9,respectively, P < 0.01 in both). This result showed that the combination of low dose flavopiridol and AdHTVP2G5-rev-casp3 can reduce the toxicity level, while maintain the synergism against xenograft growth in vivo.
In conclusion, we generated AdHTVP2G5-rev-casp3 and used it in sequential treatment with flavopiridol in OVCAR3 cells in vitro and in mouse xenograft model. We demonstrated that the sequential combination regimen exhibited synergistic cytotoxic activities against OVCAR3 cells in vitro and significantly inhibited tumor growth in mice and markedly extended the survival of tumor-bearing mice. Our findings indicate that the sequential combination regimen should be further explored as a potentially clinically useful treatment.
Acknowledgements
We thank Professor Izumi for the pBTdel-279 plasmid and useful information, Professor Michael Garey for the PBCVP2G5-lucNSN plasmid, Professor Geoffrey I for flavopiridol, Professor Ke-tong Wang for help in preparing the adenoviral vectors.
Grant support
This work was supported by National Nature Science Function of China grant 30600746.
Competing interests
The authors declare that they have no competing interests.
Authors’ contributions
AB, participated in the sequence alignment and drafted the manuscript. JY MT participated in the sequence alignment. ES FG All authors read and approved the final manuscript. Yue Song, corresponding author, the director of the project, conceived of the study, and participated in its design and coordination and drafted the manuscript. XX, carried out the molecular genetic studies. XZ, carried out the immunoassays. ZX, participated in the design of the study and performed the statistical analysis. KS, helped to conceive of the study and participated in its design. All authors read and approved the final manuscript.