Background
Lung cancer affects more than 1.2 million patients a year. The prognosis of lung cancer is very poor and long-term survival is obtained in only 5-10% of the patients. Non-small cell lung cancer (NSCLC) constitutes approximately 85% of all lung cancers and is the leading cause of tumor-related death worldwide highlighting the need for more effective treatment strategies [
1,
2]. NSCLC are inherently resistant and are generally not responsive to initial chemotherapy [
3].
Treatment of advanced NSCLC with anthracyclines, exemplified by doxorubicin, provides an overall response rate of only 30–50%. Unfortunately, its acute and cumulative dose-related toxicity poses a major problem in therapeutic outcomes. In addition to toxicity, the development of inducible drug resistance is a paramount problem in which patient fails to respond to cancer drugs.
There are various novel therapeutic strategies currently under consideration for lung cancer, as the clinical use of cytotoxic drugs is limited due to intrinsic or acquired resistance and toxicity. A better understanding of the molecular mechanisms of cytotoxic drug action has shed light on the treatment of lung cancer, and novel agents that target specific intracellular pathways related to the distinctive properties of cancer cells continue to be developed [
4]. Most DNA-damaging agents, including doxorubicin, trigger cell death via activation of p53 [
5]. The apoptotic role of p53 likely resides in its ability to disrupt the balance between antiapoptotic proteins (such as Bcl-XL, Bcl-2, and Mcl-1) and pro-apoptotic proteins (such as Bax and Bak) [
6]. Mee et al. (2008) have shown that p53 is upstream of NF-κB-mediated pathways of doxorubicin resistance and p53 is required for NF-κB mediated resistance to doxorubicin in NSCLC.
Since dysfunctions in the regulation of the cell cycle were found in almost all human cancers including NSCLC, agents targeting proteins involved in the regulation of cell cycle progression were developed [
7]. Disruption of the Rb pathway is a frequent event in NSCLC and plays an important role in tumorigenesis of NSCLCs [
8]. It has been hypothesized that the aberrant expression of cyclin D1 has strong oncogenic activity independently of pRb and p16, and may override the suppressive effects of pRb and p16. Other groups have also shown that the deregulation of cyclin D1 expression is an important characteristic of this disease [
9]. These data further strengthen the argument that cyclin D1 might be an effective lung cancer therapeutic target.
P276-00 is a novel small molecule inhibitor of cyclin-dependent kinases (Cdks) and has demonstrated synergism with different chemotherapeutic agents and is in Phase I/II clinical trials in combination with such agents/radiation for various cancers [
10]. In this study, we investigated the effect of P276-00, doxorubicin and their combination in three NSCLC cell lines differing in their p53 status and their
in vivo antitumor efficacy against a human NSCLC (H-460) xenograft.
Methods
Cell culture and reagents
Human NSCLC cell lines H-460 (p53-positive), H1299 (p53-null) and H23 (p53-mutant) were obtained from ATCC (Rockville, MD, USA) and cultured in RPMI-1640 medium containing 10% fetal bovine serum (FBS) (Hyclone, UT, USA), 2 mmol/L L-glutamine (Gibco, Grand Island, NY, USA), 100 U/mL penicillin and 100 mg/mL streptomycin (Gibco). Cells were maintained at 37°C in a humidified atmosphere containing 5% CO2. P276-00 was synthesized at Piramal Healthcare Limited, Mumbai, India and doxorubicin was purchased from Sigma. Both drugs were dissolved in dimethyl sulfoxide (DMSO) at a concentration of 10 mmol/L (10 mM) and stored at −20°C until use; diluted in culture medium RPMI-1640 immediately before use and used within 4 h. All reagents were purchased from Sigma (St. Louis, MO, USA) unless stated otherwise.
In vitrocytotoxicity assay
Cells were plated in 96-well plates and allowed to attach overnight. Each concentration of doxorubicin and P276-00 was represented by 3 wells per experiment and each experiment was repeated three times. Treated cells were maintained at 37°C in 5% CO
2 for times indicated in the legends to the figures. A modified propidium iodide (PI) assay was used to assess the effect of the compounds on the growth of the human tumor cell lines [
11]. IC
50 values were determined by plotting compound concentration versus cell viability. The combination index (CI) was calculated by the Chou-Talalay equation, which takes into account both the potency and the shape of the dose-effect curve taking advantage of the Compusyn software (ComboSyn, Inc. NY, USA). The combination index is used for the quantification of synergism or antagonism for two drugs where CI< 1, =1, and >1 indicate synergism, additive effect, and antagonism, respectively.
Analysis of cell cycle distribution by flow cytometry
H-460 cells were seeded in T-25 tissue culture flasks at a density of 1.0 × 10
6/mL and incubated overnight at 37°C. Next day the cells were treated with compounds. At the end of treatment period the cells were harvested and processed for flow cytometry as described previously [
11].
Annexin V staining
Annexin V staining was performed as described by the manufacturer (BD Biosciences). Briefly, 1 × 105 cells were washed twice in PBS and resuspended in binding buffer (10 mM HEPES, NaOH (pH 7.4), 140 mM NaCl, 2.5 mM CaCl2) at a concentration of 1 × 106 cells/ml. 5 μl of FITC-Annexin V (BD Biosciences) and 10 μl of PI (500 μg/ml in 38 mM sodium citrate) were added, and the cells incubated for 15 min in the dark at room temperature. A total of 400 μl of binding buffer was then added and the cells analyzed by flow cytometry.
Preparation and analysis of cell lysates by immunoblotting
Cells were seeded, treated with or without P276-00 or doxorubicin or their combination and were harvested at desired time points and western blotting was carried out as previously described [
12]. Antibodies used in this study were: Bcl-2, Bax, p53, Cdk-1, cyclin D1 (Santacruz Biotechnology, CA, USA), Cox-2 (Cell signaling technology, USA), anti-rabbit–HRP and anti-mouse-HRP secondary antibodies (Santacruz Biotechnology, CA, USA).
Clonogenic assay
H-460 cells were seeded at a density of 750–1000 cells per 35 mm tissue culture grade plate and incubated overnight at 37°C for the cells to attach. The cells were treated with the cytotoxic drug doxorubicin for 24 h followed by removal of medium and addition of fresh medium containing P276-00 for 96 h. At the end of the treatment, the medium was replaced by fresh complete medium and incubated for 7–14 days for colony formation. When visible colonies appeared on the plate the medium was removed and colonies were fixed with methanol: acetic acid mixture in the ratio of 2:1 for 5 min. The plates were washed with water and the fixation procedure was repeated. The plates were dried and the colonies were stained with 0.1% crystal violet stain for 3–5 min. The plates were rinsed carefully with water and dried and the colonies counted.
Tumor xenograft model
Approximately 5 × 106 H-460 cells were subcutaneously injected into severe combined immunodeficient (SCID) mice in 0.2 ml volume on the right flank and observed daily for tumor appearance. When the tumors attained a diameter of ~50 mm3, they were randomized into four groups: Group I, control vehicle; Group II, doxorubicin 2 mpk ip. once a week for 2 weeks; Group III, P276-00 20 mpk ip. every day for 5 days a week for 2 weeks; Group IV, combination of doxorubicin and P276-00, doxorubicin was followed by P276-00 after an interval of 6 h, followed everyday with P276-00 for a total of five days which comprised of one cycle. The treatment comprised of total of two cycles. Body weight was recorded everyday. Tumor measurements i.e. the length and width of the tumors were measured using the vernier caliper. Tumor weight (mg) was estimated according to the formula for a prolate ellipsoid: {Length (mm) x [width (mm)2] x 0.5} assuming specific gravity to be one and π to be three. Tumor growth in compound treated animals is calculated as T/C (Treated/Control) x 100% and growth inhibition percent (GI %) was [100-T/C%]. Animals were maintained and experiments were carried out as per the institutional animal ethical committee in compliance with the guidelines of the Committee for the Purpose of Control and Supervision on Experiments on Animals (CPCSEA), India.
Statistical analysis
Statistical comparison was made using GraphPad Prism software (version 5.0) in which one-way analysis of variance and Tukey’s multiple comparison post tests were used to determine significant differences between several treatment groups. Student’s paired t-test was used when only two groups were compared. Data are presented as mean ± S.E.M. of at least three independent experiments with triplicate. Statistical significance was evaluated by calculating P-values. Differences where P < 0.05 were considered statistically significant (*
P < 0.05; **
P < 0.01; ***
P < 0.001).
Discussion
We evaluated the cytotoxic effects of either doxorubicin or P276-00 or the combination of both the compounds in three NSCLC cell lines viz. H-460 (p53-positive), H1299 (p53-null) and H23 (p53-mutant). A sequential drug treatment strategy was chosen based on previous studies demonstrating sequence-specific synergistic effects with administration of combination of chemotherapy (doxorubicin) and Cdk inhibitor P276-00 (data not shown). The combination was synergistic in the p53 positive and p53 mutant cell line but not p53 null cell line indicating that p53 may possibly have a role in the synergistic interaction. Similar results have been observed with combined treatment of doxorubicin and another Cdk inhibitor roscovitine in human sarcoma cell lines [
17]. It has been shown earlier that doxorubicin mediated cell cycle arrest can occur either at G0/G1 or G2 check points and is thought to be mediated by the multifunctional transcription factor p53 [
18]. Doxorubicin is more effective in p53 wild type cancers. Our results were in line with this finding – the best synergism between the two drugs was observed in H-460 (p53-positive) cell line and hence it was selected for all further studies.
Cell cycle analysis demonstrated that doxorubicin alone markedly increased the percentage of cells in the G2/M phase. Same response was seen earlier by other groups in another doxorubicin sensitive human lung carcinoma cell line DLKP-SQ [
19]. It has been proposed that the G1 checkpoint is disabled in majority of cancers as a prerequisite for tumorigenesis. The G2 checkpoint, however, appears to remain functional in the majority of cancer cells. Many conventional cancer therapeutic agents exert their effect by causing DNA damage and thus retaining a functional G2 is believed to confer resistance to many such therapeutic agents. To circumvent this, attempts have been made to develop abrogators of this arrest in the G2 phase. Doxorubicin is also known to cause G2 arrest in several cell lines and the target for the G2 checkpoint pathway is Cdk1 [
20]. Combination treatment was found to downregulate Cdk1 expression levels which could account for abrogation of G2/M arrest and induction of apoptosis.
The checkpoint inhibitor p16 is practically always silenced in NSCLC due to methylation of its promoter [
21]. Loss of p16 expression leads to Rb phosphorylation by the cyclin D- cyclin-dependent kinase 4, 6 complex releasing E2F with the onset of the S phase of the cell cycle. Gain of the 11q13.1-11q14.1 region has been shown to be present in > 50% of the lung cancer cell lines. Cyclin D1 is located at this loci and the amplification of this gene is an important event in tumorigenesis [
22]. P276-00 is a potent Cdk4 and Cdk1 inhibitor [
10] and therefore, downregulation of Cdk4, Cdk1, cyclin D1 and cyclin B1 was observed by P276-00 alone and in combination. This could be a potential factor in the increased sensitivity of H-460 to the combination compared to either drug alone.
Many anticancer agents increase Bax protein and/or decrease Bcl-2 protein during the apoptotic process. Similarly, doxorubicin and P276-00-induced apoptosis in H-460 cells was accompanied by an elevation of the Bax to Bcl-2 ratio due to the downregulation of Bcl-2. p53 induces cell cycle arrest or apoptosis in response to DNA damage and regulates Bax and Bcl-2 protein expression [
4]. In response to the combination treatment, p53 levels were significantly upregulated, which could have lead to modulation of Bax and Bcl-2 expression.
One of the targets currently being evaluated in the treatment of lung cancer belongs to the cyclooxygenase (COX) class of enzymes. COX-2 overexpression is seen in many malignancies including lung cancer [
1]. Recently, it was shown by O’Kane et al. 2010 [
23] that COX-2 specific inhibitors enhance the cytotoxic effect of conventional drugs. Doxorubicin causes the activation of NF-κB in cancer cells, which in turn inhibits apoptosis induced by doxorubicin; cells with increased activity of NF-κB are thus resistant to doxorubicin [
24]. P276-00 when added after doxorubicin treatment significantly inhibited COX-2 protein levels which were increased in response to doxorubicin treatment. The synergy observed
in vitro was also seen in
in vivo antitumor efficacy studies at well-tolerated doses and schedules. Anticancer efficacy was more pronounced in combination (
P ≤ 0.05) as compared to either drug alone.
Conclusions
There are various novel therapeutic strategies under consideration, as the clinical use of cytotoxic drugs is limited due to intrinsic or acquired resistance and toxicity. Recent efforts have focused on identifying novel combinations of anticancer agents with non-overlapping mechanisms of action to obtain enhanced anticancer efficacy and reduced toxicity. Our results provide substantial evidence for the hypothesis that Cdk inhibitor P276-00 enhances doxorubicin-induced killing of NSCLC cells in vitro as well as in vivo without any significant toxicity especially in p53 positive tumors. This study will potentially provide new approaches to combination anticancer therapy for p53 positive NSCLC.
Acknowledgements
The work has been supported and carried out at Piramal Healthcare Limited, Goregaon, Mumbai. We extend our thanks for the support.
Competing interests
The author(s) declare that they have no competing interests.
Authors’ contributions
MJR designed the experiments and edited the manuscript. HK performed cell cycle studies, western blotting and in vivo studies. KJ carried out cell culture, in vitro combination studies and RT-PCR. SMM analyzed the data and wrote the manuscript. KSJ has conceptualized the project, written the discussion and edited the manuscript. All the authors except KJ, gave the final approval of the version to be submitted. Unfortunately Ms. KJ is no more.