2017 Volume 65 Issue 8 Pages 768-775
Antimetabolite drugs, including the adenosine deaminase inhibitor cladribine, have been shown to induce apoptosis in a variety of cancer cells, and have been widely used in clinical trials of various cancers in conjunction with tyrosine kinase inhibitors (TKIs). Combination treatment with cladribine and gefitinib or dasatinib is expected to have a synergistic inhibitory effect on breast cancer cell growth. Our results demonstrated that the combination treatment had synergistic activity against human breast cancer (MCF-7) cells, enhanced G2/M cell arrest and reactive oxygen species (ROS) generation, and increased the loss of mitochondrial membrane potential and cell apoptosis. In addition, the combination treatment decreased Bcl-2 expression. Our results demonstrated that cladribine in combination with gefitinib or dasatinib exerted synergistic anticancer effects on MCF-7 cells by inducing cell cycle arrest, ROS production and apoptosis through the mitochondria-mediated intrinsic pathway.
Breast cancer is the most common malignant tumor and the leading cause of cancer-related deaths in women.1,2) Current treatment for breast cancer includes surgery, radiotherapy and drug therapy, with drug therapy including hormone therapy, chemotherapy and immunotherapy.3,4) The pathogenesis of breast cancer has been studied in-depth,5) and many drugs have emerged for the treatment of breast cancer, with chemotherapy remaining the mainstay of treatment for breast cancer in addition to surgery. In the clinic, the main chemotherapeutic agents used to treat breast cancer include cisplatin and paclitaxel, docetaxel, curcumin, navelbine, and oxaliplatin. Although these chemotherapy drugs are widely used in the treatment of breast cancer, they often have severe side effects.6)
Recent studies have found that multiple signaling pathways, such as phosphatidylinositol 3-kinase (PI3K) and mitogen-activated protein kinase (MAPK), are involved in breast cancer cell proliferation, and targets of these pathways, such as epidermal growth factor receptor (EGFR), Akt, mammalian target of rapamycin (mTOR), insulin-like growth factor 1 receptor (IGF-1R), human epidermal growth factor receptor-2 (HER2), have become anti-breast cancer drug targets.7,8) We are known to target anti-breast cancer drugs trastuzumab, lapatinib and so on, but the development of drug resistance in patients has limited their clinical use. With the integration of various disciplines of drug research, the role of multiple targets in biological signaling networks and pathways to achieve appropriate physiological and pathological outcomes has been well studied.9) A growing body of literature has also shown that drug combinations have synergistic anti-cancer effects through the inhibition of these targets.10–14) In the clinic, combination chemotherapy for breast cancer is widely used, including drugs such as dasatinib and adriamycin, and has shown excellent results.15)
The clinical application of combination chemotherapy covers a wide range of treatments and chemotherapy-related antimetabolite drugs combined with anti-tumor drugs have also had a significant impact, for example, the synergistic anticancer effect of a combination of capecitabine and docetaxel in the treatment of metastatic breast cancer.16) Antimetabolites are compounds that are structurally similar to normal metabolites or coenzymes, and their primary role is to interfere with the enzymes necessary for the synthesis of normal metabolites, which in turn interferes with nucleic acid synthesis and inhibits the growth and proliferation of tumor cells. The antimetabolite drug cladribine is an adenosine deaminase inhibitor used to treat hairy cell leukemia and multiple sclerosis.17)
Tyrosine kinase inhibitors have been intensively studied as a form of targeted therapy. For example, gefitinib is an EGFR tyrosine kinase inhibitor, which interrupts EGFR signaling in target cells and has been approved by the U.S. Food and Drug Administration (FDA) as a third line therapy for non-small cell lung cancer (NSCLC). A previous study has shown that gefitinib combined with the antimetabolite drugs gemcitabine or capecitabine had significant activity against breast cancer.18–20) Moreover, dasatinib, as a second-generation c-abl oncogene (ABL) tyrosine kinase inhibitor (TKI) has been approved by the FDA for the treatment of chronic myelogenous leukemia. A combination of dasatinib and capecitabine or gemcitabine has been shown to have potential for the treatment of breast cancer.21,22) Taking into account the phase 2 clinical trials of gefitinib and dasatinib against breast cancer, we speculated that there would be a synergistic effect of dasatinib or gefitinib combined with the antimetabolite drug cladribine in the treatment of breast cancer.
The dose–effect relationship of cladribine, gefitinib and dasatinib, alone or in combination, was evaluated on the growth of human breast cancer (MCF-7) cells after the 48 h of treatment (Fig. 1). The median-effect analysis was completed according to Chou’s method.23)
MCF-7 cells were treated with cladribine (A), gefitinib (B), and dasatinib (C) alone or in combination claribine and gefitinib (1 : 1, D), cladribine: dasatinib (5 : 1, E), for 48 h. Synergistic anticancer effects of combinations of cladribine and gefitinib, and cladribine and dasatinib in MCF-7 cells. The CI (F, G) and DRI (H, I) of the combination of cladribine and gefitinib, or cladribine and dasatinib were calculated as described. The data represent the mean±S.D. from three replicate wells. p<0.05, compared with the control group.
In MCF-7 cells, all three drugs (cladribine, gefitinib and dasatinib) showed dose-dependent cytotoxic effects, with medium-effect dose (Dm) (IC50) values of 84.37, 63.30 and 17.08 µM, respectively. Since Chou–Talalay model required fixed dose ratios, we combined cladribine with gefitinib as 1 : 1 and cladribine with dasatinib as 5 : 1 based on the ratios of their Dm values. The antitumor effect of the drug combinations was carried out by the combination index (CI); the plots of CI versus fraction affected (Fa) are shown in Fig. 1. For CI values below 1, the combination of cladribine and gefitinib showed a synergistic effect at 30 to 50 inhibition levels, where the combination doses were 15 to 60 µM at 48 h. When the combination dose was 120 µM, with a CI (1.23) greater than 1, the combination of the two drugs produced an antagonistic effect. The dose-reduction index (DRI) values were also estimated, to determine the reduction in drug dose in the combination compared with the drugs alone, for a given inhibition level. As shown in Fig. 1, the DRI values for cladribine ranged from 3.12 to 4.02, whereas those for gefitinib ranged from 2.48 to 3.69.
For a CI value below 0.6, the combination of cladribine and dasatinib showed a stronger synergistic effect, where the combination doses were 9 to 36 µM at 48 h. The combination of cladribine and dasatinib also showed a synergistic effect, where the combination doses were 36 to 72 µM, and overall they showed a synergistic effect, where the combination doses were 9 to 72 µM (Fig. 1). As shown in Fig. 1, the DRI values for cladribine ranged from 1.95 to 4.01, whereas those for gefitinib ranged from 2.76 to 5.42. Overall, the results of the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay suggested that cladribine, dasatinib and gefitinib inhibited the growth of MCF-7 cells, and that the combinations of cladribine and gefitinib (1 : 1) and cladribine–dasatinib (5 : 1) exhibited synergistic and cytotoxic effects on MCF-7 cells.
Induction of Apoptosis in MCF-7 Cells by Cladribine, Gefitinib and DasatinibNext, we determined whether the cytotoxic effect of gefitinib, cladribine and dasatinib alone, as well as the synergistic effect of the combination treatment was associated with apoptosis by flow cytometry. As shown in Figs. 2A and B, cladribine, gefitinib and dasatinib alone increased the proportion of apoptotic cells after 48 h of treatment (p<0.05). Treatment with the cladribine and gefitinib combination resulted in a significant increase in the percentage of apoptotic cells compared with those treated with cladribine or gefitinib alone (33.67% vs. 13.87% or 9.03%, respectively, p<0.05), the percentage of apoptotic cells also increased when they were treated with the cladribine and dasatinib combination compared to that of cladribine or dasatinib alone (62.40% vs. 15.45% or 8.95%, respectively, p<0.05) (Figs. 2A, B). And nuclear morphology of apoptotic cells with condense/fragmented nuclei was shown in Fig. 2E by propidium iodide (PI) staining. Figure 2E shows that apoptotic cells also increased when they were treated with the two drugs combination compared to that of alone. In all, these experiments indicated that the apoptotic effect was increased significantly by the drug combinations.
Cell apoptosis (A, B, E) was induced by gefitinib (15 µM), dasatinib (3 µM) or cladribine (15 µM) alone or in combination for 48 h. More cells were accumulated at G2/M phase after combinational treatment (C, D). Nuclear morphology of apoptotic cells with condense/fragmented nuclei (shown with arrows) (E). The bar is 20 µm. Numerical data are presented as mean±S.D. (n=3). * Indicates a significant difference (p<0.05) by one-way ANOVA.
Flow cytometry was used to examine cell cycle arrest in MCF-7 cells following treatment with gefitinib, cladribine and dasatinib, alone or in combination. As shown in Figs. 2C and D, cladribine significantly decreased the percentage of G1 cells, and also increased the percentage of G2/M phase cells, as well as those in S phase (p<0.05), compared with the control. Gefitinib and dasatinib had no significant effect on the cell cycle of MCF-7 cells, but when combined with cladribine and gefitinib, gefitinib enhanced the effect of cladribine, causing a remarkable accumulation of G2/M phase cells at the expense of G1 phase cells. The combination of dasatinib and cladribine had similar effects to that of cladribine on cell cycle distribution (p<0.05).
Effect of Cladribine, Gefitinib and Dasatinib on Intracellular Reactive Oxygen Species (ROS) GenerationWe used the fluorescent probe 2′,7′-dichloro-fluorescin diacetate (DCFH-DA) to measure intracellular ROS generation to investigate whether ROS is involved in mediating apoptosis induced by cladribine, dasatinib and gefitinib. DCFH-DA is deacetylated and becomes the non-fluorescent DCFH after being taken up by cells and DCFH is converted to the green fluorescent DCF by intracellular ROS. Treatment with each drug alone or in combination enhanced ROS generation in MCF-7 cells compared with untreated cells (p<0.05). Compared with the effect of dasatinib or cladribine alone, ROS generation at 12 h in cells treated with a combination of the two compounds was much higher (the mean fluorescence intensity (FI)=171.18 vs. 111.97 or 109.51, p<0.05). ROS generation in cells treated with a combination of gefitinib and cladribine was also much higher (the mean FI=191.02 vs. 129.38 or 109.51, p<0.05), indicating that combined treatment with cladribine and dasatinib or gefitinib significant increased ROS generation (Fig. 3).
ROS production was monitored in MCF-7 cells with DCF after treatment with cladribine (15 µM), gefitinib (15 µM) and dasatinib (3 µM), alone and in combination, for 12h (A, B). ΔΨm changes were detected with Rhodamine 123 in MCF-7 cells after treatment with cladribine (15 µM), gefitinib (15 µM) and dasatinib (3 µM), alone and in combination, for 12 h (C, D). FI was measured by laser scanning confocal microscopy. The bar is 20 µm. Numerical data are presented as mean±S.D. (n=3). * Indicates a significant difference (p<0.05) by one-way ANOVA.
Changes in ΔΨm have been associated with apoptosis24); thus, we examined the influence of cladribine, gefitinib and dasatinib alone or in combination on the ΔΨm of MCF-7 cells with rhodamine 123 (Rh 123). Rh 123 is a mitochondria-specific cationic fluorescent dye, whose staining is proportional to the ΔΨm. Treatment with cladribine, gefitinib and dasatinib alone or in combination for 12 h resulted in a significant loss of ΔΨm in MCF-7 cells, as seen by the reduction of rhodamine 123 FI, compared with the untreated cells (p<0.05) (Fig. 3). Compared with the effect of dasatinib or cladribine alone, treatment of the cell with the combination with dasatinib and cladribine showed a sharp decline in Rh 123 FI at 12 h (FI of combination=59.39, dasatinib=114.92, cladribine=97.16). The gefitinib and cladribine combination treatment also showed a sharp decline in Rh 123 FI to 44.36 compared with the control (137.19), or gefitinib (99.91) or cladribine (97.16) alone after treatment for 12 h.
Effect of Cladribine, Gefitinib and Dasatinib on the Expression of Bcl-2Bcl-2 is an inner mitochondrial membrane protein that plays an important role in preventing apoptosis. Thus, we examined the effect of cladribine, gefitinib and dasatinib on Bcl-2 expression in the MCF-7 cells. The results indicated Bcl-2 protein expression decreased in a dose-dependent manner in response to cladribine, gefitinib or dasatinib alone or in combination for 12 h compared with the control in MCF-7 cells (p<0.05) (Fig. 4). Compared with the effect of dasatinib, cladribine, or gefitinib alone, the combination of gefitinib and cladribine, or dasatinib and cladribine treatment showed a significantly reduction in Bcl-2 expression (p<0.05).
Expression of Bcl-2 in MCF-7 cells was detected by the FITC-conjugated secondary antibody (A), and FI was measured by laser scanning confocal microscopy; Expression of Bcl-2 protein was analyzed by Western blotting analysis (C). Data are presented as mean±S.D. values of three independent experiments (B, D). The bar is 30 µm. p<0.05, a significantly different compared with the control group.
In this study, we demonstrated that cladribine, gefitinib and dasatinib exhibited dose-dependent cytotoxic effects on MCF-7 cells. The combinations of cladribine (7.5 to 60 µM) with dasatinib (5 : 1 ratio) and cladribine (7.5 to 30 µM) with gefitinib (1 : 1 ratio), had synergistic cytotoxicities on MCF-7 cells.
Our results showed that the IC50 of cladribine was 84.37 µM, dasatinib was 20.91 µM and gefitinib was 63.30 µM on human MCF-7 breast cancer cells after 48 h. The results of this study showed that cladribine sensitizes dasatinib-induced and gefitinib-induced cytotoxicity in human breast cancer cells and reduces the dose of dasatinib and gefitinib by more than two fold.
The combined beneficial effect of these drugs appears to be achieved partly through cell cycle arrest, ROS production, and the induction of apoptosis in the mitochondria-mediated intrinsic pathway. Mitochondria have been considered as anti-cancer drug targets and have a key function in the regulation of apoptosis.25)
Our results indicated that cladribine caused G2/M arrest in MCF-7 cells and that the combination of cladribine and gefitinib or dasatinib led to an accumulation of cells in the G2/M phases at the expense of the G1 phase. Dasatinib induced G1 arrest in MCF-7 cells. These findings suggested that cladribine, gefitinib and dasatinib may be useful for controlling cancer cell growth, as several cancer cells have defects in cell cycle checkpoints.
Mitochondria are the main production sites of reactive oxygen species (ROS). The presence of excessive ROS in mitochondria leads to opening of the mitochondrial permeability transition pore, a decline in ΔΨm and the release of cytochrome c, which, in turn, initiates caspase activation and triggers apoptosis.26) Several studies have provided evidence that dasatinib and gefitinib exhibit antitumor activity by ROS generation and apoptosis induction.27,28) Our results demonstrated that the incubation of MCF-7 cells with cladribine, gefitinib or dasatinib alone or in combination, increased ROS generation, decreased ΔΨm, down-regulated expression of the anti-apoptosis protein Bcl-2, and induced mitochondria-mediated apoptosis.
In conclusion, the findings presented in this study demonstrated, for the first time, that cladribine and gefitinib, and cladribine and dasatinib synergistically inhibited the growth of MCF-7 cells in vitro. This inhibition was achieved partly by the generation of ROS and the induction of apoptosis. Additional studies are needed to elucidate the mechanistic interactions of these drug combinations. Moreover, the idea of combining antimetabolites with tyrosine kinase inhibitors should be given greater attention, as it has great potential in the treatment of tumors with minimal side effects.
The human breast cancer cell line MCF-7 was purchased from China Center for Type Culture Collection (Wuhan, Hubei, China). Cells were cultured in RPMI-1640 medium (Gibco) supplemented with 10% fetal bovine serum (FBS) and the cells were incubated in a humidified atmosphere with 5% CO2 at 37°C. Gefitinib, cladribine and dasatinib (purity >98%) were purchased from Haoyuan Chemexpress Co., Ltd. (Shanghai, China).
Cell Viability AssayCell viability was tested by the MTT assay (Sigma-Aldrich, U.S.A.).29) MCF-7 cells were seeded in 96-well plates at 6×103 cell/well. After overnight incubation, the cells were exposed to gefitinib, cladribine or dasatinib alone or in the combination of cladribine and gefitinib, or cladribine and dasatinib. Forty-eight hours after drug treatment, 10 µL of a 5 mg/mL MTT solution was added to each well and incubated for another 4 h in a humidified atmosphere with 5% CO2 at 37°C. Subsequently, the medium was removed and 100 µL of dimethyl sulfoxide (DMSO) was added to each well, incubated for ten minutes, and the resulting absorbance at wavelength of 492 nm was detected using a Thermo Multiskan MK3 (Thermo Fisher, U.S.A.). According to the absorbance values, the inhibitory concentration 50% (IC50) was determined. The ratios of drugs used in each combination were determined using the IC50 ratios of each drug. All samples were measured in five technical replicates, with the experiments repeated in at least three biological replicates.
Interactions between the two drugs were analyzed according the median-effect principle proposed by Chou and Talalay.23,30) The CI was calculated by CompuSyn software and a CI value below 1, equal to 1, and greater than 1 represented synergism, additive effect, and antagonism, respectively. Chou used the DRI to measure the fold-number of reduction in a dose that may be produced by the synergistic combination of two drugs at a given effect level compared with the doses of each drug alone.23)
Nuclear StainingMCF-7 Cells were detached from 6-well plates by trypsinization, collected by centrifugation, washed in phosphate buffered saline (PBS) and fixed with 4% paraformaldehyde overnight at 4°C. Then the cells were washed with PBS and stained with 50 µg/mL PI (Sigma) containing 20 µg/mL RNase A for 30 min at 37°C in the dark. Nuclear morphology of apoptotic cells with condense/fragmented nuclei was detected by laser scanning confocal microscopy.
Cell Cycle and Cell Apoptosis by Flow CytometryCellular apoptosis and cell cycle stage were determined by flow cytometry after 48 h drug exposure. The cells were seeded into 6-well plates and cultured with gefitinib (15 µM), cladribine (15 µM), and dasatinib (3 µM) alone or in combination for 48 h. After treatment, the attached and floating cells were collected by centrifugation. Quantitative analysis of apoptosis was determined by double staining with Annexin V-fluorescein isothiocyanate (FITC)/PI according to the Apoptosis Detection Kit instruction. Cells for cell cycle analysis were washed with cold PBS and fixed with ice-cold 70% ethanol at −20°C overnight. Afterwards, the cells were treated with 20 µg/mL RNase A for 30 min and stained with PI for 30 min at 37°C in the dark. Finally, the DNA content of the cells was analyzed by a FACSCalibur flow cytometer equipped with the CellQuest and Modifit software (Becton Dickinson, U.S.A.).
ROS DetectionWe used the fluorescent probe DCFH-DA to measure intracellular ROS. ROS generation was evaluated according to the method of Engelmann et al.31) Briefly, the cells (1×105/mL) were grown on sterile cover slips and were treated with 15 µM of gefitinib, 15 µM of cladribine or 3 µM of dasatinib alone or in combination for 12 h. The cells were then washed with PBS and incubated with 200 µL DCFH-DA (10 µM) for 30 min in a humidified atmosphere at 37°C. Subsequently, the cells were washed with serum-free cell culture medium. Fluorescence was detected by a confocal microscope (Leica TCS SP2) with 100× objective at an excitation wavelength 488 nm and an emission wavelength of 530 nm. The fluorescence intensity (FI) of 50 cells from each sample was quantified using Leica software and Image J software.
Mitochondrial Transmembrane Potential AssessmentRhodamine 123 selectively enters mitochondria in living cells and is widely used to detect changes in mitochondrial membrane potential by confocal scanning microscopy.32) The cells (1×105/mL) grown on the aseptic cover slips were treated with 15 µM of gefitinib, 15 µM of cladribine or 3 µM of dasatinib alone or in combination for 12 h. The cells were then washed with PBS and incubated with 200 µL rhodamine 123 (2 µM) for 30 min in a humidified atmosphere at 37°C. The fluorescence images were recorded by a confocal microscope (Leica TCS SP2) with 100×objective and excitation and emission settings of 505 and 520 nm, respectively. The FI of 50 cells from each sample was quantified with Leica software and Image J software.
ImmunofluorescenceCells (1×105/mL) growing on aseptic cover slips in 24-well plates and were treated with 15 µM of gefitinib, 15 µM of cladribine or 3 µM of dasatinib alone or in combination for 12 h. After treatment, the cells were washed with PBS and fixed with 4% paraformaldehyde for 40 min, following which the cells were permeabilized with 2% Tween20 in PBS for 30 min at 37°C. Subsequently, cells were blocked with bovine serum albumin (BSA) for 30 min at 37°C and incubated with anti-Bcl-2 (1 : 200) (Cell Signaling Technology) antibodies at 4°C overnight. Proteins were then visualized on a confocal microscope after incubation with FITC-conjugated secondary anti-mouse immunoglobulin G (IgG) antibody. The FI of cells from each sample was quantified with Leica software and Image J software.
Western BlottingThe protein was separated on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gels at 14% polyacrylamide and blotted onto a polyvinylidene fluoride (PVDF) nitrocellulose membrane (Biosharp, China). Membranes were blocked in 5% skim milk powder in TBST for 1 h, and probed with a first antibody (Bcl-2) overnight at 4°C. They were incubated with horseradish peroxidase-conjugated IgG for 1 h at room temperature, and then visualized with enhanced chemiluminescence reagent (Millipore, U.S.A.).
Statistical AnalysisAll data are presented as mean±standard deviation (S.D.) from more than three biological replicates. SPSS16.0 and one-way ANOVA were used to analyze the data, and p<0.05 was considered as statistically significant.
This work was supported by the National Natural Science Foundation of China (No. 31100987) and the Natural Science Foundation of Shandong Province (No. ZR2016CM46).
The authors declare no conflict of interest.
