Abstract
The tyrosine kinase inhibitor sunitinib was recently approved for use against gastrointestinal stromal tumors and advanced renal cell carcinoma. Yet, the protective effect of sunitinib against breast cancer has been poorly investigated. In this study, we investigated the antiproliferative and apoptogenic effects of sunitinib and the possible mechanism involved against the MCF7 human breast cancer cell line. Treatment of MCF7 cells with sunitinib caused concentration-dependent cell growth suppression due to apoptosis. Apoptotic death induced by sunitinib in MCF7 cells was mediated by activation of caspase-3 and p53 mRNA and protein expression and an increase in the percentage of apoptotic cells (40%) as determined by flow cytometry. Apoptosis was associated with a significant inhibition of nuclear factor-kappa B mRNA and protein expression. Mechanistically, blocking of de novo RNA synthesis by actinomycin D significantly inhibited sunitinib-induced expression of p53 mRNA, but not that of caspase-3, indicating involvement of a transcriptional mechanism. This apoptosis-mediated inhibition of MCF7 cell growth was attributed to inhibition of cell cycle-related genes (cyclin D1 and cyclin E2) and arrest of MCF7 cells in the G2/M phase in the cell cycle, allowing up-regulation of expression of DNA repair genes such as x-ray repair cross-complementing protein 1. In addition, sunitinib exhibited concentration-dependent induction of oxidative stress genes (heme oxygenase 1 and glutathione transferase A1) through the nuclear factor erythroid 2–related factor 2 pathway. These findings lead us to propose that sunitinib suppressed the proliferation of MCF7 cells via cell-cycle arrest and apoptotic- and oxidative stress-mediated pathways.
Breast cancer is one of the most widespread and lethal forms of cancer, with almost one million new cases of breast cancer identified each year worldwide. The incidence of breast cancer morbidity and mortality has increased more than 20% since 2008 (1). Although a definitive predictor for breast cancer development is not available, many factors have been identified that facilitate the classification of women with predisposed risk to this disease (2-4). Although the etiology of breast cancer is still unclear, several signaling pathways that control cell proliferation are thought to be involved in disease progression and hence maybe targets for therapy. At an early stage of diagnosis, therapy can reduce the mortality of breast cancer (5-7).
One of those promising therapies for breast cancer is sunitinib (Figure 1), a multitargeted tyrosine kinase inhibitor. Sunitinib possesses antitumor and anti-angiogenic activity against several types of cancer, mainly gastrointestinal stromal tumors and advanced renal cell carcinoma (8). Sunitinib exerts its effect through inhibition of several receptor tyrosine kinases that are implicated in breast cancer growth and metastasis, such as vascular endothelial growth factor (VEGF) receptor, platelet-derived growth factor receptor, and colony-stimulating factor-1 receptor (9, 10). A previous study has reported that sunitinib targeted the paracrine and autocrine effects of VEGF on breast cancer to suppress tumor angiogenesis, proliferation and migration in a mouse estrogen receptor-positive breast cancer model (11). In addition, sunitinib combined with chemotherapeutic agents, such as docetaxel, fluorouracil or doxorubicin, enhanced the antitumor activity and increased the survival in patients with human epidermal growth factor receptor 2/neu-negative advanced breast cancer (10).
Several previous reviews and studies have reported an association between cell proliferation, cell-cycle control and apoptosis with carcinogenesis (12-14). Activation of apoptosis has been proposed as a potential mechanism for the effects of chemotherapeutic agents. Recent data have shown that sunitinib inhibited tumorgenesis and carcinogenesis in basal-like triple-negative breast cancer cells through inhibiting proliferation and migration, and increasing apoptosis (15).
Although studies have shown that sunitinib exerts protective effects against experimentally-induced carcinogenesis, the effects of sunitinib on breast cancer cell lines, particularly MCF7 cells, and the molecular mechanism(s) involved are still very limited. Hence, the present study was designed to test the hypothesis that sunitinib induces MCF7 cell growth and proliferation suppression through apoptotic, cell-cycle, and oxidative stress pathways.
Materials and Methods
Materials. Sunitinib malate [(Z)-N-(2-(diethylamino) ethyl)-5-((5-fluoro-2-oxoindolin-3-ylidene) methyl)-2,4-dimethyl-1H-pyrrole-3-carboxamide)] was obtained from LC Laboratories (Woburn, MA, USA). Dulbecco's modified Eagle's medium (DMEM), IgG peroxidase secondary antibody, protease inhibitor cocktail, and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) were purchased from Sigma Chemical Co. (St. Louis, MO, USA). TRIzol reagent, annexinV–fluorescein isothiocyanate (FITC), and propidium iodide (PI) were purchased from Invitrogen Co. (Grand Island, NY, USA). High Capacity cDNA Reverse Transcription kit and SYBR® Green PCR Master Mix were purchased from Applied Biosystems® (Foster city, CA, USA). Actinomycin D (Act-D) was purchased from Calbiochem® (San Diego, CA, USA). Caspase-3 colorimetric activity kit was purchased from Biovision® (Mountain View, CA, USA). Resveratrol was obtained from Toronto Research Chemicals (Toronto, ON, CANADA). Nitrocellulose membrane was obtained from Bio-Rad Laboratories (Hercules, CA, USA). Primary antibodies against target proteins were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA). Chemiluminescence western blot detection kit (Luminate forte) was obtained from Merck Millipore (Billerica, MA, USA). All other chemicals were purchased from Fisher Scientific Co. (Toronto, ON, Canada).
Cell culture and treatments. MCF7 Human breast cancer cells, obtained from the American Type Cutler Collection (Manassas, VA, USA), were maintained in DMEM with phenol red supplemented with 10% fetal bovine serum, 1X antibiotic-antimycotic (100 units/ml of penicillin, 100 μg/ml of streptomycin, and 250 ng/ml of amphotericin B), Gibco® (Grand Island, NY, USA). Cells were grown in 75 cm2 tissue culture flasks at 37°C under a 5% CO2 humidified environment.
Chemical structure of sunitinib.
The cells were seeded onto 96-, 12-, and 6-well cell culture plates in DMEM culture medium for enzyme activity, mRNA, and protein assays, respectively. In all experiments, the cells were treated for different time intervals in serum-free medium with different concentrations of sunitinib (2.5, 5, and 10 μM). Stock solutions of sunitinib was prepared in dimethylsulfoxide (DMSO) and stored at −20°C. The DMSO concentration did not exceed 0.05% (v/v).
Cell proliferation assay. The effect of sunitinib on MCF7 cell proliferation and growth was determined by measuring the capacity of reducing enzymes present only in viable cells to convert MTT to colored formazan crystals as described previously (16). The percentage of cell viability was calculated relative to control wells designated as 100% viable cells using the following formula: cell viability=(Atreated)/(Acontrol) ×100%.
Total RNA extraction and cDNA synthesis. Total cellular RNA was isolated using TRIzol reagent (Invitrogen®) according to the manufacturer's instructions and quantified by measuring the absorbance at 260 nm. RNA quality was determined by measuring the 260/280 ratio (~2.0). First-strand cDNA was synthesized using the High-Capacity cDNA reverse transcription kit (Applied Biosystems®), according to the manufacturer's instructions and as described previously (16). Briefly, 1.5 μg of total RNA from each sample was added to a mixture of 2.0 μl of 10× reverse transcriptase buffer, 0.8 μl of 25× dNTP mix (100 mM), 2.0 μl of 10× reverse transcriptase random primers, 1.0 μl of MultiScribe reverse transcriptase, and 3.2 μl of nuclease-free water. The final reaction mixture was kept at 25°C for 10 min, heated to 37°C for 120 min, heated for 85°C for 5 min, and finally cooled to 4°C.
Quantification of mRNA expression by real-time polymerase chain reaction (RT-PCR). Quantitative analysis of specific mRNA expression was performed by RT-PCR by subjecting the resulting cDNA to PCR amplification using 96-well optical reaction plates in the ABI 7500 Fast RT-PCR System (Applied Biosystems®) using SYBR Green Universal Mastermix as described previously (17). Human primers for target genes (Table I) were purchased from Integrated DNA technologies (IDT, Coralville, IA, USA). The fold change in the level of gene expression between treated and untreated cells were corrected by the levels of β-actin expression Assay controls were incorporated onto the same plate, namely, no-template controls to test for the contamination of any assay reagents. The RT-PCR data were analyzed using the relative gene expression (i.e. ΔΔ Ct) method, as described and explained previously (18). The average ΔCt from untreated MCF7 cells was used as a calibrator for each gene tested. The fold change in the level of target genes between treated and untreated cells, corrected by the level of β-actin, was determined using the following equation: fold change=2−Δ(ΔCt), where ΔCt=Ct(target) −Ct(β-actin) and Δ(ΔCt)=ΔCt(treated) −ΔCt(untreated).
Primer sequences used for real-time polymerase chain reactions.
Determination of caspase-3 activity. Caspase-3 activity was measured colorimetrically using the CaspACE assay system purchased from Biovision® (Mountain View, CA, USA) according to the manufacturer's instructions as described previously (19). Approximately 30 μg protein was incubated with 200 μM enzyme-specific colorimetric caspase-3 substrate I, acetyl-Asp-Glu-Val-Asp p-nitroanilide (Ac-DEVD-pNA) at 37°C for 2 h. Caspase-3 activity was assessed by measuring absorbance at a wavelength of 405 nm with a plate reader BioTek (Winooski, VT, USA).
Western blot analysis. Western blot analysis was performed using a previously described method (20). Briefly, 25-35 μg of protein from each treatment group, isolated from MCF7 cells as described previously (20), was separated by 10% sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis, and then electrophoretically transferred to nitrocellulose membrane. Protein blots were then blocked overnight at 4°C in blocking solution then washed several times with Tris-buffered saline (TBS)-Tween-20 before being incubated with primary antibodies against p53, cyclin D1, and cyclin E1 proteins over night at room temperature in TBS solution followed by incubation with a peroxidase-conjugated IgG secondary antibodies for 2 h at room temperature. The bands were visualized and then quantified by C-DiGit® Blot Scanner, LI-COR Biosciences (Lincoln, NE, USA) using the enhanced chemiluminescence method according to the manufacturer's instructions, Merck Millipore (Billerica, MA, USA). Membranes were subsequently stripped in a solution containing 62.5 mM Tris-HCl pH 6.7, 100 mM 2-mercaptoethanol, and 2% SDS for 30 min at 50°C and thereafter re-probed with goat anti-human β-actin primary antibody, which was used as loading control, as described above.
Flow cytometric analysis of apoptosis with annexin V staining. The percentage of cells undergoing apoptosis/necrosis was determined by flow cytometry using annexin V staining as described previously (21). Briefly, MCF7 cells were treated for 24 h with 5 μM sunitinib thereafter, the medium was removed, and cells were washed with cold phosphate-buffered saline (PBS) before trypsinization. The collected cells were centrifuged at 300 × g for 5 min and then re-suspended with 0.5 ml PBS. Cells were then stained with annexinV–FITC/PI and immediately analyzed on a Beckman Coulter FC500 Cytomics Benchtop flow cytometer, Beckman Coulter Life Sciences (Indianapolis, IN, USA) for determination of apoptotic and necrotic populations.
Analysis of cell-cycle phase distribution and progression. MCF7 cell-cycle distribution was analyzed using flow cytometery as described previously (21). Briefly, MCF7 cells seeded in a 6-well culture plates were treated for 24 h with different concentrations of sunitinib (2.5, 5, and 10 μM). Thereafter, cells were trypsinized, harvested, and fixed in 1 ml of 70% cold ethanol in test tubes and incubated at −20°C for 30 min. After incubation, cells were centrifuged at 450 × g for 5 min and the cell pellets were re-suspended in 500 μl PI (10 μg/ml) containing 300 μg/ml RNase. Cells were then incubated on ice for 30 min and filtered with a 53 μm nylon mesh. Cell-cycle distribution was calculated from 10,000 cells with ModFit LT® software using FACScaliber (Becton Dickinson, CA, USA).
Statistical analysis. The comparative analysis of the results from various experimental groups with their corresponding controls was performed using SigmaStat® for Windows, Systat Software, Inc, (San Jose, CA, USA). One-way analysis of variance (ANOVA) followed by Student–Newman–Keul's test was carried out to assess which treatment groups showed a significant difference from the control group. The differences were considered significant when p<0.05.
Effect of sunitinib (SUN) on MCF7 cell proliferation and growth. MCF7 cells were treated for 24 h with different concentrations of sunitinib. Cell viability was determined using the MTT assay. Values are presented as a percentage of the control (mean±SEM, n=6). *Significantly different at p<0.05 compared to control (0 μM).
Results
Sunitinib treatment induces inhibition of cell growth and proliferation. The concentration of sunitinib that inhibited MCF7 cell proliferation and growth was determined by the MTT assay after exposure of the MCF7 cells for 24 h to a wide range concentrations of sunitinib. Our results showed that concentrations up to 2.5 μM did not significantly affect cell viability or proliferation (Figure 2). However, 5 and 10 μM sunitinib significantly reduced cell viability by approximately 25% and 30%, respectively, whereas, higher concentrations (20 μM and 40 μM) markedly inhibited cell proliferation by 70% and 80%, respectively. Based on these findings, sunitinib concentrations of 2.5, 5, and 10 μM were utilized in all subsequent experiments on MCF7 cells (Figure 2).
Effects of sunitinib treatment on apoptosis in MCF7 cells. To examine whether the inhibitory effect of sunitinib on MCF7 cell proliferation and growth is an apoptosis-mediated mechanism, a series of independent experiments were conducted as follows. In order to determine the capacity of sunitinib to modulate the expression of apoptotic and anti-apoptotic genes, MCF7 cells were incubated for 24 h with either vehicle (DMSO) or increasing concentrations of sunitinib (2.5, 5, and 10 μM), thereafter caspase-3 and B-cell lymphoma 2 (BCL2) mRNA expressions were determined by RT-PCR. Figure 3A shows that sunitinib significantly induced caspase-3 but not BCL2 mRNA expression in a concentration-dependent manner. The maximum induction was observed at the highest concentration tested (10 μM) by approximately 3-fold.
In order to further investigate whether the induction of caspase-3 mRNA by sunitinib is also translated into a functional activity, we tested the effect of sunitinib on caspase-3 activity. Figure 3B shows that sunitinib increased caspase-3 activity in a dose-dependent manner in a fashion similar to that observed at the mRNA level.
Next, we questioned whether the induction of human caspase-3 mRNA by sunitinib (Figure 3A) is regulated at the transcriptional level. Therefore, we tested the hypothesis that sunitinib increases the de novo synthesis of caspase-3 mRNA. For this purpose, MCF7 cells were treated for 24 h with a single concentration of sunitinib (10 μM), which showed maximal induction, in the presence and absence of 5 μg/ml Act-D, an inhibitor of RNA synthesis. Thereafter, caspase-3 mRNA expression was quantified by RT-PCR. If sunitinib increased the amount of caspase-3 mRNA through increasing its de novo RNA synthesis, we would expect to observe a decrease in their mRNA content after inhibition of RNA synthesis. Figure 3C shows that pretreatment of the cells with RNA synthesis inhibitor Act-D did not alter expression of caspase-3 mRNA. On the other hand, co-treatment of MCF7 cells with 20 μM resveratrol, a well-known chempreventive compound and caspase-3 activator (22), significantly potentiated sunitinib-induced caspase-3 mRNA expression (Figure 3D).
Sunitinib-induced caspase-3 gene expression is associated with inhibition of NF-kB signaling pathway. Constitutive activity of the NF-κB transcription factor in different cancer cells appears to be important for their growth or resistance to induced apoptosis (23). To investigate whether the induction of apoptosis by sunitinib was associated with inhibition of NF-κB pathway, MCF7 cells were treated with sunitinib (2.5, 5, and 10 μM) and thereafter mRNA and protein expression levels of NF-κB were determined by RT-PCR and western blot analyses. Our results show that sunitinib significantly inhibited NF-κB mRNA and protein expression in a concentration-dependent manner to approximately 15% that of control levels at the highest concentration (10 μM) (Figure 3E and F).
Sunitinib treatment increases the percentage of apoptotic MCF7 cells. To further explore whether the sunitinib-induced reduction in cell viability is due to increased apoptotic or necrotic cell populations, the percentage of cells undergoing apoptosis/necrosis in response to a single concentration of sunitinib (5 μM) was determined by staining with annexinV–FITC and PI using flow cytometry. Figure 4 shows that 5 μM sunitinib significantly increased the percentage of cells undergoing early apoptosis from 8% to 12% and the percentage of cells undergoing late apoptosis to approximately 30% of the total cell population, as compared to the control. Therefore, approximately 42% of the total cell population became apoptotic with sunitinib treatment.
Effects of sunitinib (SUN) on the expression of apoptosis-related genes in MCF7 cells. A: MCF7 cells were treated for 24 h with different concentrations of sunitinib. Total RNA was isolated using TRIzol reagent and mRNA of caspase-3 and B-cell lymphoma 2 (BCL2) genes was quantified by real-time polymerase chain reaction (RT-PCR) and expression normalized to that of β-actin housekeeping gene. Duplicate reactions were performed for each experiment and the values represent mean of fold change±SEM, n=6. B: MCF7 cells were treated for 24 h with sunitinib, and thereafter caspase-3 activity was determined calorimetrically using the CaspACE assay system (Biovision®). Values are presented as mean±SEM (n=6). C and D: MCF7 cells were treated for 24 h with sunitinib (10 μM) in the presence and absence of either actinomycin D (Act-D) or resveratrol (RES). Caspase-3 was quantified by RT-PCR and normalized to β-actin housekeeping gene. Duplicate reactions were performed for each experiment and the values represent mean fold change±SEM, n=6. E: MCF7 cells were treated for 24 h with different concentrations of sunitinib. Total RNA was isolated using TRIzol reagent and mRNA of nuclear factor kappa B (NF-κB) gene was quantified by RT-PCR and expression normalized to that of β-actin housekeeping gene. F: MCF7 cells were treated for 24 h with sunitinib. Total protein was isolated and NF-κB protein expression was determined by western blot analysis. The intensity of protein bands was quantified relative to the signals obtained for β-actin protein using C-DiGit® blot scanner (LI-COR Biotechnology, Lincoln, NE, USA). One of three representative experiments is shown. Values are presented as the mean±SEM, n=3. *Significantly different at p<0.05 compared to control (0 μM). NS: Not significantly different.
Effect of sunitinib (SUN) on the percentage of apoptotic MCF7 cells. MCF7 cells were treated for 24 h with sunitinib, thereafter the percentage of cells undergoing apoptosis/necrosis pathway was determined. Cells were stained with annexin V – Fluorescein isothiocyanate (FITC)/propidium iodide (PI) and immediately analyzed on a Beckman Coulter FC500 Cytomics Benchtop Flow Cytometer. *Significantly different at p<0.05 compared to control (0 μM).
Sunitinib treatment induces the expression of p53 tumor-suppressor gene at the transcriptional level. Since the tumor-suppressor gene, p53, slows down cell proliferation and mediates apoptosis (24), we examined the effect of sunitinib on p53 gene expression. Figure 5A shows that sunitinib significantly induced p53 mRNA expression only at higher concentrations, 5 and 10 μM by approximately 1.7-and 2.1-fold, respectively. Similarly at the protein level, sunitinib caused significant induction of p53 protein expression at 2.5, 5 and 10 μM by approximately 3.5-, 3-, and 2-fold, respectively (Figure 5B).
Next, we questioned whether the induction of human p53 gene by sunitinib (Figure 5A) is regulated at the transcriptional level. Therefore, we tested the hypothesis that sunitinib increases de novo p53 RNA synthesis. For this purpose, MCF7 cells were treated for 24 h with a single concentration of sunitinib (10 μM), which showed maximal induction, in the presence and absence of 5 μg/ml Act-D. Thereafter, p53 mRNA expression was quantified by RT-PCR. If sunitinib increased the amount of p53 mRNA through increasing its de novo RNA synthesis, we would expect to observe a decrease in the content of mRNA after the inhibition of RNA synthesis. Figure 5C shows that pretreatment of the cells with Act-D, an RNA synthesis inhibitor, completely blocked sunitinib-induced p53 mRNA expression.
Sunitinib treatment induces MCF7 cell-cycle arrest. To examine whether the inhibitory effect of sunitinib on MCF7 cell proliferation and growth is attributed to cell-cycle arrest, we addressed this hypothesis by two approaches. Firstly, we measured the mRNA and protein expression levels of two cell-cycle markers, cyclin D1 and cyclin E1, in response to increasing concentrations of sunitinib (2.5, 5, and 10 μM). Figure 6A shows that sunitinib at 2.5 μM significant induced cyclin D1 and cyclin E1 mRNA expression by approximately 35% and 60%, respectively. In contrast, higher concentrations of 5 and 10 μM significantly reduced the mRNA expression in a concentration-dependent manner by approximately 65% and 80%, respectively for cyclin D1 and by approximately 50% and 65%, respectively for cyclin E1. Similarly, sunitinib concentration-dependent inhibition of cyclin D1 and cyclin E1 protein expression was observed (Figure 6B). These results correlated with the inhibition of cell proliferation induced by sunitinib in MCF7 cells.
The second approach was to examine the effect of sunitinib on the cell-cycle profile by flow cytometry. After 24 h treatment with sunitinib cells in the G2/M population increased in a concentration-dependent manner (Figure 6C). The increase of MCF7 cell population at the G2/M phase was accompanied by a concentration-independent decrease of cells in the G0/G1 phase (Figure 6C). On the other hand; the population of cells at the S phase was not significantly changed by sunitinib treatment.
Effects of sunitinib treatment on the expression of DNA repair genes. Induction of cell-cycle arrest allows more time for DNA repair activation (25). To further examine whether induction of cell-cycle arrest and inhibition of cell proliferation was associated with an increased expression of DNA repair genes, we quantified the mRNA expression of human apurinic endonuclease 1 (APE1) and x-ray repair cross-complementing protein 1 (XRCC1) genes in response to increasing concentrations of sunitinib. Figure 7 shows that sunitinib significantly induced APE1 and XRCC1 mRNA expression at the higher concentrations by approximately 50%.
Effect of sunitinib (SUN) on the tumour-suppressor gene p53. A: MCF7 cells were treated for 24 h with different concentrations of sunitinib. Total RNA was isolated using TRIzol reagent and p53 mRNA was quantified by RT-PCR and normalized to β-actin housekeeping gene. Duplicate reactions were performed for each experiment and the values represent mean of fold change±SEM, n=6. B: MCF7 cells were treated for 24 h with sunitinib. p53 protein expression was determined by western blot analysis as described in the Materials and Methods section. One of three representative experiments is shown. Values are presented as the mean±SEM, n=3. C: MCF7 cells were treated for 6 h with sunitinib (10 μM) in the presence and absence of actinomycin D (Act-D). p53 mRNA level was quantified by RT-PCR as described before. Values represent mean of fold change±SEM. (n=6). Significantly different at *p<0.05 compared to control (0 μM).
Effect of sunitinib (SUN) on the mRNA expression of cell-cell cycle markers and arrest. A: MCF7 cells were treated for 24 h with different concentrations of sunitinib. Cylin D1 (CCND1) and cyclin E1 (CCNE1) mRNA levels were quantified by real-time polymerase chain reaction as described in the Materials and Methods section. Values represent mean fold change±SEM (n=6). B: MCF7 cells were treated for 24 h with sunitinib. Cyclin D1 and cyclin E1 protein expression levels were determined by western blot analysis as described in the Materials and Methods section. One of three representative experiments is shown. Values are presented as the mean±SEM, n=3. C: MCF7 cells were treated with sunitinib for 24 h, thereafter changes in cell-cycle distribution were determined by flow cytometry. *Significantly different at p<0.05 compared to control (0 μM).
Effect of sunitinib (SUN) on DNA repair genes. MCF7 cells were treated for 24 h with different concentrations of sunitinib. Total RNA was isolated using TRIzol reagent and the x-ray repair cross-complementing protein 1 (XRCC1) and human apurinic endonuclease 1 (APE1) mRNA levels were quantified by real-time polymerase chain reaction and normalized to β-actin housekeeping gene. Duplicate reactions were performed for each experiment and the values represent mean fold change±SEM (n=6). *Significantly different at p<0.05 compared to control (0 μM).
Effects of sunitinib treatment on the expression of oxidative stress genes. The role of oxidative stress in the sunitinib inhibition of MCF7 cell proliferation and growth was examined by measuring the capacity of sunitinib to modulate the expression of oxidative and antioxidant genes. Thus, MCF7 cells were incubated for 24 h with increasing concentrations of sunitinib, thereafter heme oxygenase 1 (HO1), glutathione transferease A1 (GSTA1), and NAD(P)H:quinone oxidoresuctase 1 (NQO1) mRNA levels were measured by RT-PCR. Figure 8 shows that sunitinib induced HO1 and GSTA1 mRNA expression only at higher concentrations (5 and 10 μM). For example, 10 μM sunitinib induced HO1 and GSTA1 expression by approximately 2- and 3-fold, respectively (Figure 8A). In contrast, sunitinib did not significantly alter NQO1 mRNA levels at any tested concentrations (data not shown).
To examine the role of redox-sensitive transcription factors, nuclear factor erythroid 2–related factor 2 (NRF2), in sunitinib-induced modulation of oxidative stress genes, cells were incubated for 24 h with increasing concentrations of sunitinib, thereafter NRF2 mRNA level was quantified by RT-PCR. Figure 8B shows that sunitinib significantly induced NRF2 mRNA expression by approximately 1.7-fold only at the highest concentration (10 μM).
Effect of sunitinib (SUN) on the mRNA expression of oxidative stress genes. MCF7 cells were treated for 24 h with different concentrations of sunitinib. Total RNA was isolated using the TRIzol reagent and the heme oxygenase 1 (HO1), glutathione transferease A1 (GSTA1), and nuclear factor erythroid 2–related factor 2 (NRF2) mRNA levels were quantified by real-time polymerase chain reaction and normalized to β-actin housekeeping gene. Duplicate reactions were performed for each experiment and the values represent mean fold change±SEM (n=6). *Significantly different at p<0.05 compared to control (0 μM).
Discussion
The present study demonstrates strong evidence that the tyrosine kinase inhibitor sunitinib induces MCF7 human breast cancer cell growth inhibition through apoptosis, cell-cycle arrest, and oxidative stress mechanisms. This is supported by the following findings; a) induction of apoptotic gene markers at the mRNA, protein, and activity levels associated with increased percentage of apoptotic cells; b) inhibition of cell proliferation-related genes and arrest of MCF7 cells in the G2/M phase in the cell-cycle, allowing activation of DNA repair genes; and c) induction of oxidative stress markers and mediated transcription factors (Figure 8).
The in vitro MCF7 cell model was utilized in the current study to evaluate the antiproliferative effects of sunitinib on human breast cancer cells and explore the mechanisms involved since they are largely unknown in breast cancer. The in vitro concentrations of sunitinib used in this study were maintained within the therapeutic range of plasma concentration reported in human. For example, humans given 80 mg sunitinib for the treatment of advanced renal cell carcinoma had mean plasma concentrations ranging from 0.5 to 1 μM (26). Although the current in vitro sunitinib concentrations were above the feasible therapeutic plasma level in human, higher in vitro concentrations could be attributed to the excessive concentrations of insulin, glucose, FBS, and growth factor in culture media that stimulate cell growth, and promote breast cancer aggression (27). In addition, the presence of oncogenic mutation in the cell-culture system may account for the reduced efficacy and higher concentration of sunitinib required to elicit cellular responses in vitro in order to achieve the anticancer effects of sunitinib in cell-culture systems (28). This provides a high degree of in vivo relevance to the results arising from the concentrations of sunitinib (1-5 μM) that might be attained during cancer treatment. Therefore, the present results are still clinically relevant.
Activation of caspase-3 is well known to play a central role in the initiation of apoptosis (29). The ability of sunitinib to induce the expression of caspase-3 mRNA and activity, but not BCL2, suggests a death receptor-mediated pathway (29). This is supported by the finding that treatment of MCF7 cells with various inducers of apoptosis significantly inhibited the cell growth without causing morphological changes (30). Similarly to our observations, it has been reported that resveratrol inhibited MCF7 cell proliferation through induction of apoptosis (22). In addition, the increase in the percentage of apoptotic cell population (annexinV/PI-positive cell) by 40% in response to sunitinib as compared to the control clearly confirms that sunitinib is a potent inducer of apoptosis and triggers events leading to apoptotic cell death. It is generally believed that apoptosis can be induced by the activation of pro-apoptotic signaling or inhibition of survival signaling, such as transcription factor, NF-κB. In this context, we report here that sunitinib-induced apoptosis was associated with NF-κB inhibition at the mRNA and protein expression levels. These results are in agreement with the fact that caspase increases NF-κB cleavage and this inhibits its survival activity (31).
The cell cycle is a critical process that a cell undergoes in order to copy itself exactly. Deregulation of the cell cycle by abnormal expression of one or several cell-cycle regulatory proteins is a common finding in malignant tumors and cancer development. Thus, it is well reported that cell-cycle arrest and apoptosis are closely related events in which disruption of cell-cycle progression may ultimately lead to apoptotic death (12, 13). In the current study, ability of sunitinib to induce cell-cycle arrest is evidenced by three observations; firstly, sunitinib induced expression of tumor-suppressor gene, p53, at mRNA and protein levels in a concentration-dependent manner at the transcriptional level through increasing its de novo RNA synthesis. Secondly, sunitinib-induced apoptosis was associated with a concentration-dependent inhibition of cell cycle-related genes cyclin D1 and cyclin E1 at the mRNA and protein expression levels, which are definitely the most widely studied cyclins in breast tumors (12, 32). Thirdly, sunitinib arrested MCF7 cell-cycle progression in the G2/M phase through the suppression of cyclin D1 and E1, which may contribute to the apoptotic cell death process. Our results are in agreement with previous studies showed that over expression of cyclin D1 gene and hormone receptors were observed in breast tumors (33, 34) and were involved in cell cycle and estrogen receptor activation, which induces mitogenic growth factors by consecutive activation of cell cycle-dependent cyclin kinase complexes (13, 35). Thus, down-regulation of cyclin genes by anticancer drugs may be helpful in estrogen receptor-positive breast cancer cells, such as MCF7 cells. Similarly, previous studies showed that gefitinib, a tyrosine kinase inhibitor, induces G2/M arrest in breast cancer (36) and other cancer cell lines (37), whereas resveratrol was able to induce S-phase cell-cycle arrest in different breast cancer cell lines (38).
Cells which are highly proliferating, such as cancer cells, have less time to repair their damaged DNA. Thus, inhibition of cell proliferation-related genes and induction of cell-cycle arrest will allow more time to repair damaged DNA lesions. In this context, we report here that sunitinib treatment significantly induced the expression of the DNA repair genes (XRCC1 and APE1), which are involved in the base excision repair (BER) and single-strand break repair pathways (39). Our results are in agreement with a recent study on breast cancer showing that loss of XRCC1 activity by non-homologous end-joining pathway was an independent predictor of poor clinical outcome (40).
The ability of sunitinib to induce oxidative stress in a concentration-dependent manner as evidenced by the increased mRNA expression of HO1 and GSTA1 could be another mechanism mediating sunitinib-induced apoptosis and inhibition of cell proliferation. Mechanistically, this increase in the oxidative stress in the cells in response to sunitinib is attributed to increase the expression of redox-sensitive transcription factor NRF2 which subsequently induces antioxidants and promotes detoxifying enzymes such as GSTA1 (41). These results are supported by previous observations showing that induction of oxidative stress genes in breast carcinoma cells inhibits their proliferation and induces apoptosis and cell-cycle arrest (1, 42) through activation of ubiquitin-dependent proteasome degradation of cyclin D1 (43). In a manner similar to our observations, we recently reported that sunitinib inhibits proliferation of triple-negative MDA-MB231 breast cancer cells with induction of apoptosis through activation of Forkhead box type O (FOXO3A) transcription factor (44). Silencing of FOXO3A mRNA using siRNA significantly rescued MDA-MB231 cells from sunitinib-induced cell-proliferative arrest (44).
Conclusion
In the light of our findings, the current report provides first evidence that sunitinib causes breast cancer cell growth suppression through induction of apoptosis, oxidative stress, and cell-cycle arrest. Our findings increase our understanding of the possible therapeutic effect of sunitinib in breast cancer and open doors for further study.
Acknowledgements
This work was funded by the National Plan Science, Technology and Innovation (MAARIFAH), King Abdulaziz City for Science and Technology, Kingdom of Saudi Arabia, Award Number (12-MED3131-02).
Footnotes
Conflicts of Interest
There are no financial or other interests with regard to this manuscript that might be construed as a conflict of interest. All of the Authors are aware of and agree to the content of the manuscript and their being listed as an author on the manuscript.
- Received June 8, 2017.
- Revision received July 5, 2017.
- Accepted July 6, 2017.
- Copyright© 2017, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved
