Background
Among gynecologic cancers worldwide, epithelial ovarian carcinoma is the leading cause of death and the fifth most frequent cause of cancer related death across all cancers in women in the United States. Because ovarian carcinoma presents nonspecific symptoms and is often asymptomatic until late stages, the majority of patients are not diagnosed with ovarian carcinoma until they suffer from advanced stages of disease development [
1],[
2]. Platinum/taxane chemotherapy and cytoreductive surgery have proven effective as primary treatments in patients with advanced stage ovarian carcinoma, with a positive initial response in approximately 75-80% of patients. However, most patients relapse with lethal, chemo-resistant ovarian carcinoma [
3]. Rapid relapse and the development of drug resistance are the major challenges in ovarian cancer treatment that mandate the development of new adjuvant therapy for epithelial ovarian cancer.
Genetic alterations and deregulation of oncogene and tumor suppressor gene expressions are known to correlate with and promote the carcinogenesis of ovarian carcinoma. Deregulation of the expression of oncogene c-Myc is one of the most frequently encountered events present in epithelial ovarian carcinoma [
4]. Myc proteins are key regulators of cell proliferation, apoptosis, and differentiation and are thus active across multiple cellular pathways [
5]. Recent studies have provided strong evidence that c-Myc proteins combine with Max, a common Myc partner protein, to form heterodimers that can both bind to DNA and induce transactivation. The transcriptionally active c-Myc-Max dimer promotes proliferation, cell adhesion, apoptosis, and angiogenesis in cancer cells through its control on the transcription of Myc target genes [
5],[
6]. The concurrent disruption of c-Myc-Max heterotetramerization interferes with the function/expression of all subsequent downstream target genes, suggesting that the c-Myc-Max interaction is a promising molecular target for cancer therapeutics. Small-molecule c-Myc inhibitor, 10058-F4, is a cell-permeable thiazolidinone that specifically disrupts the formation and function of the c-Myc-Max heterodimer and prevents transactivation of c-Myc target genes [
7]. 10058-F4 exhibits potent anticancer activities towards liver, prostate, kidney, neuroblastoma, multiple myeloma, and lymphoma cells [
8]-[
13]. However, there is no evidence regarding the effect of 10058-F4 on ovarian carcinoma in vitro or in vivo. Understanding the molecular mechanisms of 10058-F4 in ovarian carcinoma cells may facilitate the development of improved therapeutic strategies for ovarian carcinoma.
In the present study, we aimed to elucidate the specific role of 10058-F4 in ovarian cancer cell growth. We used 10058-F4 to inhibit the c-Myc-Max interaction in two ovarian cancer cell lines expressing c-Myc in order to determine if the inhibition of c-Myc-Max subsequently inhibits cellular proliferation. This study provides cellular and molecular evidence for the impact of 10058-F4 on ovarian carcinoma cells through its inhibition of cell proliferation, and the induction of apoptosis and cell cycle arrest, and it offers the targeting of the c-Myc-Max interaction as a potential and viable strategy in ovarian cancer chemotherapy.
Materials and methods
Materials and reagents
10058-F4 (F4, Sigma, St. Louis, MO, USA) was dissolved in dimethylsulfoxide (DMSO) according the manufacturer's instructions and further diluted to indicated concentrations in culture medium before use. SKOV3 cells were maintained in DMEM RPMI-1640 culture medium supplemented with 10% fetal calf serum (FCS), 100 U/mL penicillin, and 100 mg/mL streptomycin. Hey cells were cultured in RPMI-1640 medium with 5% FBS. Fetal bovine serum (FBS) was from Invitrogen (Carlsbad, CA, USA). The Annexin V-FITC Apoptosis Detection kit and Caspase3 Activity Assay kit were purchased from Biovision (Mountain View, CA, USA). All antibodies were purchased from Cell Signaling (Boston, MA, USA). All other materials were obtained from Life Technologies or from Sigma-Aldrich.
MTT assays
Cells (4 × 103) were seeded in 96-well microplates treated with 10058-F4 as indicated. At 72 hours after incubation, 10 μL/well of 5 mg/mL 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) solution was added to the microplates. Two hours after MTT treatment, the medium was removed, and formazan crystals were dissolved by adding 100 μL dimethylsulfoxide (DMSO) per well. Cell viability was analyzed by measuring the absorbance at 575 nm using spectrometer plate reader (Tecan, Morrisville, NC). The number of viable cells was assessed by the percentage of absorbance of treated cells relative to that of solvent controls.
Colony formation assays were performed as described [
14]. Briefly, Hey and SKOV3 cells growing in log phase were seeded (3000 cells/well in a 6-well plate) in complete regular growth medium. Cells were allowed to adhere for 24 hours, and medium was replaced with fresh complete regular growth medium containing the indicated concentrations of 10058-F4. Cells were cultured at 37°C for 10 days, with medium changes every third or fourth day. Cells were stained with 0.5% crystal violet, and colonies were counted under microscope.
Cell cycle analysis using image cytometry
Hey and SKOV3 cells were treated with 10058-F4 for 24 hours, and cells were harvested and fixed with 70% (w/v) ice cold ethanol at 4°C for 1 hour. Fixed cells were washed twice with phosphate-buffered saline (PBS) and stained with PBS containing 50 μg/mL propidium iodide (PI) and 100 μg/mL RibonucleaseA (RNase A). Following a 30 minute incubation in darkness at 37°C, the number of cells in each cell cycle stage was analyzed using the Cellometer Vision CBA image cytometer (Nexcelom Bioscience, Lawrence, MA, USA)and FCS Express 4 (De Novo Software, Los Angeles, CA, USA). The fluorescence data was captured using FL1 with filter combination VB-660-502 (EX: 540 nm, EM: 660 nm).
Image cytometric analysis of apoptosis
The annexin V/propidium iodide assay was performed according to the manufacturer's recommendation. Briefly, SKOV3 and Hey cells were plated into 6-well plates and incubated for 24 hours with 10058-F4 at indicated doses. The cells were harvested, rinsed with cold PBS, re-suspended in 40 ul of binding buffer and then add 5ul FITC conjugated Annexin-V and 5ul 100 ug/ml PI and incubated for 15 min. at room temperature in the dark. A total of at least 2000 cells were collected and analyzed by image cytometry (Nexcelom Bioscience). We used FCS Express 4 software on the collected data to perform the image analysis. The fluorescence data was captured using VB-535-402 for annexin V-FITC (EX: 470 nm, EM: 535 nm) and VB-660-502 EX: 540 nm, EM: 660 nm for PI.
ATP determination assay
The ATP concentration was assessed quantitatively using the CellTiter-Glo Luminescent cell viability assay purchased from Promega Corp. (Madison, WI, USA) according to the manufacturer's instructions. Exponentially growing SKOV3 and Hey cells were seeded at a density of 4 × 103 cells per well in white opaque 96-well plates in 100 ul of fresh culture medium containing DMEM without phenol red for 24 hours at 37°C in 5% CO2. Different concentrations of 10058-F4 were added to the culture medium and SKOV3 and Hey cells were incubated at 37°C for 24 hours. Then, 100 ul CellTiter-Glo Reagent was added into each well. Finally, the luminescence was read with a microplate reader with an integration time of 1 second per well. The experiments were independently performed three times and each 10058-F4 treatment was performed in triplicate.
Reactive oxygen species (ROS) assay
ROS generation was assessed using an ROS-sensitive fluorescence indicator DCFH-DA and MitoSox red. To determine intracellular and mitochondria ROS scavenging activity, SKOV3 and Hey cells (1.0 × 104 cells/well) were seeded in black 96-well plates. After 24 hours, the cells were treated with 10058-F4 for 8 hours to induce ROS generation. After the cells were incubated with DCFH-DA (20 μM) or MitoSox (10 um) for 30 min., the fluorescence intensity was measured at an excitation wavelength of 485 nm and an emission wavelength of 530 nm for DCFH-DA and emission 590 nm for MitoSox using a Tecan spectrometer plate reader.
Caspase-activity assay
Caspase-3 activity was detected by using caspase-3 activity assay kit (Biovision). This assay is a Fluorometric assay which measures caspase-3 activities. The level of caspase-3 activity is measured by detection of cleavage of substrate DEVD-AFC. Briefly, Primary culture cells (1.5 × 105 cells/well) or ovarian cancer cells (2.5 × 105 cells/well) were seeded in 12 well plates or 6 well plates and incubated for 24 hours at 37°C. The cells were treated with 10058-F4 at different concentration for 24 hours and then lysated with lysis buffer. 50 ug cell lysates was used for measuring caspase-3 activity following manufacture instructions. The samples were reading in 96 well plates at a fluorometric plate reader.
Western blot analysis
Total proteins from SKOV3 and Hey cells were extracted in lysis buffer (Thermo Fisher Scientific, Rockford, IL) and quantified using the BCA method. Thirty micrograms of protein were separated by 12% SDS–PAGE gel. After transfer in cold room, the polyvinylidene fluoride (PVDF) membranes (Millipore, Billerica, MA, USA) were incubated overnight at 4°C with the following antibodies: beta-actin(1:3000; Santa Cruz Biotechnology, Santa Cruz, CA), cyclin D1(1:1000), CDK4(1:1000), CDK6(1:1000), p27 (1:1000) and p21(1:1000) (Cell Signaling, Boston, MA, USA). After incubation with peroxidase-coupled anti-mouse IgG and anti-rabbit IgG (Santa Cruz Biotechnology) at 37°C for 1 hour, bound proteins were visualized using ECL (Thermo Fisher Scientific) and detected using x films. The relative protein levels were calculated based on β-actin as the loading control.
Primary cell culture of ovarian carcinoma
Primary ovarian carcinoma samples were collected in the operating room of the Department of Gynecologic Oncology, Beijing Obstetrics and Gynecology Hospital, Capital Medical University. Beijing, China. A specific written informed consent was obtained from patients, and the study was approved by the Institutional Ethics Committee of Beijing Obstetrics and Gynecology Hospital, Capital Medical University, Beijing, China. In brief, the freshly obtained ovarian carcinoma tissues were washed three times in sterile, cold Hank's Buffered Salt Solution (HBSS) to remove blood and secretions, and then gently minced by scissors in DMEM/F12 medium containing 10% fetal calf serum (FCS). These tumor cells were then incubated for 1–2 hours at 37°C in 10 ml DMEM/F12 supplemented with 0.5% collagenase IA, 0.1% DNase, 100 U/ml penicillin and streptomycin with occasional shaking. After two centrifugations with PBS wash, the tumor cells were suspended in DMEM/F12 medium and then diluted to 1 × 105 cells/ml. Aliquots (100 μl) of tumor cell suspension were plated into 96 well tissue culture plates resulting in approximately 2 × 104 cells per well. The cells were incubated at 37°C for 24 hours in a 5% CO2 incubator, and then were treated with various concentrations of 10058-F4 or DMSO. Cell proliferation was assessed using an MTT assay.
Discussion
Accumulating evidence suggests that the Myc family is an excellent target for anti-cancer therapeutics due to its involvement in cell growth, metabolism, proliferation, apoptosis, and differentiation. Numerous c-Myc targeting strategies, including the inhibition of c-Myc expression or the interruption c-Myc and its downstream effects, are currently being used in experimental therapeutics for several types of cancer [
5],[
18]. Most of these approaches continue to be hampered by technical difficulties pertaining largely to delivery and the fact that many c-Myc target genes are functionally redundant and/or cell type specific [
19]. Over the last several years, several groups have developed small molecular inhibitors that interfered with the interaction between c-Myc and Max. These inhibitors restrain not only c-Myc-Max heterodimerization but also all subsequent downstream functions of c-Myc. 10058-F4 is a small molecular inhibitor that is composed of a six-member ethylbenzylidine ring and a five-member thioxothiazolidin-4-one. It specifically inhibits the dimerization of Myc and Max and prevents the transactivation and expression of the c-Myc target genes in doses up to 100 uM (13, 19). Several studies have confirmed that 10058-F4 significantly inhibited the growth of cancer cells in vitro through the induction of apoptosis and cell cycle G1 arrest [
8]-[
13],[
20]. A recent animal study has shown treated mice with 10058-F4 for 14 days was sufficient to delay tumor growth in a neuroblastoma transgenic mouse model and a xenograft mouse model, These data indicated that delaying tumor growth in vivo can be achieved by using a small molecule inhibitor that targets the c-Myc-Max interaction and targeting c-Myc-Max heterodimerization might be a good strategy for development of cancer therapy [
20]. Although clinical development of c-Myc-Max inhibitors, including 10058-F4, is limited by their relatively low potencies [
19], we needed to explore the effect of targeting the interaction between c-Myc and Max by 10058-F4 on ovarian cancer cells because little or no advancements have been made in ovarian cancer treatment over the last thirty years. The present study is the first to demonstrate that blocking the interaction between c-Myc and Max by 10058-F4 reduces the viability of ovarian carcinoma cells by reducing intracellular ATP and ROS production, downregulating c-Myc expression and inducing apoptosis and cell cycle arrest in ovarian cancer cells. Such inhibitions in ovarian cancer cells, caused by 10058-F4, are ultimately consistent with the results of other study groups [
8]-[
13],[
20]. Our primary result of general toxicity in mice showed 10058-F4 did not cause obvious toxic effects when injected 1008-F4 at a dose of 25 mg/kg daily for 5 days (data not shown). These results present further evidence that the c-Myc-Max signaling pathway is essential to ovarian cancer development and progression and that the c-Myc-Max interaction may be used as an effective molecular target for ovarian cancer therapy [
7],[
19],[
21].
One of the most striking observations in this study is the sensitivity of the ovarian cancer cells to the anti-proliferative effects of 10058-F4 despite the variations in c-Myc protein expression across the ovarian carcinoma cell lines and primary ovarian carcinoma cells, which is consistent with Holien's results in study of multiple myeloma [
13]. These results clearly pose an interesting possibility: that the concurrent disruption of c-Myc-Max heterotetramerization might also interfere with target gene expression in other ways in various cell types [
19],[
21]. This result could also account for the heterogeneity of primary cultures of ovarian cancer, showing small portions of stromal cells in some cases in our primary culture system, and this presence of stromal cells may affect the viability of and c-Myc function in the ovarian primary cultures.
Increased metabolic activity and glucose consumption in ovarian cancer patients have been linked to ovarian cancer aggressiveness [
22]-[
24]. Cellular ATP levels are a valuable predictor for chemo-resistance in cancer cells [
25]. The c-Myc oncogene is a major controller of many aspects of cellular metabolism through the regulation of genes related to glucose metabolism, glutamine metabolism, and mitochondrial biogenesis [
6]. Transgenic mice with an overexpression of c-Myc in the liver had increased glycolytic enzyme activity and production of lactic acid in the liver [
26]. Inhibition of c-Myc by SiRNA or small molecular inhibitors led to the reduction of glucose uptake and activities of glucose glycolytic enzymes (HKII, LDHA), all of which ultimately reduce ATP production [
27]-[
29]. Overexpression of Myc induces nuclear encoded mitochondrial gene expression and biogenesis and increases the production of mitochondrial ROS [
16]. c-Myc knockdown is associated with a reduction in ROS production and inhibition of autophagy in cancer cells [
30]. In agreement with previous studies on ATP reduction through the inhibition of c-Myc, the metabolic and therapeutic stress induced by 10058-F4 led to an acute ATP depletion, which was accompanied by decreased intracellular and mitochondrial ROS and ultimately led to the inhibition of cell growth in ovarian carcinoma cells.
Assessment of chemotherapeutic drug sensitivities using primary culture cancer cells provides clinically relevant information for the optimization of the cancer patient's treatment [
31]-[
33]. In the panel of 18 primary cell cultures of primary epithelial ovarian carcinoma, inhibition of cell proliferation was observed in 15 primary cultures after 72 hours of treatment. The level of c-Myc protein expression in untreated cells from primary cultures was not associated with sensitivity to 10058-F4 [
13]. However, caspase-3 activity correlated strongly with cellular response to 10058-F4 in 10 primary cultures of ovarian cancer, suggesting that the induction of apoptosis is a major action for the small molecular inhibitors that target the interaction of c-Myc-Max. The results indicated that suppression of c-Myc through the targeting of the c-Myc-Max heterodimer has broad therapeutic applications in cancers including ovarian cancer [
7],[
19],[
20].
Funding
This work was generously supported by the Beijing municipal commission of education Surface project (11320023). The project described was also supported by NIH/NCI 1K23CA143154-01A1 and the Steelman fund (Bae-Jump VL). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
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Competing interests
The authors have declared that no competing interests exist.
Authors' contributions
Manuscript editing: CZ VBJ ZW. Conceived and designed the experiments: CZ VBJ WJ. Performed the experiments: WJ MX SF HMJ LC. Analyzed the data: CZ WJ ZW. Contributed reagents/materials/analysis tools: WJ CZ VBJ LC. Wrote the paper: CZ WJ VBJ. All authors read and approved the final manuscript.