Background
Nasopharyngeal carcinoma (NPC) is an epithelial squamous cell carcinoma endemic in Southeast Asia and parts of Mediterranean and northern Africa [
1]. Radiotherapy alone cures more than 90% of cases of stage I NPC; however, patients with advanced disease tend to experience therapy failure. Several groups have shown that the 5-year survival rate for concurrent chemotherapy and radiotherapy is higher than that for radiotherapy alone in patients with advanced disease [
2,
3]. Currently, cisplatin combined with 5-fluorouracil is the first-line chemotherapeutic regimen for NPC. Although this regimen has manageable toxic effects and has yielded response rates ranging from 65% to 75% [
4], an urgent need for inpatient administration of chemotherapy has accelerated the development of newer, more tolerable and potent platinum-based regimens. We previously showed that ApoG2 in particular could potently kill NPC cells and had a synergic effect with cisplatin to induce cell death [
5]. In this study, we further investigated the effect of ApoG2 on cell cycle regulator proteins and cell cycle progression.
Gossypol and its derivates reportedly induce apoptosis by inhibiting the antiapoptotic function of the Bcl-2 family of proteins [
5,
6]. Also, authors have found cell cycle arrest in gossypol-treated cells. Several cell cycle-related molecules are involved in gossypol-induced cell cycle arrest. For example, researchers have reported that gossypol-induced cell death was coupled with upregulation of c-Fos expression and biphasic c-Myc expression in rat spermatocytes [
7]. Furthermore, transforming growth factor-β is activated by gossypol in prostate cancer cells, and gossypol upregulates p21 expression and downregulates cyclin D1 and Rb expression in colon cancer cells [
8,
9]. Modifications of these cell cycle-related molecules result in cancer cell arrest at G0/G1 phase of the cell cycle. However, Chang et al. found that gossypol did not affect cell cycle progression or the p53 or p21/WAF signaling pathway in A549 human alveolar lung cancer cells [
10]. Different oncogenic pathways are activated in different types of cancer, and treatment with gossypol may have various biochemical and molecular impacts on different cancers with specific biological behaviors.
NPC is associated with Epstein-Barr virus (EBV) infection and genetic susceptibility. EBV-encoded latent membrane protein 1 (LMP1) is a principal oncogene in cases of NPC; it can activate a number of signaling pathways, including nuclear factor-κb, mitogen-activated protein kinase, and phosphoinositide 3-kinase [
11]. Besides the LMP1-induced oncogenic pathways, dysregulation of factors such as p16, cyclin D1, and cyclin E leads to aberrations in the cell cycle in NPC cells. Therefore, NPC has multiple unique abnormalities that are potential targets for novel treatments. In this study, we examined the effect of ApoG2 on cell cycle distribution and the involved signal pathways in NPC cells. The results demonstrated that ApoG2 potently arrested cells at S phase of the cell cycle. We also observed that suppression of the c-Myc signaling pathway was responsible for the ApoG2-induced cell cycle arrest.
Materials and methods
Cells, Drugs, and Reagents
Poorly differentiated human NPC cell lines CNE-2 and HONE-1 were originally obtained from NPC patients and maintained in our laboratory in RPMI-1640 (Gibco/BRL, Gaithersburg, MD) supplemented with 10% heat-inactivated fetal bovine serum (Thermo Scientific HyClone, Logan, UT). Cells were incubated in a humidified 5% CO2 atmosphere at 37°C. ApoG2, which was supplied by Dajun Yang (Ascenta Therapeutics Incorporation, Malvern, Pennsylvania), was dissolved in pure dimethyl sulfoxide (DMSO) at the stock concentration of 20 mmol/l and stored at -20°C. 3-(4,5 dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium (MTT) were purchased from Sigma-Aldrich (St. Louis, MO). In in vivo experiments, for intraperitoneal (i.p.) injection, ApoG2 was suspended in 0.5% sodium carboxymethylcellulose and prepared on the day of use.
MTT Assay
NPC cell viability was assessed using an MTT assay based on mitochondrial conversion of MTT from soluble tetrazolium salt to an insoluble colored formazan precipitate, which was dissolved in DMSO and quantitated using a spectrophotometer (Thermo Multiskan MK3; Thermo Fisher Scientific, Waltham. MA) with optical density (OD) values [
12]. NPC cells were plated in 96-well culture clusters (Costar, Cambridge, MA) at a density of 15,000 to 25,000 cells/ml. Serial dilutions of ApoG2 were prepared from a stock solution to the desired concentrations. The final DMSO concentration was less than 0.1% (v/v). All experimental concentrations of ApoG2 were prepared in triplicate. Cells were treated with ApoG2 for 24, 48 and 72 h. Before termination of treatment, cells were incubated with 10 μl of 10 mg/ml MTT for 4 h. Then MTT and medium were depleted, and 100 μl of DMSO was added to the plates. The percent absorbance of Apog2-treated cells relative to the control (DMSO treated cells, DMSO concentration was less than 0.1%) was plotted as a linear function of the drug concentration. The antiproliferative effect of ApoG2 on NPC cells was measured as the percent of viable cells relative to the control using the equation 100% × OD
T/OD
C, in which OD
T is the mean OD value of the ApoG2-treated treated samples and OD
C is the mean OD value of the control samples. The 50% inhibitory concentration of ApoG2 was defined as the concentration of the drug required to achieve 50% growth inhibition relative to control populations.
Cell Cycle Analysis
Untreated control and ApoG2-treated CNE-2 cells were harvested, washed twice with phosphate-buffered saline (PBS), and fixed dropwise with 2 ml of 70% ice-cold ethanol. After cells fixed overnight at 4°C, cells were then washed twice with PBS; cells were then incubated in RNase (20 μg/ml) at 37°C for 30 min to avoid staining the RNA. Next, the cells were washed once with PBS; PI was added to samples at a final concentration of 15 μmol/l, and after 5 min of incubation, the cells were analyzed using flow cytometry (Beckman Coulter, Fullerton, CA). The percentages of the nuclei in CNE-2 cells at each phase of the cell cycle (G1, S, G2/M) were calculated using the MultiCycle software program (Phoenix Flow Systems, San Diego, CA).
Immunoblot Analysis
Protein analysis using immunoblotting and immunoprecipitation was performed with primary antibodies against p53 (sc-126; Santa Cruz Biotechnology, Santa Cruz, CA), p21 (sc-6246; Santa Cruz Biotechnology), c-Myc (sc-42; Santa Cruz Biotechnology), cyclin E (sc-481; Santa Cruz Biotechnology), cyclin D1 (sc-8396; Santa Cruz Biotechnology), and actin (clone AC-15; Sigma-Aldrich) as described previously [
13]. Total cell lysates were harvested, electrophoresed using 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and transferred to polyvinylidene difluoride membranes (Roche, Grenzacherstrasse, Basel, Switzerland). Immunoblotting was performed using the primary antibodies described above followed by detection of protein expression using secondary antibodies conjugated with horseradish peroxidase (Cell Signaling Technology, Danvers, MA), and blots were developed using ECL chemiluminescent reagent (Cell Signaling Technology).
RNA Interference
Transient small interfering RNA (siRNA) transfection was performed using Lipofectamine 2000 (Invitrogen, San Diego, CA) and 50 nM siRNA oligonucleotides. Commercially purchased siRNAs (Ribobio, Guangzhou, People's Republic of China) were scrambled (nontargeting), glyceraldehyde-3-phosphate dehydrogenase siRNA, and c-Myc siRNA. The three independent oligonucleotides designed for the c-Myc siRNA sequences were 5'-CAGAAATGTCCTGAGCAAT-3', 5'-AAGGTCAGAGTCTGGATCACC-3', and 5'-AAGGACTATCCTGCTGCCAAG-3'. The siRNA duplexes were introduced into CNE-2 cells according to the siRNA manufacturer's protocol. After transfection with siRNA for 48 h, cells were harvested for immunoblots and cell cycle analysis. The scrambled siRNA construct was used as a negative control.
In vivo treatment and immunohistochemistry assay
Four-week-old athymic nude (nu/nu) mice obtained from the Animal Center of Southern Medical University (Guangdong, China) received subcutaneous injection of 1 × 107 CNE-2 cells in each axillary area. When subcutaneous tumors developed to more than 1,500 mg, mice were euthanized and tumors were dissected and mechanically dissociated into equal pieces to be transplanted into the flank areas of a new group of mice. When xenograft tumors became palpable (about 0.1 mm3), mice were randomly divided into control (0.5% sodium carboxymethylcellulose solution) and ApoG2 (120 mg/kg of body weight given by intraperitoneal injection daily) groups. Each group contained 8 mice, and there was no difference in tumor size between groups. Based on our lab's policy, when xenograft tumors developed to more than 1,000 mg, mice were euthanized and tumors were dissected and weighed. Immunohistochemical analysis was performed on tissue-sample sections of CNE-2 xenografts obtained from control and ApoG2. All samples were stained with hematoxylin and eosin and microscopically examined to confirm the CNE-2 cell origin. Sections were then stained with c-Myc (#; Santa Cruz) at 4°C overnight and then visualized using diaminobenzidine (DAB) (DAKO Liquid DAB, Dako, Carpinteria, CA) as peroxidase substrates.
Statistical analysis
All analyses to compare the significance of measured levels were completed using the unpaired t-test by SPSS 16.0 software.
Discussion
ApoG2 is the oxidation product of gossypol and has two aromatic hydrocarbon quinone groups. Authors have reported that aromatic hydrocarbon quinone stimulates ROS production in hepatic cells [
17]. As we known, elevated ROS levels may damage cellular DNA, inducing generation of oxidized bases, DNA strand breaks, and stop of DNA replication, in ApoG2-treated CNE-2 cells. Recent studies provided evidence that multiple chemopreventive agents can cause generation of ROS to trigger signal transduction, culminating in cell cycle arrest and/or apoptosis [
18,
19]. However, Van Poznak et al. and Zhang et al. suggested that gossypol-induced cell cycle arrest is associated with alterations of p21, cyclin D1, and p53 and showed that p21 is the first target of gossypol to inhibit cell growth
in vivo [
9,
20]. Our data indicated that ApoG2 induced massive cells arrest at S phase of the cell cycle not only in ApoG2-sensitive NPC cells but also in ApoG2-insensitive HONE-1 cells (Fig.
3). Results of signaling pathway analysis showed that downregulation of c-Myc protein expression was the major upstream event in ApoG2-induced cell cycle arrest in NPC cells (Fig.
4). Basically, the effect of c-Myc on cell cycle is to drive quiescent cells into the cell cycle, and shortening G1 and promoting S phase entry thereby. The down-regulation of c-Myc should cause a preferential G1/S arrest rather than S arrest. However, in NPC cells, although p53 was highly expressed and its expression was never downregulated by ApoG2 in this study, p53 was mutated and functionally impaired by Epstein-Barr virus nuclear antigen 5 and deltaN-p63 in NPC cells [
21,
22]. In this scenario of malfunction of G1-S checkpoint p53, c-Myc was a main factor accounting for ApoG2-induced S phase arrest. P21 and cyclins were followed by downregulation of c-Myc expression.
c-Myc is not only a central regulator of cell proliferation but also induces cells to undergo apoptosis, unless specific signals provided by oncogenes block the apoptosis pathway [
23]. Notably, NPC cells consistently harbor EBV DNA and express EBV proteins, LMP1 and BARF1; these proteins stimulate oncogenic antiapoptotic Bcl-2 proteins to protect host cancer cells from apoptosis [
24‐
27]. ApoG2 is a potent inhibitor of antiapoptotic Bcl-2 proteins and its treatment could remove the protective effect of Bcl-2 proteins and facilitate apoptosis. In this case, downregulation of c-Myc expression by ApoG2 on one hand could let cells away from c-Myc-induced apoptosis and on other hand led to cell cycle arrest. However, by inhibiting Bcl-2 proteins, ApoG2 still helped release pro-apoptotic proteins, such as Bax and Bak, and irreversibly damaged mitochondria and induced cell apoptotic [
5].
Gossypol is clinically used in China to treat adenomyosis and hysteromyoma because of its ability to inhibit estrogen and progesterone by competitively binding to the estrogen receptor and progesterone receptor [
28]. c-Myc is a well-established target of estrogen action and plays a role in controlling cell cycle progression. Anti-estrogen treatment is reported to be able to cause an acute decrease in c-Myc expression, a subsequent decline in cyclin D1 expression, and, ultimately, inhibition of DNA synthesis and arrest of cells in a quiescent state [
29]. Estrogen receptor and progesterone receptor are known to be highly expressed in NPC cells, and their expression is considered a sign of distant metastasis and a poor prognosis [
30]. Based on our findings, we suggest that ApoG2-induced cell cycle arrest is dependent on ApoG2's downregulation of c-Myc expression. Use of ApoG2 to treat NPC may suppress the activity of estrogen and progesterone and reduce the incidence of distant metastasis and local relapse.
The concept of targeted biological therapy for cancer has emerged over the past decade. Clinical trials studying the efficacy and tolerability of these targeted agents has shown that most tumors depend on more than one signaling pathway for their growth and survival. Therefore, investigators pursue different strategies to inhibit multiple signaling pathways by developing multitargeted agents [
31]. The recent U.S. Food and Drug Administration approval of sorafenib and sunitinib, which target vascular endothelial growth factor receptor, platelet-derived growth factor receptor, FLT-3, and c-Kit, marks the use of a new generation of multitarget anticancer drugs [
32]. Our study show that ApoG2 is one such multitarget agent that targets both the antiapoptotic and cell cycle progression pathway in NPC cells by blocking antiapoptotic Bcl-2 proteins and the c-Myc oncogenic pathway. These findings provide an entirely new concept for the use of ApoG2 in cancer therapy.
Competing interests
The authors declare that they have no competing interests.
Authors' contributions
YXZ was responsible for study design. DY and XFZ performed the experiments and drafted the manuscript. JS participated in the data analysis and western-blot. All authors read and approved the final manuscript.