Sulforaphane induced cell cycle arrest in the G2/M phase via the blockade of cyclin B1/CDC2 in human ovarian cancer cells

Background Malignant tumors are the single most common cause of death and the mortality rate of ovarian cancer is the highest among gynecological disorders. The excision of benign tumors is generally followed by complete recovery; however, the activity of cancer cells often results in rapid proliferation even after the tumor has been excised completely. Thus, clinical treatment must be supplemented by auxiliary chemotherapy or radiotherapy. Sulforaphane (SFN) is an extract from the mustard family recognized for its anti-oxidation abilities, phase 2 enzyme induction, and anti-tumor activity. Methods This study investigated the cell cycle arrest in G2/M by SFN and the expression of cyclin B1, Cdc2, and the cyclin B1/CDC2 complex in PA-1 cells using western blotting and co-IP western blotting. Results This study investigated the anticancer effects of dietary isothiocyanate SFN on ovarian cancer, using cancer cells line PA-1. SFN-treated cells accumulated in metaphase by CDC2 down-regulation and dissociation of the cyclin B1/CDC2 complex. Conclusion Our findings suggest that, in addition to the known effects on cancer prevention, SFN may also provide antitumor activity in established ovarian cancer.


Background
Isothiocyanates (ITCs) are naturally occurring components of vegetables that have demonstrated biological activity against carcinogenesis as well as chemopreventive properties [1]. It has been suggested that in conjunction with chemotherapy, ITCs may enhance drug sensitivity [2]. Sulforaphane (SFN), a potent cancer preventive agent, is a dietary isothiocyanate found as a precursor glucosinolate in cruciferous vegetables such as Brussels sprouts, cauliflower and broccoli [3]. Interest in this agent has grown in recent years due to its putative beneficial pharmacological effects as an antioxidant [4], anti-inflammatory [5] and antitumor agent [6]. SFN is also a potent scavenger of reactive oxygen species (ROS), including superoxide anions and hydroxyl radicals [7]. Many studies have indicated an inverse correlation between the consumption of cruciferous vegetables and a decrease in the incidence of various tumors, including those of the prostate [8], cervical [9], colorectal [10], and lung [11]. In addition to inhibiting cell proliferation and increasing apoptosis [10], other mechanisms have also been proposed to explain the anti-carcinogenic effects of SFN. These include anti-inflammatory and antioxidative activities, the induction of phase 2 detoxification enzymes, the inhibition of cyclooxygenase 2 (COX-2) [12], and the effect on protein kinases [13].
This study investigated the influence of SFN on ovarian cancer cell lines (PA-1) with regard to the antiproliferation of PA-1 cells and induced cell cycle arrest in the G2/M phase. These results may provide support for the chemoprevention of ovarian cancer.
Cell culture PA-1 cells were purchased from the Food Industry Research and Development Institute (Hsinchu, Taiwan). The cells were maintained in flasks containing MEM supplemented with 10% (v/v) FBS and cultured in an incubator at 37°C with an atmosphere containing 5% CO 2 .

Cell proliferation assay
Cells were seeded into 96-well culture plates at 10,000 cells/well and treated with SFN. One to three days (0 μM SFN was the control group.) MTT dye (1 mg/ml) was added to each well 4 hours following treatment. The reaction was stopped by the addition of DMSO, and OD 540 was measured using a multi-well plate reader (Powerwave XS, Biotek). In the absence of cells, the background absorbance of the medium was subtracted. Results were expressed as a percentage of the control, which was considered to be 100%. Each assay was performed in triplicate and the results were expressed as the mean (+/−SEM).

Measurement of apoptosis
PA-1 cells were first seeded in 6-well plates (Orange Scientific, E.U.). Following treatment with SFN for four hours, the supernatant was discarded and cells were harvested and re-centrifuged. Cells were subsequently resuspended/incubated in 1X annexin-binding buffer  were quantified using an Li-COR near infrared imaging system. Statistical analysis was performed using a t-test; the symbols *, & and # in each group of bars indicate that the differences resulting from treatment with SFN are statistically significant at P < 0.05.
Following incubation, the stained cells were analyzed using flow cytometry (FACSCalibur, BD, USA). Data was analyzed using WinMDI 2.8 free software (BD, USA).

Cell cycle analysis
Cell cycle analysis was performed using fluorescent nucleic acid dye and propidium iodide (PI) to identify the proportion of cells in each of the three stages of interphase. Cells were treated with SFN for 24 hours, and subsequently harvested and fixed in 1 ml of cold 70% ethanol for at least eight hours at −20°C. DNA was stained in PI/RNaseA solution and the DNA content was detected using flow cytometry. Data was analyzed using WinMDI 2.8 free software (BD, USA).

Western blot assay
A total of 30-50 μg of proteins were separated by 10% SDS-PAGE and transferred to PVDF membranes (Millipore, USA). The membranes were blocked with blocking buffer (Odyddey, USA) overnight and subsequently incubated with anti-β-actin (Sigma-Aldrich, St. Louis, MO, USA), anti-CDC2, anti-caspase 3, and anti-cyclin B1 (Santa Cruz BioTechnology, USA) antibodies for 1.5~2 hours. The blots were then washed and incubated with a second antibody (IRDye Li-COR, USA) at a dilution of 1/20,000 for 30 minutes. Finally, the antigen was visualized using a near infrared imaging system (Odyssey LI-COR, USA) and data was analyzed using Odyssey 2.1 software.

Co-immunoprecipitation (Co-IP)
Co-IP is an effective means of quantifying protein-protein interaction in cells. Briefly, 500 μg of cellular proteins were labeled using anti-cyclin B1 (Santa Cruz BioTechnology, USA) following overnight incubation at room temperature. The protein-antibody immunoprecipitates were collected by protein A/G plus-agarose (Santa Cruz Bio-Technology, USA). Following the final wash, the samples were boiled and centrifuged to pellet the agarose beads. Western blot analysis of the CDC2 protein in the supernatant was then conducted. Antigens were visualized using a near infrared imaging system (Odyssey LI-COR, USA) and data was analyzed using Odyssey 2.1 software.

Statistical analysis
All data was reported as the mean (±SEM) of at least three separate experiments. A t-test or one-way ANOVA with post-hoc test was employed for statistical analysis, with significant differences determined as P < 0.05.

SFN inhibits proliferation of PA-1 cells
We hypothesized that SFN could mediate the survival of PA-1 cells and thus inhibit their growth. To explore the anti-tumor activity of SFN against PA-1 cells, an in vitro cell viability study was conducted in which

SFN repressed the cell viability of PA-1 cells without apoptosis induction
A study on apoptosis was performed to further elucidate anti-cancer mechanisms of SFN in PA-1 cells. After treating the cells with various doses of SFN, the percentage of apoptotic cells was assessed using Annexin V-FITC and propidium iodide staining, followed by flow cytometric analysis ( Figure 1B). A dot-plot of Annexin V-FITC fluorescence versus PI fluorescence also indicated a non-significant increase in the percentage of apoptotic cells treated with SFN. At SFN concentrations of 6.25 to 12.5 μM, no significant increase was observed in the percentage of cells undergoing necrosis and apoptosis ( Figure 1C) or caspase 3 activation ( Figure 1D). The results summarized in Figure 1 indicate that SFN may mediate the survival of PA-1 cells and thus inhibit their proliferation without apoptosis induction.

SFN treatment induced the accumulation of G 2 /M phase in PA-1 cells
The cell-cycle distribution of SFN-treated PA-1 cells was analyzed using flow cytometry. Prior to processing and analysis, cells were exposed to SFN for a total of 24 hours. As shown in Figure 2A, the cells exposed to SFN showed an increase in the number of cells in the G 2 /M phase (& p < 0.05 vs SFN 0 μM), compared with the number of untreated cells. This could imply that the PA-1 cells had undergone cell cycle arrest. We also found that treatment with SFN simultaneously reduced the number of cells in the G 1 phase (* p < 0.05 vs SFN 0 μM) ( Figure 2B).
Cell cycle arrest by SFN in PA-1 cells via CDC2 down regulation and dissociation of the cyclin B1/CDC2 complex Figure 3 illustrates the immunoblotting of cellular proteins from PA-1 cells treated with SFN, revealing a decrease in CDC2 following incubation with SFN ( Figure 3A). Cyclin B1 and CDC2 protein expression was quantified by measuring relative intensities. We found that CDC2 levels were significantly lower in cells incubated with SFN concentrations of 12.5 μM ( Figure 3B). Moreover, the activity of the cyclin B1/CDC2 complex (important for G2-M transition during the cell cycle) was determined by Co-IP ( Figure 4A) and quantified by measuring relative band intensities. We found that cyclin B1/CDC2 complex activity was significantly suppressed in cells incubated with an SFN concentration of 12.5 μM ( Figure 4B).
These results indicate an increase of the cell population in G 2 /M phase via a down regulation of CDC2 and dissociation of the cyclin B1/CDC2 complex following incubation with SFN in PA-1 cells.

Discussion
The results collected in this study using cell lines of human ovarian cancer provide experimental evidence indicating that SFN may induce irreversible cell cycle arrest during the G2/M phase. These dietary constituents demonstrate chemopreventive and chemotherapeutic potential through their ability to ameliorate the side effects of conventional chemotherapy [14].
This study investigated the in vitro expression of cyclin B1 and Cdc2 in PA-1 cells. The cells possess mechanisms to maintain genomic stability through cell cycle arrest [15]. At least two cell cycle checkpoints play a role in the cellular response, allowing the DNA to be repaired prior to DNA duplication (G1/S checkpoint) or mitosis (G2/M checkpoint) [16]. Cdc2 regulates mitosis and binds to cyclin B to form mitosis-promoting factor (MPF) [17]. The activity of MPF is regulated by the phosphorylation/dephosphorylation of Cdc2 as well as the accumulation of cyclin B protein and p53; GADD45 is also involved in a G2/M checkpoint and may participate in the regulation of Cdc2 kinase activity [18,19].
Other lines of evidence suggest that DNA damage excludes cyclin B1 from the nucleus, which promotes G2 arrest [20]. It is possible that cell cycle checkpoints delay cell cycle progression to allow additional time for the repair of DNA damage; however, our study found no direct evidence that DNA was repaired while the cells were arrested at the checkpoint.
Recent studies have shown that SFN inhibits the growth of tumor precursors and tumors in mice models when treatment is initiated at the time of carcinogen administration [10]. The co-inhibition of PI3K/AKT and ERK pathways activates FOXO transcription factor and enhances SFN-induced FOXO transcriptional activity, leading to cell cycle arrest and apoptosis [21].

Conclusions
We conclude that G2 delay is a common response of tumor cells to chemotherapy with SFN. We further propose that mechanisms of this delay may be reduced expression of CDC2 and dissociation of the cyclin B1/ CDC2 complex. Therefore, although certain chemopreventive effects of SFN and related isothiocyanate compounds have already been established with regard to ovarian cancer cells, SFN should be investigated further to confirm the additional antitumor properties proposed by our study.