Sulforaphane induces cell cycle arrest by protecting RB-E2F-1 complex in epithelial ovarian cancer cells

Sulforaphane (Sigma chemicals, St. Louis, MO) was dissolved in DMSO and Stock solutions were freshly prepared and added to the cell cultures to obtain the indicated final concentrations. The DMSO concentration was used at 0.01% and the same concentration was used as a vehicle. DMSO alone (0.01%) was found to have no significant effect on cellular function. Cell proliferation assays were conducted using Cell Counting Kit-8 (CCK-8) (Dojindo, Gaithersburg, MD). Cell cycle analysis was conducted using Cell Cycle Phase Determination Kit (Cayman Chemical Company, Ann Arbor, MI). Human fibroblasts were similarly treated as cancer cells to display differential cytotoxicity at any given dose. Retinoblastoma and E2F-1 antibodies were purchased from Millipore (Danvers, MA). Cyclins, CDKs, poly (ADP-ribose) polymerase (PARP) and β actin antibodies were purchased from Santa Cruz Biotechnology, (Santa Cruz, CA).

Ovarian cancer cell lines, MDAH 2774 and SkOV-3 (American Type Culture Collection, Manassas, VA) were propagated in McCoy's 5A medium supplemented with 10% fetal bovine serum, 2 mM L-glutamine, 100 units/ml penicillin, and 100 mg/ml streptomycin (Thermo Fisher Scientific, Pittsburgh, PA). Cells were cultured in a humidified atmosphere with 5% CO₂ at 37°C. Trypsin (0.25%)/EDTA solution was used to detach the cells from the culture flask for passing the cells.

Standard prototype growth curves and number of viable cells were determined for each cell line (treated and control groups) in triplicate experiments according to the CCK-8 (Dojindo, Gaithersburg, MD) manufactures' instructions. Growth curves were plotted as a percentage of the value of DMSO-treated controls minus the value of untreated cells on day 0. Day 3 values were considered for the determination of the 50% cell proliferation inhibition (IC50) for a given treatment. In some cases parallel manual count was also performed with trypan blue and counting by exclusion method using a Hemocytometer. The findings confirmed CCK-8 assay results.

Apoptotic cells were analyzed by using Annexin V FITC apoptosis detection kit (Calbiochem, Gibbstown, NJ) according to the manufacturers instructions. The DNA content of the cells was analyzed by flow cytometer and sub G1 population was considered to represent apoptotic cells. For fluorescent microscopic image analysis of apoptotic fraction of the cells, treated and control (1 × 10⁶ cells/ml) were mixed with annexin V-biotin and medium-binding reagent, and incubated in the dark for 15 min at room temperature. Cells were then centrifuged and medium was replaced with 1× Binding Buffer containing FITC-streptavidin. Propidium iodide was added to discriminate early apoptotic cells from late apoptotic or necrotic cells. A portion of cell suspension (50 μl) was placed onto a glass slide, covered with a cover slip, and viewed immediately using a fluorescence microscope equipped with FITC (green) and propidium iodide (red) Filters (Zeiss, AXio CamMRm Observer. A1).

MDAH-2774 cells were seeded at 10⁶ cells in 10 cm dishes and the culture medium changed to serum-free medium for 24 h to facilitate cell cycle synchronization. Cell cycle analysis was conducted using Cell cycle phase determination kit according to the manufacturer's instructions (Cayman chemical company, Ann Arbor, MI). Samples were analyzed in FL2 channel of flow cytometer with a 488 nm excitation laser.

These assays were performed according to our earlier publication[17]. Antibody reactions were visualized using enhanced chemiluminescence western blotting detection reagents (Amersham Pharmacia, Uppsala, Sweden).

Briefly, PANC-1 cells untreated or treated with 15 μM ritonavir for 48 h, were harvested and total RNA was isolated utilizing an RNeasy kit (Qiagen Inc., Valencia, CA) as described by the manufacturer. Total RNA was sent to MOgene company (MOgene, LC, Saint Louis, MO) for GEP analysis.

To determine if SFN had potential to induce growth arrest in ovarian cancer cells, we conducted cell proliferation assays using two human ovarian cancer cell lines, MDAH-2774 and SkOV-3. Cells were grown as sub-confluent monolayer cultures and propagated under standard conditions. Cell lines were treated with serial dilutions of SFN dissolved in DMSO. Human fibroblasts were similarly treated to display differential cytotoxicity (data not shown). SFN treatment resulted in a concentration-dependent inhibition of the proliferation of MDAH 2774 (Fig 1A) and SkOV-3 (Fig 1B) with an IC50 of ~8 μM. The extent of growth inhibition increased as a function of time in all the tested doses ranging from 5 to 20 μM. The extent of cell death with SFN increased as a function of dosage in 48 h as observed by phase contract microscopy in MDAH-2774 cell line (Fig. 1C). Since chemotherapy of ovarian cancer usually consists of paclitaxel alone or in combination with platinum based drugs, we further evaluated the influence of SFN combined with paclitaxel in treating MDAH-2774 cell line. Cells treated with paclitaxel with or without SFN and viability was assessed for 3 days. As shown in fig. 1D, 8 μM SFN which is IC50 for MDAH-2774 or 2 μM paclitaxel alone produced ~40% and ~55% cell death, respectively; however combination treatment resulted in ~70% cell death.

Inhibition of cell cycle progression in tumor cells may be associated with a concomitant activation of cell death pathways such as apoptosis. We, therefore, examined the contribution of apoptosis in SFN-treated MDAH-2774 cells. Apoptosis induces changes on the cell surface, which results in translocation of phosphatidylserine (PS) from the inner layer of the plasma membrane to the outer layer [18, 19]. Annexin V is a Ca²⁺ -dependent protein with high affinity for PS [19] that was used to assess apoptotic cells. Flow-cytometric analysis was performed to quantify apoptotic changes after treatment with SFN. Control and treated cells were prepared for bivariate analysis using annexin V stain that detects cells undergoing apoptosis and propidium iodide (PI) to detect nonviable cells. The number of pro-apoptotic cells after 48 h of 10 μM SFN treatment was substantially higher as compared with control. We observed that the ratio of apoptotic cells increased with the increased dose of SFN treatment: approximately 8% (5 μM treatment) and 20% (10 μM treatment) (Fig. 2A) of the cells over 48-hour period. Further we analyzed cells by immunofluorescence staining with Annexin V for apoptosis after SFN treatment. After 48 hr exposure to 10 μM SFN, 60% of the cells stained positive for annexin V, whereas untreated were less than 5% annexin V positive (Fig. 2B). As shown in Fig. 2C, western blotting analysis revealed the activation of PARP and of caspases-9 from counterpart pro-caspase. Further we document a dose-dependent increase in pro-apoptotic BAK and decrease in the expression of the anti-apoptotic protein Bcl-2.

In order to determine the causes of the growth inhibition observed by cell proliferation assays, we assessed the cell cycle progression of the cells in the presence of SFN. Serum starved MDAH-2774 cells were treated with either vehicle or 10 μM SFN for 12 or 24 hrs with complete growth medium. The cells were then washed, fixed, and cell cycle phase determination was performed utilizing flow cytometry and a cell cycle phase determination kit. The results demonstrated that SFN induces G1 arrest in a time-dependent manner (Fig. 3D, E and 3F) in comparison with control cells (Fig. 3A, B and 3C). There is a negligible increase of S phase cells after 12 hrs (0.7%) and 24 hrs (2.7%) with SFN treatment compared with 40% of cells in S phase for control cells at 24 hrs. Further we observed an overall increase in the cells at G1 phase of the cell cycle with SFN treatment.

Since we observed cell cycle inhibition with SFN treatment at G0/G1 phase of the cells, we evaluated genes that influence the cell cycle progression into S phase. Under phosphorylated tumor suppressor retinoblastoma proteins (RB, p107 and p130) sequesters cell cycle promoting E2F-1 transcription factors. Gene expression analysis of RB proteins revealed a 1.5 and 2.0 fold decrease of RB and p130, respectively; as well as, a 2 folds increase in p107 levels (Fig. 4A). The E2F family of proteins, E2F-1, 2 and 3 which interact with RB, demonstrated 1.0 and 1.5 fold reduction in the expression levels of E2F-1 and 2, respectively, and 1.0 fold increase in the levels of E2F-3 (Fig. 4B). Cyclins and cyclin dependent kinases (CDKs) and their inhibitors exhibit distinct expression patterns, which contribute to the temporal coordination of each event in cell cycle progression. SFN treatment resulted in the decreased expression of G1 phase cyclins and CDKs while increasing the expression of cyclin dependent kinase inhibitors (CKIs), which bind and inhibit the activity of cyclin/Cdk complexes and negatively regulate cell cycle progression (Fig. 4C, 4D & 4E).

Under-phosphorylated active retinoblastoma protein (RB) family of proteins, RB, p107 and p130 tumor suppressor proteins control cell cycle progression through the late G1 phase to the S phase by inhibiting E2F family of transcription factors [15, 20, 21]. We observed progressive conversion of phosphorylated RB to non-phosphorylated RB after 48 hrs with when MDAH-2774 cells were treated with 5 to 15 μM SFN treatment (Fig. 5B upper panel). Further E2F-1 is known to be elevated in many tumor cell lines and responsible for the expression of S phase genes that drives cell cycle progression. We observed decrease in the E2F-1 protein levels with SFN treatment corroborating cell cycle analysis results of decreased number of cells in S phase (Fig. 5A). Cyclins and cyclin-dependent kinases (CDKs) regulate the activity of RB by phosphorylation resulting in control of progression through G1 [16]. Since we observed elevated levels of under-phosphorylated RB, as expected, we observed lower levels of CDK4 and CDK6, the proteins responsible phosphorylation of RB in response to SFN in dose dependent manner (Fig. 5A). To assess if SFN treatment increases the RB-E2F-1 interaction resulting low levels of E2F-1, we conducted immunoprecipitation of cell extracts with E2F-1 polyclonal antibodies and probed with RB monoclonal antibodies. We observed a dose dependent increase in the RB levels in E2F-1 immunoprecipitates (Fig. 5B).

Cell motility following wound generation showed a greater cell migration in control cells compared with SFN treated cells. After 20 hrs, we observed almost complete closure of the wound in control cells which was inhibited by 50% and 75% by 5 μM and 10 μM SFN, respectively (Fig 6A). Cell migration was also inhibited when treated with SFN in a modified Boyden chamber. A progressive decrease in the cell migration through membrane with the SFN treatment from up to 15 μM was observed (Fig. 6B).