Sulforaphane induces cell cycle arrest by protecting RB-E2F-1 complex in epithelial ovarian cancer cells
- Christopher S Bryant2, 3,
- Sanjeev Kumar2, 3,
- Sreedhar Chamala1, 3,
- Jay Shah1,
- Jagannath Pal1,
- Mahdi Haider2,
- Shelly Seward2,
- Aamer M Qazi1, 3,
- Robert Morris2, 3,
- Assaad Semaan2,
- Masood A Shammas1,
- Christopher Steffes1, 3,
- Ravindra B Potti1,
- Madhu Prasad1,
- Donald W Weaver1 and
- Ramesh B Batchu1, 3Email author
© Bryant et al; licensee BioMed Central Ltd. 2010
Received: 1 September 2009
Accepted: 2 March 2010
Published: 2 March 2010
Sulforaphane (SFN), an isothiocyanate phytochemical present predominantly in cruciferous vegetables such as brussels sprout and broccoli, is considered a promising chemo-preventive agent against cancer. In-vitro exposure to SFN appears to result in the induction of apoptosis and cell-cycle arrest in a variety of tumor types. However, the molecular mechanisms leading to the inhibition of cell cycle progression by SFN are poorly understood in epithelial ovarian cancer cells (EOC). The aim of this study is to understand the signaling mechanisms through which SFN influences the cell growth and proliferation in EOC.
SFN at concentrations of 5 - 20 μM induced a dose-dependent suppression of growth in cell lines MDAH 2774 and SkOV-3 with an IC50 of ~8 μM after a 3 day exposure. Combination treatment with chemotherapeutic agent, paclitaxel, resulted in additive growth suppression. SFN at ~8 μM decreased growth by 40% and 20% on day 1 in MDAH 2774 and SkOV-3, respectively. Cells treated with cytotoxic concentrations of SFN have reduced cell migration and increased apoptotic cell death via an increase in Bak/Bcl-2 ratio and cleavage of procaspase-9 and poly (ADP-ribose)-polymerase (PARP). Gene expression profile analysis of cell cycle regulated proteins demonstrated increased levels of tumor suppressor retinoblastoma protein (RB) and decreased levels of E2F-1 transcription factor. SFN treatment resulted in G1 cell cycle arrest through down modulation of RB phosphorylation and by protecting the RB-E2F-1 complex.
SFN induces growth arrest and apoptosis in EOC cells. Inhibition of retinoblastoma (RB) phosphorylation and reduction in levels of free E2F-1 appear to play an important role in EOC growth arrest.
Epithelial ovarian cancer is the leading cause of mortality from gynecological malignancies, often undetectable in early stage. The difficulty of detecting the disease in its early stages and the propensity of ovarian cancer cells to develop resistance to known chemotherapeutic treatments dramatically decreases the overall survival [1, 2]. Cytoreductive surgery followed by adjuvant combination chemotherapy is the standard treatment for advanced stage disease. However, despite these interventions, long-term survival rates remain low underscoring the need for new effective drugs or drug combinations with tolerable side effects [3, 4].
Epidemiological observations indicate that cruciferous vegetables such as broccoli, cabbage, cauliflower and brussels sprouts have been shown to offer protection against various cancers [5, 6]. Phytochemicals such as glucosinolates are abundant in these vegetables that are converted into isothiocyanates such as sulforaphane (SFN) [6–8].
Recently it has been shown that SFN inhibits the growth of the epithelial ovarian cancer cell (EOC) line SkOV-3 by down-regulating AKT activity . Related compounds such as synthetic isothiocyanate derivative ethyl 4-isothiocyanatobutanoate (E-4IB) and phenylisothiocyanates have been shown to induce apoptosis in A2780 and OVCAR-3 EOC cell lines [10–13]. Cytotoxic activity has also been demonstrated in SkOV-3 after treatment with indole-3-ethyl isothiocyanate (NB7 M) resulted in the inhibition of PIK3/AKT pathway . Although upstream mechanism of action at the cell surface has been linked to the inhibition of PIK-3 and AKT pathways in earlier studies, the various down stream signaling pathways responsible for SFN activity are not thoroughly understood.
The retinoblastoma protein (RB) is a well-known regulator of G1-S phase cell cycle transition . Negative regulation of the cell cycle is due to the ability of active, under phosphorylated RB to bind the transcription factor E2F-1 and repress transcription required for S phase progression [15, 16]. Here we show that SFN enhances the RB-E2F-1 interaction in MDAH-2774 ovarian cancer cell line linking for the first time the upstream AKT inhibition to the downstream blocking of cell cycle by activating RB protein. Further we show the inhibition of invasive ability of cells and reduced migration. Also we document the increased activity of caspases-9 and also cleavage of PARP protein indicating inhibition of cell proliferation and induction of cellular apoptosis.
Materials and methods
Reagents and antibodies
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).
Cell lines and culture
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.
Cell proliferation assays
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.
Analysis of apoptotic cells
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).
Cell cycle phase determination
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.
Western blotting and Immunoprecipitations
These assays were performed according to our earlier publication. Antibody reactions were visualized using enhanced chemiluminescence western blotting detection reagents (Amersham Pharmacia, Uppsala, Sweden).
Gene Expression Profiling (GEP)
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.
SFN induces growth arrest in ovarian cancer cell lines
SFN induces apoptosis MDAH-2774 cells
SFN inhibits S phase entry of MDAH-2774 cells in cultures
SFN induced modulation of cell cycle regulatory genes
Inhibition of the phosphorylation of Retinoblastoma protein (RB) and protection of RB-E2F-1 complex by SFN
SFN inhibits cell motility and invasiveness
SFN mediating cell growth arrest has been documented in colon, prostate and several other cancers [6–8]. Chaudhuri et al, recently demonstrated that SFN inhibits the growth of the ovarian cancer cells and the inhibition of the AKT pathway is one of the upstream molecular events . In this report we investigate downstream molecular mechanisms at the level of cell cycle control in the nucleus.
Combination therapy with paclitaxel is known to increase overall survival , but contributes to the development of resistance phenotype resulting in eventual relapse of the disease . Unlike traditional chemotherapy drugs, natural therapeutics like SFN may not contribute to the development of chemo-resistant phenotype. Our results indicate a synergistic effect of cell death when SFN was used in combination with paclitaxel (Fig. 1D).
SFN treatment resulted in cleavage of Poly (ADP-ribose) polymerase-1 (PARP-1) in dose dependent manner indicating the induction of apoptosis that was further confirmed by annexin V staining. Induction of apoptosis by SFN in different cellular systems is associated with Bax protein expression . Similarly the present study indicates a dose-dependent inhibition of anti-apoptotic protein Bcl-2 and concomitant increase in the expression of the pro-apoptotic protein Bak protein.
Phosphorylated RB cannot interact with E2F-1, thus leaving large pool of free E2F-1 transcription factors driving the G1/S cell cycle transition. E2F-1 has been shown to have growth promoting activity in EOC and is over expressed in roughly half of the ovarian cancers . Similarly, studies with histopathology grade 2 ovarian cancers demonstrated that transcription factors E2F-1 and E2F-2 play a critical role in promoting tumor metastasis . In our study, gene expression profiling has demonstrated down regulation of E2F-1 and 2 but not 3 . Although we observed down regulation of RB transcript with SFN treatment in gene profile analysis, over 90% of the translated protein product is in the active, under phosphorylated form. This supports our hypothesis that the appearance of under phosphorylated RB with SFN treatment results in the inhibition of cell cycle progression by the reduction in free E2F-1. Co-immunoprecipitation experiments also demonstrated the increased expression of the RB-E2F-1 complex with SFN treatment potentially adding to the reduction of E2F-1 levels.
Gene profile analysis showed down regulation of these CDKs with SFN treatment corroborating the under phosphorylated status of RB. Although SFN treatment is known to causes induction of p21 in prostate cancer , in our gene profile analysis, we observed a twofold decrease in p21. It is possible that in the case of ovarian cancers, CDK inhibition works through the induction of p15, p18 and p19 following up regulation with SFN treatment.
Invasion followed by degradation of basal membrane is hallmark of tumor metastasis where proliferating tumor cells infiltrate into other tissues . Wound healing assays and Boyden chamber assays provide evidence that the cell motility and invasiveness are inhibited by SFN. These findings suggested that SFN has very good potential for use in the treatment against invasion and metastasis of EOC.
SFN induces growth arrest and apoptosis in EOC cells by inhibiting RB phosphorylation and reduction in the levels of free E2F-1. In summary, we have provided evidence that SFN suppresses growth of EOC cells in vitro by contributing to the modulation of cell cycle regulatory proteins and by increasing the apoptosis. These effects may be correlated to the observed inhibition of cell migration. These observations highlight the possibility that SFN may be a good candidate for combination therapy of EOC with paclitaxel.
List of Abbreviations
Epithelial Ovarian Cancer
This work was supported in part by Department of Surgery, Wayne State University and Karmanos Cancer Institute (KCI), Detroit, MI.
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