Re:브로콜리 새싹의 효과들 - 항암효과

[문형철]

BEYOND REASON

브로콜리 새싹에는 설포라판 항암성분이 브로콜리에 비해 20배 넘게 들어있음. 

Transl Oncol. 2010 Dec; 3(6): 389–399. 

Published online 2010 Dec 1. doi: 10.1593/tlo.10235

PMCID: PMC3000464

PMID: 21151478

Anticancer Activity of a Broccoli Derivative, Sulforaphane, in Barrett Adenocarcinoma: Potential Use in Chemoprevention and as Adjuvant in Chemotherapy1

Aamer Qazi,*,2 Jagannath Pal,†‡,2 Ma'in Maitah,*,2 Mariateresa Fulciniti,†‡ Dheeraj Pelluru,†‡ Puru Nanjappa,†‡Saem Lee,‡ Ramesh B Batchu,* Madhu Prasad,* Christopher S Bryant,* Samiyah Rajput,† Sergei Gryaznov,§David G Beer,¶ Donald W Weaver,* Nikhil C Munshi,†‡ Raj K Goyal,‡ and  Masood A Shammas†‡

Author information Article notes Copyright and License information Disclaimer

This article has been cited by other articles in PMC.

Go to:

Abstract

INTRODUCTION: The incidence of Barrett esophageal adenocarcinoma (BEAC) has been increasing at an alarming rate in western countries. In this study, we have eval‎uated the therapeutic potential of sulforaphane (SFN), an antioxidant derived from broccoli, in BEAC. 

METHODS: BEAC cells were treated with SFN, alone or in combination with chemotherapeutic, paclitaxel, or telomerase-inhibiting agents (MST-312, GRN163L), and live cell number determined at various time points. The effect on drug resistance/chemosensitivity was eval‎uated by rhodamine efflux assay. Apoptosis was detected by annexin V labeling and Western blot analysis of poly(ADP-ribose) polymerase cleavage. Effects on genes implicated in cell cycle and apoptosis were determined by Western blot analyses. To eval‎uate the efficacy in vivo, BEAC cells were injected subcutaneously in severe combined immunodeficient mice, and after the appearance of palpable tumors, mice were treated with SFN. 

RESULTS: SFN induced both time- and dose-dependent decline in cell survival, cell cycle arrest, and apoptosis. The treatment with SFN also suppressed the expression‎ of multidrug resistance protein, reduced drug efflux, and increased anticancer activity of other antiproliferative agents including paclitaxel. A significant reduction in tumor volume was also observed by SFN in a subcutaneous tumor model of BEAC. Anticancer activity could be attributed to the induction of caspase 8 and p21 and down-regulation of hsp90, a molecular chaperon required for activity of several proliferation-associated proteins. 

CONCLUSIONS: These data indicate that a natural product with antioxidant properties from broccoli has great potential to be used in chemoprevention and treatment of BEAC.

Go to:

Introduction

Various epidemiologic studies have indicated that consumption of broccoli is associated with a lower risk of cancer [1], including breast [2], prostate [3], lung, stomach [4], and colon [5] cancers. The anticancer effect of broccoli has been attributed to sulforaphane (SFN), an isothiocyanate formed by hydrolysis of a precursor glucosinolate called “glucoraphanin” [1]. Although glucoraphanin is found in varying amounts in all cruciferous vegetables, the highest concentration of this compound is found in broccoli and its sprouts [6]. Among various parts of mature broccoli, the florets have the maximum amount of SFN. The amount of SFN in 1 g of dry broccoli florets ranges from 507 to 684 µg [7]. The sprouts of broccoli seem to have 20 to 30 times higher concentration of glucoraphanin, an SFN precursor [8,9], indicating that 1 oz of sprouts may have the amount of antioxidant present in 20 oz of mature broccoli. Glucoraphanin is converted to SFN by myrosinase, an enzyme released from broccoli during its consumption and also found in our stomach [1]. The reduced risk of cancer after consumption of broccoli is associated with the ability of SFN to inhibit phase 1 enzymes (implicated in the conversion of procarcinogens to carcinogens) and induce phase 2 enzymes (implicated in detoxification and excretion of carcinogens from body) [1,6].

However, the anticancer activity of SFN is not limited to its ability to promote detoxification and removal of carcinogens. SFN has been shown to inhibit cell cycle progression, induce apoptotic cell death, and inhibit angiogenesis in a variety of cancer cell types [1,6]. Exposure to SFN (20 µM) has been shown to induce a chk2 kinase-dependent arrest at the G2/M phase of cell cycle in prostate cancer (PC3) cells [10]. A G2/M arrest after treatment with SFN has also been demonstrated in human colon cancer (HCT116) cells [10]. Although cell cycle arrest at G2M has been frequently observed after treatment of cancer cells with SFN, arrest at other phases of cell cycle has also been reported for many cell lines. For example, the treatment of colon cancer (HT-29) cells with this agent led to the induction of p21, down-regulation of cyclin D1 and cyclin A, and a G1 cell cycle arrest [11], whereas prostate cancer (LnCap) cells arrested at G1/S after treatment with 10 µM drug [12,13].

SFN has also been shown to induce apoptosis, or programmed cell death, in cancer cells. Treatment with 15 µM SFN induced apoptosis in both the p53-positive and p53-negative human colon cancer cell lines [14]. Similarly, exposure to 10 µM SFN caused apoptosis in prostate cancer DU145 cells [15]. The mechanisms of SFN-induced apoptosis in these cell lines include activation of caspase 7 and 9 [14] and/or release of cytochrome C from mitochondria [16].

Anticancer activity of SFN has also been demonstrated in vivo. SFN has been shown to inhibit growth of human pancreatic cancer [17], human prostate cancer [18], murine osteosarcoma xenografts [19], and prevent intestinal polyposis [11], UV light-induced skin tumors [20], carcinogen-induced skin tumors [21], and carcinogen-induced stomach tumors [22] in vivo. SFN has also been reported to increase natural killer cell activity and antibody-dependent cellular cytotoxicity in both the control and tumor-bearing mice [23]. These and other modulations of immune system by SFN have been shown to play important role in the inhibition of metastatic spread of melanoma in mice [24].

Barrett esophageal adenocarcinoma (BEAC) develops in Barrett esophagus, a precancerous condition associated with chronic esophageal reflux. Because the cancer starts to spread before onset of clinical symptoms, BEAC patients usually have a dreary outcome with a poor survival rate [25]. Moreover, the incidence of this cancer has been increasing at a disturbing rate in Europe and United States [26]. Because the effect of SFN on BEAC cells has not been demonstrated, we studied the effect of this natural food ingredient, either alone or in combination with other anticancer agents including paclitaxel, on cell cycle and cell viability in BEAC cell lines. We have shown that SFN induces cell cycle arrest and apoptosis in BEAC cells at concentrations (3–7 µM) lower than (10–20 µM) those of SFN required to kill other cancer cell lines. Moreover, the SFN also suppressed multidrug resistance protein (MRP), reduced drug efflux, and significantly enhanced the anticancer activity of telomerase inhibitors (MST-312, GRN163L) and paclitaxel, a chemotherapeutic currently used to treat BEAC, in BEAC cell lines tested. The treatment with SFN was also associated with induction of caspase 8 and p21 and suppression of hsp90, a molecular chaperone required for activity of several proliferation related proteins. A significant anticancer activity of SFN was also observed in a subcutaneous tumor model in vivo. These data indicate a marked anticancer activity of SFN in BEAC and provide a rationale for clinical eval‎uation.

Go to:

Materials and Methods

Chemicals

l-Sulforaphane (SFN) and paclitaxel were purchased from Sigma-Aldrich (St Louis, MO) and dissolved in phosphate-buffered saline. MST-312 [N,N′-bis(2,3-dihydroxybenzoyl)-1,3-phenylenediamine] was purchased from Calbiochem/EMD Biosciences (Madison, WI) and dissolved in DMSO. GRN163L is a palmitoyl (C16) lipid—attached N3′-P5′ phosphoramidate oligonucleotide, targeting the template region of RNA subunit of telomerase (hTR) and was obtained from Geron Corporation (Menlo Park, CA). GRN140833 mismatch oligonucleotide was also obtained from Geron Corporation and used as a negative control.

Barrett Adenocarcinoma Cell Lines and Normal Cells

BEAC cell line FLO-1 and a lung adenocarcinoma cell line H460 have been described previously [27]. BEAC cell line OE33, from European Collection Of Cell Cultures, was purchased through Sigma-Aldrich and has been described previously [28,29]. Normal primary human esophageal epithelial cells (HEEC) were purchased from ScienCell Research Laboratories (Carlsbad, CA) and have been described previously [30]. FLO-1 cells were cultured in Dulbecco modified Eagle medium (Sigma) supplemented with 10% fetal bovine serum (HyClone, South Logan, UT). OE33 cells were cultured in RPMI-1640 supplemented with 2 mM l-glutamine and 10% fetal bovine serum. Normal HEEC (human esophageal epithelial cells) were cultured in epithelial cell medium-2 (ScienCell Research Laboratories). Cells were maintained in a state of logarithmic growth at 37°C in humidified air with 5% CO2, as described previously [30]. For RNA and protein analyses, cultures were harvested at the same final cell density (5 x 105/ml) and immediately processed.

Treatment and Growth of Cells

Cells (5 x 105) were plated in 100-mm dishes and treated with SFN alone or in combination with paclitaxel (PAC) and MST-312, at concentrations described in the figure legends. Substrate-attached viable cell number was counted, and cell viability was confirmed by trypan blue exclusion or cell proliferation assays at various time points. Cell proliferation assays were performed using Cell Counting Kit-8 (Dojindo Molecular Technologies, Inc, Gaithersburg, MD) according to the manufacturer's protocol. The method provides a highly sensitive colorimetric assay for the determination of viable cell number and is based on the production of a yellow product (formazan) after reduction of a highly water-soluble tetrazolium salt by dehydrogenases in viable cells. The amount of the formazan dye generated is measured by a plate reader and is directly proportional to the number of viable cells.

Rhodamine Efflux Assay

FLO-1 cells, plated in a six-well plate, were pretreated with SFN (3 or 5 µM) for 5 hours or with cyclosporin A (10 µM; a known inhibitor of MDR1 gene product) for 1 hour. Rhodamine 123 (3 µM) was then added into the same medium, and the cells were incubated for an additional 1.5 hours. Rhodamine-containing medium was then removed and cells were washed with PBS and incubated overnight at 37°C, 5% CO2 with serum-free medium containing the same concentrations of drugs without rhodamine. Cells were harvested, washed with PBS, and eval‎uated for rhodamine 123 florescence by flow cytometry.

Cell Cycle Analysis

The effect of SFN and PAC on progression of the cell cycle was determined using the Cell Cycle Phase Determination Kit (Cayman Chemical Company, Ann Arbor, MI). Briefly, the control and treated cells were washed and resuspended in assay buffer at 106/ml. Fixative (1 ml) was then added, and cells were allowed to permeabilize for at least 2 hours. The cells were centrifuged, the fixative was removed, and cells were resuspended in 0.5 ml of staining solution containing propidium iodide. After 30 minutes of incubation at room temperature, the cells were analyzed in the FL2 channel of a flow cytometer with a 488-nm excitation filter.

Apoptosis Assay

Apoptotic cells were detected using the Annexin V-Biotin Apoptosis Detection Kit (Oncogene Research Products, San Diego, CA). Untreated or treated BEAC cells (1 x 106 cells/ml) were mixed with annexin V-biotin and medium-binding reagent and incubated in the dark for 15 minutes at room temperature. Cells were then centrifuged, and the medium was replaced with 1x binding buffer (Oncogene Research Products) containing fluorescein isothiocyanate (FITC)-streptavidin (Amersham Life Sciences, Inc, Arlington Heights, IL). A portion of the cell suspension (50 µl) was placed onto a glass slide, covered with a coverslip, and viewed immediately using a fluorescence microscope equipped with FITC (green) filter. Two hundred cells, representing at least five distinct microscopic fields, were analyzed to assess the fraction of FITC-labeled cells for each sample.

Western Blot Analysis

Approximately 50 mg of protein was suspended in Laemmli sample buffer (0.1 M Tris-HCl buffer pH 6.8, 1% SDS, 0.05% β-mercaptoethanol, 10%glycerol, and 0.001% bromphenol blue), boiled for 2 minutes, and electrophoresed on 4% to 20% glycerol gradient SDS-polyacrylamide gel for 4 hours at 120 V. Gels were electroblotted onto Trans-Blot nitrocellulose membrane (Bio-Rad Laboratories, Hercules, CA) at 40 V for 3 hours in a Tris-glycine buffer system. Incubation with indicated antibodies was performed for 2 hours in PBS-Tween 20 (PBST) containing 1% BSA with constant rocking. Blots were washed with PBST and incubated in either antirabbit or antimouse horseradish peroxidase (Santa Cruz Biotechnology, Inc, Santa Cruz, CA) conjugates for 2 hours in PBST containing 3% nonfat dry milk. After washing, specific proteins were detected using an enhanced chemiluminescence, according to the instructions provided in the manual (Amersham Life Sciences, Inc).

In Vivo Study

The in vivo efficacy of SFN was tested in a murine xenograft model of BEAC in which FLO-1 cells were injected subcutaneously in severe combined immunodeficient (SCID) mice. After detection of tumors, mice were treated with either 0.75 mg of SFN or 10% DMSO subcutaneously daily for 2 weeks. Tumor growth was measured in two perpendicular dimensions once every 3 days using a caliper and the following formula: V = (a2 x b) / 2, where a is the width of the tumor (smaller diameter) and b is the length (larger diameter).

Go to:

Results

SFN Induces Time- and Dose-Dependent Decline in Survival of BEAC Cells

FLO-1 and OE33 cells were cultured in the presence or absence of SFN at various concentrations and for variable length of time, substrate-attached viable cell number was counted, and cell viability was confirmed by trypan blue exclusion or CCK 8 assays (Figure 1, A and B). SFN induced both time- and dose-dependent decline in survival of BEAC cells (Figure 1). Exposure to 7 µM SFN led to a complete cell death in both BEAC cell lines tested, in a period of 3 to 5 days (Figure 1, A and B). A substantial antiproliferative activity was also seen by SFN at 5 µM, which killed 100% of OE33 cells in 3 days and 83% ± 4% of FLO-1 cells in 5 days. The treatment of FLO-1 cells with 3 µM SFN led to 74% ± 4% cell death in 5 days. At 1 µM, SFN was less affective and killed 54% of OE33 cells in 3 days and 39% of FLO-1 cells in 5 days (Figure 1). These data indicate that SFN inhibits the proliferation of BEAC cells. To eval‎uate the effect of SFN on the morphology of treated cells, both the cancer (FLO-1, OE33) and normal (fibroblasts, primary esophageal epithelial) cells were treated with 3 µM SFN for 72 hours and photographed under a phase-contrast microscope. As shown in Figure 1 (C and D), whereas the cancer (FLO-1 and OE33) cells started to detach and/or reduce in size and number, both types of normal cells (fibroblasts and esophageal epithelial cells) remained unaffected by the treatment.

Figure 1

Effect of SFN on BEAC cell survival. BEAC cells were cultured in the medium containing no SFN or various concentrations of SFN. Cells were harvested at different time points as indicated and proliferative potential was assessed by trypan blue exclusion and/or proliferation assay, based on the production of a yellow product (formazan) after reduction of a highly water-soluble tetrazolium salt by dehydrogenases in viable cells. The growth curves show the mean of three independent experiments, with SEM. (A) Barrett adenocarcinoma (FLO-1) cells treated with various concentrations of SFN. (B) BEAC (OE33) cells treated with various concentrations of SFN. (C) Photomicrograph of BEAC (FLO-1 and OE33) cells treated with 3 µM SFN for 72 hours. (D) Photomicrograph of normal diploid fibroblasts and primary normal esophageal epithelial cells (ScienCell Research Laboratories) treated with 3 µM SFN for 72 hours. (E) FLO-1 cells were treated with SFN for 48 hours, detached floating cells from the medium and the attached cells (by trypsinization) were collected separately and eval‎uated for number and viability using trypan blue exclusion. The number of cells detached after treatment with various concentrations of SFN is expressed as percent of untreated FLO-1 cells. “Total” represents the total number of detached cells whereas “Dead” reflects the fraction of dead cells in detached cell population. (F) Panel (I): FLO-1 cells were incubated with various concentrations of SFN for 48 hours, and the expression‎ of caspase 8 was detected by Western blot analysis, using anti-caspase 8 mouse monoclonal antibody (Cell Signaling, Danvers, MA). Panel (II): Bar graph showing caspase 8 expression‎ relative to β-actin.

To eval‎uate the nature of detached cells, FLO-1 cells were treated with SFN for 48 hours. Detached floating cells from the medium and the attached cells (by trypsinization) were collected separately and eval‎uated for the number and viability using trypan blue exclusion. Figure 1E shows the relative proportion of cells detached after treatment with various concentrations of SFN, expressed as percent of untreated FLO-1 cells. The total number of detached cells increased with increasing concentrations of SFN. Although majority (∼60%) of detached cells were dead, approximately 40% appeared alive, indicating detachment before complete death (Figure 1E).

Consistent with decline in cell viability and attachment, Western blot analysis indicated that treatment of BEAC cells with SFN leads to a marked induction of caspase 8, an initiator caspase implicated in death receptor-induced apoptosis (Figure 1F).

SFN Increases Intracellular Accumulation of Drug in BEAC Cells

To eval‎uate if SFN can increase intracellular drug accumulation and chemosensitivity by inhibiting drug efflux, we used rhodamine efflux assay. Rhodamine 123 is a substrate of MRP and P-glycoprotein, a product of multidrug resistance gene (MDR), implicated in the extrusion of drugs outside the cell. We therefore exposed BEAC cells, pretreated with SFN or cyclosporin A (broad-spectrum inhibitor of multidrug resistance gene products), to rhodamine 123. Rhodamine-containing medium was then removed, and cells were washed with PBS and incubated overnight in the presence of corresponding drugs without rhodamine. Cells were harvested, washed with PBS, and eval‎uated for rhodamine 123 florescence by flow cytometry. Figure 2 (A and B) shows a dose dependent increase in the accumulation of intracellular rhodamine 123 in FLO-1 cells treated with different concentrations of SFN. These data indicate that SFN inhibits the drug efflux, thus increasing the intracellular concentration and chemosensitivity. Consistent with these data, Western blot analysis showed down-regulation of MRP in SFN-treated, relative to untreated FLO-1 cells (Figure 2C).

Figure 2

SFN increases intracellular accumulation of drug and enhances antiproliferative activity of other anticancer agents in BEAC cells. (A and B) Effect of SFN on intracellular drug accumulation. BEAC (FLO-1) cells, pretreated with SFN or cyclosporin A, were exposed to rhodamine 123. Rhodamine was then removed, and cells were incubated overnight in the presence of corresponding drugs as described. Cells were harvested, washed, and eval‎uated for rhodamine 123 florescence by flow cytometry. (A) The amount of rhodamine 123 fluorescence retained was measured using a fluorescence-activated cell flow analyzer. (B) Bar graph showing dose-dependent increases in the accumulation of intracellular rhodamine 123 in FLO-1 cells treated with different concentrations of SFN. (C) FLO-1 cells were treated with various concentrations of SFN for 24 hours, and the expression‎ of MRP was monitored by Western blot analysis using MRP (E-19) antibody (Santa Cruz Biotechnology, Inc).

SFN Significantly Enhances the Antiproliferative Effect of Chemotherapeutic and Telomerase-Inhibiting Agents in BEAC Cells

We next eval‎uated if SFN can enhance the antiproliferative affect of other anticancer agents. For this purpose, we chose paclitaxel (a chemotherapeutic agent used for treatment of BEAC), MST-312 (a chemical inhibitor of telomerase), and GRN163L (a lipidated oligonucleotide targeting RNA component of telomerase). For SFN-PAC combination study, we first cultured the FLO-1 and OE33 cells in the presence of paclitaxel at various concentrations and for variable length of time and eval‎uated for cell viability as described (not shown). Concentrations of SFN and paclitaxel inducing similar cell death at a given time point were used for combination study. As shown in Figure 3A, the treatment of FLO-1 cells with 1 µM paclitaxel and 3 µM SFN for 3 days led to 57% ± 6% and 61% ± 6% cell death, respectively. However, a combination of both drugs led to 92% ± 3% cell death, indicating a significant (P > .0003) 35% increase in cell death relative to paclitaxel alone (Figure 3A). Similarly, in OE33 cells, paclitaxel alone induced 42% ± 5% cell death, whereas a combination of paclitaxel and SFN led to 79% ± 4% cell death, showing a significant (P < .01) 37% increase in cell death relative to paclitaxel alone (Figure 3B). These data show that SFN can significantly enhance the antiproliferative effect of paclitaxel in BEAC cells.

Figure 3

Effect of SFN on the antiproliferative activity of other agents. BEAC cells, FLO-1 (A) or OE33 (B), were cultured in the medium containing no drug, 1 µM paclitaxel, 3 µM SFN, or a combination of both 1 µM paclitaxel and 3 µM SFN. Cells were harvested at indicated time points, substrate-attached live cell number was determined, and cell viability was confirmed by trypan blue exclusion and/or proliferation assays described. Bar graphs show the mean of three independent experiments, with SEM. (C and D) Effect of SFN on antiproliferative activity of FLO-1 and H460 adenocarcinoma cells pretreated with telomerase inhibitors. FLO-1 cells (C), pretreated with MST-312 (1 µM) for 10 days, were treated with SFN (3 or 5 µM) for 48 hours, and live cell number was determined. H460 lung adenocarcinoma cells (D), treated with GRN163L or mismatch oligonucleotide at 2 µM for 10 days, were exposed to SFN (2 µM) and eval‎uated for live cell number everyday for the next 3 days. Error bars represent SEMs of triplicate experiments.

To eval‎uate the effect of SFN on antiproliferative effect of telomerase inhibiting agents, FLO-1 cells were pretreated with a telomerase inhibitor MST-312 (1 µM) for 10 days to initiate telomere shortening and were then treated with SFN (3 or 5 µM) for 48 hours. As shown in Figure 3C, the treatment of FLO-1 cells pretreated with MST-312 to SFN led to a significant (P ≥ .002) decrease in cell growth, relative to cells treated with MST-312 or SFN alone. SFN at 3 and 5 µM increased the cell death by 36% and 48%, respectively, in FLO-1 cells pretreated with MST-312. Similar observations were also made in H460 lung adenocarcinoma cells treated with telomerase inhibitor GRN163L, a lipid-attached oligonucleotide-targeting RNA component of telomerase. H460, pretreated with GRN163L or mismatch oligonucleotide at 2 µM for 10 days, was exposed to SFN (2 µM) and eval‎uated for live cell number everyday for next 3 days. Addition of SFN to cells pretreated with 163L reduced the live cell number to 16.3% ± 3.2% in 3 days, whereas ∼40% cells were still alive when drugs were used separately (Figure 3D). These data indicate that SFN significantly enhances the anticancer activity of chemotherapeutic and other antiproliferative agents.

SFN Inhibits Cell Cycle Progression and Enhances the Ability of Paclitaxel to Induce Cell Cycle Arrest

Paclitaxel is a microtubule inhibitor and is known to induce cell cycle arrest at G2-M. In this study, we eval‎uated the effect of SFN on cell cycle progression and on paclitaxel-induced cell cycle arrest in BEAC cells. FLO-1 cells were treated with SFN (3 µM), paclitaxel (1 µM), or both for 3 days, and the effect on cell cycle progression was determined using the Cell Cycle Phase Determination Kit (Cayman Chemical Company). Briefly, the control and treated cells were fixed, stained with propidium iodide, and analyzed using a flow cytometer. Although 41% of control cells were in the S-phase, the fraction of cells in the S-phase after treatment with paclitaxel and SFN was only 18% and 24%, respectively (Figure 4A). Consistent with cell viability data, a more powerful inhibition of the S-phase was observed when paclitaxel was combined with SFN (Figure 4A, panel IV). Cells treated with paclitaxel were arrested at G2-M (55%) or G1(27%), whereas those treated with SFN were mostly at G1. However, when SFN was added along with paclitaxel, majority of cells were arrested at the G1 phase of the cell cycle (Figure 4A).

Figure 4

Effect of SFN, paclitaxel, and combination treatments on cell cycle in BEAC cells. (A) Barrett adenocarcinoma (FLO-1) cells, untreated or treated with SFN (3 µM), paclitaxel (1 µM), or both for 72 hours, were processed for eval‎uation of cell cycle using the Cell Cycle Phase Determination Kit (Cayman Chemical Company). Phases of cell cycle were analyzed in the FL2 channel of a flow cytometer with a 488-nm excitation filter. Panels: (I) untreated BEAC (FLO-1) cells, (II) BEAC (FLO-1) cells treated with SFN, (III) BEAC (FLO-1) cells treated with paclitaxel, and (IV) BEAC (FLO-1) cells treated with both the paclitaxel and SFN. (B) Induction of p21 by SFN. Panels: (I) Western blot showing protein levels of p21 after the above treatments and (II) bar graph showing fold induction in p21 protein levels in those treated relative to control cells, after normalization with β-actin. (C) Suppression of hsp90 by SFN: Transcript levels of hsp90 in FLO-1 cells after treatments described above. Panels: (I) Protein levels of hsp90 after the above treatments and (II) bar graph showing fold reduction in hsp90 protein levels in treated relative to control cells after normalization with β-actin.

Consistent with cell cycle arrest, the treatment with paclitaxel, SFN, and the combination of both drugs led to 5-fold, 9-fold, and 57-fold induction of cell cycle inhibiting protein, p21 (Figure 4B, panels I and II). These data not only confirm‎ that p21 was induced after SFN treatment but also demonstrate a marked effect of the combination treatment in BEAC cells. SFN treatment also led to the down-regulation of hsp90, a molecular chaperone required for activity of several proliferation-associated proteins (Figure 4C). The suppression of hsp90 was also the strongest when SFN was used in combination with paclitaxel.

SFN Induces Apoptotic Cell Death and Enhances the Proapoptotic Activity of Paclitaxel

BEAC cells (FLO-1 and OE33) were treated with SFN (3 µM) and paclitaxel (1 µM) and analyzed for apoptotic cell death. Both untreated or BEAC cells were sequentially treated with annexin V-biotin and FITC-streptavidin and apoptotic cells were eval‎uated by a fluorescence microscope. Approximately 200 cells, representing at least five distinct microscopic fields, were analyzed to assess the fraction of annexin-positive cells for each sample. After a 3-day exposure, 43% ± 6% of cells treated with paclitaxel, 50% ± 7% of cells treated with SFN, and 80% ± 8% of cells treated with both paclitaxel and SFN were annexin V-positive (Figure 5A). These data indicate that both paclitaxel and SFN induce apoptosis in BEAC cells, and the addition of SFN significantly increases the fraction of cells undergoing apoptosis. To further eval‎uate the effect of these treatments and their combination on apoptosis, we analyzed poly(ADP-ribose) polymerase (PARP), a protein which undergoes caspase 3-mediated cleavage during apoptosis. At an earlier time point of 48 hours, the cells treated with paclitaxel or SFN alone had only 5% to 6% of cleaved PARP product, whereas a substantial fraction (35%) of PARP was found to be cleaved in the cells treated with combination of both drugs (Figure 5B).

Figure 5

Effect of SFN, paclitaxel, and combination treatments on apoptosis in BEAC cells. (A) BEAC (FLO-1) cells, untreated or treated with SFN (3 µM), paclitaxel (1 µM), or both for 72 hours, were analyzed for apoptosis using the Annexin V-Biotin Apoptosis Detection Kit. Cells were sequentially treated with annexin V-biotin and FITC-streptavidin. FITC-streptavidin-labeled apoptotic cells within the same microscopic field were viewed and photographed by phase-contrast (PC) or by fluorescence emitted at 518 nm (FITC filter). Using the FITC filter, apoptotic cells appear bright green. Panels: (I) Annexin labeling of FLO-1 cells, untreated or treated as described and (II) bar graph showing percent apoptotic cells after each treatment. (B) FLO-1 cells were treated as described for panel A but for a duration of 48 hours and analyzed for cleavage of PARP, a marker for apoptosis. Panels: (I) PARP was identified by Western blot analysis using a rabbit polyclonal antibody against PARP (Santa Cruz Biotechnology, Inc) and (II) bar graph showing percentage of cleaved 89-kDa PARP fragment.

In Vivo Efficacy of SFN

In vivo efficacy of SFN was demonstrated in a murine model in which SCID mice were subcutaneously inoculated in the interscapular area with human Barrett adenocarcinoma (BEAC; FLO-1) cells. After detection of tumors, mice were treated with either 0.75 mg of SFN or 10% DMSO subcutaneously daily, for 2 weeks. Tumor growth was measured in two perpendicular dimensions, once every 3 days, using a caliper. As shown in Figure 6A, the tumor size was significantly reduced in the mice treated with SFN, relative to control mice. In Figure 6B, the tumor sizes in individual mice show that tumors in three of four treated mice were smaller than any control mice. These data show that SFN has significant antitumor activity in vivo.

Figure 6

In vivo efficacy of SFN in BEAC. SCID mice were inoculated subcutaneously in the interscapular area with FLO-1 (BEAC) cells, and after the appearance of tumors, mice were treated intraperitoneally with PBS alone or SFN 25 mg/kg (5 times per week). Average tumor sizes in all treated and control mice (A) and tumor sizes in individual mice (B) are shown.

Go to:

Discussion

In this study, we have eval‎uated the therapeutic potential of SFN, an antioxidant derived from broccoli, in BEAC. We have found that: 1) SFN induces both the cell cycle arrest and apoptosis in BEAC cell lines tested and inhibits tumor growth in a subcutaneous model of BEAC in vivo. 2) The concentration of SFN (3–7 µM) required to induce cell cycle arrest and apoptosis in BEAC cells is lower than that (10–20 µM) reported for similar affects in several other cancer cell types. 3) Treatment of FLO-1 cells with SFN is associated with down-regulation of MRP and reduction in drug efflux. 4) SFN significantly enhances the antiproliferative activity of other antiproliferative agents (paclitaxel, a chemotherapeutic used in the treatment of BEAC) and telomerase inhibitors MST-312 and GRN163L. 5) Addition of SFN with paclitaxel leads to a marked increase in the expression‎ of p21, cell cycle arrest, and apoptosis in BEAC cells. 6) Anticancer activity of SFN could be attributed, at least in part, to the induction of caspase 8 and p21 and the suppression of MRP and hsp90, a molecular chaperone required for activity of several proliferation associated proteins.

Anticancer effects of SFN have been demonstrated in several malignancies including human colon, bladder, prostate, ovarian, lymphoblastoid, pancreatic, cervical cancer, and lung cancers [15,17,31–36]. However, to our knowledge, this is the first report demonstrating the ability of SFN to induce apoptotic cell death, increase chemosensitivity, and significantly enhance the antiproliferative effects of chemotherapeutic and telomerase-inhibiting agents in BEAC cells. Moreover, the SFN could induce apoptosis in BEAC cell lines at 3 to 7 µM, a concentration lower than 10 to 40 µM, reported to induce apoptotic cell death in most other cancers including colon, ovarian, non-small lung, cervical, breast, lung adenocarcinoma, hepatoma, and prostate [10,15,18,31–39], indicating a greater sensitivity of this agent for BEAC.

SFN also significantly enhanced the antiproliferative activity of other anticancer agents including a chemotherapeutic agent, paclitaxel, used for the treatment of BEAC. A combination of SFN and paclitaxel led to a significant increase in the fraction of apoptotic cells, as indicated by annexin labeling (Figure 5). To further confirm‎ the apoptosis and the increased efficacy of combination treatment, we analyzed the treated cells for PARP cleavage at an earlier time point of 48 hours. In the cells treated with paclitaxel, SFN, or both drugs, the fraction of PARP cleaved into the 89-kDa fragment was 6%, 5%, and 35%, respectively. These data indicate that the combination of both the drugs leads to a remarkable increase in apoptotic activity and is consistent with the significant increase in cell death and annexin labeling after exposure of BEAC cells to both the paclitaxel and SFN.

SFN also significantly enhanced the antiproliferative activity of a chemical inhibitor of telomerase (MST-312) in BEAC cells. A similar effect was also observed in lung adenocarcinoma H460 cells in which SFN significantly enhanced the antiproliferative activity of GRN163L, an oligonucleotide targeting the RNA component of telomerase. Consistent with these observations, we have found that SFN downregulates MRP and reduces drug efflux in BEAC cells. Because MRPs have been implicated in the export of chemotherapeutic drugs including paclitaxel [40], it is possible that SFN enhances the chemosensitivity of other antiproliferative agents, at least in part, by increasing intracellular drug concentration.

Exposure of BEAC cells to SFN was associated with both the cell cycle arrest and apoptosis, and it is consistent with the observations made in other cancer cell types including, osteosarcoma, ovarian carcinoma, colon, prostate, and lung cancers [1,14,19,32,41,42]. As shown in Figure 4, the treatment with SFN led to the accumulation of FLO-1 cells at the G1 phase of the cell cycle. Although a G2/M cell cycle arrest is more commonly seen after exposure of cancer cells to SFN [15,18,43], arrest at G1 has been observed for human colon cancer (HT-29) cells [44]. Consistent with our observations in BEAC cells, the G1 arrest in colon HT-29 cells was also associated with induction of p21.

Several mechanisms have been proposed for induction of apoptosis after treatment of cancer cells with SFN. SFN-induced apoptosis in colon cancer (HT-29) cells is attributed to up-regulation of Bax and release of cytochrome C from mitochondria [16]. In both the prostate and colon cancer cells, SFN treatment was associated with inhibition of histone deacetylase, leading to increased histone acetylation and apoptosis [42,45]. SFN has also been reported to induce apoptosis through activation of AP-1 [46], activation of MAPK pathways (ERK, JNK, p38) [44,47], and down-regulation of nuclear factor κB [48]. In our study, the apoptosis in BEAC cells could be attributed, at least in part, to the induction of caspase 8 and p21 and suppression of hsp90. Exposure of BEAC cells to SFN led to a marked induction of caspase 8, an initiator caspase implicated in death receptor-mediated apoptosis [45]. Consistent with this, the transcript levels of caspase 8 and several death receptors were also elevated in SFN-treated BEAC cells (not shown). SFN, paclitaxel, and the combination of both led to 9-fold, 5-fold, and 57-fold induction of p21 in FLO-1 cells, respectively. These data indicate that there could be a remarkable effect of adding SFN with paclitaxel in treating BEAC.

In summary, these studies demonstrate that SFN inhibits the proliferation of BEAC cells at a nontoxic concentration, reduces tumor growth in vivo, and significantly enhances the anticancer activity of other chemotherapeutic and antiproliferative agents in BEAC. These data therefore indicate that a natural product with antioxidant properties from broccoli has a specific activity against BEAC, making it an ideal compound for therapy and possible chemoprevention of this disease.

Go to:

Footnotes

1This work was supported by grants from R01CA125711 to MAS, from the Department of Veterans Affairs Merit Review Awards and from the National Institutes of Health grant RO1-124929 to NCM and grants P50-100007 and PO1-78378 to NCM and Dr. K. C. Anderson and by funds from Karmanos Cancer Institute and Department of Surgery, Wayne State University, Detroit, MI.

Go to:

References

1. Clarke JD, Dashwood RH, Ho E. Multi-targeted prevention of cancer by sulforaphane. Cancer Lett. 2008;269:291–304. [PMC free article] [PubMed] [Google Scholar]

2. Ambrosone CB, McCann SE, Freudenheim JL, Marshall JR, Zhang Y, Shields PG. Breast cancer risk in premenopausal women is inversely associated with consumption of broccoli, a source of isothiocyanates, but is not modified by GST genotype. J Nutr. 2004;134:1134–1138. [PubMed] [Google Scholar]

3. Joseph MA, Moysich KB, Freudenheim JL, Shields PG, Bowman ED, Zhang Y, Marshall JR, Ambrosone CB. Cruciferous vegetables, genetic polymorphisms in glutathione S-transferases M1 and T1, and prostate cancer risk. Nutr Cancer. 2004;50:206–213. [PubMed] [Google Scholar]

4. van Poppel G, Verhoeven DT, Verhagen H, Goldbohm RA. Brassica vegetables and cancer prevention. Epidemiology and mechanisms. Adv Exp Med Biol. 1999;472:159–168. [PubMed] [Google Scholar]

5. Chung FL, Conaway CC, Rao CV, Reddy BS. Chemoprevention of colonic aberrant crypt foci in Fischer rats by sulforaphane and phenethyl isothiocyanate. Carcinogenesis. 2000;21:2287–2291. [PubMed] [Google Scholar]

6. Zhang Y, Talalay P, Cho CG, Posner GH. A major inducer of anticarcinogenic protective enzymes from broccoli: isolation and elucidation of structure. Proc Natl Acad Sci USA. 1992;89:2399–2403.[PMC free article] [PubMed] [Google Scholar]

7. Campas-Baypoli ON, Sanchez-Machado DI, Bueno-Solano C, Ramirez-Wong B, Lopez-Cervantes J. HPLC method validation for measurement of sulforaphane level in broccoli by-products. Biomed Chromatogr. 2010;24:387–392. [PubMed] [Google Scholar]

8. Fahey JW, Zhang Y, Talalay P. Broccoli sprouts: an exceptionally rich source of inducers of enzymes that protect against chemical carcinogens. Proc Natl Acad Sci USA. 1997;94:10367–10372.[PMC free article] [PubMed] [Google Scholar]

9. Zhang Y, Talalay P. Anticarcinogenic activities of organic isothiocyanates: chemistry and mechanisms. Cancer Res. 1994;54:1976s–1981s. [PubMed] [Google Scholar]

10. Singh SV, Herman-Antosiewicz A, Singh AV, Lew KL, Srivastava SK, Kamath R, Brown KD, Zhang L, Baskaran R. Sulforaphane-induced G2/M phase cell cycle arrest involves checkpoint kinase 2-mediated phosphorylation of cell division cycle 25C. J Biol Chem. 2004;279:25813–25822. [PubMed] [Google Scholar]

11. Hu R, Khor TO, Shen G, Jeong WS, Hebbar V, Chen C, Xu C, Reddy B, Chada K, Kong AN. Cancer chemoprevention of intestinal polyposis in ApcMin/+ mice by sulforaphane, a natural product derived from cruciferous vegetable. Carcinogenesis. 2006;27:2038–2046. [PubMed] [Google Scholar]

12. Chiao JW, Chung FL, Kancherla R, Ahmed T, Mittelman A, Conaway CC. Sulforaphane and its metabolite mediate growth arrest and apoptosis in human prostate cancer cells. Int J Oncol. 2002;20:631–636. [PubMed] [Google Scholar]

13. Wang L, Liu D, Ahmed T, Chung FL, Conaway C, Chiao JW. Targeting cell cycle machinery as a molecular mechanism of sulforaphane in prostate cancer prevention. Int J Oncol. 2004;24:187–192.[PubMed] [Google Scholar]

14. Pappa G, Lichtenberg M, Iori R, Barillari J, Bartsch H, Gerhauser C. Comparison of growth inhibition profiles and mechanisms of apoptosis induction in human colon cancer cell lines by isothiocyanates and indoles from Brassicaceae. Mutat Res. 2006;599:76–87. [PubMed] [Google Scholar]

15. Cho SD, Li G, Hu H, Jiang C, Kang KS, Lee YS, Kim SH, Lu J. Involvement of c-Jun N-terminal kinase in G2/M arrest and caspase-mediated apoptosis induced by sulforaphane in DU145 prostate cancer cells. Nutr Cancer. 2005;52:213–224. [PubMed] [Google Scholar]

16. Gamet-Payrastre L, Li P, Lumeau S, Cassar G, Dupont MA, Chevolleau S, Gasc N, Tulliez J, Tercé F. Sulforaphane, a naturally occurring isothiocyanate, induces cell cycle arrest and apoptosis in HT29 human colon cancer cells. Cancer Res. 2000;60:1426–1433. [PubMed] [Google Scholar]

17. Pham NA, Jacobberger JW, Schimmer AD, Cao P, Gronda M, Hedley DW. The dietary isothiocyanate sulforaphane targets pathways of apoptosis, cell cycle arrest, and oxidative stress in human pancreatic cancer cells and inhibits tumor growth in severe combined immunodeficient mice. Mol Cancer Ther. 2004;3:1239–1248. [PubMed] [Google Scholar]

18. Singh AV, Xiao D, Lew KL, Dhir R, Singh SV. Sulforaphane induces caspase-mediated apoptosis in cultured PC-3 human prostate cancer cells and retards growth of PC-3 xenografts in vivo. Carcinogenesis. 2004;25:83–90. [PubMed] [Google Scholar]

19. Matsui TA, Murata H, Sakabe T, Sowa Y, Horie N, Nakanishi R, Sakai T, Kubo T. Sulforaphane induces cell cycle arrest and apoptosis in murine osteosarcoma cells in vitro and inhibits tumor growth in vivo. Oncol Rep. 2007;18:1263–1268. [PubMed] [Google Scholar]

20. Dinkova-Kostova AT, Jenkins SN, Fahey JW, Ye L, Wehage SL, Liby KT, Stephenson KK, Wade KL, Talalay P. Protection against UV-light-induced skin carcinogenesis in SKH-1 high-risk mice by sulforaphane-containing broccoli sprout extracts. Cancer Lett. 2006;240:243–252. [PubMed] [Google Scholar]

21. Xu C, Huang MT, Shen G, Yuan X, Lin W, Khor TO, Conney AH, Kong AN. Inhibition of 7,12-dimethylbenz(a)anthracene-induced skin tumorigenesis in C57BL/6 mice by sulforaphane is mediated by nuclear factor E2-related factor 2. Cancer Res. 2006;66:8293–8296. [PubMed] [Google Scholar]

22. Fahey JW, Haristoy X, Dolan PM, Kensler TW, Scholtus I, Stephenson KK, Talalay P, Lozniewski A. Sulforaphane inhibits extracellular, intracellular, and antibiotic-resistant strains of Helicobacter pylori and prevents benzo[a]pyrene-induced stomach tumors. Proc Natl Acad Sci USA. 2002;99:7610–7615.[PMC free article] [PubMed] [Google Scholar]

23. Thejass P, Kuttan G. Augmentation of natural killer cell and antibodydependent cellular cytotoxicity in BALB/c mice by sulforaphane, a naturally occurring isothiocyanate from broccoli through enhanced production of cytokines IL-2 and IFN-gamma. Immunopharmacol Immunotoxicol. 2006;28:443–457.[PubMed] [Google Scholar]

24. Thejass P, Kuttan G. Modulation of cell-mediated immune response in B16F-10 melanoma-induced metastatic tumor-bearing C57BL/6 mice by sulforaphane. Immunopharmacol Immunotoxicol. 2007;29:173–186. [PubMed] [Google Scholar]

25. Blot WJ, McLaughlin JK. The changing epidemiology of esophageal cancer. Semin Oncol. 1999;26:2–8. [PubMed] [Google Scholar]

26. Devesa SS, Blot WJ, Fraumeni JF., Jr Changing patterns in the incidence of esophageal and gastric carcinoma in the United States. Cancer. 1998;83:2049–2053. [PubMed] [Google Scholar]

27. Aggarwal S, Taneja N, Lin L, Orringer MB, Rehemtulla A, Beer DG. Indomethacin-induced apoptosis in esophageal adenocarcinoma cells involves upregulation of Bax and translocation of mitochondrial cytochrome C independent of COX-2 expression‎. Neoplasia. 2000;2:346–356. [PMC free article][PubMed] [Google Scholar]

28. Ogunwobi OO, Beales IL. Statins inhibit proliferation and induce apoptosis in Barrett's esophageal adenocarcinoma cells. Am J Gastroenterol. 2008;103:825–837. [PubMed] [Google Scholar]

29. Tselepis C, Morris CD, Wakelin D, Hardy R, Perry I, Luong QT, Harper E, Harrison R, Attwood SE, Jankowski JA. Upregulation of the oncogene c-myc in Barrett's adenocarcinoma: induction of c-myc by acidified bile acid in vitro. Gut. 2003;52:174–180. [PMC free article] [PubMed] [Google Scholar]

30. Yoshida N, Katada K, Handa O, Takagi T, Kokura S, Naito Y, Mukaida N, Soma T, Shimada Y, Yoshikawa T, et al. Interleukin-8 production via protease-activated receptor 2 in human esophageal epithelial cells. Int J Mol Med. 2007;19:335–340. [PubMed] [Google Scholar]

31. Andelova H, Rudolf E, Cervinka M. In vitro antiproliferative effects of sulforaphane on human colon cancer cell line SW620. Acta Medica (Hradec Kralove) 2007;50:171–176. [PubMed] [Google Scholar]

32. Chuang LT, Moqattash ST, Gretz HF, Nezhat F, Rahaman J, Chiao JW. Sulforaphane induces growth arrest and apoptosis in human ovarian cancer cells. Acta Obstet Gynecol Scand. 2007:1–6. [PubMed] [Google Scholar]

33. Jin CY, Moon DO, Lee JD, Heo MS, Choi YH, Lee CM, Park YM, Kim GY. Sulforaphane sensitizes tumor necrosis factor-related apoptosisinducing ligand-mediated apoptosis through downregulation of ERK and Akt in lung adenocarcinoma A549 cells. Carcinogenesis. 2007;28:1058–1066. [PubMed] [Google Scholar]

34. Mi L, Wang X, Govind S, Hood BL, Veenstra TD, Conrads TP, Saha DT, Goldman R, Chung FL. The role of protein binding in induction of apoptosis by phenethyl isothiocyanate and sulforaphane in human non-small lung cancer cells. Cancer Res. 2007;67:6409–6416. [PubMed] [Google Scholar]

35. Park SY, Kim GY, Bae SJ, Yoo YH, Choi YH. Induction of apoptosis by isothiocyanate sulforaphane in human cervical carcinoma HeLa and hepatocarcinoma HepG2 cells through activation of caspase-3. Oncol Rep. 2007;18:181–187. [PubMed] [Google Scholar]

36. Shan Y, Sun C, Zhao X, Wu K, Cassidy A, Bao Y. Effect of sulforaphane on cell growth, G(0)/G(1) phase cell progression and apoptosis in human bladder cancer T24 cells. Int J Oncol. 2006;29:883–888.[PubMed] [Google Scholar]

37. Chaudhuri D, Orsulic S, Ashok BT. Antiproliferative activity of sulforaphane in Akt-overexpressing ovarian cancer cells. Mol Cancer Ther. 2007;6:334–345. [PMC free article] [PubMed] [Google Scholar]

38. Pledgie-Tracy A, Sobolewski MD, Davidson NE. Sulforaphane induces cell type-specific apoptosis in human breast cancer cell lines. Mol Cancer Ther. 2007;6:1013–1021. [PubMed] [Google Scholar]

39. Yeh CT, Yen GC. Effect of sulforaphane on metallothionein expression‎ and induction of apoptosis in human hepatoma HepG2 cells. Carcinogenesis. 2005;26:2138–2148. [PubMed] [Google Scholar]

40. Vanhoefer U, Cao S, Minderman H, Tóth K, Scheper RJ, Slovak ML, Rustum YM. PAK-104P, a pyridine analogue, reverses paclitaxel and doxorubicin resistance in cell lines and nude mice bearing xenografts that overexpress the multidrug resistance protein. Clin Cancer Res. 1996;2:369–377. [PubMed] [Google Scholar]

41. Mi L, Xiao Z, Hood BL, Dakshanamurthy S, Wang X, Govind S, Conrads TP, Veenstra TD, Chung FL. Covalent binding to tubulin by isothiocyanates: a mechanism of cell growth arrest and apoptosis. J Biol Chem. 2008;283:22136–22146. [PMC free article] [PubMed] [Google Scholar]

42. Myzak MC, Hardin K, Wang R, Dashwood RH, Ho E. Sulforaphane inhibits histone deacetylase activity in BPH-1, LnCaP and PC-3 prostate epithelial cells. Carcinogenesis. 2006;27:811–819.[PMC free article] [PubMed] [Google Scholar]

43. Herman-Antosiewicz A, Xiao H, Lew KL, Singh SV. Induction of p21 protein protects against sulforaphane-induced mitotic arrest in LNCaP human prostate cancer cell line. Mol Cancer Ther. 2007;6:1673–1681. [PubMed] [Google Scholar]

44. Shen G, Xu C, Chen C, Hebbar V, Kong AN. p53-independent G1 cell cycle arrest of human colon carcinoma cells HT-29 by sulforaphane is associated with induction of p21CIP1 and inhibition of expression‎ of cyclin D1. Cancer Chemother Pharmacol. 2006;57:317–327. [PubMed] [Google Scholar]

45. Myzak MC, Karplus PA, Chung FL, Dashwood RH. A novel mechanism of chemoprotection by sulforaphane: inhibition of histone deacetylase. Cancer Res. 2004;64:5767–5774. [PubMed] [Google Scholar]

46. Jeong WS, Kim IW, Hu R, Kong AN. Modulation of AP-1 by natural chemopreventive compounds in human colon HT-29 cancer cell line. Pharm Res. 2004;21:649–660. [PubMed] [Google Scholar]

47. Jakubikova J, Sedlak J, Mithen R, Bao Y. Role of PI3K/Akt and MEK/ERK signaling pathways in sulforaphane- and erucin-induced phase II enzymes and MRP2 transcription, G2/M arrest and cell death in Caco-2 cells. Biochem Pharmacol. 2005;69:1543–1552. [PubMed] [Google Scholar]

48. Xu C, Shen G, Chen C, Gelinas C, Kong AN. Suppression of NF-κB and NF-κB-regulated gene expression‎ by sulforaphane and PEITC through IκBα, IKK pathway in human prostate cancer PC-3 cells. Oncogene. 2005;24:4486–4495. [PubMed] [Google Scholar]