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Activation of several concurrent proapoptic pathways by sulforaphane in human colon cancer cells SW620
Food and Chemical Toxicology 47 (2009) 2366–2373
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Activation of several concurrent proapoptic pathways by sulforaphane in human colon cancer cells SW620 E. Rudolf *, H. Andeˇlová, M. Cˇervinka Department of Medical Biology and Genetics, Charles University in Prague, Faculty of Medicine in Hradec Kralove, Simkova 870, Hradec Kralove 500 38, Czech Republic
a r t i c l e
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Article history: Received 4 March 2009 Accepted 22 June 2009
Keywords: Sulforaphane Apoptosis DNA-damage response Caspases Colon cancer, p53
a b s t r a c t Despite the reported cytotoxicity and apoptosis-inducing properties of sulforaphane (SF) in colon cancer cells, the details concerning individual mechanisms and signaling cascades underlying SF-mediated apoptosis remain unclear. To understand different aspects of SF-induced proapoptic signaling in advanced colon carcinoma, we investigated its mechanisms in metastatic SW620 cell line. Our results indicate that in SW620 cells SF acts to induce multivariate cascades including DNA-damage response pathway whose proapoptotic signaling is nevertheless reduced owing to the mutant status of p53 and caspase-2-JNK pathway which seems to complement and enhance p53-dependent signaling, however only in wild-type p53. Furthermore, both pathways require the active role of mitochondria and do not depend on generation of ROS, making SF an attractive chemopreventive agent whose antitumor properties should be further investigated in colon cancer. Ó 2009 Elsevier Ltd. All rights reserved.
1. Introduction Colon cancer represents one the most important causes of premature death in developed countries (Potter, 1999). Due to the fact that successful treatment modalities for this malignancy are still limited, significant attention is currently being paid to preventive programs which may interfere with the process of colon carcinogenesis at all its stages, thereby improving patients’ survival and enhancing possible impact of medical interventions. The chemopreventive strategy in the case of colon cancer is based on the consumption of various agents including phytochemicals which have been shown (1) to protect the normal cells and tissues of the gastrointestinal tract (2) to interfere with the activity and metabolism of the recognized carcinogenic and mutagenic factors and (3) to selectively exercise cytostatic, cytotoxic and proapoptotic activity towards premalignant and malignant cell populations (Mason, 2002). In particular the last mentioned property of chemopreventive compounds has attracted considerable interest among scientists as it is known that often colon cancer is diagnosed at advanced stages characterized by generalized spread of malignant cells throughout the body, making any medical intervention notoriously difficult (Sharma et al., 2001). Abbreviations: CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid; DCF, dichlorofluorescein; DFCH/DA, 20 -70 -dichlorodihydrofluorescein diacetate; DMEM, Dulbecco’s modified Eagle’s medium; DMSO, dimethylsulfoxide; DTT, dithiotreitol; FBS, fetal bovine serum; JNK, c-Jun N-terminal kinase; MAPK, mitogen activated protein kinases; NAC, N-acetylcysteine; NFjB, nuclear factor kappa-B; ROS, reactive oxygen species; SF, sulforaphane. * Corresponding author. E-mail address: [email protected] (E. Rudolf). 0278-6915/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.fct.2009.06.034
Sulforaphane (SF) is an isothiocyanate derived from cruciferous vegetables such as broccoli, cauliflower, cabbage and kale (Zhang et al., 1992). Since its identification as a potential chemopreventive chemical, SF has been widely investigated and numerous studies demonstrated versatile and multiple natures of its anticancer effects. Thus depending on the type of study model, SF was reported to target cancer initiation and cancer progression both in vitro and in vivo while showing antiproliferative and cytotoxic potential in tumor cells (Juge et al., 2007). The cytotoxicity of SF in colon cancer models is linked with the production of reactive oxygen species (ROS) and selective targeting of key signaling molecules and events responsible for transcriptional regulation and deregulation of differentiation, cell cycle and cell death-apoptosis (Clarke et al., 2008). In addition, SF has been reported to increase global histone acetylation at the Bax and p21 promoters associated with cell cycle arrest (both G2/M and G1) and apoptosis (Jakubikova et al., 2005; Myzak et al., 2004; Singh et al., 2004). This activation of apoptosis appears to be p53-independent but proceeds via mitochondria-mediated cytochrome c release, subsequent activation of caspases and the specific PARP cleavage (Gamet-Payrastre et al., 2000; Pappa et al., 2006). Despite the reported apoptosis-inducing properties of SF in malignant colonocytes, the details concerning individual mechanisms and signaling cascades underlying SF-mediated apoptosis remain unclear. One reason is a wide range of effects induced by SF in tumor cells; i.e. on the one hand activation of proapoptic signaling via AP-1, mitogen activated protein kinases (MAPK), death receptors and mitochondria and on the other hand active suppression of prosurvival signals as exemplified by SF-mediated
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inhibition of the nuclear factor kappa-B (NFjB) activation (Jeong et al., 2004; Shen et al., 2006). To understand different aspects of SF-induced proapoptic signaling in advanced colon carcinoma, we employed metastatic SW620 cell line and treated it with the dose of SF and during the time periods which we recently reported to be associated with apoptosis (Andelova et al., 2007). Our results indicate that in SW620 cells SF acts to induce multivariate cascades including DNA-damage response pathway whose proapoptotic signaling is nevertheless reduced owing to the mutant status of p53 and caspase-2-JNK pathway which seems to complement and enhance p53-dependent signaling, however only in wild-type p53. Furthermore, both pathways require the active role of mitochondria and appear not to depend on generation of ROS. 2. Materials and methods 2.1. Cell line and treatment Human colon cancer cell line SW620 (ATCC No. CCL-227TM, San Diego, USA) was cultivated in Dulbecco’s modified Eagle’s medium (DMEM, Gibco, Prague, Czech Republic) supplemented with 10% fetal bovine serum (FBS) (Gibco, Prague, Czech Republic). Cells were maintained in 37 °C and 5% CO2 in an incubator. Passaging took place twice a week upon 90% cells’ confluence using 0.05% EDTA/trypsin. Cells were treated with 20 lM of SF or berberine (25 lM) for the indicated time points up to 48 h. The caspase inhibitors, cyclosporin A, Pifithrin and SP600125 were dissolved in DMSO as stock solutions. Stock solution of N-acetylcysteine (NAC) was prepared in serum free DMEM. The working concentrations of individual chemicals were achieved by diluting their stock solutions in treatment medium and were as follows: NAC (antioxidant, 1 mM – added to cells 24 h prior to SF exposure), cyclosporin A (cytochrome c release inhibitor, 5 lM – supplemented to cells 30 min before exposure to SF), Pifithrin (p53 inhibitor, 30 lM – added to cells 24 h prior to SF treatment), SP600125 (JNK inhibitor, 10 lM – supplemented to cells 30 min before exposure to SF), z-VAD-fmk (pancaspase inhibitor) and z-VDVAD-fmk (caspase-2 inhibitor) (both 10 lM – administered simultaneously with SF), z-DEVD-fmk (caspase-3 inhibitor, 5 lM – added to cells simultaneously with SF), z-LEHD-fmk (caspase-9 inhibitor) and z-IETD-fmk (caspase-8 inhibitor) (both 10 lM – added to cells simultaneously with SF) and Ac-DEVD-CHO (caspase-7 inhibitor, 15 lM added to cells 1 h prior to SF treatment).
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resuspended in DMEM (pH adjusted to 7.2) and 5 lmol DFCH/DA was added (5 min, 37 °C). Changes in the fluorescence intensity (485 nm excitation; 538 nm emission) were measured by Shimadzu UV–Visible spectrophotometer UV – 1601 (SHIMADZU DEUTSCHLAND GmbH, Germany). The data were expressed as a percentage of fluorescence intensity increase per 106 cells. 2.5. ELISA assay of, ERK, p38 and JNK activities Treated and control cells were harvested and collected by centrifugation. Whole cell extracts were prepared by lysis in cell extraction buffer (10 mM Tris, 100 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1 mM NaF, 20 mM Na4P2O7, 2 mM Na3VO4, 1% Triton X-100, 10% glycerol, 0.1% SDS and 0.5% deoxycholate with 1 mM protease inhibitor cocktail) for 30 min, on ice, with vortexing at 10 min intervals. ERK, p38 and c-Jun N-terminal kinase (JNK) activities were measured using Elisa kits (Sigma–Aldrich, St. Luis, MO, USA and Calbiochem, San Diego, CA, USA) specific for total ERK and phospho-ERK (pTpY185/187), total and phospho-p38 (pTpY180/182) and total and phospho-JNK (pTpY183/183) according to manufacturer’s instructions. The assays were performed in 96-well plate format and samples were read against standard curves obtained from ERK and phospho-ERK, p38 and phospho-p38, JNK and phospho-JNK standards. Results were normalized to micrograms of protein in the cell extract and expressed as the ratio of phospho to total kinase in the same sample. The typical ratios in the control samples were as follows: ERK – 0.2, p38 – 0.15 and JNK – 0.24. The results of SF treatment were expressed as percentage of control values. 2.6. Cell lysis and Western blot analysis Total cell extracts were prepared by lysis in an ice-cold lysis buffer (137 mM NaCl, 10% glycerol, 1% n-octyl-b-D-glucopyranoside, 50 mM NaF, 20 mM Tris, 1 mM sodium orthovanadate, Complete TMMini). Samples were loaded onto a 12% SDS/polyacrylamide gel. Each lysate contained equal amount of protein (30 lg) as determined by BCA assay. After electrophoresis, proteins were transferred to a PVDF membrane (100 V, 60 min) and incubated at 25 °C for 1.5 h with a solution containing 5% nonfat dry milk, 10 mmol Tris–HCl (pH 8.0), 150 mmol sodium chloride, and 0.1% Tween 20 (TBST). Membranes were incubated with primary antibodies (anti-ATM, 1:1000; anti-p-ATM, 1: 500; anti-Chk2 1:600; anti p-Chk2, 1:1000; anti-p-p53, both 1:250; anti-p53, 1:1000 and anti-b-actin, 1:750) at 4 °C overnight followed by five 6 min washes in TBST. Next, the blots were incubated with secondary peroxidase-conjugated antibodies (1:1000, 1 h, 25 °C), washed with TBST and the signal was developed with a chemiluminescence (ECL) detection kit (Boehringer Mannheim-Roche, Basel, Switzerland). Relative quantifications of protein expression were measured using GelQuant Ver 2.7 software (DNR Bio-Imaging Systems, Jerusalem, Israel).
2.2. Chemicals 2.7. Mitochondrial transmembrane potential (Dwm) analysis Sulforaphane (SF) was obtained from Alexis biochemicals (Axxora Corporation, San Diego, USA). 20 -70 - dichlorodihydrofluorescein diacetate, JC-1, Annexin V-FITC, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid (CHAPS); dithiotreitol (DTT), dimethylsulfoxide (DMSO), propidium iodide, N-acetylcysteine (NAC), JNK-specific inhibitor SP600125 and b-actin were purchased from Sigma–Aldrich (Prague, Czech Republic). Primary antibodies against ATM, Chk2, Thr-68 phospho-Chk2, Ser-15 and Ser-46 phospho-p53 and p53 were purchased from Cell Signaling Technology, Inc. (Danvers, MA, USA). Secondary antibodies were from Alexis Corporation (Lausen, Switzerland). p53-specific inhibitor Pifithrin and primary antibody against Ser-1981 phospho-ATM was acquired from Calbiochem (EMD Biosciences, Inc., La Jolla, Ca, USA). Caspase inhibitors were from ICN Biomedicals Inc. (Irvine, USA). All other chemicals were of highest analytical grade. 2.3. Transient plasmid transfection Normal human p53 sequence obtained from blood of a healthy donor via RT– PCR was cloned into the mammalian expression vector pcDNA3.1/HygroÓ (Invitrogen, Prague, Czech Republic). SW620 cells were transiently transfected with pcDNA3.1/HygroÓ-p53 as follows: 3 106 cells were suspended in 1 ml DMEM medium (+10% FBS) mixed with 20 lg of plasmid in Lipofectamine plusTM reagent and left for 12 h. Transfected cells were placed immediately into prewarmed DMEM medium (+10% FBS) and incubated at 37 °C, 12 h prior to SF treatment. Control (mock-transfected) cells were transfected with the control vector pcDNA3.1/ HygroÓ/lacZ. The transfection efficiency was assessed by measurement of b-galactosidase activity in cells, and was typically 50–60% (as measured by flow cytometry 24 h post transfection). 2.4. Oxidative stress Generation of hydrogen peroxide and/or hydroxyl radical in treated and control SW620 cells was measured by intracellular conversion of 20 -70 -dichlorodihydrofluorescein diacetate (DFCH/DA) into a fluorescent product dichlorofluorescein (DCF). Briefly, cells were detached by a cell scraper and collected by centrifugation (50g, 5 min, 4 °C – JOUAN M21, Trigon, Prague, Czech Republic). Thereafter, the cells were
Cell cultures were seeded into cultivation flasks and allowed to grow overnight. After treatment with SF, cells were rinsed in PBS and stained with JC-1 dye for 15 min at 37 °C. Mitochondrial membrane potential was assessed by microfluorimetry analysis using TECAN SpectraFluor Plus (TECAN Austria GmbH, Grödig, Austria). Mitochondrial transmembrane potential changes were indicated as an increase in fluorescence intensity at 528 nm. 2.8. Cytochrome release assay FunctionElisa cytochrome c kit (Active Motif, Rixensart, Belgium) which is based on colorimetric detection of horseradish peroxidase-conjugated Streptavidin complex of cytochrome c and capture antibody was used to measure translocation of cytochrome c into cytoplasm of control and SF-treated cells. Briefly, SW620 cells were grown to 75% confluence and treated to SF during 48 h. Cells were harvested after 6, 12, 18, 24, 30, 36 and 48 h of induction and mitochondrial and cytosolic extracts were prepared using the Mitochondria Fractionation Kit (Active Motif, Rixensart, Belgium). The amount of cytochrome c in prepared lysates was spectrophotometrically measured at 450 nm using a scanning multiwell spectrophotometer Titertek Multiscan MCC/340 (ICN Biochemicals, Frankfurt, Germany). Data were expressed as increase in absorbance at 450 nm/lg of lysate/well. TM
2.9. Measurement of caspases activity SF-treated and control cultures at 12, 24, 36 and 48 h were harvested by centrifugation (600g, 5 min, JOUAN MR 22, Trigon, Prague, Czech Republic) and lysed on ice for 20 min in a lysis buffer containing 50 mM HEPES, 5 mM CHAPS and 5 mM DTT. The lysates were centrifuged at 14,000g, 10 min, 4 °C, and the supernatants were collected and stored at 80 °C. The enzyme activity was measured in a 96well microplate using a kinetic fluorometric method based on the hydrolysis of the fluorogenic caspase-specific substrate (DEVD-AFC for caspase-3, Ac-LEHD-AFC for caspase-9, IETD-AFC for caspase-8 VDVAD-AFC for caspase-2 and Ac-DEVDpNA for caspase-7, 37 °C, 1 h) by individual caspases. Specific inhibitors of caspase-9, -8, -2, -3 and -7 were used to confirm the specificity of the cleavage
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reaction. Fluorescence was recorded at 460/40 nm after excitation at 360/40 nm using TECAN SpectraFluor Plus (TECAN Austria GmbH, Grödig, Austria). Results are shown as fold increase in activity relative to untreated cells. 2.10. Detection of apoptosis by phosphatidyl serine translocation The extent of apoptosis in SF-treated and control cells was determined by Annexin V-FITC/propidium iodide assay as described before (Rudolf and Cervinka, 2006). 2.11. Statistics Statistical analysis was carried out with a statistical program GraphPad Prism 4 (GraphPad Software, Inc., San Diego, USA). We used one way-Anova test with Dunnett’s post test for multiple comparisons. Results were compared with control samples, and means were considered significant if P < 0.05.
3. Results 3.1. Sulforaphane-induced apoptosis in SW620 cells is mitochondriadependent and involves activation of several caspases Sulforaphane-induced time-dependent mitochondrial apoptosis in metastatic SW620 colon cancer cells as measured by phosphatidyl serine translocation. To verify the role of mitochondria in this process, mitochondrial activity was first examined. As shown in Fig. 1, SF-induced time-dependent depolarization of mitochondrial membrane potential, with first significant decrease observed at 24 h of treatment. Concomitant with the SF-induced loss of mitochondrial membrane potential, cytochrome c was released into cytoplasm of exposed cells as exemplified by enrichment of cytochrome c cytoplasmic pools and corresponding decrease in cytochrome c content in mitochondria. The involvement of permeability transition pore in this process was further demonstrated by a significant prevention of cytochrome c loss and abrogation of apoptosis upon employment of the specific inhibitor cyclosporin A.
In the next step, kinetics of activity of selected initiator as well as executioner caspases was examined. Results presented in Fig. 2A demonstrate time-dependent activation of caspase-9, caspase-3 and caspase-7 whose activities first became significantly increased at 24 and 30 h of exposure, respectively, and remained so during entire 48 h treatment. On the other hand, no caspase-8 activation was observed in SF-treated cells. This lack of caspase-8 activity was further verified by additional experiments where this activity was measured in the same cells treated with SF and berberine to rule out the possibility of false negative results. Fig. 2B demonstrates that unlike SF berberine significantly activated caspase-8 as early as at 12 h of exposure and this activity remained markedly elevated until the end of experiment. Furthermore, an increased activity of caspase-2 following the treatment with SF was noted too albeit with a different time-profile; e.g. the first significant activation was seen at 12 h of exposure, with maximum activity being detected at 24 h of treatment whereas in case of caspase-9, -3 and -7 the peak activity was reached at 48 h of exposure. 3.2. Sulforaphane activates DNA-damage pathway with central role of p53 To understand the role of the specific SF-targets which are responsible for activation of apoptosis in SW620 cells and with respect to available literature data, DNA-damage signaling was investigated using immunoblotting analysis. Fig. 3 clearly indicates the specific activation of ATM kinase and its substrate Chk2 kinase at 24 h of treatment with SF. On the other hand, no changes in expression and specific activity of p53 were detected. To verify whether the observed lack of response was due to the mutant nature of p53, transfection of SW620 cells with wild-type p53 was carried out. Fig. 4 shows a significantly increased expression of p53 in transfected cells along with its specific phosphorylation at Ser-15 but not at Ser-46 following the treatment with SF. Furthermore,
Fig. 1. Apoptosis induced by sulforaphane (SF) in SW620 colon cancer cells. (A) Kinetics of SF-induced apoptosis and the effect of cyclosporine A during 48 h of treatment as measured by phosphatidylserine externalization. (B) Loss of mitochondrial membrane potential (Dwm) in cells exposed to SF during 48 h of treatment and the effect of cyclosporine A. (C) Translocation of cytochrome c into cytosol and (D) Decreasing concentration of cytochrome c in mitochondrial fraction of SF-exposed cells during 48 h of treatment with attenuating effect of cyclosporine A. Cells were exposed to SF (20 lM) with prior pretreatment with the above-mentioned cytochrome c release inhibitor and individual endpoints were analyzed as described in Section 2. Results represent means ± SD of at least three experiments *P < 0.05 compared to untreated control cells and #P < 0.05 compared to cells exposed to SF with one way-Anova test and Dunnett’s post test for multiple comparisons.
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Fig. 2. The effect of (A) sulforaphane treatment (20 lM) on caspase-2, -9, -8, -3 and -7 activities and (B) berberine (25 lM) treatment on caspase-8 activity in SW620 cells during 48 h of cultivation. Cells were lysed and caspase activities were determined in a 96-well microplate using a kinetic fluorometric method as described Section 2. Results represent means ± SD of five experiments. Activities of caspases-9 and -3 were significantly increased at 24–48 h treatment interval; caspase-2 activity first became markedly increased at 12 h of exposure and caspase7 activity significantly increased only at 30 h of exposure. Berberine-induced activation of caspase-8 was significant from 12 h of exposure till the end.
in thus manipulated cells the rate of apoptosis observed after treatment with SF was significantly elevated and could be abrogated with p53-specific inhibitor Pifithrin. 3.3. Sulforaphane activates JNK via caspase-2 in the absence of oxidative stress Since SF is known to initiate apoptosis of tumor cells via oxidative stress and activation of the specific proapoptotic cascades including MAPK, their involvement was studied in the present model during 48 h of treatment. It was found that exposure of SW620 cells to SF resulted in a time-dependent activation of JNK although its increased activity was detectable between 15 and 28 h only. Conversely, no changes in ERK and p38 activities were recorded throughout all treatment intervals (Fig. 5). Analysis of ROS levels in SF-exposed cells further showed that SF at the employed concentration did not induce oxidative stress in colon cancer cells and to this end pretreatment of these cells with antioxidant NAC had no influence on JNK activity. On the other hand, unlike inhibition of other caspases the use of pharmacological inhibitor of caspase-2 significantly decreased JNK activity, thereby hinting at their functional connection (Fig. 5). 3.4. p53-dependent and caspase-2-JNK pathways represent two complementing cascades in sulforaphane-induced apoptosis of wildtype SW620 cells To determine the relative involvement of p53-dependent and caspase-2-JNK pathways in SF-mediated apoptosis of SW620 colon
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Fig. 3. Activation of DNA-damage signaling in 20 lM sulforaphane exposed SW620 cells as determined by Western blotting analysis described in Section 2. Relative quantifications of protein expression were measured using GelQuant Ver 2.7 software (DNR Bio-Imaging Systems, Jerusalem, Israel). Results represent means ± SD of at least three experiments *P < 0.05 compared to untreated control cells with one way-Anova test and Dunnett’s post test for multiple comparisons.
cancer cells, we compared the effect of a selected antioxidant, p53specific inhibitor, caspase-2 and pan caspase inhibitors on apoptosis of these cells which were either non-transfected with wild-type p53 or transfected. As is shown in Fig. 6 addition of antioxidant NAC had no effect on observed apoptosis induced by SF irrespective of p53 status of the cells. Similarly, pharmacological inhibition of caspases in SF-treated cells was equally efficient in abrogation of apoptosis. Inhibition of caspase-2 in SW620 cells with mutant p53 significantly reduced SF-mediated apoptosis and this reduction was markedly higher than that observed after p53-specific inhibition. Conversely, both caspase-2 and p53 inhibitors proved to be of equal efficiency in SW620 cells with wild-type p53. Concomitant inhibition of p53 and caspase-2 in SF-treated wild-type p53 SW620 cells markedly reduced the observed apoptosis rate while in mutant p53 SW620 cells this reduction was no different from inhibition of either p53 or caspase-2 alone. 4. Discussion Cytotoxicity of SF in colon cancer cells is associated with decreased proliferation, cell cycle arrest and apoptosis (Clarke et al., 2008). Initial studies on SF-induced apoptosis carried out on HCT-116 and HT-29 cells proved that this process is characterized by mitochondria-mediated-activation of caspase-9, and -7 and PARP cleavage with active involvement of Bax, Bak and Bcl-XL proteins. Furthermore, since apoptosis after treatment with SF was observed in HCT-116 p53 knockout cell line too it was suggested that
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Fig. 4. Expression and activation of p53 in 20 lM sulforaphane (SF)-treated SW620 cells during 24 h. (A) Expression levels of protein p53 in non-transfected cells with mutant p53 allele, mock-transfected cells and cells transfected with wild-type p53. (B) Phosphorylation at Ser-15 of p53 protein in non-transfected cells with mutant p53 allele, mock-transfected cells and cells transfected with wild-type p53. (C) Phosphorylation at Ser-46 of p53 protein in non-transfected cells with mutant p53 allele, mocktransfected cells and cells transfected with wild-type p53. Cells were lysed and protein expression and activation were determined by immunoblotting with subsequent band analysis as described in Section 2. (D) Changes in the rate of apoptosis reflecting p53 status in SW620 cells exposed to SF during 48 h and the effect of p53 inhibition by the specific p53 inhibitor Pifithrin as determined by phosphatidylserine externalization described in Section 2. Results represent means ± SD of at least three experiments. *P < 0.05 compared to non-transfected cells and #P < 0.05 compared to p53-transfected cells with one way-Anova test and Dunnett’s post test for multiple comparisons.
it proceeds in a p53-independent manner (Gamet-Payrastre et al., 2000; Pappa et al., 2006). Despite these reports confirming proapoptotic potential of SF in malignant colonocytes, the details concerning the early effects of SF on cell stress signaling prior to mitochondria remain unclear. This is especially true in case of colonic tumor cells representing advanced stages of carcinogenesis. In our previous report we showed cytotoxic, antiproliferative and proapoptic properties of SF in metastatic SW620 colon cancer cells. Analyses of SF-induced apoptosis revealed distinct morphological changes in dying cells such as membrane blebbing and chromatin condensation as well as biochemical and molecular events including DNA-damage sensing and increased activity of caspase-3 (Andelova et al., 2007). To characterize further SF-induced apoptosis in SW620 cells and to learn more details about possible mechanisms underlying it we first focused on mitochondria which have been shown to play a central role in SF-mediated cell death (Juge et al., 2007). Our results clearly demonstrate SF-dependent loss of mitochondrial membrane potential with concomitant translocation of cytochrome c into the cytoplasm and confirm similar observations reported in other tumor cell lines (Karmakar et al., 2006; Tang and Zhang, 2005). Mitochondria contribute to apoptosis by being an organelle where several signaling cascades including those mediated by initiator caspases converge, which in turn leads to activation of executioner caspases via released mitochondrial proteins such as cytochrome c. Thus in the next step we measured the activity of selected members of caspase family following the treatment with SF. We found that several caspases (caspase-3, -7) are activated in response to SF as was described before by us
and others (Andelova et al., 2007; Pappa et al., 2006). In addition, our data argue for the increased activity of caspase-9 and -2 in the treated cells but in the absence of caspase-8 involvement. These findings both agree with the acknowledged activation of intrinsic apoptotic pathway by SF while provide evidence of SF-mediated-activation of DNA-damage signaling. The role of DNA-damage response in SF-induced apoptosis of colon cancer cells remains unclear. SF has been reported to induce DNA-damage but the involvement of p53 in this process and downstream apoptotic signaling seems to be controversial (Fimognari et al., 2002, 2005; Gamet-Payrastre et al., 2000). Here we provide the evidence of SF-dependent activation of ATM kinase and its target Chk2 kinase which nevertheless failed to activate p53. Also, no changes in p53 levels were detected. While being in line with previous reports, this observation proves the intactness of initial DNA-damage signaling which seems to be stalled at the level of p53. To verify whether a mutant status of p53 in the studied SW620 cells is responsible for this stalling, we transfected cells with dominant wild-type p53 sequence and in these cells measured p53 response after treatment with SF. As shown in Fig. 4, in thus manipulated cells, SF-induced an increase in p53 levels with concomitant phosphorylation of p53 protein at Ser-15. In addition, these changes led to massively increased apoptosis which could be significantly inhibited by p53-specific inhibitor, thereby underlying the importance of wild-type p53 in downstream signaling of SF-induced apoptosis. As mentioned before, we detected time-dependent activation of caspase-2 in SW620 cells treated with SF. Caspase-2 is the only
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Fig. 5. Effect of 20 lM sulforaphane (SF) on intracellular signaling in SW620 cells during 48 h of treatment. (A) Activation of selected mitogen activated protein kinases (MAPKs – ERK1/2, JNK and p38) during 48 h of treatment. (B) Generation of oxidative stress in treated cells during 48 h of exposure. (C) Effect of antioxidant N-acetylcystein (NAC), caspase-2 inhibitor z-VDVAD-fmk, caspase-3 inhibitor z-DEVD-fmk, caspase-9 inhibitor z-LEHD-fmk, caspase-8 inhibitor z-IETD-fmk and caspase-7 inhibitor AcDEVD-CHO on SF-induced JNK activation. Cells were incubated and treated as described in Section 2. Kinase activities were measured using ELISA kits in 96-well plates (Section 2). Results were normalized to micrograms of protein in the cell extract and expressed as the ratio of phospho-kinase to total kinase in the same sample. Oxidative stress was determined spectrophotometrically by intracellular conversion of 20 -70 -dichlorodihydrofluorescein diacetate (DFCH/DA) into a fluorescent product dichlorofluorescein (DCF). The results were expressed as a percentage of fluorescence intensity increase per 106 cells. Results represent means ± SD of at least three experiments. *P < 0.05 compared to control cultures and #P < 0.05 compared to cultures pretreated with NAC with one way-Anova test and Dunnett’s post test for multiple comparisons.
caspase present constitutively in the nucleus and provides an important link between DNA-damage and the mitochondrial apoptotic pathway (Zhivotovsky et al., 1999). Previous studies suggested JNK as one of the possible downstream targets of this caspase (Panaretakis et al., 2005). Since SF has been shown to activate all three MAPKs (Cho et al., 2005; Hu et al., 2003; Yeh and Yen, 2005), we wanted to determine their involvement in SF-dependent
signaling in the present model. Our analyses provided evidence of elevated JNK activity but not p38 and ERK activities which is in contrast with observations obtained with HT-29 cells and may reflect degree of differences between both cell lines at different stages of colon carcinogenesis. The adherence of time-frame of JNK activation and caspase-2 activity in SF-treated SW620 cells supports the notion of their sequential linkage. Therefore the
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vent phosporylation of JNK and subsequent apoptosis indicate that ROS did not play any role in SF-induced proapoptotic signaling. It is conceivable; however, that at higher SF concentrations oxidative stress might play a role in apoptosis of tumor cells as it has been observed that this process is strictly concentration-dependent (Clarke et al., 2008). Still, the almost simultaneous activation of two DNA-damage depending cascades by SF in SW620 cells raised the question of whether there is any functional linkage between these two processes. The employed inhibitors of all caspases, caspase-2 and p53 in cells with mutant p53 and dominant wild-type p53 showed that in the presence of mutant p53 both p53-dependent and caspase-2 dependent pathways contribute to apoptosis although caspase-2 pathway seems to be of higher importance and efficiency, acting as if to substitute for defective p53 pathway. Conversely, wild-type p53 cells show equal activity and efficiency of both cascades which seem likely to mutually enhance and complement each other. To our knowledge this is the first report on the importance and relationship of p53-depnding signaling and caspase-2 pathway in SF-induced apoptosis, in particular in colon cancer cells. In summary, the present work informs about apoptotic activity of SF in metastatic colon cancer cell line SW620. SF-induced apoptosis is not associated with oxidative stress but involves DNA-damage signaling whose efficiency is likely to depend on p53 status and caspase-2 activation with important verified linkage between caspase-2 and JNK. The activity of both pathways may putatively serve to amplify the critical proapoptotic signals interacting with mitochondria which in turn activate effector caspases. The ability of SF to initiate multiple proapoptotic cascades even in advanced stages of colon cancer harboring mutant p53 render this chemical an attractive cytotoxic and chemopreventive agent whose further evaluation is therefore warranted. Conflict of interest statement Fig. 6. Effects of inhibition of oxidative stress (N-acetylcysteine, NAC), p53 signaling (Pifithrin), activation of caspases (pan caspase inhibitor z-VAD-fmk) and activation of caspase-2 (z-VDVAD-fmk) on 20 lM sulforaphane (SF)-induced apoptosis in SW620 cells during 48 h of treatment. (A) Non-transfected SW620 cells with mutant p53. (B) SW620 cells transfected with wild-type p53. Cells were preincubated or coincubated with the specified inhibitors, exposed to SF and apoptosis was determined as described in Section 2. Results represent means ± SD of at least three experiments. *P < 0.05 compared to cells treated with SF and #P < 0.05 compared to cultures pretreated with p53-inhibiting Pifithrin with one way-Anova test and Dunnett’s post test for multiple comparisons. (C) Comparison of effects of p53 inhibition, caspase-2 inhibition and simultaneous inhibition of both p53 and caspase-2 in SW620 cells with differing p53 status. In this independent experiment, cells were preincubated or coincubated with the specified inhibitors, exposed to SF and apoptosis was determined as described in Section 2. Results represent means ± SD of at least three experiments. *P < 0.05 compared to cells treated with SF and #P < 0.05 compared to cultures where either p53 or caspase-2 were inhibited with one way-Anova test and with one way-Anova test and Dunnett’s post test for multiple comparisons.
relation between caspase-2 and JNK was further evaluated by inhibiting caspase-2 activity. The fact that JNK phosphorylation was severely attenuated in cells treated the specific caspase-2 inhibitor supports a direct role of this caspase in activation of JNK while providing possible connection between caspase-2 and mitochondria via JNK and its downstream target Bak as was suggested before (Pappa et al., 2006). DNA lesions induced by SF in SW620 cells result in activation of DNA-damage signaling pathway as well as increase caspase-2 activity. Since SF is known to induce elevated levels of ROS which might be responsible for DNA damaging and subsequent phenomena we measured ROS generation in our study model (Shen et al., 2006). The observed lack of any significant oxidative stress at all treatment intervals along with failure of antioxidant NAC to pre-
The authors declare that there are no conflicts of interest. Acknowledgment This work was supported by Czech Republic Ministry of Education Research Project MSM 0021620820. References Andelova, H., Rudolf, E., Cervinka, M., 2007. In vitro antiproliferative effects of sulforaphane on human colon cancer cell line SW620. Acta Med. (Hradec Kralove) 50, 171–176. Cho, S.D., Li, G., Hu, H., Jiang, C., Kang, K.S., Lee, Y.S., Kim, S.H., Lu, J., 2005. 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 52, 213–224. Clarke, J.D., Dashwood, R.H., Ho, E., 2008. Multi-targeted prevention of cancer by sulforaphane. Cancer Lett. 269, 291–304. Fimognari, C., Nusse, M., Berti, F., Iori, R., Cantelli-Forti, G., Hrelia, P., 2002. Cyclin D3 and p53 mediate sulforaphane-induced cell cycle delay and apoptosis in nontransformed human T lymphocytes. Cell. Mol. Life Sci. 59, 2004–2012. Fimognari, C., Sangiorgi, L., Capponcelli, S., Nusse, M., Fontanesi, S., Berti, F., Soddu, S., Cantelli-Forti, G., Hrelia, P., 2005. A mutated p53 status did not prevent the induction of apoptosis by sulforaphane, a promising anti-cancer drug. Invest. New Drugs 23, 195–203. Gamet-Payrastre, L., Li, P., Lumeau, S., Cassar, G., Dupont, M.A., Chevolleau, S., Gasc, N., Tulliez, J., Terce, F., 2000. Sulforaphane, a naturally occurring isothiocyanate, induces cell cycle arrest and apoptosis in HT29 human colon cancer cells. Cancer Res. 60, 1426–1433. Hu, R., Kim, B.R., Chen, C., Hebbar, V., Kong, A.N., 2003. The roles of JNK and apoptotic signaling pathways in PEITC-mediated responses in human HT-29 colon adenocarcinoma cells. Carcinogenesis 24, 1361–1367. Jakubikova, J., Sedlak, J., Mithen, R., Bao, Y., 2005. 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. 69, 1543–1552.
E. Rudolf et al. / Food and Chemical Toxicology 47 (2009) 2366–2373 Jeong, W.S., Kim, I.W., Hu, R., Kong, A.N., 2004. Modulatory properties of various natural chemopreventive agents on the activation of NF-kappaB signaling pathway. Pharm. Res. 21, 661–670. Juge, N., Mithen, R.F., Traka, M., 2007. Molecular basis for chemoprevention by sulforaphane: a comprehensive review. Cell. Mol. Life Sci. 64, 1105–1127. Karmakar, S., Weinberg, M.S., Banik, N.L., Patel, S.J., Ray, S.K., 2006. Activation of multiple molecular mechanisms for apoptosis in human malignant glioblastoma T98G and U87MG cells treated with sulforaphane. Neuroscience 141, 1265–1280. Mason, J.B., 2002. Nutritional chemoprevention of colon cancer. Semin. Gastrointest. Dis. 13, 143–153. Myzak, M.C., Karplus, P.A., Chung, F.L., Dashwood, R.H., 2004. A novel mechanism of chemoprotection by sulforaphane: inhibition of histone deacetylase. Cancer Res. 64, 5767–5774. Panaretakis, T., Laane, E., Pokrovskaja, K., Bjorklund, A.C., Moustakas, A., Zhivotovsky, B., Heyman, M., Shoshan, M.C., Grander, D., 2005. Doxorubicin requires the sequential activation of caspase-2, protein kinase Cdelta, and c-Jun NH2-terminal kinase to induce apoptosis. Mol. Biol. Cell 16, 3821–3831. Pappa, G., Lichtenberg, M., Iori, R., Barillari, J., Bartsch, H., Gerhauser, C., 2006. Comparison of growth inhibition profiles and mechanisms of apoptosis induction in human colon cancer cell lines by isothiocyanates and indoles from Brassicaceae. Mutat. Res. 599, 76–87. Potter, J.D., 1999. Colorectal cancer: molecules and populations. J. Natl. Cancer Inst. 91, 916–932.
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Rudolf, E., Cervinka, M., 2006. Cytoskeletal changes in non-apoptotic cell death. Acta Med. (Hradec Kralove) 49, 123–128. Sharma, R.A., Manson, M.M., Gescher, A., Steward, W.P., 2001. Colorectal cancer chemoprevention: biochemical targets and clinical development of promising agents. Eur. J. Cancer 37, 12–22. Shen, G., Xu, C., Chen, C., Hebbar, V., Kong, A.N., 2006. 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. 57, 317–327. Singh, S.V., Herman-Antosiewicz, A., Singh, A.V., Lew, K.L., Srivastava, S.K., Kamath, R., Brown, K.D., Zhang, L., Baskaran, R., 2004. Sulforaphane-induced G2/M phase cell cycle arrest involves checkpoint kinase 2-mediated phosphorylation of cell division cycle 25C. J. Biol. Chem. 279, 25813–25822. Tang, L., Zhang, Y., 2005. Mitochondria are the primary target in isothiocyanateinduced apoptosis in human bladder cancer cells. Mol. Cancer Ther. 4, 1250– 1259. Yeh, C.T., Yen, G.C., 2005. Effect of sulforaphane on metallothionein expression and induction of apoptosis in human hepatoma HepG2 cells. Carcinogenesis 26, 2138–2148. Zhang, Y., Talalay, P., Cho, C.G., Posner, G.H., 1992. A major inducer of anticarcinogenic protective enzymes from broccoli: isolation and elucidation of structure. Proc. Natl. Acad. Sci. USA 89, 2399–2403. Zhivotovsky, B., Samali, A., Gahm, A., Orrenius, S., 1999. Caspases: their intracellular localization and translocation during apoptosis. Cell Death Differ. 6, 644–651.
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Activation of several concurrent proapoptic pathways by sulforaphane in human colon cancer cells SW620 E. Rudolf *, H. Andeˇlová, M. Cˇervinka Department of Medical Biology and Genetics, Charles University in Prague, Faculty of Medicine in Hradec Kralove, Simkova 870, Hradec Kralove 500 38, Czech Republic
a r t i c l e
i n f o
Article history: Received 4 March 2009 Accepted 22 June 2009
Keywords: Sulforaphane Apoptosis DNA-damage response Caspases Colon cancer, p53
a b s t r a c t Despite the reported cytotoxicity and apoptosis-inducing properties of sulforaphane (SF) in colon cancer cells, the details concerning individual mechanisms and signaling cascades underlying SF-mediated apoptosis remain unclear. To understand different aspects of SF-induced proapoptic signaling in advanced colon carcinoma, we investigated its mechanisms in metastatic SW620 cell line. Our results indicate that in SW620 cells SF acts to induce multivariate cascades including DNA-damage response pathway whose proapoptotic signaling is nevertheless reduced owing to the mutant status of p53 and caspase-2-JNK pathway which seems to complement and enhance p53-dependent signaling, however only in wild-type p53. Furthermore, both pathways require the active role of mitochondria and do not depend on generation of ROS, making SF an attractive chemopreventive agent whose antitumor properties should be further investigated in colon cancer. Ó 2009 Elsevier Ltd. All rights reserved.
1. Introduction Colon cancer represents one the most important causes of premature death in developed countries (Potter, 1999). Due to the fact that successful treatment modalities for this malignancy are still limited, significant attention is currently being paid to preventive programs which may interfere with the process of colon carcinogenesis at all its stages, thereby improving patients’ survival and enhancing possible impact of medical interventions. The chemopreventive strategy in the case of colon cancer is based on the consumption of various agents including phytochemicals which have been shown (1) to protect the normal cells and tissues of the gastrointestinal tract (2) to interfere with the activity and metabolism of the recognized carcinogenic and mutagenic factors and (3) to selectively exercise cytostatic, cytotoxic and proapoptotic activity towards premalignant and malignant cell populations (Mason, 2002). In particular the last mentioned property of chemopreventive compounds has attracted considerable interest among scientists as it is known that often colon cancer is diagnosed at advanced stages characterized by generalized spread of malignant cells throughout the body, making any medical intervention notoriously difficult (Sharma et al., 2001). Abbreviations: CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid; DCF, dichlorofluorescein; DFCH/DA, 20 -70 -dichlorodihydrofluorescein diacetate; DMEM, Dulbecco’s modified Eagle’s medium; DMSO, dimethylsulfoxide; DTT, dithiotreitol; FBS, fetal bovine serum; JNK, c-Jun N-terminal kinase; MAPK, mitogen activated protein kinases; NAC, N-acetylcysteine; NFjB, nuclear factor kappa-B; ROS, reactive oxygen species; SF, sulforaphane. * Corresponding author. E-mail address: [email protected] (E. Rudolf). 0278-6915/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.fct.2009.06.034
Sulforaphane (SF) is an isothiocyanate derived from cruciferous vegetables such as broccoli, cauliflower, cabbage and kale (Zhang et al., 1992). Since its identification as a potential chemopreventive chemical, SF has been widely investigated and numerous studies demonstrated versatile and multiple natures of its anticancer effects. Thus depending on the type of study model, SF was reported to target cancer initiation and cancer progression both in vitro and in vivo while showing antiproliferative and cytotoxic potential in tumor cells (Juge et al., 2007). The cytotoxicity of SF in colon cancer models is linked with the production of reactive oxygen species (ROS) and selective targeting of key signaling molecules and events responsible for transcriptional regulation and deregulation of differentiation, cell cycle and cell death-apoptosis (Clarke et al., 2008). In addition, SF has been reported to increase global histone acetylation at the Bax and p21 promoters associated with cell cycle arrest (both G2/M and G1) and apoptosis (Jakubikova et al., 2005; Myzak et al., 2004; Singh et al., 2004). This activation of apoptosis appears to be p53-independent but proceeds via mitochondria-mediated cytochrome c release, subsequent activation of caspases and the specific PARP cleavage (Gamet-Payrastre et al., 2000; Pappa et al., 2006). Despite the reported apoptosis-inducing properties of SF in malignant colonocytes, the details concerning individual mechanisms and signaling cascades underlying SF-mediated apoptosis remain unclear. One reason is a wide range of effects induced by SF in tumor cells; i.e. on the one hand activation of proapoptic signaling via AP-1, mitogen activated protein kinases (MAPK), death receptors and mitochondria and on the other hand active suppression of prosurvival signals as exemplified by SF-mediated
E. Rudolf et al. / Food and Chemical Toxicology 47 (2009) 2366–2373
inhibition of the nuclear factor kappa-B (NFjB) activation (Jeong et al., 2004; Shen et al., 2006). To understand different aspects of SF-induced proapoptic signaling in advanced colon carcinoma, we employed metastatic SW620 cell line and treated it with the dose of SF and during the time periods which we recently reported to be associated with apoptosis (Andelova et al., 2007). Our results indicate that in SW620 cells SF acts to induce multivariate cascades including DNA-damage response pathway whose proapoptotic signaling is nevertheless reduced owing to the mutant status of p53 and caspase-2-JNK pathway which seems to complement and enhance p53-dependent signaling, however only in wild-type p53. Furthermore, both pathways require the active role of mitochondria and appear not to depend on generation of ROS. 2. Materials and methods 2.1. Cell line and treatment Human colon cancer cell line SW620 (ATCC No. CCL-227TM, San Diego, USA) was cultivated in Dulbecco’s modified Eagle’s medium (DMEM, Gibco, Prague, Czech Republic) supplemented with 10% fetal bovine serum (FBS) (Gibco, Prague, Czech Republic). Cells were maintained in 37 °C and 5% CO2 in an incubator. Passaging took place twice a week upon 90% cells’ confluence using 0.05% EDTA/trypsin. Cells were treated with 20 lM of SF or berberine (25 lM) for the indicated time points up to 48 h. The caspase inhibitors, cyclosporin A, Pifithrin and SP600125 were dissolved in DMSO as stock solutions. Stock solution of N-acetylcysteine (NAC) was prepared in serum free DMEM. The working concentrations of individual chemicals were achieved by diluting their stock solutions in treatment medium and were as follows: NAC (antioxidant, 1 mM – added to cells 24 h prior to SF exposure), cyclosporin A (cytochrome c release inhibitor, 5 lM – supplemented to cells 30 min before exposure to SF), Pifithrin (p53 inhibitor, 30 lM – added to cells 24 h prior to SF treatment), SP600125 (JNK inhibitor, 10 lM – supplemented to cells 30 min before exposure to SF), z-VAD-fmk (pancaspase inhibitor) and z-VDVAD-fmk (caspase-2 inhibitor) (both 10 lM – administered simultaneously with SF), z-DEVD-fmk (caspase-3 inhibitor, 5 lM – added to cells simultaneously with SF), z-LEHD-fmk (caspase-9 inhibitor) and z-IETD-fmk (caspase-8 inhibitor) (both 10 lM – added to cells simultaneously with SF) and Ac-DEVD-CHO (caspase-7 inhibitor, 15 lM added to cells 1 h prior to SF treatment).
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resuspended in DMEM (pH adjusted to 7.2) and 5 lmol DFCH/DA was added (5 min, 37 °C). Changes in the fluorescence intensity (485 nm excitation; 538 nm emission) were measured by Shimadzu UV–Visible spectrophotometer UV – 1601 (SHIMADZU DEUTSCHLAND GmbH, Germany). The data were expressed as a percentage of fluorescence intensity increase per 106 cells. 2.5. ELISA assay of, ERK, p38 and JNK activities Treated and control cells were harvested and collected by centrifugation. Whole cell extracts were prepared by lysis in cell extraction buffer (10 mM Tris, 100 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1 mM NaF, 20 mM Na4P2O7, 2 mM Na3VO4, 1% Triton X-100, 10% glycerol, 0.1% SDS and 0.5% deoxycholate with 1 mM protease inhibitor cocktail) for 30 min, on ice, with vortexing at 10 min intervals. ERK, p38 and c-Jun N-terminal kinase (JNK) activities were measured using Elisa kits (Sigma–Aldrich, St. Luis, MO, USA and Calbiochem, San Diego, CA, USA) specific for total ERK and phospho-ERK (pTpY185/187), total and phospho-p38 (pTpY180/182) and total and phospho-JNK (pTpY183/183) according to manufacturer’s instructions. The assays were performed in 96-well plate format and samples were read against standard curves obtained from ERK and phospho-ERK, p38 and phospho-p38, JNK and phospho-JNK standards. Results were normalized to micrograms of protein in the cell extract and expressed as the ratio of phospho to total kinase in the same sample. The typical ratios in the control samples were as follows: ERK – 0.2, p38 – 0.15 and JNK – 0.24. The results of SF treatment were expressed as percentage of control values. 2.6. Cell lysis and Western blot analysis Total cell extracts were prepared by lysis in an ice-cold lysis buffer (137 mM NaCl, 10% glycerol, 1% n-octyl-b-D-glucopyranoside, 50 mM NaF, 20 mM Tris, 1 mM sodium orthovanadate, Complete TMMini). Samples were loaded onto a 12% SDS/polyacrylamide gel. Each lysate contained equal amount of protein (30 lg) as determined by BCA assay. After electrophoresis, proteins were transferred to a PVDF membrane (100 V, 60 min) and incubated at 25 °C for 1.5 h with a solution containing 5% nonfat dry milk, 10 mmol Tris–HCl (pH 8.0), 150 mmol sodium chloride, and 0.1% Tween 20 (TBST). Membranes were incubated with primary antibodies (anti-ATM, 1:1000; anti-p-ATM, 1: 500; anti-Chk2 1:600; anti p-Chk2, 1:1000; anti-p-p53, both 1:250; anti-p53, 1:1000 and anti-b-actin, 1:750) at 4 °C overnight followed by five 6 min washes in TBST. Next, the blots were incubated with secondary peroxidase-conjugated antibodies (1:1000, 1 h, 25 °C), washed with TBST and the signal was developed with a chemiluminescence (ECL) detection kit (Boehringer Mannheim-Roche, Basel, Switzerland). Relative quantifications of protein expression were measured using GelQuant Ver 2.7 software (DNR Bio-Imaging Systems, Jerusalem, Israel).
2.2. Chemicals 2.7. Mitochondrial transmembrane potential (Dwm) analysis Sulforaphane (SF) was obtained from Alexis biochemicals (Axxora Corporation, San Diego, USA). 20 -70 - dichlorodihydrofluorescein diacetate, JC-1, Annexin V-FITC, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid (CHAPS); dithiotreitol (DTT), dimethylsulfoxide (DMSO), propidium iodide, N-acetylcysteine (NAC), JNK-specific inhibitor SP600125 and b-actin were purchased from Sigma–Aldrich (Prague, Czech Republic). Primary antibodies against ATM, Chk2, Thr-68 phospho-Chk2, Ser-15 and Ser-46 phospho-p53 and p53 were purchased from Cell Signaling Technology, Inc. (Danvers, MA, USA). Secondary antibodies were from Alexis Corporation (Lausen, Switzerland). p53-specific inhibitor Pifithrin and primary antibody against Ser-1981 phospho-ATM was acquired from Calbiochem (EMD Biosciences, Inc., La Jolla, Ca, USA). Caspase inhibitors were from ICN Biomedicals Inc. (Irvine, USA). All other chemicals were of highest analytical grade. 2.3. Transient plasmid transfection Normal human p53 sequence obtained from blood of a healthy donor via RT– PCR was cloned into the mammalian expression vector pcDNA3.1/HygroÓ (Invitrogen, Prague, Czech Republic). SW620 cells were transiently transfected with pcDNA3.1/HygroÓ-p53 as follows: 3 106 cells were suspended in 1 ml DMEM medium (+10% FBS) mixed with 20 lg of plasmid in Lipofectamine plusTM reagent and left for 12 h. Transfected cells were placed immediately into prewarmed DMEM medium (+10% FBS) and incubated at 37 °C, 12 h prior to SF treatment. Control (mock-transfected) cells were transfected with the control vector pcDNA3.1/ HygroÓ/lacZ. The transfection efficiency was assessed by measurement of b-galactosidase activity in cells, and was typically 50–60% (as measured by flow cytometry 24 h post transfection). 2.4. Oxidative stress Generation of hydrogen peroxide and/or hydroxyl radical in treated and control SW620 cells was measured by intracellular conversion of 20 -70 -dichlorodihydrofluorescein diacetate (DFCH/DA) into a fluorescent product dichlorofluorescein (DCF). Briefly, cells were detached by a cell scraper and collected by centrifugation (50g, 5 min, 4 °C – JOUAN M21, Trigon, Prague, Czech Republic). Thereafter, the cells were
Cell cultures were seeded into cultivation flasks and allowed to grow overnight. After treatment with SF, cells were rinsed in PBS and stained with JC-1 dye for 15 min at 37 °C. Mitochondrial membrane potential was assessed by microfluorimetry analysis using TECAN SpectraFluor Plus (TECAN Austria GmbH, Grödig, Austria). Mitochondrial transmembrane potential changes were indicated as an increase in fluorescence intensity at 528 nm. 2.8. Cytochrome release assay FunctionElisa cytochrome c kit (Active Motif, Rixensart, Belgium) which is based on colorimetric detection of horseradish peroxidase-conjugated Streptavidin complex of cytochrome c and capture antibody was used to measure translocation of cytochrome c into cytoplasm of control and SF-treated cells. Briefly, SW620 cells were grown to 75% confluence and treated to SF during 48 h. Cells were harvested after 6, 12, 18, 24, 30, 36 and 48 h of induction and mitochondrial and cytosolic extracts were prepared using the Mitochondria Fractionation Kit (Active Motif, Rixensart, Belgium). The amount of cytochrome c in prepared lysates was spectrophotometrically measured at 450 nm using a scanning multiwell spectrophotometer Titertek Multiscan MCC/340 (ICN Biochemicals, Frankfurt, Germany). Data were expressed as increase in absorbance at 450 nm/lg of lysate/well. TM
2.9. Measurement of caspases activity SF-treated and control cultures at 12, 24, 36 and 48 h were harvested by centrifugation (600g, 5 min, JOUAN MR 22, Trigon, Prague, Czech Republic) and lysed on ice for 20 min in a lysis buffer containing 50 mM HEPES, 5 mM CHAPS and 5 mM DTT. The lysates were centrifuged at 14,000g, 10 min, 4 °C, and the supernatants were collected and stored at 80 °C. The enzyme activity was measured in a 96well microplate using a kinetic fluorometric method based on the hydrolysis of the fluorogenic caspase-specific substrate (DEVD-AFC for caspase-3, Ac-LEHD-AFC for caspase-9, IETD-AFC for caspase-8 VDVAD-AFC for caspase-2 and Ac-DEVDpNA for caspase-7, 37 °C, 1 h) by individual caspases. Specific inhibitors of caspase-9, -8, -2, -3 and -7 were used to confirm the specificity of the cleavage
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reaction. Fluorescence was recorded at 460/40 nm after excitation at 360/40 nm using TECAN SpectraFluor Plus (TECAN Austria GmbH, Grödig, Austria). Results are shown as fold increase in activity relative to untreated cells. 2.10. Detection of apoptosis by phosphatidyl serine translocation The extent of apoptosis in SF-treated and control cells was determined by Annexin V-FITC/propidium iodide assay as described before (Rudolf and Cervinka, 2006). 2.11. Statistics Statistical analysis was carried out with a statistical program GraphPad Prism 4 (GraphPad Software, Inc., San Diego, USA). We used one way-Anova test with Dunnett’s post test for multiple comparisons. Results were compared with control samples, and means were considered significant if P < 0.05.
3. Results 3.1. Sulforaphane-induced apoptosis in SW620 cells is mitochondriadependent and involves activation of several caspases Sulforaphane-induced time-dependent mitochondrial apoptosis in metastatic SW620 colon cancer cells as measured by phosphatidyl serine translocation. To verify the role of mitochondria in this process, mitochondrial activity was first examined. As shown in Fig. 1, SF-induced time-dependent depolarization of mitochondrial membrane potential, with first significant decrease observed at 24 h of treatment. Concomitant with the SF-induced loss of mitochondrial membrane potential, cytochrome c was released into cytoplasm of exposed cells as exemplified by enrichment of cytochrome c cytoplasmic pools and corresponding decrease in cytochrome c content in mitochondria. The involvement of permeability transition pore in this process was further demonstrated by a significant prevention of cytochrome c loss and abrogation of apoptosis upon employment of the specific inhibitor cyclosporin A.
In the next step, kinetics of activity of selected initiator as well as executioner caspases was examined. Results presented in Fig. 2A demonstrate time-dependent activation of caspase-9, caspase-3 and caspase-7 whose activities first became significantly increased at 24 and 30 h of exposure, respectively, and remained so during entire 48 h treatment. On the other hand, no caspase-8 activation was observed in SF-treated cells. This lack of caspase-8 activity was further verified by additional experiments where this activity was measured in the same cells treated with SF and berberine to rule out the possibility of false negative results. Fig. 2B demonstrates that unlike SF berberine significantly activated caspase-8 as early as at 12 h of exposure and this activity remained markedly elevated until the end of experiment. Furthermore, an increased activity of caspase-2 following the treatment with SF was noted too albeit with a different time-profile; e.g. the first significant activation was seen at 12 h of exposure, with maximum activity being detected at 24 h of treatment whereas in case of caspase-9, -3 and -7 the peak activity was reached at 48 h of exposure. 3.2. Sulforaphane activates DNA-damage pathway with central role of p53 To understand the role of the specific SF-targets which are responsible for activation of apoptosis in SW620 cells and with respect to available literature data, DNA-damage signaling was investigated using immunoblotting analysis. Fig. 3 clearly indicates the specific activation of ATM kinase and its substrate Chk2 kinase at 24 h of treatment with SF. On the other hand, no changes in expression and specific activity of p53 were detected. To verify whether the observed lack of response was due to the mutant nature of p53, transfection of SW620 cells with wild-type p53 was carried out. Fig. 4 shows a significantly increased expression of p53 in transfected cells along with its specific phosphorylation at Ser-15 but not at Ser-46 following the treatment with SF. Furthermore,
Fig. 1. Apoptosis induced by sulforaphane (SF) in SW620 colon cancer cells. (A) Kinetics of SF-induced apoptosis and the effect of cyclosporine A during 48 h of treatment as measured by phosphatidylserine externalization. (B) Loss of mitochondrial membrane potential (Dwm) in cells exposed to SF during 48 h of treatment and the effect of cyclosporine A. (C) Translocation of cytochrome c into cytosol and (D) Decreasing concentration of cytochrome c in mitochondrial fraction of SF-exposed cells during 48 h of treatment with attenuating effect of cyclosporine A. Cells were exposed to SF (20 lM) with prior pretreatment with the above-mentioned cytochrome c release inhibitor and individual endpoints were analyzed as described in Section 2. Results represent means ± SD of at least three experiments *P < 0.05 compared to untreated control cells and #P < 0.05 compared to cells exposed to SF with one way-Anova test and Dunnett’s post test for multiple comparisons.
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Fig. 2. The effect of (A) sulforaphane treatment (20 lM) on caspase-2, -9, -8, -3 and -7 activities and (B) berberine (25 lM) treatment on caspase-8 activity in SW620 cells during 48 h of cultivation. Cells were lysed and caspase activities were determined in a 96-well microplate using a kinetic fluorometric method as described Section 2. Results represent means ± SD of five experiments. Activities of caspases-9 and -3 were significantly increased at 24–48 h treatment interval; caspase-2 activity first became markedly increased at 12 h of exposure and caspase7 activity significantly increased only at 30 h of exposure. Berberine-induced activation of caspase-8 was significant from 12 h of exposure till the end.
in thus manipulated cells the rate of apoptosis observed after treatment with SF was significantly elevated and could be abrogated with p53-specific inhibitor Pifithrin. 3.3. Sulforaphane activates JNK via caspase-2 in the absence of oxidative stress Since SF is known to initiate apoptosis of tumor cells via oxidative stress and activation of the specific proapoptotic cascades including MAPK, their involvement was studied in the present model during 48 h of treatment. It was found that exposure of SW620 cells to SF resulted in a time-dependent activation of JNK although its increased activity was detectable between 15 and 28 h only. Conversely, no changes in ERK and p38 activities were recorded throughout all treatment intervals (Fig. 5). Analysis of ROS levels in SF-exposed cells further showed that SF at the employed concentration did not induce oxidative stress in colon cancer cells and to this end pretreatment of these cells with antioxidant NAC had no influence on JNK activity. On the other hand, unlike inhibition of other caspases the use of pharmacological inhibitor of caspase-2 significantly decreased JNK activity, thereby hinting at their functional connection (Fig. 5). 3.4. p53-dependent and caspase-2-JNK pathways represent two complementing cascades in sulforaphane-induced apoptosis of wildtype SW620 cells To determine the relative involvement of p53-dependent and caspase-2-JNK pathways in SF-mediated apoptosis of SW620 colon
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Fig. 3. Activation of DNA-damage signaling in 20 lM sulforaphane exposed SW620 cells as determined by Western blotting analysis described in Section 2. Relative quantifications of protein expression were measured using GelQuant Ver 2.7 software (DNR Bio-Imaging Systems, Jerusalem, Israel). Results represent means ± SD of at least three experiments *P < 0.05 compared to untreated control cells with one way-Anova test and Dunnett’s post test for multiple comparisons.
cancer cells, we compared the effect of a selected antioxidant, p53specific inhibitor, caspase-2 and pan caspase inhibitors on apoptosis of these cells which were either non-transfected with wild-type p53 or transfected. As is shown in Fig. 6 addition of antioxidant NAC had no effect on observed apoptosis induced by SF irrespective of p53 status of the cells. Similarly, pharmacological inhibition of caspases in SF-treated cells was equally efficient in abrogation of apoptosis. Inhibition of caspase-2 in SW620 cells with mutant p53 significantly reduced SF-mediated apoptosis and this reduction was markedly higher than that observed after p53-specific inhibition. Conversely, both caspase-2 and p53 inhibitors proved to be of equal efficiency in SW620 cells with wild-type p53. Concomitant inhibition of p53 and caspase-2 in SF-treated wild-type p53 SW620 cells markedly reduced the observed apoptosis rate while in mutant p53 SW620 cells this reduction was no different from inhibition of either p53 or caspase-2 alone. 4. Discussion Cytotoxicity of SF in colon cancer cells is associated with decreased proliferation, cell cycle arrest and apoptosis (Clarke et al., 2008). Initial studies on SF-induced apoptosis carried out on HCT-116 and HT-29 cells proved that this process is characterized by mitochondria-mediated-activation of caspase-9, and -7 and PARP cleavage with active involvement of Bax, Bak and Bcl-XL proteins. Furthermore, since apoptosis after treatment with SF was observed in HCT-116 p53 knockout cell line too it was suggested that
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Fig. 4. Expression and activation of p53 in 20 lM sulforaphane (SF)-treated SW620 cells during 24 h. (A) Expression levels of protein p53 in non-transfected cells with mutant p53 allele, mock-transfected cells and cells transfected with wild-type p53. (B) Phosphorylation at Ser-15 of p53 protein in non-transfected cells with mutant p53 allele, mock-transfected cells and cells transfected with wild-type p53. (C) Phosphorylation at Ser-46 of p53 protein in non-transfected cells with mutant p53 allele, mocktransfected cells and cells transfected with wild-type p53. Cells were lysed and protein expression and activation were determined by immunoblotting with subsequent band analysis as described in Section 2. (D) Changes in the rate of apoptosis reflecting p53 status in SW620 cells exposed to SF during 48 h and the effect of p53 inhibition by the specific p53 inhibitor Pifithrin as determined by phosphatidylserine externalization described in Section 2. Results represent means ± SD of at least three experiments. *P < 0.05 compared to non-transfected cells and #P < 0.05 compared to p53-transfected cells with one way-Anova test and Dunnett’s post test for multiple comparisons.
it proceeds in a p53-independent manner (Gamet-Payrastre et al., 2000; Pappa et al., 2006). Despite these reports confirming proapoptotic potential of SF in malignant colonocytes, the details concerning the early effects of SF on cell stress signaling prior to mitochondria remain unclear. This is especially true in case of colonic tumor cells representing advanced stages of carcinogenesis. In our previous report we showed cytotoxic, antiproliferative and proapoptic properties of SF in metastatic SW620 colon cancer cells. Analyses of SF-induced apoptosis revealed distinct morphological changes in dying cells such as membrane blebbing and chromatin condensation as well as biochemical and molecular events including DNA-damage sensing and increased activity of caspase-3 (Andelova et al., 2007). To characterize further SF-induced apoptosis in SW620 cells and to learn more details about possible mechanisms underlying it we first focused on mitochondria which have been shown to play a central role in SF-mediated cell death (Juge et al., 2007). Our results clearly demonstrate SF-dependent loss of mitochondrial membrane potential with concomitant translocation of cytochrome c into the cytoplasm and confirm similar observations reported in other tumor cell lines (Karmakar et al., 2006; Tang and Zhang, 2005). Mitochondria contribute to apoptosis by being an organelle where several signaling cascades including those mediated by initiator caspases converge, which in turn leads to activation of executioner caspases via released mitochondrial proteins such as cytochrome c. Thus in the next step we measured the activity of selected members of caspase family following the treatment with SF. We found that several caspases (caspase-3, -7) are activated in response to SF as was described before by us
and others (Andelova et al., 2007; Pappa et al., 2006). In addition, our data argue for the increased activity of caspase-9 and -2 in the treated cells but in the absence of caspase-8 involvement. These findings both agree with the acknowledged activation of intrinsic apoptotic pathway by SF while provide evidence of SF-mediated-activation of DNA-damage signaling. The role of DNA-damage response in SF-induced apoptosis of colon cancer cells remains unclear. SF has been reported to induce DNA-damage but the involvement of p53 in this process and downstream apoptotic signaling seems to be controversial (Fimognari et al., 2002, 2005; Gamet-Payrastre et al., 2000). Here we provide the evidence of SF-dependent activation of ATM kinase and its target Chk2 kinase which nevertheless failed to activate p53. Also, no changes in p53 levels were detected. While being in line with previous reports, this observation proves the intactness of initial DNA-damage signaling which seems to be stalled at the level of p53. To verify whether a mutant status of p53 in the studied SW620 cells is responsible for this stalling, we transfected cells with dominant wild-type p53 sequence and in these cells measured p53 response after treatment with SF. As shown in Fig. 4, in thus manipulated cells, SF-induced an increase in p53 levels with concomitant phosphorylation of p53 protein at Ser-15. In addition, these changes led to massively increased apoptosis which could be significantly inhibited by p53-specific inhibitor, thereby underlying the importance of wild-type p53 in downstream signaling of SF-induced apoptosis. As mentioned before, we detected time-dependent activation of caspase-2 in SW620 cells treated with SF. Caspase-2 is the only
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Fig. 5. Effect of 20 lM sulforaphane (SF) on intracellular signaling in SW620 cells during 48 h of treatment. (A) Activation of selected mitogen activated protein kinases (MAPKs – ERK1/2, JNK and p38) during 48 h of treatment. (B) Generation of oxidative stress in treated cells during 48 h of exposure. (C) Effect of antioxidant N-acetylcystein (NAC), caspase-2 inhibitor z-VDVAD-fmk, caspase-3 inhibitor z-DEVD-fmk, caspase-9 inhibitor z-LEHD-fmk, caspase-8 inhibitor z-IETD-fmk and caspase-7 inhibitor AcDEVD-CHO on SF-induced JNK activation. Cells were incubated and treated as described in Section 2. Kinase activities were measured using ELISA kits in 96-well plates (Section 2). Results were normalized to micrograms of protein in the cell extract and expressed as the ratio of phospho-kinase to total kinase in the same sample. Oxidative stress was determined spectrophotometrically by intracellular conversion of 20 -70 -dichlorodihydrofluorescein diacetate (DFCH/DA) into a fluorescent product dichlorofluorescein (DCF). The results were expressed as a percentage of fluorescence intensity increase per 106 cells. Results represent means ± SD of at least three experiments. *P < 0.05 compared to control cultures and #P < 0.05 compared to cultures pretreated with NAC with one way-Anova test and Dunnett’s post test for multiple comparisons.
caspase present constitutively in the nucleus and provides an important link between DNA-damage and the mitochondrial apoptotic pathway (Zhivotovsky et al., 1999). Previous studies suggested JNK as one of the possible downstream targets of this caspase (Panaretakis et al., 2005). Since SF has been shown to activate all three MAPKs (Cho et al., 2005; Hu et al., 2003; Yeh and Yen, 2005), we wanted to determine their involvement in SF-dependent
signaling in the present model. Our analyses provided evidence of elevated JNK activity but not p38 and ERK activities which is in contrast with observations obtained with HT-29 cells and may reflect degree of differences between both cell lines at different stages of colon carcinogenesis. The adherence of time-frame of JNK activation and caspase-2 activity in SF-treated SW620 cells supports the notion of their sequential linkage. Therefore the
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vent phosporylation of JNK and subsequent apoptosis indicate that ROS did not play any role in SF-induced proapoptotic signaling. It is conceivable; however, that at higher SF concentrations oxidative stress might play a role in apoptosis of tumor cells as it has been observed that this process is strictly concentration-dependent (Clarke et al., 2008). Still, the almost simultaneous activation of two DNA-damage depending cascades by SF in SW620 cells raised the question of whether there is any functional linkage between these two processes. The employed inhibitors of all caspases, caspase-2 and p53 in cells with mutant p53 and dominant wild-type p53 showed that in the presence of mutant p53 both p53-dependent and caspase-2 dependent pathways contribute to apoptosis although caspase-2 pathway seems to be of higher importance and efficiency, acting as if to substitute for defective p53 pathway. Conversely, wild-type p53 cells show equal activity and efficiency of both cascades which seem likely to mutually enhance and complement each other. To our knowledge this is the first report on the importance and relationship of p53-depnding signaling and caspase-2 pathway in SF-induced apoptosis, in particular in colon cancer cells. In summary, the present work informs about apoptotic activity of SF in metastatic colon cancer cell line SW620. SF-induced apoptosis is not associated with oxidative stress but involves DNA-damage signaling whose efficiency is likely to depend on p53 status and caspase-2 activation with important verified linkage between caspase-2 and JNK. The activity of both pathways may putatively serve to amplify the critical proapoptotic signals interacting with mitochondria which in turn activate effector caspases. The ability of SF to initiate multiple proapoptotic cascades even in advanced stages of colon cancer harboring mutant p53 render this chemical an attractive cytotoxic and chemopreventive agent whose further evaluation is therefore warranted. Conflict of interest statement Fig. 6. Effects of inhibition of oxidative stress (N-acetylcysteine, NAC), p53 signaling (Pifithrin), activation of caspases (pan caspase inhibitor z-VAD-fmk) and activation of caspase-2 (z-VDVAD-fmk) on 20 lM sulforaphane (SF)-induced apoptosis in SW620 cells during 48 h of treatment. (A) Non-transfected SW620 cells with mutant p53. (B) SW620 cells transfected with wild-type p53. Cells were preincubated or coincubated with the specified inhibitors, exposed to SF and apoptosis was determined as described in Section 2. Results represent means ± SD of at least three experiments. *P < 0.05 compared to cells treated with SF and #P < 0.05 compared to cultures pretreated with p53-inhibiting Pifithrin with one way-Anova test and Dunnett’s post test for multiple comparisons. (C) Comparison of effects of p53 inhibition, caspase-2 inhibition and simultaneous inhibition of both p53 and caspase-2 in SW620 cells with differing p53 status. In this independent experiment, cells were preincubated or coincubated with the specified inhibitors, exposed to SF and apoptosis was determined as described in Section 2. Results represent means ± SD of at least three experiments. *P < 0.05 compared to cells treated with SF and #P < 0.05 compared to cultures where either p53 or caspase-2 were inhibited with one way-Anova test and with one way-Anova test and Dunnett’s post test for multiple comparisons.
relation between caspase-2 and JNK was further evaluated by inhibiting caspase-2 activity. The fact that JNK phosphorylation was severely attenuated in cells treated the specific caspase-2 inhibitor supports a direct role of this caspase in activation of JNK while providing possible connection between caspase-2 and mitochondria via JNK and its downstream target Bak as was suggested before (Pappa et al., 2006). DNA lesions induced by SF in SW620 cells result in activation of DNA-damage signaling pathway as well as increase caspase-2 activity. Since SF is known to induce elevated levels of ROS which might be responsible for DNA damaging and subsequent phenomena we measured ROS generation in our study model (Shen et al., 2006). The observed lack of any significant oxidative stress at all treatment intervals along with failure of antioxidant NAC to pre-
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