In vitro effects of 2-methoxyestradiol on cell numbers, morphology, cell cycle progression, and apoptosis induction in oesophageal carcinoma cells

Cell Biochem Funct 2009; 27: 205–210.
Published online 2 April 2009 in Wiley InterScience(www.interscience.wiley.com) DOI: 10.1002/cbf.1557 In vitro effects of 2-methoxyestradiol on cell numbers, morphology,cell cycle progression, and apoptosis induction in oesophagealcarcinoma cells Veneesha Thaver 1,2, Mona-Liza Lottering 2, Dirk van Papendorp 2 and Annie Joubert 2* 1Department of Physiology, University of Limpopo, Garankuwa, Pretoria, South Africa2Department of Physiology, University of Pretoria, Pretoria, South Africa The influence of 2-methoxyestradiol (2-ME) was investigated on cell numbers, morphology, cell cycle progression, and apoptosis induction inan oesophageal carcinoma cell line (WHCO3). Dose-dependent studies (1 Â 10À9M–1 Â 10À6M) revealed that 2-ME significantly reducedcell numbers to 60% in WHCO3 after 72 h of exposure at a concentration of 1 Â 10À6M compared to vehicle-treated cells. Morphologicalstudies entailing light-, fluorescent-, as well as transmission electron microscopy (TEM) confirmed 2-ME’s antimitotic effects. These resultsindicated hallmarks of apoptosis including cell shrinkage, hypercondensation of chromatin, cell membrane blebbing, and apoptotic bodies intreated cells. Flow cytometric analyses demonstrated an increase in the G2/M-phase after 2-ME exposure; thus preventing cells fromproceeding through the cell cycle. b-tubulin immunofluorescence revealed that 2-ME caused spindle disruption. In addition, increasedexpression of death receptor 5 protein was observed further supporting the proposed mechanism of apoptosis induction via the extrinsicpathway in 2-ME-exposed oesophageal carcinoma cells. Copyright # 2009 John Wiley & Sons, Ltd.
key words — oesophageal carcinoma; 2-methoxyestradiol; metaphase block; apoptosis phosphorylation and inactivation of Bcl-2, a pro-apoptoticprotein, thus contributing to apoptotic induction.8,9 Never- 2-Methoxyestradiol (2-ME), a 17-beta estradiol metabolite, theless, apoptotic induction via the extrinsic and intrinsic is a mitogen antagonist and tubulin poison that hinders cell pathways appears to be dependent on cell type.13–15 proliferation and induces apoptosis in a large diversity of It is also known that 2-ME displays a dose-dependent non-tumor and tumor cells.1–4 2-ME implements both its biphasic pattern on cell proliferation at concentrations antiangiogenic and antitumor influence regardless of the ranging from 10À8 to 10À5 M. Stimulatory effects have been cell’s hormone receptor status and is accountable for demonstrated at low concentrations of 2-ME and inhibitory abnormal mitotic spindle formation and mitotic accumu- effects were observed at high concentrations.3,11,16 Corre- lation in both estrogen receptor (ER) positive- and ER- spondingly, in vivo studies have demonstrated stimulation negative cells.5–7 Accordingly, this endogenous estradiol and inhibition of tumor growth by 2-ME depending on metabolite has manifested as a potential anticancer agent.5 dosage.17,18 In concert, these research studies imply a Current evidence has suggested that 2-ME is the causative multifaceted nature of the action of 2-ME. Data illustrated agent leading to an increase in Cdc2 kinase activity, the that, in addition to the established signaling pathways there activation of c-Jun NH2-terminal kinase signaling, gener- may be supplementary pathways that have not been ation of reactive oxygen species and an altered ratio of Bax/ Bcl-2 in favor of Bax, ultimately culminating into ME.13,14,16,19–21 This biphasic effect may also be cell line apoptosis.8–11 Cell division cycle (Cdc) 2 kinase activity is a cell cycle regulatory component essential for Preclinical data illustrated that 2-ME might be considered commencement of mitosis, whereas Cdc2 inactivation is in the treatment of multiple myeloma, sarcoma and other needed for mitotic exit. Prolonged Cdc2 activity can sustain solid tumors, therefore portraying it as a possible anticancer the cell in mitosis for an indefinite period until particular agent when compared to conventional chemotherapeutic conditions are met for mitotic exit.12 JNK is involved in the treatments.5,16,22,23 Phase I and phase II clinical trials with 2-ME revealed its therapeutic potential when administered topatients with metastatic breast cancers and prostate cancers * Correspondence to: A. Joubert, Department of Physiology, P.O. Box 2034, with only minor side-effects in some of the patients namely Pretoria 0001, South Africa. Tel: þ27 12 3192246; Fax: þ27 12 3211679.
E-mail: annie.joubert@up.ac.za hot flushes, reversible liver enzyme elevations, fatigue, and Copyright # 2009 John Wiley & Sons, Ltd.
diarrhoea.5,16,19,22 Clinical studies employing 2-ME in (v/v). Controls included showed that 0.1% had no toxic cancer patients revealed that treatment is linked with effects on these cells in experiments conducted.
clinical advantages including prolonged, stable disease,partial or complete responses, and an exceptional safety Since the mechanism of action of 2-ME is multifaceted Exponentially growing WHCO3 cells were seeded in 24- and appears to vary according to cell type2,5,11,24 the aim of well culture plates at a density of 20 000 viable cells per well this study was to investigate the mechanism of action of 2- and exposed to a dilution series of 2-ME with a final ME in an oesophageal carcinoma cell line by determining its concentration of 10À6, 10À7, 10À8, 10À9 M respectively for influence on cell numbers, morphology, cell cycle pro- 72 h at 378C. The experiment was terminated by replacing the growth medium with 300ml of 1% glutaraldehyde in PBSfor 15 min. Crystal violet (1%, in PBS) was added for30 min. The culture wells were subsequently immersed in running tap water for 15 min. After the plates had dried, 500 ml of 0.2% Triton X-100 was added to each well. Plateswere incubated for 90 min and 200 ml of the liquid content 2-ME, Eagles’ Minimum Essential Medium with Earle’s was transferred to 96-well plates.25 The absorbance salts, L-glutamine and NaHCO3 (MEM), Trypsin-EDTA, (measured at 570 nm) of the samples was analyzed using trypan blue, thymidine, hydroxyurea, anti-human Bcl-2 antibody, mouse monoclonal antibody against human b- X800 Universal Microplate Reader (Bio-Tek Instru- ments Inc., Analytical Diagnostic Products, Weltevreden tubulin (Clone 2-28-33) biotin-conjugated anti-mouse IgG SA). Results shown are representative of three independent (Fab-specific, developed in goat), FITC-conjugate diluent experiments (each conducted in triplicate).
and ExtrAvidin1-FITC conjugate were supplied by SigmaChemical Co. (St. Louis, MO, USA). Hematoxylin, eosin, ethanol, xylol, and Entellan fixative were purchased from Merck (Darmstadt, Germany). Propidium iodide was Propidium iodide and hoechst 33342 staining. To study the supplied by DAKO Chemical Supplies (Glostrup, Den- viability and presence of apoptotic cells after 2-ME mark). DAKO LSAB Kit was purchased from Dako treatment, 500 000 WHCO3 cells were seeded onto heat- Corporation (Santa Barbara, CA, USA). The death receptor sterilized cover slips and exposed to 1 Â 10À6 M 2-ME for five antibody and human anti-goat IgG were purchased from 24 h. An exposure time of 24 h was chosen since significant Calbiochem (Darmstadt, Germany). Heat-inactivated fetal reductions in cell number were visible after 24 h of treatment calf serum (FCS), sterile cell culture flasks and plates were with 2-ME. Medium was removed, the cells were gently obtained though Sterilab Services (Kempton Park, Johan- rinsed with PBS and 2 ml of 0.5 mg mlÀ1 Hoechst 33342 nesburg, SA). Phosphate buffered saline (PBS), penicillin, (HO) in PBS was added to each well. Samples were streptomycin, and fungizone were obtained from Highveld Biological (Sandringham, SA). Quetol, Reynolds’ lead 0.5 ml of propidium iodide (PI) solution (40 mg mlÀ1 in citrate, aqueous uranyl acetate, and toludine blue were PBS) was added. Within 5 min cover slips were mounted on purchased from Merck Co. (Johannesburg, South Africa).
microscope slides with mounting fluid (90% glycerol, 4% N- All other chemicals were of analytical grade and supplied by propyl-gallate, 6% PBS) and examined under a fluorescence Sigma Chemical Co. (St. Louis, MO, USA).
microscope. Photographs were taken with 400 ASA film ona Nikon Optiphot microscope (Nikon, Tokyo, Japan) withUV-light and a blue filter. Viable and apoptotic (although having irregular appearances) cells will stain light blue. The WHCO3 cells were a gift from Professors Veale and latter phenomenon illustrates that these cells have functional Thornley (Department of Zoology, University of Witwa- cell membranes capable of excluding PI. However, cells tersrand, Johannesburg, South Africa). These cells were with compromised membrane integrity will stain bright obtained though a biopsy from a patient with squamous oesophageal carcinoma and are described as a poorlydifferentiated, non-keratinizing Hematoxylin and eosin staining (H and E staining) propagated as monolayers in MEM at 378C in a humidifiedatmosphere containing 5% CO2. Media were supplemented WHCO3 cells (250 000) were seeded onto heat-sterilized with 10% heat-inactivated fetal calf serum, penicillin cover slips in 6-well plates. Cells were exposed to (100 mg LÀ1), streptomycin (100 mg LÀ1), and fungizone 1 Â 10À6 M 2-ME for a period of 24 h at 378C. Many cells (250 mg LÀ1). Non-viable cells were excluded with the were not adherent to cover slips after exposures of 24–48 h trypan blue staining procedure. Stock solutions of 2-ME and had disintegrated to become floating debris. Thus, it was were prepared in dimethyl sulphoxide (DMSO) at concen- not possible to observe morphological changes occurring trations of 2 Â 10À3M and stored at room temperature. The during this period when studying the influence of 2-ME on DMSO content of the final dilutions never exceeded 0.1% WHCO3 cells by means of H and E staining. Cells were Copyright # 2009 John Wiley & Sons, Ltd.
Cell Biochem Funct 2009; 27: 205–210.
INFLUENCE OF 2-ME IN OESOPHAGEAL CARCINOMA CELLS fixed in Bouin’s fixative for 60 min after exposure to 2-ME 1 h (1:100). Following washing, cells were finally incubated and stained by standard hematoxylin and eosin staining with ExtrAvidin1-FITC conjugate (1:200 in FITC-con- jugate diluent) for 1 h. The cover slips were mounted with aglycerol-based mounting fluid after the final 3 Â 5-min washstep. The cells were examined with a Nikon Optiphot microscope equipped with an episcopic-fluorescence attach- Exponentially growing cells (500 000) were seeded in ment and an excitation-emission filter with an average 25 cm2 flasks and exposed to 0.1% DMSO (vehicle control) and 1 Â 10À6 M 2-ME for 24 h respectively. Cells werewashed with PBS (3x) and scraped off the bottom of theflask. Subsequently, ultra-thin sections of cells were Immunofluorescent detection of death receptor 5 (DR5) prepared. Cells were fixed in 2.5% glutaraldehyde in Cells (500 000) were seeded onto heat-sterilized glass cover 0.075 M phosphate buffer, (pH 7.4–7.6) for 1 h and rinsed slips in 6-well plates and exposed to 2-ME or DMSO 3 times for 5 min each with 0.075 M phosphate buffer.
controls for a period of 24 h at 378C. The cells were then Thereafter sections were fixed in 0.25% aqueous osmium fixed in 10% formalin (2 mM EGTA in PBS) for 10 min and tetroxide and rinsed (3x) in distilled water in a fume hood.
permeabilized in ice-cold 97% methanol containing 2 mM Samples were dehydrated in ethanol (70, 100%), infiltrated EGTA at -208C for 10 min. Cells were subsequently washed with 30% quetol in acetone for 1 h and furthermore in PBS (3 Â 5 min) before incubation for 1 h with a mouse infiltrated with 60% quetol in ethanol for 1 h, and thereafter monoclonal antibody against human Death Receptor with pure quetol for 4 h. Sections were polymerized at 658C 5 (Clone 2-28-33; 1:1000). After washing with PBS, the for 24–36 h. Ultra-thin sections were mounted on grids, cells were incubated with biotin-conjugated anti-mouse IgG contrasted for 10 min in 4% aqueous uranyl acetate and (Fab-specific, developed in goat) in FITC-conjugate diluent rinsed in water. Enhancement of contrast was obtained by as secondary antibody for 1 h (1:100). Following washing, placing the samples in Reynolds’ lead citrate for 2 min and cells were finally incubated with ExtrAvidin1-FITC rinsing the samples in water. Samples were cut into 0.5 mm conjugate (1:200 in FITC-conjugate diluent) for 1 h. The monitor sections, stained with toluidine blue, and immersed cover slips were mounted with a glycerol-based mounting fluid after the final 3 Â 5-min wash step. Cells wereexamined with a Nikon Optiphot microscope equipped with an episcopic-fluorescence attachment and an exci-tation-emission filter with an average wavelength of 495 mm WHCO3 cells were seeded into 25 cm2 flasks as described above. Cell cycle analyses were performed after 24 h ofexposure to 1 Â 10À6 M 2-ME at 378C. Cells weretrypsinized in equal volumes of trypsin (0.25%) and EDTA (1 mM), fixed in 99.5% methanol and stored at À208C.
Data obtained from three independent experiments were Methanol was removed by centrifugation at 200 Â g for statistically analyzed for significance using the two-tailed 10 min. The sediments were resuspended in 1 ml 1% CaCl2 Student t-test for samples. Means are presented in bar charts, and 50 mg mlÀ1 propidium iodide and incubated for 20 min while shaking gently. Each analysis was based on at least p-values < 0.05 were regarded statistically significant.
10 000 events employing a Coulter Epic-XS flow cytometer.
The data were analyzed using a multicycle analysis program(MulticycleAV software).
WHCO3 cell growth was expressed as a percentage of the To visualize the effect of 2-ME on spindle formation in control after exposure to different concentrations of 2-ME WHCO3 cells, indirect immunofluorescence was employed.
(10À6, 10À7, 10À8, 10À9 M) for 72 h. 2-ME reduced cell Cells (500 000) were seeded onto heat-sterilized glass cover numbers to 60% when compared to vehicle-treated controls slips in 6-well plates. After exposure to 2-ME or DMSO for after exposure to 10À6 M 2-ME for 72 h. An à indicates a 24 h at 378C, cells were fixed in 10% formalin (2 mM EGTA statistically significant p-value < 0.05 for growth inhibition in PBS) for 10 min and permeabilized in ice-cold 97% methanol containing 2 mM EGTA at À208C for 10 min.
Subsequently cells were washed in PBS (3 Â 5 min) before incubation for 1 h with a mouse monoclonal antibodyagainst human b-tubulin (Clone 2-28-33; 1:1000). After Propidium iodide and hoechst 33342 staining. PI and HO washing with PBS, cells were incubated with biotin- staining were conducted to determine the presence of conjugated anti-mouse IgG (Fab-specific, developed in apoptotic cells after treatment with 2-ME. Viable and goat) in FITC-conjugate diluent as secondary antibody for apoptotic cells have intact cell membranes and are stained Copyright # 2009 John Wiley & Sons, Ltd.
Cell Biochem Funct 2009; 27: 205–210.
Hematoxylin and Eosin staining of WHCO3 control cells exposed to 0.1% DMSO (vehicle) (A) and 1 Â 10À6 M 2-ME (B) for24 h (400 Â magnification). Clusters of rounded cells with hypercondensed Dose-dependent study of WHCO3 cells treated with a dilution chromatin, as well as apoptotic bodies are visible in the treated cells series of 2-ME (10À6, 10À7, 10À8, 10À9 M) for 72 h. Cell numbers areexpressed as a percentage of the control. A 40% decrease in cell number wasnoted at 10À6 M of 2-ME WHCO3-treated cells. Ã indicates p-value < 0.05 intact nucleoli in contrast to cells treated with 2-ME thatshowed condensed chromatin, irregular nuclear membrane, light blue, while cells that have lost their membrane integrity and increased mitochondrial aggregation toward the nucleus are stained bright red. After 2-ME treatment most cells stained light blue (indicated in black and white) and wererounded in appearance due to a metaphase block and showed apoptotic features including cytoplasmic shrinking, mem-brane blebbing, and apoptotic bodies (Figure 2A, B).
Quantitative analysis of DNA content was conducted bymeans of flow cytometry in order to determine the effects of2-ME on cycle progression after 24 h of exposure. 2-ME- Hematoxylin and eosin staining (H and E staining) treated cells revealed an increase in the sub G1/0 apoptotic The antiproliferative effect of 2-ME observed above could fraction when compared to vehicle-treated cells (Figure 5).
be attributed to either growth inhibition (cytostatic effect) or An increase in the amount of cells in the G2/M-phase was induction of cell death. Thus, morphological characteristics of the cytoplasm and nuclear components of cells treatedwith 2-ME and DMSO respectively were studied by meansof hematoxylin and eosin staining to confirm 2-ME’santimitogenic effect (Figure 3A, B). After 2-ME treatment,most cells were rounded in appearance due to a metaphaseblock and showed apoptotic features including hypercon-densed chromatin, cytoplasmic shrinking, membrane bleb-bing, and apoptotic bodies when compared to their vehicle-treated controls (Figure 3A, B).
(A) Transmission electron microscopy of WHCO3 control cells exposed to 0.1% DMSO (vehicle) (A) and 1 Â 10À6 M 2-ME (B) for 24 h.
Hypercondensed chromatin and increased mitochondrial aggregationaround the nucleus are visible (B). (Scale bar ¼ 0.5 mm) TEM was employed to view subcellular structures in twodimensions. WHCO3 control cells (Figure 4A) revealed Propidium iodide and Hoecsht 33342 staining of WHCO3 cells exposed to 0.1% DMSO (vehicle) (A) and 1 Â 10À6 M 2-ME (B) for 24 h(black and white images; 400 Â magnification). 2-ME-treated cells are The effect of 2-ME on cell cycle progression after 24 h of rounded in appearance due to a metaphase block. Apoptotic features exposure. 2-ME-treated cells presented with an increased sub G1/0 apoptotic including cytoplasmic shrinking, membrane blebbing, and apoptotic bodies fraction, as well as a G2/M-phase increase when compared to vehicle-treated Copyright # 2009 John Wiley & Sons, Ltd.
Cell Biochem Funct 2009; 27: 205–210.
INFLUENCE OF 2-ME IN OESOPHAGEAL CARCINOMA CELLS various cell lines that are affected by 2-ME at inhibitoryconcentrations ranging from 0.08 to 5 mM.
In the present study conducted, 2-ME was shown to exert antiproliferative activity in the WHCO3 oesophagealcarcinoma cell line investigated. Morphological changesoccurring during apoptosis namely cell shrinkage, mem-brane blebbing condensation of nuclear chromatin intosharply delineated masses that become marginated againstthe nuclear membrane, as well as the formation of apoptoticbodies30 were demonstrated in 2-ME-treated WHCO3 cells.
Immunofluorescent staining of b-tubulin in WHCO3 cells.
These data are consistent with previous results from our 2-ME-treated cells were rounded, accumulated in metaphase and spindle laboratory where inhibition of cell growth in breast cancer disruption with fragmented polar formations were evident when compared cells (MCF-7) was demonstrated following 2ME treatment.
2ME-treated MCF-7 cells also exhibited abnormal meta-phase cells, membrane blebbing, apoptotic bodies, anddisturbed spindle formation. However, these observationswere either absent, or less pronounced in the non-tumorigenic MCF-12A cells.11 In this study, cells treated with 2-ME revealed an increase in mitochondrial numbers aggregating around the nuclearenvelope. Mitochondria are important sensors and amplifiersin intracellular death signaling pathways and are corecomponents of the cell death machinery.14 Changes inmitochondrial membrane structure, either by disruption ofthe outer membrane or by Bax activation31 can lead toapoptosis. In addition, up-regulation of DR5 was also Immunofluorescent detection of DR5 in 2-ME-treated WHCO3 demonstrated in 2-ME-treated cells and is consistent with cells. Up-regulation of DR5 expression was observed as an increased previous data where 2-ME was shown to up-regulate DR5 occurrence of white spots when compared to vehicle-treated controls and sensitize cancer cells to TRAIL-induced apoptosis in Furthermore, we have previously demonstrated a significant increase in Cdc2 kinase activity in 2-ME-treated cells whencompared to vehicle-treated controls in WHCO3 cells.10 Cdc2 Since previous research has shown that 2-ME induces cell kinase activity was statistically significantly increased (1.7- death by causing microtubule disruption and blocking cells fold) ( p < 0.005) after 2-ME exposure when compared to in metaphase in other cell lines, the influence of 2-ME was vehicle-treated controls. Our observation contributes to the subsequently investigated on spindle formation in WHCO3 elucidating of the mechanism of action in WHCO3 cells by means of immunofluorescent staining of b-tubulin.
oesophageal carcinoma cells and reveals that 2-ME causes 2-ME-treated cells were rounded, accumulated in metaphase a metaphase arrest, disrupts mitotic spindle formation, and also showed spindle disruption with fragmented polar enhances Cdc2 kinase activity leading to persistence of the formations when compared to vehicle-treated controls spindle checkpoint, and thus prolonged metaphase arrest culminating in the induction of apoptosis in WHCO3 cells.
The observed up-regulation of DR5 further supports theproposed mechanism of apoptosis induction via the extrinsic pathway in WHCO3 oesophageal carcinoma cells.
To investigate whether the extrinsic pathway of apoptosiswas activated after treatment with 2-ME, DR5 was chosen asa marker. Immunofluorescent detection of DR5 in 2-ME- treated WHCO3 cells demonstrated an up-regulation of DR5expression when compared to vehicle-treated controls This research was supported by grants from the Medical Research Council of South Africa (AG374, AK076), theCancer Association of South Africa (AK246), and theStruwig-Germeshuysen Cancer Research Trust of South Africa (AJ038). Electron microscopy was conducted at Previous research has revealed that 2-ME plays an important the Electron microscopy Unit at the University of Pretoria role in the induction of apoptosis and especially in actively and flow cytometric analysis was performed at the Depart- proliferating cancerous cells.3,5,16,22,28,29 Pribluda et al.16 ment of Pharmacology at the Faculty of Health Sciences accounted of the antiproliferative effects of 2-ME by listing Copyright # 2009 John Wiley & Sons, Ltd.
Cell Biochem Funct 2009; 27: 205–210.
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