Bisindolylmaleimide IX facilitates extrinsic and initiates intrinsic apoptosis in TNF-a-resistant human colon adenocarcinoma COLO 205 cells
Beata Pajak Æ Agnieszka Turowska Æ
Arkadiusz Orzechowski Æ Barbara Gajkowska
Published online: 18 March 2008
© Springer Science+Business Media, LLC 2008
Abstract
Human COLO 205 colon adenocarcinoma cells are immune to extrinsic apoptosis induced by immunomodulatory cytokines. Among the antiapoptotic mechanisms responsible for the immune escape, the overexpression of the cFLIP protein seems to be critical. cFLIP appears to inhibit the TNF-a-induced death receptor signal. The application of the metabolic inhibitor bisindolylmaleimide IX (Bis-IX), known as a potent PKC repressor, sensitized COLO 205 cells to TNF-a-mediated apoptosis. The Western-blot analysis revealed that the susceptibility of human COLO 205 cells to apoptogenic stimuli resulted from time-dependent reduction in cFLIPL and TRADD protein levels. At the same time, the level of FADD protein was up-regulated. Additionally, the com- bined TNF-a and Bis-IX treatment caused cleavages of Bid and procaspase-9, as well as cytochrome c release. Thus, the evidence of this study indicates that Bis-IX facilitates the death receptor signal mediated by TNF-R1. Moreover, Bis-IX alone initiated intrinsic apoptosis, which could be abolished by Bcl-2 delivery. It heralds the involvement of mitochondria in caspase-8-independent intrinsic apoptosis. In turn, the treatment with bisindo- lylmaleimide III (Bis-III) did not assist TNF-a-dependent apoptosis.
Keywords : TNF-a · Bisindolylmaleimides · cFLIP · Immune escape · Colon cancer
Introduction
Apoptosis, or programmed cell death (PCD) is an essential mechanism used to selectively eliminate useless cells in all living organisms. The loss or decrease in cell apoptosis is associated with a wide range of disorders including cancer [1]. TNF-a is a potent apoptogenic cytokine released by T cells or macrophages, which binds to its cognate cell sur- face receptors, TNF-R1 and TNF-R2, and the resulting complex triggers apoptosis through the caspase cascade [2]. Impaired TNF-a-dependent death signaling is linked to antiapoptosis (repression of death signal) and it contributes to the excessive cell proliferation and tumor induction [3, 4]. There is strong evidence that neoplastic cells, including colon cancer, counteract apoptotic signals by expressing antiapoptotic proteins capable of suppressing the death signal. Among the proteins which have been identified as inhibiting extrinsic apoptosis, the cellular FLICE-like inhibitory protein (cFLIP) seems to play considerable role. cFLIP structurally resembles caspase-8 but it lacks prote- olytic activity, thus functioning as a dominant negative inhibitor of caspase-8 (FLICE) [2, 5]. A critical role of cFLIP in the resistance of certain cancers to death ligands has been demonstrated by several authors [6–9] with a reduction in cellular levels of cFLIP to assist elevated sensitivity of cancers to death ligands. The development of novel therapeutic agents is necessary to improve the survival rates of patients bearing tumors resistant to death ligand-mediated apoptosis.
Bisindolylmaleimide IX (Bis-IX) is a potent factor that could serve as a useful adjuvant in the treatment of meta- static colorectal cancer. Bisindolylmaleimides have been originally described as inhibitors of protein kinase C (PKC) [10–12]. Zhou et al. [13] estimated the potentiating effect of several Bis derivatives on Fas-mediated apoptosis in astrocytoma 1231N1 cells. They found that some of bis- indolylmaleimides facilitated Fas-mediated apoptosis in tumor cells. The greatest potentiation was observed with Bis-VIII and -IX; Bis-III, -X and -XI produced interme- diate potentiation, whereas Bis-I, -II and -IV did not sensitize cells to Fas-mediated cell death. The aim of this study was to examine whether certain Bis (Bis-III and Bis- IX) derivatives could be useful in the treatment of colon cancer. Apparently, Bis-IX substantially smoothened the progress of TNF-a-induced apoptosis in human colon adenocarcinoma COLO 205 cells. The amplification of TNF-a-mediated cell death seems to be at least equally important to its well recognized PKC inhibition. Further- more, Bis-IX modulated the expression of pro- and anti- apoptotic proteins involved in death signal transduction and allowed the activation of the mitochondrial apoptotic pathway.
Materials and methods
Reagents
All reagents: dimethyl sulfoxide (DMSO), Tris, 4-(2-hydroxy- ethyl)-1-piperazine-ethanesulfonic acid (HEPES), ethylenedi- aminetetraacetic acid (EDTA), (2-aminoethoxyethane)-N,N, N0,N0-tetraacetic acid (EGTA), polyoxyethylene sorbitan monolaurate (TWEEN 20), sodium chloride (NaCl), bovine serum albumin (BSA), 3-(4,5-dimethylthiazol-2-yl)-2-5- diphenyltetrazolium bromide (MTT), phenyl-methylsulpho- nylfluoride (PMSF), dithiothreitol (DTT), paraformaldehyde, cycloheximide (CHX), actinomycin D (AD), bisbenzimide (HO33342), propidium iodide (PI), tumor necrosis factor-a (TNF-a), staurosporine (STS), z-Ile-Glu(O-Me)-Thr-Asp(O- Me) fluoromethyl ketone (z-IETD-fmk), D-mannitol, sucrose were cell culture tested, of high purity, and unless otherwise stated purchased from Sigma-Aldrich Chemical Co. (St. Louis, MO, USA). Bisindolylmaleimide III (Bis-III) and bisindolyl- maleimide IX (Bis-IX) were obtained from Alexis Biochemicals (Lausen, Switzerland). N-Benzyloxycarbonyl- Val-Ala-Asp(O-Me) fluoromethyl ketone (z-VAD-fmk) was purchased from Calbiochem (San Diego, CA, USA). Reagents for experimental applications were prepared according to the manufacturer’s recommendations and if possible stored as stock solutions (1,000-fold the highest working concentration).
All primary, except mouse monoclonal anti-cytochrome c oxidase subunit IV antibody (COX-IV, Molecular Probes, Eugene, OR, USA), and secondary horseradish peroxidase (HRP) conjugated antibodies were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Other reagents were purchased as stated in the description of the respective methods (see the text). Sodium dodecyl sulphate (SDS) 100 g/l, sequi- blot polyvinylidene fluoride (PVDF) membrane 0.2 lm and all reagents for immunoblotting were obtained from Bio-Rad Laboratories (Hercules, CA, USA). Sera, media and antibiotics were obtained from Gibco Life Technologies (Paisley, United Kingdom).
Cell culture
Human colon adenocarcinoma cell line COLO 205 was purchased from American Type Culture Collection (ATCC). Cells were maintained in the exponential phase of growth in growth medium (GM, 100 ml/l Fetal Bovine Serum (FBS)/Dulbecco’s Modified Eagle Medium (DMEM) with Glutamax and antibiotic-antimycotic mix- ture (Penicillin G sodium salt 50 IU/ml, Streptomycin sulphate 50 lg/ml, Gentamycin sulphate 20 lg/ml, Fun- gizone—Amphotericin B 1 lg/ml)). The cells were grown at 37°C, in a controlled, humidified 50 ml/l CO2 atmo- sphere, on 96-well flat-bottomed or tissue culture Petri dishes (100 mm diameter, BD Biosciences).
Experimental procedure
During propagation, the medium was changed every other day until cultures reached 100% confluence. One-day (24 h) prior to the experiment, confluent cells (cells of the same cell density fully covering the surface of the dish) were then switched to post-mitotic status to induce quies- cence (withdrawal from cell cycle) by replacing GM with 20 g/l BSA/DMEM designated as a control medium (CTRL). In the above-mentioned conditions divisions of COLO 205 cell have been completed. During the study freshly prepared media with or without experimental fac- tors had been changed according to the experimental schedule.
Cell viability
Cell viability based on mitochondrial function was assayed by the ability of cells to convert soluble MTT (3-(4,5- dimethylthiazol-2-yl)-2-5-diphenyltetrazolium bromide) into an insoluble purple formazan reaction product with minor modifications to protocol described by [14]. For this assay, during the last hour of incubation the media were replaced with MTT solution (5 mg/ml in DMEM without phenol red, sterilized by filtration). MTT solution was then aspirated and formazan in cells was instantly dissolved by the addition of 100 ll DMSO. Before the application of MTT, cells were examined under phase-contrast micros- copy to visually assess the degree of cell death. The absorbance was measured at 570 nm with ELISA reader type Infinite 200 (TECAN, Austria). Percentage of viable cells was measured by MTT conversion into purple for- mazan either in 20 g/l BSA/DMEM or 1 ml/l DMSO and was related to mitochondrial respiration or activity of mitochondrial dehydrogenases.
Apoptotic index and detection of apoptotic cells using morphological criteria
Cells were grown on Lab-Tek 4-Chamber Slide w/Cover (Permanox Slide Sterile, Nalge Nunc International, Naperville, IL, USA). Cytotoxicity with resultant cell death was evaluated by microscopic observations. Apoptosis was evaluated by in situ uptake of propidium iodide (PI) and bisbenzimide (HO33342) as described by McKeague et al. [15]. To avoid detachment the cells were then washed gently with medium. Attached cells were stained by both propidium iodide followed by Hoechst 33342 staining to distinguish live, necrotic, early- and late-apoptotic cells. Afterwards, the cells were fixed with an equal volume of methanol/acetic acid (3:1 v/v) and the cells were gently washed with ice cold PBS (Phosphate-Buffered Saline including Ca2+ and Mg2+) and mounted on slides using mounting medium prepared according to the manufac- turer’s protocol (Mowiol, CN Biosciences Inc., La Jolla, CA, USA). A BX-60 Olympus fluorescent microscope equipped with a PM20 automatic photomicrograph system was used for photographic recording. At least one hundred nuclei were counted under ultraviolet light in ten (or more if necessary) randomly chosen visual fields per slide. In the same visual field the excitation of propidium iodide should lead to the appearance of red nuclei of the necrotic dead cells (no such cells have been detected). Cells were con- sidered apoptotic if they were PI-negative and chromatin was condensed at the periphery of nuclei. Apoptotic index was calculated from the number of apoptotic nuclei versus total number of nuclei at each visual field (n = 10). Three independent experiments were performed.
Immunofluorescence
COLO 205 cells were propagated in multiwell 4 Chamber Culture Slides and treated (or untreated) with experimental factors present in the CTRL medium (20 g/l BSA/DMEM or 0.1% v/v DMSO in 20 g/l BSA/DMEM). Exactly 30 min prior to the end of the experiment the Mitotracker Red (50 ng/ml) was added if the cell mitochondria were to be visualized (Cell Signaling Technology Inc., Danvers,MA, USA). After the experiment ended, the cells were fixed as follows: washed twice with PBS+, fixed in 0.25% (v/v) formaldehyde for 15 min in room temperature, washed twice with PBS containing 10 g/l BSA/PBS, sus- pended in ice-cold 70% methanol and stored at 4°C for 0.5 h. At the end of the procedure the methanol was aspirated. Before staining, the cells were washed three times with 10 g/l BSA/PBS and then were incubated for 1 h at 37°C with primary goat polyclonal anti-caspase-9 cleaved form diluted 1:150. After incubation the cells were washed three times with 10 g/l BSA/PBS and subsequently were incubated for 1 h at 4°C with 1:500 secondary chicken anti-goat antibody conjugated to Alexa Fluor 568 (Molecular Probes Inc., Eugene, Oregon, USA). The cells were then incubated with SYTOX green (Molecular Probes Inc., Eugene, Oregon, USA) or Hoechst 33342 for 15 min at room temperature, to visualize the cell nuclei. After- wards, the cells were washed five times with 10 g/l BSA/ PBS, the chamber walls were removed and coverslips were mounted on microscope slides using an anti-fade Mowiol mounting medium (CN Biosciences Inc., La Jolla, CA, USA). As a negative control only secondary antibodies were used. Cells were visualized using confocal micro- scope FV-500 (Olympus Optical Co., Hamburg, Germany) or scanning cytometry—SCAN^R Screening System (Olympus Optical Co., Hamburg, Germany). The fluores- cence excitation was provided by 488 and 543 nm He–Ne laser beams. Fluorescences were measured using dichroic mirrors and filters for 505, 525, 560 and 610 nm wave- lengths. Acquired data were stored in a series of 12 bit grey images separately and colored artificially by software.
Ultrastructural studies
Cells were fixed in 2% para-formaldehyde and 2.5% glu- taraldehyde in 0.1 M sodium cacodylate buffer (pH 7.4) for 2 h at 4°C. Cells were washed with the same buffer and post-fixed with 1% OsO4 in 0.1 M sodium cacodylate buffer for 1 h. Cells were dehydrated in a graded ethanol alcohol series, and embedded in Epon 812. Ultrathin sec- tions were processed according to the post-embedding procedure. The sections were mounted on the formvar- coated nickel grids, air-dried, and stained for 10 min with 4.7% uranyl acetate and for 2 min with lead citrate. The sections were examined and photographed with a JEM 1011 electron microscope.
Cell subfractionation and detection of cytochrome c
Cells were first collected and washed with cold PBS once, then resuspended in 500 ll ice-cold HIM buffer (200 mM D-mannitol, 70 mM sucrose, 10 mM HEPES, 1 mM EGTA, pH 7.4) supplemented with 0.4 mM PMSF, 10 lg/ml of aprotinin, 1 mM DTT and 10 lg/ml of sodium ortho- vanadate. Cells were incubated on ice for 15 min and then passed through a gauge #25 (25G) needle 20 times to disrupt cell membranes. The cell homogenates were applied to a series of centrifugations at 1,000g for 10 min and 4°C followed by 9,000g for 20 min, 4°C to fractionate unbroken cells, and nuclear fraction, and mitochondrial fraction, respectively. After the final centrifugation the supernatant was collected as the cytosolic fraction. Equal amounts of cytosolic or mitochondrial proteins were sub- jected to Western-blot analysis to evaluate the level of cytochrome c. Membranes were also probed with goat polyclonal anti-b-actin or anti-COX-IV antibodies to nor- malize cytosolic and mitochondrial protein level, respectively.
Sample preparation for electrophoresis and immunoblotting
To obtain whole-cell lysates a 1 ml aliquot of ice-cold PBS was added and cells were immediately scraped from the plastics and collected by centrifugation (10,000g for 10 min, 4°C). An 1.0 ml aliquot of RIPA buffer (19 PBS, 10 ml/l Igepal CA-630, 5 g/l sodium deoxycholate, 1 g/l SDS) supplemented with 0.4 mM PMSF, 10 lg/ml of aprotinin and 10 lg/ml of sodium orthovanadate was added to lyse the cell pellet and cells were broke up by repetitive triturating with the syringe with attached needle (21G,0.8 mm diameter). Cell suspension was then left on ice (4°C) for 30 min, and centrifuged for another 5 min (4°C, 10,000g). The resulting viscous solution was divided into smaller volumes and transferred to fresh Eppendorf tubes and stored at -80°C until used. Soluble protein concen- tration in the lysates was determined by a protein-dye- binding method [16] with a commercial reagent (Bio-Rad Laboratories, Hercules, CA, USA).
Electrophoresis and immunoblotting
Equal amounts of sample protein (either 50 or 30 lg) isolated from the treated or untreated COLO 205 cells were then resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) followed by immuno- blotting. The electrotransfer of proteins to PVDF membranes (0.2 lm) was performed for 1.5 h at 100 V and followed by overnight blocking (4°C) in TBS buffer (20 mM Tris, 500 mM NaCl, pH 7.5) supplemented with 50 g/l non-fat powdered milk. After washing in TBST (TBS containing 0.5 ml/l Tween 20), the membranes were immunostained by standard methods provided by the manufacturer (Santa Cruz, CA, USA). They were probed with a primary antibody (mouse monoclonal anti-cFLIPL, anti-TNF-R1, anti-COX IV, anti-procaspase-8, rabbit polyclonal anti-FADD, anti-procaspase-3, anti-Bcl-2, anti- cytochrome c and goat polyclonal anti-TRADD, anti-Bid, anti-procaspase-9 antiserum) (Santa Cruz, CA, USA) for 1 h at 20°C or overnight at 4°C, washed three times in TBST and were further incubated with the secondary donkey anti-rabbit or anti-mouse or anti-goat antibody conjugated with HRP (see Reagent’s section). Membranes were also probed with goat polyclonal anti-b-actin anti- body to normalize protein levels. The blots were developed using the enhanced chemiluminescence (ECL) detection system (Amersham International, Aylesbury, UK) accord- ing to the manufacturer’s protocol. After exposure, photographs were taken with a Kodak DC 290 zoom digital camera and were scanned and analyzed using the Kodak EDAS 290/Kodak 1D 3.5 system.
Immunoprecipitation and Western-blotting
Whole-cell lysates containing 900 lg of protein were incubated overnight at 4°C with 1.5 lg (7.5 ll) rabbit polyclonal anti-FADD and an additional 3 h with 30 ll protein A/G bead slurry (Santa Cruz Biotechnology, Santa Cruz, CA, USA). Beads were then washed four times with cold RIPA buffer, boiled in sample buffer (29 Laemmli buffer) for 3–5 min, separated by 10% SDS-PAGE, trans- ferred to a PVDF membrane and probed with a mouse monoclonal anti-FLIPL IgG antibody (1 lg/ml) for detec- tion of endogenous protein associations. For detection of activated proteins, RIPA lysates from the same collections were separated by SDS-PAGE, transferred to a PVDF membrane (0.2 lm) and probed with appropriate antiserum as previously described. Finally, probing with primary antibody against immunoprecipitation antibody and sub- sequent species specific secondary antibody were used to verify equal protein loading. The enhanced chemilumi- nescence (ECL or ECL Plus) method was used for protein detection (Amersham International, Aylesbury, UK).
Bcl-2 protein cellular delivery
Recombinant biologically functional Bcl-2 protein (R&D Systems Inc., Minneapolis, MN, USA) was delivered into COLO 205 cells with SAINT-PhD [1-methyl-4-(cis-9- dioleyl)methylpyridinium-chloride (SAINT-18) 1,2-Dio- leyl-sn-glycero-3-phosphoethanolamine (DOPE)] reagent according to the producer’s instruction (Synvolux Thera- peutics, Groningen, The Netherlands). For Western blot analysis cells were cultured on 60 mm Petri dish. When cells became confluent the profection procedure was per- formed. A 20 lg of Bcl-2 protein was diluted with the HEPES buffered salt (HBS buffer, pH 7.4) to achieve a volume of 300 ll. Then, 200 ll of SAINT-PhD into pro- tein/HBS solution was added. The mixture was filled up to 2.5 ml with 10% FBS/DMEM medium. Just prior to pro- fection the medium was aspirated and cells were washed once with HBS. Then SAINT-PhD/protein complexes were overlaid onto the washed cells and incubated at 37°C for 4 h. After incubation the transfection mixture was filled up to 4 ml with extra medium. When performing profection in culture plates of various sizes the quantity of HBS and SAINT-PhD reagents were scaled in proportion to the surface area of the plate. In case of Western blot analysis the level of Bcl-2 protein was examined 24 h after trans- fection. For MTT assay, when transfection procedure was completed, cells were treated with tested compounds and cell viability was evaluated at 6, 12 or 24 h of experiment.
Statistical analysis
Each treatment was carried out in triplicate and each experiment was repeated at least twice. The results were statistically evaluated with one-way ANOVA and Tukey’s multiple range test when compared to control treatments. These analyses were performed using GraphPad PrismTM version 4.03 software (GraphPad Software Inc., San Diego, CA, USA). In order to show the quantitative differences, the percentage of initial control value was set arbitrarily as 100% (experimental value/initial control value 9 100) at each time point used. Statistical differences were inter- preted as significant at P \ 0.05 and highly significant at P \ 0.01.
Results
Bisindolylmaleimide IX potentiates TNF-a-mediated apoptosis Examination of human colon adenocarcinoma COLO 205 cells revealed that upon treatment with Bis-IX the cells became highly sensitive to the apoptotic effect of TNF-a (Fig. 1b). Irrespective to the presence of functional TNF- R1 receptors on plasma membrane of COLO 205 cells [17] it was previously reported that these cells are resistant to apoptotic stimuli induced by TNF-a [7]. Therefore it is obvious that the incubation of COLO 205 cells with a physiological concentration of TNF-a (10 ng/ml) had no detectable effect on cell morphology (Fig. 1a), apoptotic index (Fig. 1d), cell survival (Fig. 3b), or procaspase-3 level (Fig. 1e) compared to that of cells kept in control conditions. In contrast, after incubation with both TNF-a (10 ng/ml) and Bis-IX (5 lM) a significant number of cells became round, with apparent membrane blebbing and condensed nuclei (Fig. 2). Overall, these changes are typ- ical for apoptosis detected at least in the 6th-h of experiment (Fig. 1b). The apoptotic index (AI) ranged from 1.27 ± 0.32 for control to 28.67 ± 4.43,52.28 ± 3.89 and 67.67 ± 7.25 for concomitant TNF-a and Bis-IX treatment at 6, 12 and 24 h, respectively (Fig. 1d). Moreover, the MTT assay confirmed reduced viability of COLO 205 cells (Fig. 3a). Accordingly, the hallmarks of apoptosis were elevated as demonstrated by SDS-PAGE and immunoblotting which showed time- dependent reduction of procaspase-3 levels (Fig. 1e). Importantly, both cytochemistry (PI/HO33342 staining) and Western-blot analysis indicated, that even though Bis- IX (5 lM) was apparently proapoptotic, it moderately affected COLO 205 cells as well as procaspase-3 level when used individually (Fig. 1c–e). These data clearly show that combined treatment of Bis-IX and TNF-a resulted in a rapid and extensive death of COLO 205 cells suggesting the synergy of the effects of Bis-IX and TNF-a.
Fig. 1 Series of images of COLO 205 cells obtained from fluorescent microscope under UV light. Nuclear chromatin is shown after serial staining with propidium iodide/bisbenzimide (see Materials and Methods section). (a) COLO 205 cells are immune to TNF-a- dependent apoptosis. Neither chromatin condensation nor nuclei fragmentation were detected after TNF-a (10 ng/ml) treatment regardless of time (6, 12 and 24 h). (b) Bis-IX facilitates TNF-a- dependent apoptosis in COLO 205 cells. TNF-a (10 ng/ml) and Bis- IX (5 lM) co-treatment induced both chromatin condensation and fragmentation of nuclei. (c) Bis-IX (5 lM) treatment induced significant increase in both chromatin condensation and nuclei fragmentation (at least in the 12th-h of experiment). Images were acquired under 209 lens and 59 digital zoom. (d) Bar charts representing apoptotic indices (AI) calculated for TNF-a amounted to 2.95 ± 0.41, 3.27 ± 0.74 and 3.71 ± 0.59 (P [ 0.05) in the 6th, 12th and 24th-h of treatment, respectively; for TNF-a + Bis-IX amounted to 1.27 ± 0.32, 28.67 ± 4.42 and 52.28 ± 3.89 in the 6th, 12th and 24th-h of treatment, respectively; and for Bis-IX amounted to 1.27 ± 0.32, 3.60 ± 0.72 and 12.03 ± 1.51 at 6th, 12th and 24th-h of treatment, respectively. Significant differences between the treat- ment means and control value are indicated by * P \ 0.05,** P \ 0.01 and *** P \ 0.001. (e) Western-blot analyses of whole-cell lysates obtained from COLO 205 cells. Immunoblots show the expression of procaspase-3 protein after TNF-a (10 ng/ml), Bis-IX (5 lM) and TNF-a (10 ng/ml) + Bis-IX (5 lM) treatments at 6, 12 and 24 h of treatment.
Bis-IX facilitates TNF-a-mediated apoptosis in a dose-dependent manner
The dose dependency of Bis-IX on TNF-a-mediated apoptosis of COLO 205 cells was determined using Hoe- chst 33342/PI staining to quantify apoptotic cells in membrane are present, bar—1 lm. (b) The early apoptotic cell; cell exhibits nuclear condensation and fragmentation, cell shrinkage and ruffled nuclear and cell membranes; the cytoplasmic condensation with ribosome loss from rough endoplasmic reticulum is present in the 6th-h of combined treatment with TNF-a and Bis-IX. Note fragments of ultrastructurally unchanged COLO 205 cell in the vicinity to apoptotic cell, bar—2 lm. (c) Typical apoptotic cell; note pyknosis of nucleus with chromatin and cytoplasmic condensation, fragmentation to apoptotic bodies in the 12th-h of combined treatment with TNF-a and Bis-IX, bar—1 lm. (d) Late apoptotic cells; very small cells containing condensed, or ‘‘non-transparent’’ chromatin, which is segregated into crescent and caps. Some cells are disinte- grated into apoptotic bodies in the 24th-h of combined treatment with TNF-a and Bis-IX, bar—2 lm. Each experiment was repeated at least twice with similar results cultures exposed to varying concentrations of Bis-IX. In the absence of TNF-a, Bis-IX (5, 10 lM) moderately, but significantly impaired viability of COLO 205 cells though these effects were almost identical (Fig. 3a). Moreover, in the absence of Bis-IX, TNF-a did not exert convincing cytotoxic effect (Fig. 3b). In the presence of Bis-IX, however, (even at lower concentrations range) a dramatic increase in TNF-a-dependent apoptosis was noticed (Fig. 3b). This time, the potentiating effect of Bis-IX on apoptosis was both dose and time-dependent. With higher concentration of Bis-IX, there is a greater rate of apoptosis induced by TNF-a (Fig. 3b). Thus, at 10 ng/ml TNF-a, the potentiating effect of Bis-IX was clearly evident at con- centrations as low as 1.25 lM of Bis-IX, whereas nearly complete apoptosis was induced with 5 lM of Bis-IX after 24 h.
Fig. 2 Images of the same visual field of TNF-a-(10 ng/ml) and Bis- IX (5 lM)-treated COLO 205 cells in phase contrast (a) versus fluorescent UV (b) light. Arrows indicate the cells with hallmarks of apoptosis, such as cell shrinkage, membrane blebbing and fragmen- tation to apoptotic bodies shown in visual light (left). In the same cells the chromatin condensation is evident when observed under fluores- cent UV light (right). Images were acquired under 409 lens and 59 digital zoom. (c) Ultrathin sections of COLO 205 cells fixed in 2.5% glutaraldehyde and 2% paraformaldehyde, postfixed in OsO4 + K3- Fe(CN)6 and embedded in Epon 812. Electron microscopy evaluation of Bis-IX-mediated (5 lM) sensitization to TNF-a-induced (10 ng/ ml) apoptosis. From left: (a) Control cell ultrastructurally unchanged containing oval nucleus with cytoplasm enriched in organelles (mitochondria, endoplasmic reticulum, Golgi complex). In cytoplasm, abundant small mitochondria are mainly distributed in perinuclear region and show tendency to fuse. Numerous microvilli in plasma.
Fig. 3 (a) TNF-a and Bis-IX treatment diminishes viability of COLO 205 cells. MTT assay showing dose-response effect of Bis-IX (5 lM ( ) or 10 lM (u)) or TNF-a (10 ng/ml) and Bis-IX (5 lM) (h) co- treatment (6, 12, 24 h) on viability of COLO 205 cells. Significant differences between the treatment means and control values at respective times are indicated by * P \ 0.05, ** P \ 0.01 and *** P \ 0.001. Values represent means from two identical experi- ments carried out in quadruplicates ±SEM. (b) Dose dependencies of Bis-IX and TNF-a (10 ng/ml) in potentiation of apoptosis. COLO 205 cells were incubated for 6, 12 and 24 h without (d) or with TNF-a (e) (10 ng/ml), and with 1.25 lM (.), 2.5 lM (h), or 5 lM (■) of Bis-IX. Apoptosis was determined by propidium iodide/bisbenzimide staining. Values shown are mean ± SEM of AI calculated as described in Materials and Methods. Significant differences between the treatment means and control value are indicated by * P \ 0.05,** P \ 0.01 and *** P \ 0.001.
Apoptosis with bisindolylmaleimide IX is not PKC-dependent
Marked Bis-IX potentiation of TNF-a-induced apoptosis prompted us to determine whether bisindolylmaleimide III (Bis-III) exerts a similar effect. In particular it was of interest to determine whether this effect is related to the inhibition of PKC. Thus, we compared the effect of Bis-III versus another potent PKC inhibitor, staurosporine (STS), on TNF-a-dependent apoptosis. The capability of Bis-III to inhibit PKC activity was previously well documented, for example by Han et al. [18] and Brehmer et al. [19]. Sim- ilarly, STS is a well known powerful inhibitor of the PKC family [20]. Moreover, both inhibitors are often used to repress PKC activity in routine studies. Based on the MTT assay, we found that Bis-III (20 lM) inhibited proliferation rather than sensitized COLO 205 cells to TNF-a-dependent apoptosis (Fig. 4a). The latter was confirmed by Western- blot analysis, which did not provide any evidence of the reduction in the procaspase-3 protein level (Fig. 4b and c). Correspondingly, another PKC inhibitor (STS) structurally unrelated to bisindolylmaleimide failed to potentiate TNF- a-mediated apoptosis (Fig. 4d). To verify the role of PKC inhibitors, COLO 205 cells were pretreated with 20 lM of Bis-III for 60 min and then 5 lM of Bis-IX and 10 ng/ml of TNF-a were added to the cell culture and incubation continued for 20 h. The ability of Bis-IX to sensitize COLO 205 cells to TNF-a-dependent apoptosis was not affected by the presence of Bis-III (data not shown), sug- gesting that Bis-IX interacts with a distinct cellular target(s) than Bis-III. These results indicate that the inhibition of PKC cannot substantiate the extensive cell death induced by both TNF-a and Bis-IX co-treatment.
Fig. 4 Bis-III does not potentiate TNF-a-induced apoptosis. (a) MTT assay. The effect of combined TNF-a (10 ng/ml) and Bis-III (20 lM) treatment (.) on viability of COLO 205 cells (6, 12, 24 h). Significant differences between the treatment means and control values at respective times are indicated by * P \ 0.05, ** P \ 0.01 and *** P \ 0.001. Values represent means from two identical experiments carried out in quadruplicates ±SEM. (b) Western-blot analyses of whole-cell lysates obtained from COLO 205 cells. Immunoblots showing the expression of procaspase-3 protein after TNF-a (10 ng/ml) and Bis-III (20 lM) co-treatment at 6, 12 and 24 h.
Bisindolylmaleimide IX reduces the cFLIP protein level and affects the expression of other TNF-R1 signalosome components in COLO 205 cell line
Based on our previous observations that the sensitization of COLO 205 cells to TNF-a-mediated apoptosis by metabolic inhibitors, such as cycloheximide (CHX) [7] or sodium butyrate (NaBt) [21] is associated with the reduced level of antiapoptotic protein, cFLIPL, we performed both Western- blot and immunoprecipitation studies to examine the influ- ence of Bis-IX on the level of cFLIPL protein. Additionally, we determined the level of other TNF-R1 signalosome components, such as TNF-R1, TRADD and FADD. As shown on Fig. 5, the combined TNF-a and Bis-IX treatments resulted in a time-dependent decline in cFLIPL protein level. Furthermore, the immunoprecipitation analysis confirmed that the cFLIPL level was diminished in DISC complex of treatment. (c) Bar charts represent relative integrated optical density values of the respective blots. Each experiment was repeated at least twice with similar results. Data are representative values. (d) PKC inhibitor staurosporine (STS) does not sensitize COLO 205 cells to TNF-a-induced apoptosis. MTT assay showing viability of COLO 205 cells after STS (5 lM) and TNF-a (10 ng/ml) co-treatment ( ) (6, 12, 24 h). Significant differences between the treatment means and control values at respective times are indicated by * P \ 0.05,** P \ 0.01 and *** P \ 0.001. Values represent means from two identical experiments carried out in quadruplicates ±SEM (Fig. 5c). Concomitant treatment with TNF-a and Bis-IX influenced the expression of other TNF-R1 signalosome proteins, either. As shown on Fig. 5a and b TNF-R1 and TRADD proteins were down-regulated, especially after 24 h of incubation, whereas FADD level was elevated. These results suggest that Bis-IX promotes DISC complex (TNF- R1-TRADD-FADD-FLICE) formation and it impairs the formation of TRADD-TRAF2-RIP. In a consequence, apoptosis was facilitated and cell death occurred.
Bisindolylmaleimide-IX initiates intrinsic apoptotic pathway
The effect of the TNF-a and bisindolylmaleimide-IX on mitochondrial integrity was studied by examining the processing of Bid, procaspase-8 and procaspase-9 proteins and the release of cytochrome c from mitochondria to the cytoplasm. We used Western-blot and/or scanning cytom- etry to evaluate the levels of procaspase-9 and active form of caspase-9. From data shown in Fig. 6a it is evident that had also diminished. Simultaneously, the level of cleaved form of caspase-9 increased highly significantly in relation to control cells (Fig. 7a). The presence of caspase-8 inhibitor z-IETD-fmk (20 lM) (Fig. 6b) resulted in lack of procaspase-8 activation and Bid cleavage. However, the cytochrome c was still released and procaspase-9 level declined. As shown of Fig. 6c, the presence of pan-caspase inhibitor z-VAD-fmk (10 lM) abrogated TNF-a and Bis- IX-induced apoptosis. As a consequence there was neither reduction in procaspase-3 and -9, nor Bid cleavage and cytochrome c release. It is noteworthy that z-IETD-fmk postponed the reduction of procaspase-3 level induced by TNF-a and Bis-IX co-treatment until the 24th-h of treat- ment (Fig. 6b). The effects of z-IETD-fmk and z-VAD- fmk inhibitors on Bis-IX or TNF-a and Bis-IX-induced cell death were also evaluated by MTT assay. It appeared that the viability of Bis-IX-treated cells was diminished.
Fig. 5 The effect of Bis-IX treatment on the expression of TNF-R1 signalosome components. (a) Western-blot analyses of whole-cell lysates obtained from COLO 205 cells. Immunoblots show the expression of cFLIP, TNF-R1, FADD and TRADD proteins after TNF-a (10 ng/ml) and Bis-IX (5 lM) co-treatment at 6, 12 and 24 h. (b) Bar charts represent relative integrated optical density values of the respective blots. Each experiment was repeated at least twice with similar results. Data are representative values. (c) The analysis of protein–protein interactions analysed by immunoprecipitation. The whole-cell lysates obtained from COLO 205 cells treated simulta- neously with TNF-a (10 ng/ml) and Bis-IX (5 lM) at 6, 12 and 24 h versus untreated cells (CTRL) were immunoprecipitated with anti- FADD antibody absorbed on A/G agarose beads. cFLIP protein was detected by immunoblotting following electrophoresis of the cell lysates. Each experiment was repeated at least twice with similar results. Data are representative values.
Fig. 6 Bis-IX and TNF-a-mediated activation of intrinsic apoptotic pathway in COLO 205 cells. Western-blot analyses of whole-cell or cytosolic and mitochondrial lysates obtained from COLO 205 cells. Immunoblots showing the expression of procaspase-8, procaspase-9, Bid, procaspase-3 and cytochrome c proteins after TNF-a (10 ng/ml) and Bis-IX (5 lM) treatment (6, 12 and 24 h) without (a) or with the
TNF-a and Bis-IX co-treatment caused time-dependent cleavage of Bid and procaspase-8 proteins and the release of cytochrome c. At the same time, the procaspase-9 level presence of (b) dominant negative caspase-8 inhibitor (z-IETD-fmk) (20 lM) or (c) pan-caspase inhibitor (z-VAD-fmk) (10 lM). Mem- branes were also probed with goat polyclonal anti-b-actin or COX-IV antibodies to normalize cytosolic (c) and mitochondrial (mt) protein levels, respectively.
Fig. 7 (a) Top: Gallery of representative COLO 205 cells relocated by the SCAN^R system from the cytogram region of high active caspase-9 fluorescence in TNF-a and Bis-IX-treated cells, in comparison to control (CTRL) cells. Gallery clearly shows that in almost all of TNF-a and Bis-IX-treated cells the level of caspase-9 was elevated. Caspase-9 was visualized by Alexa Fluor 568 (red fluorescence), and cell nuclei were stained with SYTOX green (green fluorescence). In COLO 205 treated cells the nuclei condensation was evident by intensive green fluorescence. Lens magnification 409. Bottom: Quantitative evaluation of caspase-9 level based on SCAN^R scanning cytometry. Bar charts represent percentage of cells with high caspase-9 fluorescence in comparison to total number of cells,3.06 ± 3.04 for CTRL and 54.28 ± 4.49 for TNF-a and Bis-IX-co- treated cells. More than 5,000 cells were analyzed in each visual filed. Values represent means from two identical experiments ±SEM. (b) MTT assay showing viability of COLO 205 cells after Bis-IX (5 lM) (D) or Bis-IX (5 lM) and z-IETD-fmk (20 lM) (■) or Bis-IX (5 lM) and z-VAD-fmk (10 lM) (s) treatment. (c) MTT assay showing viability of COLO 205 cells after Bis-IX (5 lM) (D) or TNF-a (10 ng/ml) and Bis-IX (5 lM) (h) or TNF-a (10 ng/ml) and Bis-IX (5 lM) and z-IETD-fmk (20 lM) (■) or TNF-a (10 ng/ml) and Bis- IX (5 lM) and z-VAD-fmk (10 lM) (s) treatments (6, 12, 24 h). Means ± SEM bearing different small letters within particular treatment differ at least significantly P \ 0.05. Significant differences between the treatment means at respective times are indicated by letters. Values represent means from two identical experiments carried out in quadruplicates. (d) Western-blot analyses of whole-cell lysates obtained from COLO 205 cells. Immunoblots showing the expression of Bcl-2 protein at 24th-h after mock- or Bcl-2-transfec- tion. Membranes were also probed with goat polyclonal anti-b-actin antibody to normalize protein level. (e) MTT assay showing viability of mock- (■) or Bcl-2-transfected COLO 205 cells. Additionally, the effect of elevated Bcl-2 protein level on Bis-IX (5 lM) (D) or TNF-a (10 ng/ml) and Bis-IX (5 lM) (h) or TNF-a (10 ng/ml) and Bis-IX (5 lM) and z-IETD-fmk (20 lM) (s) (6, 12, 24 h) action was examined. Means ± SEM bearing different small letters within particular treatment differ at least significantly P \ 0.05. Significant differences between the treatment means at respective times are indicated by letters. Values represent means from two identical experiments carried out in quadruplicates particular at 12 and 24 h of experiment (Fig. 3a). As shown on Fig. 7b the presence of z-VAD-fmk ended Bis-IX cytotoxic action, whereas this effect was not observed when z-IETD-fmk was used to block caspase-8 activity. When Bis-IX was added together with TNF-a (Fig. 3a) the percentage of viable cells dramatically decreased. Z-IETD- fmk, which inhibited extracellular apoptosis, partially protected COLO 205 cells (Fig. 7c) from TNF-a and Bis- IX cytotoxicity. The viability of z-VAD-fmk-treated cells upon TNF-a and Bis-IX stimulation was almost similar to control cells (Fig. 7c). These results suggested that acti- vation of intrinsic apoptotic pathway resulted from TNF-a- independent Bis-IX action and that TNF-a further aug- mented cell death. To verify this assumption, we delivered biologically functional Bcl-2 protein into COLO 205 in order to evaluate whether intrinsic antiapoptotic protein protects tumor cells from Bis-IX-induced apoptosis. Wes- tern-blot analysis confirmed the efficient Bcl-2 profection (protein-transfection) of COLO 205 cells (Fig. 7d). Addi- tionally, the MTT assay showed that elevated level of Bcl- 2 inhibited Bis-IX-induced cell death, whereas it did not affect TNF-a and Bis-IX-induced extrinsic apoptosis (Fig. 7e). Thus, Bcl-2-transfected cells were totally pro- tected from TNF-a and Bis-IX-mediated cytotoxicity when caspase 8 inhibitor z-IETD-fmk was present (Fig. 7e). We conclude that Bis-IX is a potent proapoptotic factor for colon adenocarcinoma cells, in particular when combined with TNF-a.
Discussion
One promising tool for treating tumor cells immune to apoptosis is to make use of the cytotoxic drugs which should sensitize cancer cells to death-inducing ligands [22, 23]. From this point of view, certain bisindolylmaleimides are believed to modulate the resistance of human colon adenocarcinoma COLO 205 cells to TNF-a-induced apoptosis [7, 17]. Alternatively, cytotoxic drugs can induce cell death via the up-regulation of death receptors [24]. In this case, however, we found that the expression of TNF- R1 receptors was down-regulated after 24 h co-treatment with TNF-a and Bis-IX (Fig. 5a). These results are in agreement with observations reported by Rokhlin et al. [25], who found that Bis-IX alone did not affect the expression of receptors such as: CD95, TRAIL-R1 and TRAIL-R2. We provide evidence that in contrast to the cytotoxic drugs which up-regulate the expression of cell surface death receptors [24, 26], Bis-IX mediates its effect by a novel mechanism. It became apparent that Bis-IX mediated sensitization of COLO 205 cells to TNF-a- dependent apoptosis (Figs. 1–3). It most likely resulted from the repression of factor(s) which inhibited death signal cascade. Some of our previous results demonstrated that COLO 205 cells are insensitive to TNF-a-induced apoptosis because caspase-8 activation is inhibited by the cFLIP protein [7, 21]. Moreover, the knock-down of cFLIP restored the susceptibility of COLO 205 cells to the apoptotic signal from TNF-R1 (data not published). To verify this hypothesis, we performed Western-blot and immunoprecipitation studies to evaluate the levels of TNF- R1 signalosome components after Bis-IX or Bis-III administration. As shown on Fig. 5, the presence of Bis-IX resulted in a time-dependent down-regulation of cFLIP and TRADD proteins with concomitant increase in FADD protein. The modulation of cFLIP protein by bis- indolylmaleimides was previously reported by Willems et al. [27], who found that in dendritic cells (DC) Bis-III administration down-regulates the cFLIPL level and sen- sitizes DC cells to Fas-mediated apoptosis. On the other hand, Rokhlin et al. [25] did not observe any changes in the expression of cFLIPS/L, IAP1, IAP2 and XIAP antiapo- ptotic proteins upon Bis-IX treatment of LNCaP prostate cancer cells. It suggests, that Bis derivatives could act differently. Our data clearly demonstrated that Bis-IX but not Bis III allowed TNF-a to induce apoptosis. Thus, in contrast to Willems et al. [27], Bis-III did not potentiate TNF-a-dependent cell death in COLO 205 cells (Figs. 1, 3 and 4a, b). Correspondingly, we conclude that the cytotoxic effects of Bis IX and Bis III are dependent on the cell type. In our cellular model, combined TNF-a and Bis-III treat- ment resulted in retarded growth of colorectal cancer cells. This observation is in agreement with Han et al [18] who have shown, that Bis-III, Bis-II and Bis-I evoke merely cytostatic effects on HL-60 acute myeloid leukaemic cells, whereas Bis-IX (Ro-31-8220) did induce apoptosis. In addition, the Western-blot analyses of Bis-III-treated COLO 205 cells showed that in contrast to Bis-IX, Bis-III did not affect the TNF-R1 signalosome components (data not shown). Our results support the observations of Zhou et al. [13] who found that among nine tested Bis deriva- tives ultimately Bis-VIII and Bis-IX promoted Fas-induced apoptosis rather then Bis-I, Bis-II, Bis-III, Bis-IV, Bis-V, Bis-X and Bis-XI. It is worth to note, that Bisindolylma- leimides were initially described as PKC inhibitors [10, 11, 28]. In particular, Bis-III is believed to block PKC- dependent activation of c-Raf-1, the known downstream target of PKC [18]. Recently, several reports have indi- cated that Bisindolylmaleimides also act on other cellular targets. It is now apparent that they might affect gene transcription [25], the activity of MKP-1 and JNK [29], or that they reverse MDR activity [30]. Some authors claim that the Bis derivatives’ cytotoxic effects are exclusively PKC-independent [13]. According to Fig. 4, another PKC inhibitor (STS), does not seem to facilitate the TNF-a- dependent apoptosis. Similarly, the blockage of PKC by Bis-III did not affect TNF-a and Bis-IX-induced apoptosis (data not shown). It is concluded that the observed effect of Bis-IX did not result from PKC inhibition but it occurred at another level of cell regulation.
Rokhlin et al. [25] demonstrated that Bis-IX could act as a transcription factor in LNCaP prostate cancer cells. The authors evaluated the levels of p21/WAF-1, p53 and MDM-2 proteins after individual treatment with actino- mycin D (AD), cycloheximide (CHX) or Bis-IX. The results of immunoblotting have shown that the expression of certain proteins was affected similarly by AD or Bis-IX but rather differently from CHX. Surprisingly, in COLO 205 colon cancer cells, CHX but not AD sensitized cells to TNF-a-dependent apoptosis [7]. Moreover, Bis-IX and CHX, but not AD, reduced cFLIP protein levels in the 24-h experiment. Therefore, Bis-IX is likely to modulate the translation process of short-lived proteins; however the exact mechanism of how Bis-IX smoothened the progress of TNF-a-dependent apoptosis needs further examination. The present study also illustrates the effect of Bis-IX in conjuction with the mitochondria-dependent apoptotic pathway. As shown on Fig. 6, the combined Bis-IX and TNF-a treatment led to both procaspase-8 and Bid cleav- ages. At the same time, the cytochrome c was released from mitochondria and caspase-9 was activated. The pos- sible explanation for caspase-9 activation was suggested by Rokhlin et al. [25]. He and his colleagues noticed that Bis- IX is an innate fluorochrome and fluoresces within the cells. Detailed scrutiny revealed that mitochondria accu- mulated Bis-IX as early as 2 h after Bis-IX administration. Furthermore, Snowden et al. [31] found out that Bis-IX is able to induce intrinsic apoptotic pathway by conforma- tional changes and subcellular redistribution of Bax, followed by the activation of caspase-9 in chronic lym- phocytic leukaemic (CLL) cells. In CLL cells Bis-IX- induced apoptosis was also accompanied by a rapid caspase-mediated cleavage of Mcl-1 antiapoptotic protein. Interestingly, previous studies [32] performed on CLL cells revealed that Bis-IX does not sensitize CLL cells to TRAIL-induced apoptosis neither it affects the cFLIP protein level. It confirms our assumption that Bis-IX effects are cell type specific. Presumably, Bid and procaspase-9 cleavages resulted from the intrinsic apoptotic cascade activation in COLO 205 cells. However, it should be kept in mind that Bid cleavage could occur by caspase-8 acti- vation in DISC complex (type II cell death) [33, 34]. The truncated Bid (tBid) is able to translocate to the mito- chondrial membrane where it is complexed with Bax and a voltage-dependent anion channel (VDAC). Such com- plexes allow the release of mitochondrial components of apoptosome (dATP, cytochrome c). Consequently, apop- tosome triggers procaspase-9 conversion into active caspase-9. To verify whether the observed changes in Bid, procaspase-9 and cytochrome c levels are associated with intrinsic apoptosis, the caspase-8 inhibitor (z-IETD-fmk) has been used. Z-IETD-fmk prevented Bid and procasapse- 8 cleavage, whereas it did not affect procaspase-9 con- version into caspase-9 or cytochrome c release. In the presence of caspase-8 inhibitor, however, the TNF-a and Bis-IX co-treatment caused a delay in caspase-3 activation (Fig. 6b). Additionally, cell viability was significantly elevated with regard to TNF-a and Bis-IX co-treatment. Moreover, viability was not significantly different when compared with Bis-IX alone (Fig. 7c). Furthermore, the combined TNF-a and Bis-IX treatment did not reduced cell viability when z-VAD-fmk was used (Fig. 7d). However, it is noteworthy, that in these circumstances cell growth was inhibited and the Bis-IX biological effect was similar to Bis-III action. The presented results corroborate the idea, that Bis-IX facilitated TNF-a-induced apoptosis by both extrinsic (caspase-8-dependent) and mitochondria-depen- dent PCD (Fig. 6a). However, Bis-IX strongly stimulates mitochondria to release cytochrome c with subsequent activation of caspase-9 in TNF-a-independent manner. It was confirmed when Bcl-2 delivery was able to inhibit intrinsic but not extrinsic apoptosis (Fig. 7e). Taken these results together, it is apparent that both TNF-a and Bis-IX induce extrinsic apoptosis, and that this effect is amplified by Bis-IX who targeted mitochondria. We draw the picture which shows the molecular mechanism of Bis-IX death- promoting action (Fig. 8). Further approach is to determine the proteins specifically targeted by this inhibitor. It would be advantageous if Bis-IX does not induce apoptosis in normal cells. The latter point was supported by the results of Zhou et al. [13] who injected rats with 250 lg of Bis- VIII every other day using five doses without any detect- able side effects. These authors also demonstrated that Bis- VIII did not exert any substantial effect on activated T cells while having little effect on non-activated T cells.
Fig. 8 Schematic illustration of distinct signaling pathways induced by TNF-a and Bis-IX co-treatment in COLO 205 cells. Bis-IX- mediated down-regulation of cFLIP sensitizes COLO 205 cells to TNF-a-induced apoptosis. DISC complex facilitates caspase-8 for- mation and subsequent procaspase-3 activation. At the same time the caspase-8-dependent Bid cleavage occurs. Then, tBid forms com- plexes with Bax and VDAC to release cytochrome c. As a consequence, the procaspase-9 is activated to induce caspase-3. The presence of specific dominant-negative caspase-8 inhibitor (z-IETD- fmk) prevented extrinsic apoptosis. On the other hand, Bis-IX initiates intrinsic apoptotic pathway abrogated next to the overexpression of Bcl-2 protein which inhibits mitochondrial apoptotic pathway. Finally, the pan-caspase z-VAD-fmk inhibitor protected COLO 205 cells from both extrinsic and intrinsic apoptosis.
Conclusion
Our results suggest that Bis-IX may serve as a good can- didate for colon anti-cancer therapy. Bis-IX is able to potentiate TNF-a-induced apoptosis in COLO 205 cells by eliminating the antiapoptotic protein (cFLIP). Bis-IX treatment activates intrinsic apoptotic pathway with no contribution of TNF-a. It is expected that the inhibition of gene expression (transcription or translation) by Bis-IX would not induce apoptosis in normal cells. Our data suggests that Bis-IX should be seriously considered as the alternative adjuvant in therapeutic anti-cancer strategies.
Acknowledgments Support for this work was provided by grant No. N312 012 32/0761 from the State Committee for Scientific Research in Poland. We thank Dr. Patrycja Pawlikowska (Cochin Institute, INSERM, Paris, France) for the procedure of subcellular fractionation. We appreciate Dr. Elizabeth M. Sajdel-Sulkowska, Department of Psychiatry, Brigham and Women’s Hospital, Assistant Professor of Psychiatry, Harvard Medical School, USA, and Dr. Vic Narurkar, Davis and Bay Area Laser Institute, San Francisco, Cali- fornia, USA, for their help in preparation of the manuscript. Beata Pajak dedicates this paper to Iwona Szymanska for her outstanding support and friendly attitude.
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