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Critical role for c-FLIPL on Fas resistance in colon carcinoma cell line HT-29
Cell Biology International 32 (2008) 329e336 www.elsevier.com/locate/cellbi
Critical role for c-FLIPL on Fas resistance in colon carcinoma cell line HT-29 Fenglin Zang a,b,1, Baocun Sun a,c,d,*,1, Xiulan Zhao d, Ruifang Niu a,e, Shiwu Zhang a,c, Man Yu f, Xiyin Wei a,e, Lin Zhang a,e a
Key Laboratory of Cancer Prevention and Therapy, Tianjin Cancer Institute and Hospital, Tianjin Medical University, Tianjin 300060, PR China b Department of Breast Pathology and Research, Tianjin Cancer Institute and Hospital, Tianjin Medical University, Tianjin 300060, PR China c Department of Pathology, Tianjin Cancer Institute and Hospital, Tianjin Medical University, Tianjin 300060, PR China d Department of Pathology, Tianjin Medical University, Tianjin 300070, PR China e Core Laboratory, Tianjin Cancer Institute and Hospital, Tianjin Medical University, Tianjin 300060, PR China f Department of Cellular and Molecular Medicine, University of Ottawa, 451 Smyth Road, Ottawa, ON K1H 8M5, Canada Received 22 July 2007; revised 18 September 2007; accepted 9 December 2007
Abstract The cellular Fas-associated death domain-like interleukin-1b-converting enzyme inhibitory protein-long form (c-FLIPL) is a key regulator of Fas signaling, although owing a dominant-negative homologue of caspase-8, the role of c-FLIPL remains controversial. In the present study, two pairs of small interfering RNA (siRNA) directed against c-FLIPL were used to assess the effect of c-FLIPL on Fas-mediated apoptosis of colon carcinoma in vitro. HT-29 cell line was selected for overexpression of c-FLIPL and Fas with RTePCR and flow cytometry analyses. After electroporation, the mRNA level of c-FLIPL was significantly decreased (control siRNA versus c-FLIPL siRNA, 77.97 5.61% versus 26.22 3.79%) and the maximum interfering efficiency was around 66.49% using semi-quantitative RTePCR analysis. Knockdown of c-FLIPL with the specific siRNA sensitized colon carcinoma cells to Fas-mediated apoptosis (control siRNA versus c-FLIPL siRNA, 5.68 2.11% versus 29.50 2.27%) using DNA content analysis and Annexin VeFITC analysis. In conclusion, our study indicated that c-FLIPL might be a suppressor of Fas-mediated apoptosis in Fas antigen expressing colon carcinoma and therefore a potential target for novel anticancer therapies. Ó 2007 International Federation for Cell Biology. Published by Elsevier Ltd. All rights reserved. Keywords: c-FLIPL; Colon carcinoma; Fas; RNA interference; Apoptosis
1. Introduction During the process of neoplastic transformation, tumor cells gradually obtain resistance to environmental apoptotic Abbreviations: c-FLIPL, cellular Fas-associated death domain-like interleukin-1b-converting enzyme inhibitory protein-long form; FasL, Fas ligand; DISC, death-inducing signaling complex; FADD, Fas-associated death domain; DED, death effector domain; TRAIL, TNF-related apoptosis-inducing ligand; siRNA, small interfering RNA. * Corresponding author: Department of Pathology, Tianjin Cancer Institute and Hospital, Tianjin Medical University, Tianjin 300060, PR China. Tel./ fax: þ86 22 2334 0123x5221. E-mail address: [email protected] (B. Sun). 1 These authors contributed equally to this work.
signaling and escape immune surveillance. The shift to an apoptosis-insensitive phenotype is one of the most important mechanisms for chemoresistance. At the molecular level, malignant cells up-regulate apoptosis-inhibiting genes and/or down-regulate apoptosis-promoting genes. Colon is a rich source of immune cells and aging epithelia are strictly monitored and promptly cleared out by the immune system. Colon carcinoma cells have decreased levels of Fas, tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) receptor1/2 and other death receptors, allowing them to escape immune killing by the cytotoxic T lymphocytes and other activated cells (O’Connell et al., 2000; Van Geelen et al., 2004). In addition, a subgroup of colon carcinoma cells expressing moderate-to-high levels of Fas are still resistant to apoptosis
1065-6995/$ - see front matter Ó 2007 International Federation for Cell Biology. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.cellbi.2007.12.002
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induced by Fas ligand (FasL) or agonistic antibody, implying that the level of Fas expression is not in correspondence with apoptotic sensitivity (Lee and Shacter, 2001). In fact, many colon carcinoma cells simultaneously express their own FasL, which clearly has no negative effect on their survival and may confer them a protective immunomodulatory effect (von Reyher et al., 1998). It was reported that inhibition of protein synthesis with cycloheximide may sensitize colon carcinoma cells to TRAIL-mediated apoptosis, indicating that a short-lived inhibitory protein is involved (Hernandez et al., 2001). Binding of FasL to Fas induces a series of proteineprotein interactions that lead to assembly of a death-inducing signaling complex (DISC). Upon ligation, Fas recruits an adaptor molecule Fas-associated death domain (FADD) via a cytoplasmic domain of nearly 70 amino acid residues (Schulze-Osthoff et al., 1998). FADD contains a death effector domain (DED) that allows the recruitment of the proenzyme form of caspase-8, also known as FADD-like interleukin-1b-converting enzyme, resulting in proteolytic self-activation of caspase-8 (Muzio et al., 1998). In brief, the DISC is formed by the cross-linked receptor Fas, FADD, and caspase-8, which in turn activates downstream effector caspases and induces apoptosis. Such an effect on Fas-mediated caspase-8 activation and apoptosis has been inhibited in many other models by cellular Fas-associated death domain-like interleukin-1b-converting enzyme inhibitory protein (c-FLIP) (Irmler et al., 1997). At the mRNA level, c-FLIP can be found as multiple splice variants, whereas at the protein level only three isoforms have been reported: a 55 kDa long form (c-FLIPL), a 26 kDa short form (c-FLIPS) and a 24 kDa form (c-FLIPR). These three isoforms contain two N-terminal DEDs with homology to caspase-8 and c-FLIPL also bears a caspase-like domain devoid of proteolytic ability (Krueger et al., 2001; Golks et al., 2005). After FasL triggering, c-FLIP binds with the DED of FADD and the resultant Fas-FADD-c-FLIP complex lower the level of DISC (Thome and Tschopp, 2001). An inhibitory role of c- FLIPS and a c-FLIPR has been well documented, whereas the role of c-FLIPL in apoptosis is still controversial. Some researchers believe that high expression of c-FLIPL desensitizes several solid and hematologic tumors to FasL/TRAIL-induced apoptosis (Grambihler et al., 2003; Mathas et al., 2004; Zhang et al., 2004). Others maintain that the c-FLIPL/caspase-8 heterodimers have the same substrate specificity as casapse-8/caspase-8 homodimers and activate the death executioner caspase-3 (Micheau et al., 2002; Donepudi et al., 2003). Another study showed that c-FLIPL recruitment contributes to the first cleavage of caspase-8 in the DISC, where the c-FLIPL intermediate form inhibits further cleavage of caspase-8 (Yang et al., 2003). In the present study, we investigated the role of c-FLIPL in Fas-mediated apoptosis in colon carcinoma. To achieve this objective, a colon carcinoma cell line that expressed high level of endogenous c-FLIPL and underwent apoptosis via the Fas signaling was selected. By silencing c-FLIPL transcription with genetic inhibitors, the cell line turned from apoptosis-
resistant into apoptosis-sensitive phenotype. Our study shows that c-FLIPL is an apoptosis suppressor in Fas expressing colon carcinoma and specific siRNA targeting of c-FLIPL might be a potential gene therapy method for further development. 2. Materials and methods 2.1. Cell culture Human colon carcinoma cell lines HT-29, Colo205 and Lovo were purchased from American Type Culture Collection. SW620 cell line was a kind gift from Dr. Joe O’Connell (National University of Ireland, Cork, Ireland). All cell lines were grown in RPMI-1640 medium (Gibco, USA) supplemented with 10% heat-inactivated standard fetal bovine serum (FBS, Hyclone, USA), 100 units/ml penicillin, and 100 mg/ml streptomycin in a humidified 5% CO2 atmosphere in air at 37 C. Cells were harvested with 0.25% trypsine0.02% EDTA at 37 C for 2e5 min and were washed once in supplemented medium. 2.2. Fas immunofluorescence measurement A total of 5 105 cells were suspended in 100 ml PBS and incubated with 1 mg FITC-conjugated mouse mAb against human Fas (clone UB2, Immunotech, USA) in the dark at room temperature for 20 min. Cells were washed to remove excess antibody and resuspended in 500 ml PBS. Isotype-matched mAb was carried out as a control in all cell lines and non-specific binding was excluded. Immunofluorescence detection was performed by flow cytometry using an EPICS-XL System IIÔ analysis program (Beckman Coulter, USA). Ten thousand cells were examined for each sample and data are shown as the percentage of specific positive cells: Fas expression (%) ¼ Positive Cells with Fas mAb (%) Positive Cells with Isotype-matched mAb (%). 2.3. RTePCR detection of c-FLIPL Total RNA was isolated from 106 colon carcinoma cells using Trizol Reagent (Gibco, USA) according to the manufacturer’s protocol. cDNA was synthesized using AMV reverse transcriptase (Promega, USA) with random hexanucleotide primers (TaKaRa, Japan). A total of 5 ml of cDNA was used in a 25 ml PCR reaction containing 0.4 mM of each of the relevant primers and 12.5 ml of Premix Taq (TaKaRa). The sense and antisense primers were as follows respectively: c-FLIPL, 50 -ACCGAGACTACGACAGCTTTGTG-30 and 50 -CAATGTGAAGATCCAGGAGTGGG-30 ; b-actin, 50 -ATCATGTTTGAGACCTTCAACA-30 and 50 -CATCTCTTGCTCGAAGTCCA-30 . The amplification cycles were denaturation at 94 C for 45 s, annealing at 65 C for 45 s, and extension at 72 C for 2 min for 35 cycles. The 429-bp c-FLIPL and 318-bp b-actin PCR products were analyzed by electrophoresis on 1.3% agarose gels and visualized by ethidium bromide staining with DL2000 DNA size marker (TaKaRa).
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2.4. RNA interference Double-stranded 21-mer siRNA molecules (siRNA-F1 and siRNA-F2) specific for two different c-FLIPL sites (accession no. U97074) were kindly provided by Harald Wajant (University of Stuttgart, Stuttgart, Germany) (Siegmund et al., 2002). 19-mer double-stranded siRNA molecules (siRNA-H) corresponding to the human T-cell leukemia virus tax gene (HTLV-1 Tax, accession no. AS038239) were used as a control (Williams et al., 2003). siRNA sequences and target mRNAs are listed in Table 1. No homology exists between HTLV-1 Tax target sequences and any known human genes upon BLAST confirmation. Approximately 106 HT-29 colon carcinoma cells were gently resuspended in 800 ml Hypo-osmolar (90 mOsmol/kg) buffer (Eppendorf, Germany) for 25 min at room temperature. A total of 50, 100, or 150 nM siRNAs was transfected into cells in a 4-mm cuvette using an Eppendorf Multiporator at 500 V for 7 ms. After electroporation, cells were incubated for 5e10 min and then plated in 6-well culture plates. RPMI-1640 medium with 15% FBS and without antibiotics was added into each well. Four hours later, cells were cultured in regular medium. The efficiency of RNA interference is evaluated by semi-quantitative RTePCR analysis using the Kodak Image Station 440CF (Kodak, USA). Interfering Efficiency (%) ¼ [c-FLIPL relative content of siRNA-H (%) c-FLIPL relative content of siRNA-F1/siRNA-F2 (%)]/c-FLIPL relative content of siRNA-H (%) 100. Relative Content of c-FLIPL (%) ¼ c-FLIPL Net. Int./b-actin Net. Int. 100. The c-FLIPL protein expression upon siRNA treatment was assessed with Western blot analysis as previously described (Zhang et al., 2004). The antibodies and dilutions used included anti-c-FLIPL mouse mAb (NF6, Alexis Biochemicals, USA) at 1:500 and anti-GAPDH mouse mAb (6C5, Abcam, USA) at 1:5000. Filters were subsequently incubated with horseradish peroxidase-conjugated secondary antibodies and developed using the enhanced chemiluminescence system (Pierce, USA). 2.5. Assessment of apoptosis The sensitivity of colon carcinoma cells to Fas-mediated apoptosis was detected by treating cells with the agonistic anti-Fas CH11 IgM mAb (Immunotech) at 0.1 mg/ml for 16 h. A total of 5 105 cells were washed in PBS, fixed in cold 95% ethanol overnight, washed in PBS, and incubated in PBS containing 20 mg/ml propidium iodide (PI, Sigma, USA) and 50 mg/ml DNase-free RNase A (Sigma) for Table 1 siRNA sequences and target mRNA siRNA
Sequences 0
Target mRNA Length 0
siRNA-F1 5 -ACAUUAGGUGGAACCACAUCU-3 50 -AGAUGUGGUUCCACCUAAUGU-30 siRNA-F2 50 -GUAACUUGUCCCUGCUCCUUG-30 50 -CAAGGAGCAGGGACAAGUUAC-30 siRNA-H 50 -GAUGGACGCGUUAUCGGCU-30 50 -AGCCGAUAACGCGUCCAUC-30
c-FLIPL 472-492 c-FLIPL 908-928 HTLV-1 Tax 754-772
21 21 19
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30 min at 37 C. The hypodiploid peak, adjacent to the G0/ G1 peak, was determined by flow cytometry (Beckman Coulter). In order to distinguish apoptotic changes from viability or necrosis, cells were detected by simultaneous staining with Annexin VeFITC and PI using an Annexin VeFITC Kit (Immunotech). Cells were treated and the analysis was carried out as suggested in the manufacturer’s protocols. 3. Results 3.1. Differential expression of Fas in colon carcinoma cells Our results showed that cell surface expression of Fas was heterogeneous in the four colon carcinoma cell lines (Fig. 1). The expression of Fas on HT-29 cells was highest (around 42.46%) among all the cell lines detected. The Fas level of Colo205 cells was moderate. In contrast, Fas protein was nearly not detected on the SW620 and Lovo cells surface because no difference in fluorescence was observed between cells exposed to anti-Fas mAb and cells exposed to isotypematched control mAb. For each tumor type, at least three samples are measured. 3.2. Differential expression of c-FLIPL mRNA in colon carcinoma cells To investigate the expression of c-FLIPL mRNA in human colon carcinoma cell lines, total RNA was extracted from subconfluent cell cultures and analyzed by RTePCR. As a 429-bp amplified fragment of c-FLIPL transcript was detected by ethidium bromide electrophoresis in HT-29, Colo205, and SW620 cells (Fig. 2); it was almost undetectable in Lovo cells. The results were confirmed by three independent assays. 3.3. Silencing of c-FLIPL mRNA and protein expression by specific siRNA in HT-29 cells The siRNA-F1, siRNA-F2 or siRNA-F1 and siRNA-F2 mixture were electroporated into HT-29 cells in order to evaluate the potency of RNA interference and analyze the efficiency of the two targeting sites. The unrelated siRNA-H was transfected into HT-29 cells as a control. Both siRNAF1 and siRNA-F2 effectively decreased c-FLIPL mRNA and the efficiency of siRNA-F2 (at about 65.41%) was slightly higher, compared with siRNA-H (Fig. 3A). To determine whether the interfering efficiency was dependent on the concentration of exogenous oligonucleotides, various amounts of siRNA (50, 100, or 150 nM) were tested. c-FLIPL mRNA decreased significantly after transfection with 100 nM or 150 nM siRNA-F2; and the interfering efficiency of 100 nM siRNA-F2 was highest among various concentrations (w66.49%; Fig. 3B). HT-29 cells were transfected with 100 nM siRNA-F2 and were harvested for semi-quantitative RTePCR analysis at 24, 48, or 72 h post-electroporation. At 24 h, the level of c-FLIPL mRNA expression diminished, while the maximum reduction by RNA interference was
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Fig. 1. Differential expression of Fas in the four colon carcinoma cell lines. Cells were stained with FITC-conjugated anti-Fas mAb and analyzed on a EPICS-XL flow cytometer. Scales were the same in all panels and each sample was repeated >3 times. (A) The yellow curves represent Fas expression detected by specific anti-Fas mAb and the red ones are analyzed by isotype-matched antibody. (B) The percentage of Fas expression is quantitated with the fluorescent intensity. Fas expression (%) ¼ Positive Cells with Fas mAb (%) Positive Cells with Isotype-matched mAb (%). Values are expressed as means SD.
detected after 48 h and the interfering efficiency was nearly 62.36% (Fig. 3C). HT-29 cells were treated under the optimal experimental conditions that cells were transfected with 100 nM siRNA-F2 and harvested at 48 h post-electroporation for cellular lysates. It is clear from Fig. 3D that siRNA-F2 significantly reduced the level of c-FLIPL protein without affecting the level of a control protein, GAPDH. 3.4. c-FLIPL-specific siRNA sensitize HT-29 cells to Fas-mediated apoptosis To study the potential functions of c-FLIPL on Fas-mediated apoptosis, HT-29 cells were transfected with c-FLIPLspecific siRNA and challenged with agonistic anti-Fas mAb.
Fig. 2. RTePCR analysis of c-FLIPL mRNA expression in the colon carcinoma cell lines. RTePCR was performed on total RNA extracted from the cultured cells and the PCR products were stained with DL2000 DNA size marker. The housekeeping gene b-actin was used as a control. Three independent experiments gave similar results.
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Fig. 3. The expression of c-FLIPL was down-regulated by specific siRNA. HT-29 cells were electroporated with 2 pairs of specific siRNA targeting different sites of c-FLIPL (sequences described in Section 2) or a pair of unrelated siRNA-H targeting HTLV-1 tax. The relative content of c-FLIPL mRNA and the efficiency of RNA interference were evaluated by semi-quantitative RTePCR analysis. Interfering Efficiency (%) ¼ [c-FLIPL relative content of siRNA-H (%) c-FLIPL relative content of siRNA-F1/siRNA-F2 (%)]/c-FLIPL relative content of siRNA-H (%) 100. Three independent experiments are presented. Values are expressed as means SD. (A) HT-29 cells were transfected with 100 nM siRNA-F1 or siRNA-F2 or the siRNA mixture (50 nM siRNA-F1 plus 50 nM siRNA-F2). Cells were harvested at 48 h post-electroporation for determining the efficiency of RNA interference. (B) HT-29 cells were transfected with siRNA-F2 at various concentrations (nM) as indicated and the relative content of c-FLIPL mRNA were evaluated at 48 h post-electroporation. (C) The expression of c-FLIPL mRNA was analyzed in HT-29 cells transfected with 100 nM siRNA-F2 for various time periods (h) as indicated. (D) HT-29 cells were transfected with 100 nM siRNA-F2 and harvested at 48 h post-electroporation. Cellular lysates were immunoblotted with monoclonal anti-c-FLIPL or anti-GAPDH as described.
Cells transfected with siRNA-F2 showed a significant increase in the hypodiploid peak using DNA content analysis (Fig. 4A). In contrast, the cells transfected with siRNA-H had a very low hypodiploid DNA peak. The early phase of apoptosis was further determined by Annexin VeFITC analysis (Fig. 4B). Transfection of siRNA-F2 into HT-29 cells led to a substantial shift in apoptotic sensitivity: after 16 h of anti-Fas treatment, HT-29 cells showed an increased number of apoptotic cells at approximately 29.5% compared with the cells transfected with siRNA-H. It should also be noted that siRNA-F2 is a specific transcriptional inhibitor targeting c-FLIPL that when used in combination with agonistic anti-Fas antibody (0.1 mg/ml)
instigated HT-29 cells to become Fas sensitive. Treatment of HT-29 cells with siRNA-F2 alone did not lead to significant cell death (data not shown). 4. Discussion Fas-mediated apoptosis is an effective pathway for cell-mediated cytotoxicity and participates in the immune response against neoplastic cells. The emergence of resistance to this pathway contributes to tumor cells escape from immune surveillance and is an important step during the carcinogenesis process. A more widespread mechanism of this resistance
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Fig. 4. RNA interference directed against c-FLIPL resulted in an increase in apoptotic cells. The specific siRNA-F2 (100 nM) or the control siRNA-H (100 nM) was electroporated into HT-29 cells 48 h before the agonistic anti-Fas mAb addition. The cells were harvested for evaluating apoptosis after the anti-Fas mAb (0.1 mg/ ml) treatment for 16 h. The results were representative of three separate experiments. Values are expressed as means SD. (A) HT-29 cells were labeled with PI to analyze the percentage of cells with hypodiploid DNA using flow cytometry. (B) Annexin VeFITC analysis was carried out in order to identify the apoptotic cells from viable cells and dead cells. The percentage of the apoptotic cells was indicated in the Annexin VeFITCþ/PI population and was quantified with the dot plots.
described in several models is the over-expression of antiapoptosis proteins. Data from gene expression profiling showed that c-FLIP is over-expressed in several types of carcinomas and is associated with an increased mortality in patients with prostate, breast, and lung cancers (Bhattacharjee et al., 2001; van’t Veer et al., 2002; Varambally et al., 2005). c-FLIP isoforms are characterized as a short half-life by inhibiting of protein synthesis with cycloheximide and it has been proposed that mechanisms of differential degradation of c-FLIP isoforms might be important for apoptosis sensitivity (Mitsiades et al., 2000; Schmitz et al., 2004). c-FLIPR and c-FLIPS contain only two tandem repeats of DEDs and inhibit activation of caspase-8 and apoptosis in a similar mechanism (Djerbi et al.,
2001; Golks et al., 2005). In contrast, c-FLIPL shares extensive homology with caspase-8, with a C-terminal domain that is highly homologous to the caspase-8 protease domain yet enzymatically inactive due to the lack of key active site residues. To date, the role of c-FLIPL in apoptosis remains controversial. In the present study, we observed a significant increase of the hypodiploid peak in colon carcinoma cells in which cFLIPL was down-regulated with specific siRNA. Further studies showed that such cells underwent early phase of apoptosis to Fas signaling for the appearance of phosphatidylserine on the cell surface. It has been previously reported that the expression of c-FLIPL in colon adenocarcinomas is frequently elevated at both mRNA and protein level compared with
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matched normal tissues and adenomatous polyps (Ryu et al., 2001). Our results indicated that c-FLIPL promotes colon carcinoma cells to escape Fas-mediated apoptosis in vitro, shedding slight light on the mechanism of such frequent event in vivo during the malignant transformation or progression of colon carcinoma. The mechanism of cell death attenuation by c-FLIPL has not been completely elucidated. Early experiments suggested that c-FLIPL, as a potential competitive inhibitor, preclude recruitment of caspase-8 to the DISC and thereby prevent its activation. This idea was supported by the fact that upon over-expression of the viral homolog v-FLIP-E8, c-FLIPL is cleaved into a p43 subunit that remains at the DISC and a releasing p12 subunit (Thome et al., 1999). Loss-of-function experiments suggested that depleted c-FLIPL results in more complete processing of the caspase and faster turnover of the intermediate (Sharp et al., 2005). However, caspase-8 can not be processed to form active tetramer, which executes the key proteolytic function in the apoptotic pathway (Krueger et al., 2001). The partial processing of active caspase-8 in caspase-8/c-FLIPL heterodimers retains active caspase-8 at the DISC, preventing its release into the cytosol (Chang et al., 2003). More interestingly, not only full-length c-FLIPL but also p43 subunit can inhibit FasL induced apoptosis, implying both full-length and cleaved c-FLIPL possess strong apoptosisresistant ability (Boatright et al., 2004). In addition, chemotherapeutic drug cisplatin inhibits phosphorylation of c-FLIPL without down-regulating its expression levels and sensitizes melanoma cells to TRAIL-induced apoptosis, indicating that phosphorylation may play an essential role in the regulation of c-FLIPL anti-apoptosis activity (Higuchi et al., 2003; Song et al., 2003). However, in other carcinomas, c-FLIPL can promote caspase-8 activation by acting directly on the protease domain (Han et al., 1997; Inohara et al., 1997; Guseva et al., in press). Purified caspase-8 is shown to heterodimerize with c-FLIPL, and these heterodimers have a higher affinity for Fas-FADD complexes and considerable proteolytic activity compared with caspase-8/capase-8 homodimers (Boatright et al., 2004). Further studies show that c-FLIPL is a dual function regulator for caspase-8 activation and apoptosis dependent on its environmental conditions and expression level. In the case of T cell antigen receptor-mediated signaling, c-FLIPL likely plays an important role in caspase-8 activation (Micheau et al., 2002; Misra et al., 2007). In contrast, when Fas is ligated, capase-8 activation can be inhibited by c-FLIPL-mediated competition with caspase-8 for recruitment to FADD (Hu et al., 2000). Moreover, at the levels comparable with the endogenous protein, c-FLIPL may enhance procaspase-8 activation in the DISC and Fas-mediated apoptosis, whereas at very high non-physiological levels achieved by transient overexpression, it inhibits apoptosis (Chang et al., 2002). In the present study, we assessed the specific function of endogenous c-FLIPL by a selective siRNA knockdown approach and found that depletion of endogenous c-FLIPL augmented apoptosis induction by agonistic antibody in colon carcinoma cell line. Thus, c-FLIPL functions primarily as an inhibitor, rather
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than a promoter, of Fas-mediated apoptosis. These results, which seem to contradict the dual-function studies, might be reconciled by the possibility that the HT-29 cell line expresses intermediate or high level of c-FLIPL, which is enough to inhibit formation of DISC and death receptor-mediated apoptosis. Based on the previous research and our experiments, c-FLIPL and caspase-8 expression may not fully explain independently the apoptotic fate of the cells. A recent study shows that in thyroid carcinomas, the proteolytic cleavage and activation of caspase-8 depends on the balance between expression levels for caspase-8 and c-FLIPL, and correlates with favorable clinical prognosis (Mitsiades et al., 2006). It should be noted that both the death receptor pathway and the mitochondrial pathway are involved in apoptosis. In some cells, apoptosis that is initiated through the death receptor pathway still requires amplification through the mitochondrial pathway of caspases activation (Kuwana et al., 1998; Rosen et al., 2001). Our results indicate that the four colon carcinoma cell lines escape apoptosis at different points in different pathways. HT-29 cells which express high levels of Fas and c-FLIPL develop an apoptosis-resistant phenotype perhaps mainly by down-regulation of c-FLIPL. SW620 and Colo205 cells express lower levels of Fas and high or moderate levels of c-FLIPL respectively. These two cell lines are supposed to escape Fas-mediated apoptosis by either up-regulating c-FLIPL or down-regulating Fas. Lovo cells show minimal Fas and c-FLIPL and are reported to high express Bcl-2 and Bcl-XL, suggesting that apoptosis can be prevented by inhibiting the mitochondrial pathway (Nita et al., 1998). Further studies are needed to assess the relative contribution of these two pathways to apoptosis. In summary, our data suggest that the over-expression of cFLIPL in colon carcinoma suppresses Fas-mediated apoptosis. We propose that inhibiting c-FLIPL expression may be a useful strategy to investigate as a possible approach for treatment of malignant cells.
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Nita ME, Nagawa H, Tominaga O, Tsuno N, Fujii S, Sasaki S, et al. 5-Fluorouracil induces apoptosis in human colon cancer cell lines with modulation of Bcl-2 family proteins. Br J Cancer 1998;78:986e92. O’Connell J, Bennett MW, Nally K, Houston A, O’Sullivan GC, Shanahan F. Altered mechanisms of apoptosis in colon cancer: Fas resistance and counterattack in the tumor-immune conflict. Ann N Y Acad Sci 2000;910: 178e92. discussion 193e195. Rosen K, Coll ML, Li A, Filmus J. Transforming growth factor-alpha prevents detachment-induced inhibition of c-Src kinase activity, Bcl-XL down-regulation, and apoptosis of intestinal epithelial cells. J Biol Chem 2001;276:37273e9. Ryu BK, Lee MG, Chi SG, Kim YW, Park JH. Increased expression of cFLIPL in colonic adenocarcinoma. J Pathol 2001;194:15e9. Schmitz I, Weyd H, Krueger A, Baumann S, Fas SC, Krammer PH, et al. Resistance of short term activated T cells to CD95-mediated apoptosis correlates with de novo protein synthesis of c-FLIPshort. J Immunol 2004;172:2194e200. Schulze-Osthoff K, Ferrari D, Los M, Wesselborg S, Peter ME. Apoptosis signaling by death receptors. Eur J Biochem 1998;254:439e59. Sharp DA, Lawrence DA, Ashkenazi A. Selective knockdown of the long variant of cellular FLICE inhibitory protein augments death receptor-mediated caspase-8 activation and apoptosis. J Biol Chem 2005;280:19401e9. Siegmund D, Hadwiger P, Pfizenmaier K, Vornlocher HP, Wajant H. Selective inhibition of FLICE-like inhibitory protein (FLIP) expression with small interfering RNA oligonucleotides (siRNAs) is sufficient to sensitize tumor cells for TRAIL-induced apoptosis. Mol Med 2002;8:725e32. Song JH, Song DK, Herlyn M, Petruk KC, Hao C. Cisplatin down-regulation of cellular Fas-associated death domain-like interleukin-1b-converting enzyme-like inhibitory proteins to restore tumor necrosis factor-related apoptosis-inducing ligand-induced apoptosis in human melanoma cells. Clin Cancer Res 2003;9:4255e66. Thome M, Tschopp J. Regulation of lymphocyte proliferation and death by FLIP. Nat Rev Immunol 2001;1:50e8. Thome M, Martinon F, Hofmann K, Rubio V, Steiner V, Schneider P, et al. Equine herpesvirus-2 E10 gene product, but not its cellular homologue, activates NF-kappaB transcription factor and c-Jun N-terminal kinase. J Biol Chem 1999;274:9962e8. Van Geelen CM, de Vries EG, de Jong S. Lessons from TRAIL-resistance mechanisms in colorectal cancer cells: paving the road to patient-tailored therapy. Drug Resist Updat 2004;7:345e58. van’t Veer LJ, Dai H, van de Vijver MJ, He YD, Hart AA, Mao M, et al. Gene expression profiling predicts clinical outcome of breast cancer. Nature 2002;415:530e6. Varambally S, Yu J, Laxman B, Rhodes DR, Mehra R, Tomlins SA, et al. Integrative genomic and proteomic analysis of prostate cancer reveals signatures of metastatic progression. Cancer Cell 2005;8:393e406. von Reyher U, Strater J, Kittstein W, Gschwendt M, Krammer PH, Moller P. Colon carcinoma cells use different mechanisms to escape CD95-mediated apoptosis. Cancer Res 1998;58:526e34. Williams NS, Gaynor RB, Scoggin S, Verma U, Gokaslan T, Simmang C, et al. Identification and validation of genes involved in the pathogenesis of colorectal cancer using cDNA microarrays and RNA interference. Clin Cancer Res 2003;9:931e46. Yang BF, Xiao C, Roa WH, Krammer PH, Hao C. Calcium/calmodulin-dependent protein kinase II regulation of c-FLIP expression and phosphorylation in modulation of Fas-mediated signaling in malignant glioma cells. J Biol Chem 2003;278:7043e50. Zhang X, Jin TG, Yang H, DeWolf WC, Khosravi-Far R, Olumi AF. Persistent c-FLIP(L) expression is necessary and sufficient to maintain resistance to tumor necrosis factor-related apoptosis-inducing ligand-mediated apoptosis in prostate cancer. Cancer Res 2004;64:7086e91.
Critical role for c-FLIPL on Fas resistance in colon carcinoma cell line HT-29 Fenglin Zang a,b,1, Baocun Sun a,c,d,*,1, Xiulan Zhao d, Ruifang Niu a,e, Shiwu Zhang a,c, Man Yu f, Xiyin Wei a,e, Lin Zhang a,e a
Key Laboratory of Cancer Prevention and Therapy, Tianjin Cancer Institute and Hospital, Tianjin Medical University, Tianjin 300060, PR China b Department of Breast Pathology and Research, Tianjin Cancer Institute and Hospital, Tianjin Medical University, Tianjin 300060, PR China c Department of Pathology, Tianjin Cancer Institute and Hospital, Tianjin Medical University, Tianjin 300060, PR China d Department of Pathology, Tianjin Medical University, Tianjin 300070, PR China e Core Laboratory, Tianjin Cancer Institute and Hospital, Tianjin Medical University, Tianjin 300060, PR China f Department of Cellular and Molecular Medicine, University of Ottawa, 451 Smyth Road, Ottawa, ON K1H 8M5, Canada Received 22 July 2007; revised 18 September 2007; accepted 9 December 2007
Abstract The cellular Fas-associated death domain-like interleukin-1b-converting enzyme inhibitory protein-long form (c-FLIPL) is a key regulator of Fas signaling, although owing a dominant-negative homologue of caspase-8, the role of c-FLIPL remains controversial. In the present study, two pairs of small interfering RNA (siRNA) directed against c-FLIPL were used to assess the effect of c-FLIPL on Fas-mediated apoptosis of colon carcinoma in vitro. HT-29 cell line was selected for overexpression of c-FLIPL and Fas with RTePCR and flow cytometry analyses. After electroporation, the mRNA level of c-FLIPL was significantly decreased (control siRNA versus c-FLIPL siRNA, 77.97 5.61% versus 26.22 3.79%) and the maximum interfering efficiency was around 66.49% using semi-quantitative RTePCR analysis. Knockdown of c-FLIPL with the specific siRNA sensitized colon carcinoma cells to Fas-mediated apoptosis (control siRNA versus c-FLIPL siRNA, 5.68 2.11% versus 29.50 2.27%) using DNA content analysis and Annexin VeFITC analysis. In conclusion, our study indicated that c-FLIPL might be a suppressor of Fas-mediated apoptosis in Fas antigen expressing colon carcinoma and therefore a potential target for novel anticancer therapies. Ó 2007 International Federation for Cell Biology. Published by Elsevier Ltd. All rights reserved. Keywords: c-FLIPL; Colon carcinoma; Fas; RNA interference; Apoptosis
1. Introduction During the process of neoplastic transformation, tumor cells gradually obtain resistance to environmental apoptotic Abbreviations: c-FLIPL, cellular Fas-associated death domain-like interleukin-1b-converting enzyme inhibitory protein-long form; FasL, Fas ligand; DISC, death-inducing signaling complex; FADD, Fas-associated death domain; DED, death effector domain; TRAIL, TNF-related apoptosis-inducing ligand; siRNA, small interfering RNA. * Corresponding author: Department of Pathology, Tianjin Cancer Institute and Hospital, Tianjin Medical University, Tianjin 300060, PR China. Tel./ fax: þ86 22 2334 0123x5221. E-mail address: [email protected] (B. Sun). 1 These authors contributed equally to this work.
signaling and escape immune surveillance. The shift to an apoptosis-insensitive phenotype is one of the most important mechanisms for chemoresistance. At the molecular level, malignant cells up-regulate apoptosis-inhibiting genes and/or down-regulate apoptosis-promoting genes. Colon is a rich source of immune cells and aging epithelia are strictly monitored and promptly cleared out by the immune system. Colon carcinoma cells have decreased levels of Fas, tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) receptor1/2 and other death receptors, allowing them to escape immune killing by the cytotoxic T lymphocytes and other activated cells (O’Connell et al., 2000; Van Geelen et al., 2004). In addition, a subgroup of colon carcinoma cells expressing moderate-to-high levels of Fas are still resistant to apoptosis
1065-6995/$ - see front matter Ó 2007 International Federation for Cell Biology. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.cellbi.2007.12.002
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induced by Fas ligand (FasL) or agonistic antibody, implying that the level of Fas expression is not in correspondence with apoptotic sensitivity (Lee and Shacter, 2001). In fact, many colon carcinoma cells simultaneously express their own FasL, which clearly has no negative effect on their survival and may confer them a protective immunomodulatory effect (von Reyher et al., 1998). It was reported that inhibition of protein synthesis with cycloheximide may sensitize colon carcinoma cells to TRAIL-mediated apoptosis, indicating that a short-lived inhibitory protein is involved (Hernandez et al., 2001). Binding of FasL to Fas induces a series of proteineprotein interactions that lead to assembly of a death-inducing signaling complex (DISC). Upon ligation, Fas recruits an adaptor molecule Fas-associated death domain (FADD) via a cytoplasmic domain of nearly 70 amino acid residues (Schulze-Osthoff et al., 1998). FADD contains a death effector domain (DED) that allows the recruitment of the proenzyme form of caspase-8, also known as FADD-like interleukin-1b-converting enzyme, resulting in proteolytic self-activation of caspase-8 (Muzio et al., 1998). In brief, the DISC is formed by the cross-linked receptor Fas, FADD, and caspase-8, which in turn activates downstream effector caspases and induces apoptosis. Such an effect on Fas-mediated caspase-8 activation and apoptosis has been inhibited in many other models by cellular Fas-associated death domain-like interleukin-1b-converting enzyme inhibitory protein (c-FLIP) (Irmler et al., 1997). At the mRNA level, c-FLIP can be found as multiple splice variants, whereas at the protein level only three isoforms have been reported: a 55 kDa long form (c-FLIPL), a 26 kDa short form (c-FLIPS) and a 24 kDa form (c-FLIPR). These three isoforms contain two N-terminal DEDs with homology to caspase-8 and c-FLIPL also bears a caspase-like domain devoid of proteolytic ability (Krueger et al., 2001; Golks et al., 2005). After FasL triggering, c-FLIP binds with the DED of FADD and the resultant Fas-FADD-c-FLIP complex lower the level of DISC (Thome and Tschopp, 2001). An inhibitory role of c- FLIPS and a c-FLIPR has been well documented, whereas the role of c-FLIPL in apoptosis is still controversial. Some researchers believe that high expression of c-FLIPL desensitizes several solid and hematologic tumors to FasL/TRAIL-induced apoptosis (Grambihler et al., 2003; Mathas et al., 2004; Zhang et al., 2004). Others maintain that the c-FLIPL/caspase-8 heterodimers have the same substrate specificity as casapse-8/caspase-8 homodimers and activate the death executioner caspase-3 (Micheau et al., 2002; Donepudi et al., 2003). Another study showed that c-FLIPL recruitment contributes to the first cleavage of caspase-8 in the DISC, where the c-FLIPL intermediate form inhibits further cleavage of caspase-8 (Yang et al., 2003). In the present study, we investigated the role of c-FLIPL in Fas-mediated apoptosis in colon carcinoma. To achieve this objective, a colon carcinoma cell line that expressed high level of endogenous c-FLIPL and underwent apoptosis via the Fas signaling was selected. By silencing c-FLIPL transcription with genetic inhibitors, the cell line turned from apoptosis-
resistant into apoptosis-sensitive phenotype. Our study shows that c-FLIPL is an apoptosis suppressor in Fas expressing colon carcinoma and specific siRNA targeting of c-FLIPL might be a potential gene therapy method for further development. 2. Materials and methods 2.1. Cell culture Human colon carcinoma cell lines HT-29, Colo205 and Lovo were purchased from American Type Culture Collection. SW620 cell line was a kind gift from Dr. Joe O’Connell (National University of Ireland, Cork, Ireland). All cell lines were grown in RPMI-1640 medium (Gibco, USA) supplemented with 10% heat-inactivated standard fetal bovine serum (FBS, Hyclone, USA), 100 units/ml penicillin, and 100 mg/ml streptomycin in a humidified 5% CO2 atmosphere in air at 37 C. Cells were harvested with 0.25% trypsine0.02% EDTA at 37 C for 2e5 min and were washed once in supplemented medium. 2.2. Fas immunofluorescence measurement A total of 5 105 cells were suspended in 100 ml PBS and incubated with 1 mg FITC-conjugated mouse mAb against human Fas (clone UB2, Immunotech, USA) in the dark at room temperature for 20 min. Cells were washed to remove excess antibody and resuspended in 500 ml PBS. Isotype-matched mAb was carried out as a control in all cell lines and non-specific binding was excluded. Immunofluorescence detection was performed by flow cytometry using an EPICS-XL System IIÔ analysis program (Beckman Coulter, USA). Ten thousand cells were examined for each sample and data are shown as the percentage of specific positive cells: Fas expression (%) ¼ Positive Cells with Fas mAb (%) Positive Cells with Isotype-matched mAb (%). 2.3. RTePCR detection of c-FLIPL Total RNA was isolated from 106 colon carcinoma cells using Trizol Reagent (Gibco, USA) according to the manufacturer’s protocol. cDNA was synthesized using AMV reverse transcriptase (Promega, USA) with random hexanucleotide primers (TaKaRa, Japan). A total of 5 ml of cDNA was used in a 25 ml PCR reaction containing 0.4 mM of each of the relevant primers and 12.5 ml of Premix Taq (TaKaRa). The sense and antisense primers were as follows respectively: c-FLIPL, 50 -ACCGAGACTACGACAGCTTTGTG-30 and 50 -CAATGTGAAGATCCAGGAGTGGG-30 ; b-actin, 50 -ATCATGTTTGAGACCTTCAACA-30 and 50 -CATCTCTTGCTCGAAGTCCA-30 . The amplification cycles were denaturation at 94 C for 45 s, annealing at 65 C for 45 s, and extension at 72 C for 2 min for 35 cycles. The 429-bp c-FLIPL and 318-bp b-actin PCR products were analyzed by electrophoresis on 1.3% agarose gels and visualized by ethidium bromide staining with DL2000 DNA size marker (TaKaRa).
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2.4. RNA interference Double-stranded 21-mer siRNA molecules (siRNA-F1 and siRNA-F2) specific for two different c-FLIPL sites (accession no. U97074) were kindly provided by Harald Wajant (University of Stuttgart, Stuttgart, Germany) (Siegmund et al., 2002). 19-mer double-stranded siRNA molecules (siRNA-H) corresponding to the human T-cell leukemia virus tax gene (HTLV-1 Tax, accession no. AS038239) were used as a control (Williams et al., 2003). siRNA sequences and target mRNAs are listed in Table 1. No homology exists between HTLV-1 Tax target sequences and any known human genes upon BLAST confirmation. Approximately 106 HT-29 colon carcinoma cells were gently resuspended in 800 ml Hypo-osmolar (90 mOsmol/kg) buffer (Eppendorf, Germany) for 25 min at room temperature. A total of 50, 100, or 150 nM siRNAs was transfected into cells in a 4-mm cuvette using an Eppendorf Multiporator at 500 V for 7 ms. After electroporation, cells were incubated for 5e10 min and then plated in 6-well culture plates. RPMI-1640 medium with 15% FBS and without antibiotics was added into each well. Four hours later, cells were cultured in regular medium. The efficiency of RNA interference is evaluated by semi-quantitative RTePCR analysis using the Kodak Image Station 440CF (Kodak, USA). Interfering Efficiency (%) ¼ [c-FLIPL relative content of siRNA-H (%) c-FLIPL relative content of siRNA-F1/siRNA-F2 (%)]/c-FLIPL relative content of siRNA-H (%) 100. Relative Content of c-FLIPL (%) ¼ c-FLIPL Net. Int./b-actin Net. Int. 100. The c-FLIPL protein expression upon siRNA treatment was assessed with Western blot analysis as previously described (Zhang et al., 2004). The antibodies and dilutions used included anti-c-FLIPL mouse mAb (NF6, Alexis Biochemicals, USA) at 1:500 and anti-GAPDH mouse mAb (6C5, Abcam, USA) at 1:5000. Filters were subsequently incubated with horseradish peroxidase-conjugated secondary antibodies and developed using the enhanced chemiluminescence system (Pierce, USA). 2.5. Assessment of apoptosis The sensitivity of colon carcinoma cells to Fas-mediated apoptosis was detected by treating cells with the agonistic anti-Fas CH11 IgM mAb (Immunotech) at 0.1 mg/ml for 16 h. A total of 5 105 cells were washed in PBS, fixed in cold 95% ethanol overnight, washed in PBS, and incubated in PBS containing 20 mg/ml propidium iodide (PI, Sigma, USA) and 50 mg/ml DNase-free RNase A (Sigma) for Table 1 siRNA sequences and target mRNA siRNA
Sequences 0
Target mRNA Length 0
siRNA-F1 5 -ACAUUAGGUGGAACCACAUCU-3 50 -AGAUGUGGUUCCACCUAAUGU-30 siRNA-F2 50 -GUAACUUGUCCCUGCUCCUUG-30 50 -CAAGGAGCAGGGACAAGUUAC-30 siRNA-H 50 -GAUGGACGCGUUAUCGGCU-30 50 -AGCCGAUAACGCGUCCAUC-30
c-FLIPL 472-492 c-FLIPL 908-928 HTLV-1 Tax 754-772
21 21 19
331
30 min at 37 C. The hypodiploid peak, adjacent to the G0/ G1 peak, was determined by flow cytometry (Beckman Coulter). In order to distinguish apoptotic changes from viability or necrosis, cells were detected by simultaneous staining with Annexin VeFITC and PI using an Annexin VeFITC Kit (Immunotech). Cells were treated and the analysis was carried out as suggested in the manufacturer’s protocols. 3. Results 3.1. Differential expression of Fas in colon carcinoma cells Our results showed that cell surface expression of Fas was heterogeneous in the four colon carcinoma cell lines (Fig. 1). The expression of Fas on HT-29 cells was highest (around 42.46%) among all the cell lines detected. The Fas level of Colo205 cells was moderate. In contrast, Fas protein was nearly not detected on the SW620 and Lovo cells surface because no difference in fluorescence was observed between cells exposed to anti-Fas mAb and cells exposed to isotypematched control mAb. For each tumor type, at least three samples are measured. 3.2. Differential expression of c-FLIPL mRNA in colon carcinoma cells To investigate the expression of c-FLIPL mRNA in human colon carcinoma cell lines, total RNA was extracted from subconfluent cell cultures and analyzed by RTePCR. As a 429-bp amplified fragment of c-FLIPL transcript was detected by ethidium bromide electrophoresis in HT-29, Colo205, and SW620 cells (Fig. 2); it was almost undetectable in Lovo cells. The results were confirmed by three independent assays. 3.3. Silencing of c-FLIPL mRNA and protein expression by specific siRNA in HT-29 cells The siRNA-F1, siRNA-F2 or siRNA-F1 and siRNA-F2 mixture were electroporated into HT-29 cells in order to evaluate the potency of RNA interference and analyze the efficiency of the two targeting sites. The unrelated siRNA-H was transfected into HT-29 cells as a control. Both siRNAF1 and siRNA-F2 effectively decreased c-FLIPL mRNA and the efficiency of siRNA-F2 (at about 65.41%) was slightly higher, compared with siRNA-H (Fig. 3A). To determine whether the interfering efficiency was dependent on the concentration of exogenous oligonucleotides, various amounts of siRNA (50, 100, or 150 nM) were tested. c-FLIPL mRNA decreased significantly after transfection with 100 nM or 150 nM siRNA-F2; and the interfering efficiency of 100 nM siRNA-F2 was highest among various concentrations (w66.49%; Fig. 3B). HT-29 cells were transfected with 100 nM siRNA-F2 and were harvested for semi-quantitative RTePCR analysis at 24, 48, or 72 h post-electroporation. At 24 h, the level of c-FLIPL mRNA expression diminished, while the maximum reduction by RNA interference was
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Fig. 1. Differential expression of Fas in the four colon carcinoma cell lines. Cells were stained with FITC-conjugated anti-Fas mAb and analyzed on a EPICS-XL flow cytometer. Scales were the same in all panels and each sample was repeated >3 times. (A) The yellow curves represent Fas expression detected by specific anti-Fas mAb and the red ones are analyzed by isotype-matched antibody. (B) The percentage of Fas expression is quantitated with the fluorescent intensity. Fas expression (%) ¼ Positive Cells with Fas mAb (%) Positive Cells with Isotype-matched mAb (%). Values are expressed as means SD.
detected after 48 h and the interfering efficiency was nearly 62.36% (Fig. 3C). HT-29 cells were treated under the optimal experimental conditions that cells were transfected with 100 nM siRNA-F2 and harvested at 48 h post-electroporation for cellular lysates. It is clear from Fig. 3D that siRNA-F2 significantly reduced the level of c-FLIPL protein without affecting the level of a control protein, GAPDH. 3.4. c-FLIPL-specific siRNA sensitize HT-29 cells to Fas-mediated apoptosis To study the potential functions of c-FLIPL on Fas-mediated apoptosis, HT-29 cells were transfected with c-FLIPLspecific siRNA and challenged with agonistic anti-Fas mAb.
Fig. 2. RTePCR analysis of c-FLIPL mRNA expression in the colon carcinoma cell lines. RTePCR was performed on total RNA extracted from the cultured cells and the PCR products were stained with DL2000 DNA size marker. The housekeeping gene b-actin was used as a control. Three independent experiments gave similar results.
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Fig. 3. The expression of c-FLIPL was down-regulated by specific siRNA. HT-29 cells were electroporated with 2 pairs of specific siRNA targeting different sites of c-FLIPL (sequences described in Section 2) or a pair of unrelated siRNA-H targeting HTLV-1 tax. The relative content of c-FLIPL mRNA and the efficiency of RNA interference were evaluated by semi-quantitative RTePCR analysis. Interfering Efficiency (%) ¼ [c-FLIPL relative content of siRNA-H (%) c-FLIPL relative content of siRNA-F1/siRNA-F2 (%)]/c-FLIPL relative content of siRNA-H (%) 100. Three independent experiments are presented. Values are expressed as means SD. (A) HT-29 cells were transfected with 100 nM siRNA-F1 or siRNA-F2 or the siRNA mixture (50 nM siRNA-F1 plus 50 nM siRNA-F2). Cells were harvested at 48 h post-electroporation for determining the efficiency of RNA interference. (B) HT-29 cells were transfected with siRNA-F2 at various concentrations (nM) as indicated and the relative content of c-FLIPL mRNA were evaluated at 48 h post-electroporation. (C) The expression of c-FLIPL mRNA was analyzed in HT-29 cells transfected with 100 nM siRNA-F2 for various time periods (h) as indicated. (D) HT-29 cells were transfected with 100 nM siRNA-F2 and harvested at 48 h post-electroporation. Cellular lysates were immunoblotted with monoclonal anti-c-FLIPL or anti-GAPDH as described.
Cells transfected with siRNA-F2 showed a significant increase in the hypodiploid peak using DNA content analysis (Fig. 4A). In contrast, the cells transfected with siRNA-H had a very low hypodiploid DNA peak. The early phase of apoptosis was further determined by Annexin VeFITC analysis (Fig. 4B). Transfection of siRNA-F2 into HT-29 cells led to a substantial shift in apoptotic sensitivity: after 16 h of anti-Fas treatment, HT-29 cells showed an increased number of apoptotic cells at approximately 29.5% compared with the cells transfected with siRNA-H. It should also be noted that siRNA-F2 is a specific transcriptional inhibitor targeting c-FLIPL that when used in combination with agonistic anti-Fas antibody (0.1 mg/ml)
instigated HT-29 cells to become Fas sensitive. Treatment of HT-29 cells with siRNA-F2 alone did not lead to significant cell death (data not shown). 4. Discussion Fas-mediated apoptosis is an effective pathway for cell-mediated cytotoxicity and participates in the immune response against neoplastic cells. The emergence of resistance to this pathway contributes to tumor cells escape from immune surveillance and is an important step during the carcinogenesis process. A more widespread mechanism of this resistance
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Fig. 4. RNA interference directed against c-FLIPL resulted in an increase in apoptotic cells. The specific siRNA-F2 (100 nM) or the control siRNA-H (100 nM) was electroporated into HT-29 cells 48 h before the agonistic anti-Fas mAb addition. The cells were harvested for evaluating apoptosis after the anti-Fas mAb (0.1 mg/ ml) treatment for 16 h. The results were representative of three separate experiments. Values are expressed as means SD. (A) HT-29 cells were labeled with PI to analyze the percentage of cells with hypodiploid DNA using flow cytometry. (B) Annexin VeFITC analysis was carried out in order to identify the apoptotic cells from viable cells and dead cells. The percentage of the apoptotic cells was indicated in the Annexin VeFITCþ/PI population and was quantified with the dot plots.
described in several models is the over-expression of antiapoptosis proteins. Data from gene expression profiling showed that c-FLIP is over-expressed in several types of carcinomas and is associated with an increased mortality in patients with prostate, breast, and lung cancers (Bhattacharjee et al., 2001; van’t Veer et al., 2002; Varambally et al., 2005). c-FLIP isoforms are characterized as a short half-life by inhibiting of protein synthesis with cycloheximide and it has been proposed that mechanisms of differential degradation of c-FLIP isoforms might be important for apoptosis sensitivity (Mitsiades et al., 2000; Schmitz et al., 2004). c-FLIPR and c-FLIPS contain only two tandem repeats of DEDs and inhibit activation of caspase-8 and apoptosis in a similar mechanism (Djerbi et al.,
2001; Golks et al., 2005). In contrast, c-FLIPL shares extensive homology with caspase-8, with a C-terminal domain that is highly homologous to the caspase-8 protease domain yet enzymatically inactive due to the lack of key active site residues. To date, the role of c-FLIPL in apoptosis remains controversial. In the present study, we observed a significant increase of the hypodiploid peak in colon carcinoma cells in which cFLIPL was down-regulated with specific siRNA. Further studies showed that such cells underwent early phase of apoptosis to Fas signaling for the appearance of phosphatidylserine on the cell surface. It has been previously reported that the expression of c-FLIPL in colon adenocarcinomas is frequently elevated at both mRNA and protein level compared with
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matched normal tissues and adenomatous polyps (Ryu et al., 2001). Our results indicated that c-FLIPL promotes colon carcinoma cells to escape Fas-mediated apoptosis in vitro, shedding slight light on the mechanism of such frequent event in vivo during the malignant transformation or progression of colon carcinoma. The mechanism of cell death attenuation by c-FLIPL has not been completely elucidated. Early experiments suggested that c-FLIPL, as a potential competitive inhibitor, preclude recruitment of caspase-8 to the DISC and thereby prevent its activation. This idea was supported by the fact that upon over-expression of the viral homolog v-FLIP-E8, c-FLIPL is cleaved into a p43 subunit that remains at the DISC and a releasing p12 subunit (Thome et al., 1999). Loss-of-function experiments suggested that depleted c-FLIPL results in more complete processing of the caspase and faster turnover of the intermediate (Sharp et al., 2005). However, caspase-8 can not be processed to form active tetramer, which executes the key proteolytic function in the apoptotic pathway (Krueger et al., 2001). The partial processing of active caspase-8 in caspase-8/c-FLIPL heterodimers retains active caspase-8 at the DISC, preventing its release into the cytosol (Chang et al., 2003). More interestingly, not only full-length c-FLIPL but also p43 subunit can inhibit FasL induced apoptosis, implying both full-length and cleaved c-FLIPL possess strong apoptosisresistant ability (Boatright et al., 2004). In addition, chemotherapeutic drug cisplatin inhibits phosphorylation of c-FLIPL without down-regulating its expression levels and sensitizes melanoma cells to TRAIL-induced apoptosis, indicating that phosphorylation may play an essential role in the regulation of c-FLIPL anti-apoptosis activity (Higuchi et al., 2003; Song et al., 2003). However, in other carcinomas, c-FLIPL can promote caspase-8 activation by acting directly on the protease domain (Han et al., 1997; Inohara et al., 1997; Guseva et al., in press). Purified caspase-8 is shown to heterodimerize with c-FLIPL, and these heterodimers have a higher affinity for Fas-FADD complexes and considerable proteolytic activity compared with caspase-8/capase-8 homodimers (Boatright et al., 2004). Further studies show that c-FLIPL is a dual function regulator for caspase-8 activation and apoptosis dependent on its environmental conditions and expression level. In the case of T cell antigen receptor-mediated signaling, c-FLIPL likely plays an important role in caspase-8 activation (Micheau et al., 2002; Misra et al., 2007). In contrast, when Fas is ligated, capase-8 activation can be inhibited by c-FLIPL-mediated competition with caspase-8 for recruitment to FADD (Hu et al., 2000). Moreover, at the levels comparable with the endogenous protein, c-FLIPL may enhance procaspase-8 activation in the DISC and Fas-mediated apoptosis, whereas at very high non-physiological levels achieved by transient overexpression, it inhibits apoptosis (Chang et al., 2002). In the present study, we assessed the specific function of endogenous c-FLIPL by a selective siRNA knockdown approach and found that depletion of endogenous c-FLIPL augmented apoptosis induction by agonistic antibody in colon carcinoma cell line. Thus, c-FLIPL functions primarily as an inhibitor, rather
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than a promoter, of Fas-mediated apoptosis. These results, which seem to contradict the dual-function studies, might be reconciled by the possibility that the HT-29 cell line expresses intermediate or high level of c-FLIPL, which is enough to inhibit formation of DISC and death receptor-mediated apoptosis. Based on the previous research and our experiments, c-FLIPL and caspase-8 expression may not fully explain independently the apoptotic fate of the cells. A recent study shows that in thyroid carcinomas, the proteolytic cleavage and activation of caspase-8 depends on the balance between expression levels for caspase-8 and c-FLIPL, and correlates with favorable clinical prognosis (Mitsiades et al., 2006). It should be noted that both the death receptor pathway and the mitochondrial pathway are involved in apoptosis. In some cells, apoptosis that is initiated through the death receptor pathway still requires amplification through the mitochondrial pathway of caspases activation (Kuwana et al., 1998; Rosen et al., 2001). Our results indicate that the four colon carcinoma cell lines escape apoptosis at different points in different pathways. HT-29 cells which express high levels of Fas and c-FLIPL develop an apoptosis-resistant phenotype perhaps mainly by down-regulation of c-FLIPL. SW620 and Colo205 cells express lower levels of Fas and high or moderate levels of c-FLIPL respectively. These two cell lines are supposed to escape Fas-mediated apoptosis by either up-regulating c-FLIPL or down-regulating Fas. Lovo cells show minimal Fas and c-FLIPL and are reported to high express Bcl-2 and Bcl-XL, suggesting that apoptosis can be prevented by inhibiting the mitochondrial pathway (Nita et al., 1998). Further studies are needed to assess the relative contribution of these two pathways to apoptosis. In summary, our data suggest that the over-expression of cFLIPL in colon carcinoma suppresses Fas-mediated apoptosis. We propose that inhibiting c-FLIPL expression may be a useful strategy to investigate as a possible approach for treatment of malignant cells.
Acknowledgments This work was supported by grants from the Natural Sciences Foundation of Tianjin, PR China (013611511). We are grateful to Professor Harald Wajant for the kind provision of c-FLIPL-siRNA. References Bhattacharjee A, Richards WG, Staunton J, Li C, Monti S, Vasa P, et al. Classification of human lung carcinomas by mRNA expression profiling reveals distinct adenocarcinoma subclasses. Proc Natl Acad Sci USA 2001;98:13790e5. Boatright KM, Deis C, Jenault JB, Sutherlin DP, Salvesen GS. Activation of caspase-8 and -10 by FLIPL. Biochem J 2004;382:651e7. Chang DW, Xing Z, Pan Y, Algeciras-Schimnich A, Barnhart BC, YaishOhad S, et al. c-FLIP(L) is a dual function regulator for caspase-8 activation and CD95-mediated apoptosis. EMBO J 2002;21:3704e14. Chang DW, Xing Z, Capacio VL, Peter ME, Yang X. Interdimer processing mechanism of procaspase-8 activation. EMBO J 2003;22:4132e42.
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