TY - JOUR
AU - Kirschfink, M
AB - Summary Inflammation is a critical component of tumour progression. Although complement and tumour necrosis factor (TNF)-α potentially exert significant anti-tumour effects, both mediators may also promote tumour progression. It has been demonstrated that sublytic complement confers resistance on tumour cells not only against lytic complement, but also other danger molecules such as perforin. In low concentrations, TNF promotes survival of malignant cells rather than exerting cytotoxic activity. In this study, we tested if sublytic complement is able to interfere with TNF-mediated tumour cell killing. Our results demonstrate that either subcytotoxic concentrations of TNF or sublytic complement rescue prostate carcinoma cells (DU145) from TNF-α-mediated cell death. Upon pretreatment with low-dose TNF-α, but not upon pre-exposure to sublytic complement, TNF resistance was associated with the down-regulation of TNF receptor 1 (TNF-R1) expression. Complement-induced protection against TNF-mediated apoptosis accompanied the induction of anti-apoptotic proteins [B cell leukaemia/lymphoma (Bcl)-2 and Bcl-xL] at an early stage followed by inhibition of the TNF-induced decrease in the amount of Bcl-2 and Bcl-xL. Cell protection also accompanied the inhibition of caspase-8 activation, poly (ADP-ribose) polymerase (PARP)-1 cleavage and the activation of nuclear factor (NF)-κB. Our data extend our current view on the induction of tumour cell resistance against cytotoxic mediators supporting the role of the tumour microenvironment in mediating protection against the anti-cancer immune response. cell death, complement, DU145, TNF Introduction Cancer-related inflammation is associated with the presence of inflammatory cells and inflammatory mediators in the tumour microenvironment which elicit anti-tumour, but also tumour-promoting, activities [1,2]. The complement system is an essential constituent of innate immunity. It is involved actively in the host defence against infectious agents and the removal of immune complexes or apoptotic cells [3,4]. Complement activation by either of the three major pathways, the classical, alternative and lectin pathways, leads subsequently to formation of the pore-like membrane attack complex (C5b-9, MAC [5]). Despite the fact that activated complement components and the terminal MAC have been found deposited in tumour tissue [6–8], its functional relevance is still unclear and has even been disputed [9]. The failure of complement to destroy malignant cells effectively is of considerable clinical interest, as it restricts the optimal efficiency of tumour-directed antibody therapy [10]. A basal mechanism by which nucleated cells are protected from complement attack is the constitutive expression of membrane regulatory proteins, such as membrane co-factor protein (MCP; CD46), decay-accelerating factor (DAF; CD55) and CD59 [11]. It appears that, in particular, over-expression of these inhibitors on malignant cells reduces significantly their susceptibility to complement-mediated destruction [11,12]. Furthermore, ecto-proteases [13,14] and the expression of the heat shock protein mortalin [15] confer resistance to complement-mediated lysis on tumour cells by degradation of active complement components or MAC shedding. The protection of tumour cells against complement can also be induced or augmented upon stimulation with cytokines, hormones and drugs [16,17]. One of the most potent agents to increase resistance to complement attack is sublytic complement itself [18]. This complement-induced protection has been shown to require complete MAC formation, protein and RNA synthesis and free extracellular Ca2+. Induced resistance is not limited to MAC attack, but also to other cytotoxic mediators such as perforin, streptolysin O and melittin which, in turn, induce protection against complement lysis [19]. To counteract successful complement attack, sublytic complement activates a survival programme. Although a transmembrane receptor for C5b-9 is unknown, the MAC induces Ca2+ influx and mobilization and activates phospholipases protein kinase C (PKC) and extracellular regulated kinase (ERK), which results in target cell resistance to complement-mediated damage [20–27]. Tumour necrosis factor (TNF) was named for its ability to induce rapid haemorrhagic necrosis of experimental cancers [28]. However, it soon became evident that the cytokine exerts in-vivo anti-tumour activity and cytotoxicity against some, but not all, tumour cells [29]. Today TNF is considered a major player in host defence and inflammation with activities that extend far beyond its originally described anti-tumour effect [30]. TNF signalling may lead not only to target cell apoptosis and necrosis, but also to tumour progression and metastasis by induction of survival genes [31,32]. TNF exerts its multiple biological activities via interaction with TNF receptor 1 (TNF-R1) and TNF-R2 [33,34]. TNF-R1 is expressed constitutively in most tissues, whereas expression of TNF-R2 is highly regulated and is found typically on cells of the immune system. TNF binds to the death domain containing TNF-R1 to recruit TNF receptor-associated death domain (TRADD), Fas-associated death domain (FADD) and caspase-8, thereby forming the death-inducing signalling complex [35,36]. However, activated TNF-R1 also recruits receptor-interacting protein (RIP) and TNF receptor-associated factor 2 (TRAF2) and activates nuclear factor (NF)-κB, which is involved in cell survival, proliferation, anti-apoptosis and the inflammatory response [35]. RIP was also found to be essential for FAS, TRAIL and TNF-induced programmed necrosis [37]. As TNF is either produced constitutively or induced in malignant cells it may exert activities towards tumour progression in the microenvironment, even in the absence of invading inflammatory cells [38]. It has also been reported that numerous tumours are resistant to TRAIL-induced cytotoxicity; however, the reasons for this are not yet fully understood [39]. Because the development of such resistance phenomena may be induced in a microenvironment which contains multiple inflammatory mediators, we wished to determine if susceptibility of tumour cells to TNF-mediated destruction may be modulated not only by TNF itself but also by complement. In this study we demonstrate that pre-exposure of human prostate carcinoma cells (DU145) to sublytic complement decreases significantly their susceptibility to TNF-mediated killing. This indicates that limited complement activation within the tumour microenvironment may contribute to the resistance of malignant cells not only to subsequent complement attack, but also to TNF-mediated cell death. Materials and methods Cell lines, antibodies and serum DU145 human prostate carcinoma cells (American Type Culture Collection, Manassas, VA, USA) were cultured in RPMI-1640 (PAA Laboratories, Cölbe, Germany) containing 10% heat-inactivated fetal calf serum (FCS) (Gibco-Invitrogen, Eggenstein, Germany) at 37°C and 5% CO2. Polyclonal anti-serum against DU145 (αDU145) was prepared in rabbits by three intravenous injections of 3 × 106 intact DU145 cells and inactivated at 56°C for 30 min, as described previously [40]. As a source for complement, normal human serum (NHS) was collected freshly from healthy blood donors. Parts of them were heat-inactivated (30 min, 56°C) and frozen in aliquots at −70°C. Pretreatment with sublytic complement or subcytotoxic TNF DU145 cells (5 × 105/ml in RPMI-1640, supplemented with 10% heat-inactivated FCS) were cultured overnight at 37°C and 5% CO2. The cells were pretreated with 10 ng/ml TNF-α (ImmunoTools, Friesoythe, Germany) for 2 h at 37°C, which was predetermined in dose–response and kinetic experiments to be subcytotoxic (5–10% cell death). Another batch of cells was pretreated with αDU145 antibody in 10% NHS at sublytic concentration [SLC, producing 5–10% cell lysis by 2,3-bis-(2-methoxy-4-nitro-5-sulphophenyl)-2H-tetrazolium-5-carboxanilide inner salt (XTT) for 30 min at 37°C, as described previously [16]. Cells treated with αDU145 antibody and heat-inactivated 10% NHS [heat-inactivated sublytic complement (SLCia)] served as control. All cells were then exposed to cytotoxic concentrations of TNF (500 ng/ml). Flow cytometry analysis of TNF-R1 expression DU145 cells, pretreated with either subcytotoxic concentrations of TNF or sublytic complement, were washed [phosphate-buffered saline (PBS), 1% bovine serum albumin (BSA)] and incubated with mouse monoclonal antibody (mAb) anti-TNF-R1 (R&D Systems, Wiesbaden, Germany) for 30 min at 4°C, washed again and treated with fluorescein isothiocyanate (FITC)-conjugated goat anti-mouse IgG for 30 min at 4°C. Cells were fixed with 1% paraformaldehyde in PBS before cytofluorometric analysis (FACScan; Becton Dickinson, Heidelberg, Germany). Expression of TNF-R1 was quantified using Quifikit (Dako, Hamburg, Germany), where a series of beads coated with different but well-defined quantities of mouse monoclonal immunoglobulin (Ig)G molecules are used for the construction of the calibration curve, according to the supplier's instructions. Analysis of TNF-mediated cytotoxicity XTT assay DU145 cells were incubated for 48 h with increasing concentrations of TNF-α. Then, 50 µl XTT solution (Roche Diagnostics, Mannheim, Germany) was added and incubated for 4 h at 37°C and 5% CO2. Colour development [optical density (OD) value] was measured on an enzyme-linked immunosorbent assay (ELISA) reader (Sunrise™; Tecan, Crailsheim, Germany) at λ = 490 nm/650 nm. All cytotoxicity assays were performed in the presence of 1 µg/ml actinomycin D (Sigma, Steinheim, Germany), as the cytotoxic potential of TNF is revealed and survival mechanisms are inactivated only in combination with metabolic inhibitors, allowing apoptosis to proceed [41]. Cytotoxicty was calculated as follows: cytotoxicity (%) =  (ODcontrol − ODsample)/(ODcontrol − ODblank) × 100% (controls: cells only with culture medium; blank: only culture medium). Annexin-V/propidium iodide assay DU145 cells (1 × 106) were incubated for 8, 12 or 24 h with 500 ng/ml TNF in the presence of 1 µg/ml actinomycin D. After washing and centrifugation (200 g, 5 min), the cells were resuspended in 100 µl of annexin-V-FLUOS/propidium iodide (PI) labelling solution (Roche Diagnostics), incubated for 10–15 min at 15–25°C and analysed by flow cytometry. Annexin-V-positive/PI-negative cells were defined as early apoptotic cells, annexin V-positive/PI-positive as late apoptotic or necrotic cells. Western blot Cells were lysed with radioimmunoprecipitation assay (RIPA) buffer [30 mM Tris-HCl, pH 7·4, 0·15 M NaCl, 1% Nonidet P-40, 0·1% sodium dodecyl sulphide (SDS), 0·5% sodium deoxycholate, 1 mM ethylenediamine tetraacetic acid (EDTA), 1 mM dithiothreitol (DTT), 2 mM MgCl2, 1 mM NaVO4, 0·5 mM phenylmethylsulphonyl fluoride (PMSF), 100 µg/ml aprotinin and 100 µg/ml leupeptin]. An equal amount of protein from each cell lysate was boiled for 5 min at 95°C, separated on a 10% SDS-polyacrylamide gel and electroblotted to a nitrocellulose membrane. The membranes were blocked for 1 h with Tris-buffered saline (TBS), 5% skimmed milk, and then immunoblotted for 1 h with mouse anti-caspase-8 IgG (BD Pharmingen, Heidelberg, Germany), mouse anti-poly (ADP-ribose) polymerase (PARP)-1 IgG1 (BD Pharmingen), mouse anti-Bcl-2 IgG1 (Santa Cruz Biotechnology, Heidelberg, Germany), rabbit anti-Bcl-xL IgG, rabbit anti-Mcl-1 IgG (all from Cell Signaling, New England Biolabs, Frankfurt, Germany) or rabbit anti-β actin IgG (Sigma). After washing with TBS, 0·05% Tween-20, the membranes were incubated with peroxidase-conjugated affinity purified goat anti-mouse or goat anti-rabbit IgG (Dianova, Hamburg, Germany) for 1 h. Protein bands were visualized by chemiluminescence applying the enhanced chemiluminescence (ECL) Western blotting reagent (GE Healthcare, Munich, Germany). NF-κB p65 activity assay Cells were lysed according to the manufacturer's protocol (nuclear extract kit, Active Motif; THP Medical Products, Wien, Austria). Nuclear extract (2·5 µg) from each cell lysate was analysed by ELISA (λ = 450 nm/655 nm) for NF-κB p65 activity (TransAM NF-κB p65 kit, Active Motif). Statistical analysis Data are expressed as mean ± standard deviation. Statistical differences were analysed by two-sided unpaired Student's t-test. A P-value < 0·05 was considered statistically significant. Results Sublytic complement confers resistance to TNF-mediated cytotoxicity To determine whether pretreatment with subcytotoxic concentrations of TNF has any impact on the susceptibility of the prostate carcinoma cells to TNF-mediated cytotoxicity, DU145 cells were incubated with subcytotoxic TNF or medium for 2 h, followed subsequently by exposure to cytotoxic doses of TNF in the presence of 1 µg/ml actinomycin D for 48 h. DU145 became significantly more resistant to TNF-induced tumour cell killing upon pretreatment with low doses of TNF (Fig. 1a). Next, we analysed if TNF resistance could be induced by complement. After pre-exposure to sublytic complement (SLC: αDU145 antibody in 10% NHS at sublytic concentration) or heat-inactivated sublytic complement (SLCia: NHS replaced by 10% heat-inactivated NHS) as control, the prostate cancer cells also became significantly resistant to TNF-mediated cytotoxicity (Fig. 1b). αDU145 antibody, NHS alone or heat-inactivated sublytic complement did not affect TNF-mediated cytotoxicity in DU145 (not shown). Fig. 1 Open in new tabDownload slide Tumour necrosis factor (TNF) and complement both decrease the sensitivity of DU145 prostate carcinoma cells to TNF-mediated cell death. (a) DU145 cells were pretreated with either 10 ng/ml TNF (a), sublytic complement (SLC) or heat-inactivated sublytic complement (SLCia) as control (b). Cells pre-incubated with buffer were taken as reference in both experimental series. After washing, cells were exposed to different concentrations of TNF (1, 8, 62·5, 500 ng/ml) in the presence of 1 µg/ml actinomycin D for 48 h at 37°C. Cell death was determined by 2,3-bis-(2-methoxy-4-nitro-5-sulphophenyl)-2H-tetrazolium-5-carboxanilide (XTT) assay (n ≥ 8 independent experiments; *P < 0·05; **P < 0·01; ***P < 0·001). Fig. 1 Open in new tabDownload slide Tumour necrosis factor (TNF) and complement both decrease the sensitivity of DU145 prostate carcinoma cells to TNF-mediated cell death. (a) DU145 cells were pretreated with either 10 ng/ml TNF (a), sublytic complement (SLC) or heat-inactivated sublytic complement (SLCia) as control (b). Cells pre-incubated with buffer were taken as reference in both experimental series. After washing, cells were exposed to different concentrations of TNF (1, 8, 62·5, 500 ng/ml) in the presence of 1 µg/ml actinomycin D for 48 h at 37°C. Cell death was determined by 2,3-bis-(2-methoxy-4-nitro-5-sulphophenyl)-2H-tetrazolium-5-carboxanilide (XTT) assay (n ≥ 8 independent experiments; *P < 0·05; **P < 0·01; ***P < 0·001). These data indicate that pretreatment with subcytotoxic concentrations of either TNF or sublytic complement protects tumour cells against TNF-mediated cell killing. Sublytic complement has no impact on TNF-R1 expression As TNF exerts its cytotoxic effect mainly via the death domain containing TNF-R1, we analysed the expression of the cytokine receptor to explore the mechanism underlying the impaired sensitivity to TNF induced by sublytic complement. Interestingly, the expression of TNF-R1 was down-regulated significantly only by low-dose TNF (Fig. 2a), whereas no effect was observed upon exposure to sublytic complement (Fig. 2b), indicating that other mechanisms are responsible for complement-mediated protection against TNF. Fig. 2 Open in new tabDownload slide In contrast to low-dose tumour necrosis factor (TNF), sublytic complement has no impact on TNF factor receptor 1 (TNF-R1) expression. DU145 cells were treated with either 10 ng/ml TNF for 2 h (a) or sublytic complement (SLC) or heat-inactivated sublytic complement (SLCia) as control (b) [αDU145 antibody for 30 min, then 10% normal human serum (NHS) (10% heat-inactivated NHS as control) for 1 h]. TNF-R1 expression was detected by mouse monoclonal antibody (mAb) anti-TNF-R1 [mouse immunoglobulin (Ig)G1 as isotype control] by fluorescence activated cell sorter (FACS) analysis. From a calibration curve, set up with mouse IgG-coupled beads of defined quantities, the number of TNF-R1 molecules per cell was quantified (n ≥ 3 independent experiments; *P < 0·05; **P < 0·01; ***P < 0·001). Fig. 2 Open in new tabDownload slide In contrast to low-dose tumour necrosis factor (TNF), sublytic complement has no impact on TNF factor receptor 1 (TNF-R1) expression. DU145 cells were treated with either 10 ng/ml TNF for 2 h (a) or sublytic complement (SLC) or heat-inactivated sublytic complement (SLCia) as control (b) [αDU145 antibody for 30 min, then 10% normal human serum (NHS) (10% heat-inactivated NHS as control) for 1 h]. TNF-R1 expression was detected by mouse monoclonal antibody (mAb) anti-TNF-R1 [mouse immunoglobulin (Ig)G1 as isotype control] by fluorescence activated cell sorter (FACS) analysis. From a calibration curve, set up with mouse IgG-coupled beads of defined quantities, the number of TNF-R1 molecules per cell was quantified (n ≥ 3 independent experiments; *P < 0·05; **P < 0·01; ***P < 0·001). Sublytic complement protects tumour cells against TNF -induced apoptosis As TNF mediates both cell necrosis and apoptosis, we next analysed the possible impact of sublytic complement on TNF-induced apoptosis in DU145. As expected, 500 ng/ml TNF induced apoptosis (early apoptosis: annexin V-positive and PI-negative; late apoptosis: annexin V and PI double-positive), which was also reflected by caspase-8 activation (see below, Fig. 4a). After pretreatment with sublytic complement, TNF-induced apoptosis (either early or late apoptosis) decreased over the time compared to the control (pretreatment with inactive sublytic complement) (Fig. 3). We conclude from these data that sublytic complement confers resistance to TNF-induced apoptosis to tumour cells. Fig. 3 Open in new tabDownload slide Sublytic complement confers onto DU145 cells resistance to tumour necrosis factor (TNF)-induced cell death. DU145 cells were pretreated either with sublytic complement (SLC), heat-inactivated sublytic complement (SLCia) or medium as controls. After washing, cells were treated with 500 ng/ml TNF in the presence of 1 µg/ml actinomycin D for 8, 12 and 24 h at 37°C. Cells were collected and washed, stained with annexin V and propidium iodide (PI) and analysed by flow cytometry (annexin V-FL1H and PI–FL2H) (n ≥ 3 independent experiments; *P < 0·05; **P < 0·01; ***P < 0·001). Fig. 3 Open in new tabDownload slide Sublytic complement confers onto DU145 cells resistance to tumour necrosis factor (TNF)-induced cell death. DU145 cells were pretreated either with sublytic complement (SLC), heat-inactivated sublytic complement (SLCia) or medium as controls. After washing, cells were treated with 500 ng/ml TNF in the presence of 1 µg/ml actinomycin D for 8, 12 and 24 h at 37°C. Cells were collected and washed, stained with annexin V and propidium iodide (PI) and analysed by flow cytometry (annexin V-FL1H and PI–FL2H) (n ≥ 3 independent experiments; *P < 0·05; **P < 0·01; ***P < 0·001). Sublytic complement inhibits TNF-mediated caspase-8 activation and PARP-1 cleavage Upon TNF binding, TNF-R1 recruits TRADD via its death domain and TRADD binds FADD, which then activates caspase-8 and caspase-3, leading subsequently to cell apoptosis. To analyse the molecular mechanisms by which sublytic complement impaired the sensitivity to TNF-induced apoptosis, we determined the activation of caspase-8. As shown in Fig. 4a,b, pre-exposure to sublytic complement inhibited significantly the activation of caspase-8 at different time-points. This accompanied a reduced cleavage of the caspase substrate PARP-1 throughout the entire observation period (Fig. 4c,d). Fig. 4 Open in new tabDownload slide Sublytic complement inhibits caspase-8 activation and poly (ADP-ribose) polymerase-1 (PARP-1) cleavage. DU145 cells were pretreated with sublytic complement (SLC) or heat-inactivated sublytic complement (SLCia) as control. After washing, cells were treated with 500 ng/ml tumour necrosis factor (TNF) in the presence of 1 µg/ml actinomycin D for 8, 12 and 24 h at 37°C; 2 × 105 cells were lysed and an equal amount of protein from each cell lysate was used for Western blot detection of caspase-8 (a,b) and PARP-1 (c,d), with β-actin as control (one of n ≥ 3 independent experiments). Fig. 4 Open in new tabDownload slide Sublytic complement inhibits caspase-8 activation and poly (ADP-ribose) polymerase-1 (PARP-1) cleavage. DU145 cells were pretreated with sublytic complement (SLC) or heat-inactivated sublytic complement (SLCia) as control. After washing, cells were treated with 500 ng/ml tumour necrosis factor (TNF) in the presence of 1 µg/ml actinomycin D for 8, 12 and 24 h at 37°C; 2 × 105 cells were lysed and an equal amount of protein from each cell lysate was used for Western blot detection of caspase-8 (a,b) and PARP-1 (c,d), with β-actin as control (one of n ≥ 3 independent experiments). Sublytic complement induces the expression of anti-apoptotic proteins To determine whether or not anti-apoptotic and pro-apoptotic proteins would be affected by sublytic complement we analysed expression of the anti-apoptotic proteins Bcl-2, Bcl-xL and Mcl-1. We found that in response to TNF (and in the presence of inactivated SLC), Bcl-2 and Bcl-xL decreased gradually over time and Mcl-1 disappeared. Pretreatment with sublytic complement blocked the inhibitory effect of TNF on Bcl-2 and Bcl-xL expression and even appeared to increase expression at 8 h in comparison to non-TNF-treated cells (Fig. 5a–c). Actinomycin D at a low concentration (1 µg/ml) did not affect the expression of anti-apoptotic proteins (data not shown). Fig. 5 Open in new tabDownload slide Sublytic complement induces anti-apoptotic protein expression. DU145 cells were pretreated with sublytic complement (SLC) or heat-inactivated sublytic complement (SLCia) as control. After washing, cells were treated with 500 ng/ml TNF in the presence of 1 µg/ml actinomycin D for 8, 12 and 24 h at 37°C; 2 × 105 cells were lysed and an equal amount of protein from each cell lysate was used for Western blot detection (a). Densitometric evaluation of the results of (a) of Bcl-2 (b) and Bcl-xL (c) is shown in comparison to β-actin as control (n ≥ 3 independent experiments; *SLC- compared with SLCia-pretreated cells; #SLC-pretreated compared with untreated cells; * or #P < 0·05; ** or ##P < 0·01; ***P < 0·001). Fig. 5 Open in new tabDownload slide Sublytic complement induces anti-apoptotic protein expression. DU145 cells were pretreated with sublytic complement (SLC) or heat-inactivated sublytic complement (SLCia) as control. After washing, cells were treated with 500 ng/ml TNF in the presence of 1 µg/ml actinomycin D for 8, 12 and 24 h at 37°C; 2 × 105 cells were lysed and an equal amount of protein from each cell lysate was used for Western blot detection (a). Densitometric evaluation of the results of (a) of Bcl-2 (b) and Bcl-xL (c) is shown in comparison to β-actin as control (n ≥ 3 independent experiments; *SLC- compared with SLCia-pretreated cells; #SLC-pretreated compared with untreated cells; * or #P < 0·05; ** or ##P < 0·01; ***P < 0·001). However, sublytic complement failed to affect TNF-mediated inhibition of Mcl-1 expression (Fig. 5a). These data indicate that sublytic complement counteracts TNF-mediated suppression of the anti-apoptotic proteins Bcl-2 and Bcl-xL. Sublytic complement activates NF-κB Bcl-2 and Bcl-xL are regulated in an NF-κB-dependent manner [42,43]. We therefore analysed if sublytic complement activates NF-κB, providing a possible explanation for the up-regulation of both the anti-apoptotic proteins. Indeed, NF-κB activation was induced by sublytic complement alone but also enhanced the effect of TNF (Fig. 6), suggesting that sublytic complement-induced NF-κB activation may contribute to the impaired sensitivity of tumour cells towards TNF-mediated apoptosis. Fig. 6 Open in new tabDownload slide Sublytic complement induces nuclear factor (NF)-κB activation. DU145 cells were pretreated with sublytic complement (SLC) or heat-inactivated sublytic complement (SLCia), anti-DU145 antibody alone or with medium as controls. After washing, cells were exposed to 500 ng/ml tumour necrosis factor (TNF) in the presence of 1 µg/ml actinomycin D or medium as control for 1 or 2 h at 37°C. To exclude a direct effect of Cells were lysed and 2·5 µg nuclear extract from each cell lysate was analysed for NF-κB p65 activity. Data are presented as optical density (OD) value for NF-κB p65 activity (n ≥ 3 independent experiments, *P < 0·05; **P < 0·01; ***P < 0·001). Fig. 6 Open in new tabDownload slide Sublytic complement induces nuclear factor (NF)-κB activation. DU145 cells were pretreated with sublytic complement (SLC) or heat-inactivated sublytic complement (SLCia), anti-DU145 antibody alone or with medium as controls. After washing, cells were exposed to 500 ng/ml tumour necrosis factor (TNF) in the presence of 1 µg/ml actinomycin D or medium as control for 1 or 2 h at 37°C. To exclude a direct effect of Cells were lysed and 2·5 µg nuclear extract from each cell lysate was analysed for NF-κB p65 activity. Data are presented as optical density (OD) value for NF-κB p65 activity (n ≥ 3 independent experiments, *P < 0·05; **P < 0·01; ***P < 0·001). Discussion TNF plays an important role in the regulation and co-ordination of multiple immune and inflammatory reactions. Upon activation, TNF-R1 can engage either apoptotic or survival pathways. In this study, we demonstrate that DU145 prostate carcinoma cells become resistant to TNF-mediated apoptosis if they have been pre-exposed to TNF in sublethal concentrations. The reduced susceptibility of the tumour cell was associated with a significant down-regulation of TNF-R1. Our observations are in line with previous reports, indicating that subcytotoxic TNF supports the survival of at least some malignant cell lines [44] and decreases apoptosis of neutrophils [45] and that resistance to apoptosis occurs in dendritic cells which lack TNF-R1 [46]. Induction of protective mechanisms by TNF enables cells to resist its cytotoxic activity [47], and metabolic inhibitors, such as actinomycin D or mitomycin C [48,49] or the additional impact of interferon (IFN)-γ[50], appear to abolish these protective mechanisms. Inducible mechanisms of resistance of tumour cells have also been described for complement [12,18,51]. The assembly of C5b-9 macromolecules induces cell death by a lytic dose of complement [52]. In contrast, upon low-grade activation, sublytic complement protects cells from complement-mediated cell death [18]. As cross-protection was shown for other dangerous molecules, such as perforin [19], we wanted to determine whether or not tumour cells were able to resist the cytotoxic action of TNF if they were pre-exposed to sublytic complement. Our results demonstrate that complement protects tumour cells from TNF-induced cell death. Interestingly, sublytic complement failed to affect the expression of TNF-R1 (as did TNF) but inhibited caspase-8 activation and PARP-1 cleavage, pointing to an interference with TNF-mediated apoptotic signalling. The substrate of the caspases, PARP1, can be cleaved into two fragments, a 24 kDa N-terminal peptide that retains the DNA binding domains and a C-terminal 89 kDa fragment that has reduced catalytic activity; its detection is usually taken as a sensitive marker for apoptosis [53,54]. TNF-induced apoptosis is regulated by anti- and pro-apoptotic proteins [55,56]. The Bcl-2 family contains a group of proteins which can govern mitochondrial outer membrane permeabilization and can be either pro-apoptotic (Bax, Bad, Bak and Bok) or anti-apoptotic (Bcl-2, Bcl-xL and Bcl-w). Of those anti-apoptotic proteins, Bcl-2, Bcl-xL and Mcl-1 were reported to counteract TNF-α-induced apoptosis [57,58]. In this study we show that sublytic complement not only induces Bcl-2 and Bcl-xL expression, but also blocks TNF-induced suppression of these anti-apoptotic molecules, in accordance with previous reports. Sublytic C5b-9 has been shown to rescue Schwann cells from apoptosis via Bad phosphorylation at Ser 136 and increases expression of Bcl-xL [59]. Furthermore, in oligodendrocytes, C5b-9 inhibits caspase-8 activation, up-regulates FADD-like ICE (FLICE) inhibitory protein (FLIP) and increases Bcl-2 protein with Bad phosphorylation at Ser112 and Ser136 and its dissociation from Bcl-xL [25,26,60]. In our study it appears that complement counteracts the inhibitory action of TNF on Bcl-2 or Bcl-xL expression, which may also contribute to the protection of tumour cells against TNF-induced apoptosis. Expression of Bcl-2 and Bcl-xL is NF-κB-dependent [42,61], and inhibition of NF-κB-dependent Bcl-xL expression promotes cell apoptosis [42,43,61]. Furthermore, tumour cells, in which NF-κB is activated constitutively, have been shown to be resistant to TNF-mediated cytotoxicity [62]. We therefore hypothesized that sublytic complement may up-regulate anti-apoptotic molecule expression via employment of NF-κB. Our data indicate that activation of NF-κB is induced by sublytic complement and also protects tumour cells from TNF-α-mediated apoptosis by up-regulation of anti-apoptotic molecules such as Bcl-2 and Bcl-xL. Sublytic C5b-9 induces Ca2+ influx and mobilization [63], with subsequent activation of PKC [64] that is related closely to NF-κB activation. C5b-9 has been shown to interact with G-proteins, leading to activation of PI3K/Akt [25]. By regulation of this pathway, TNF-α-mediated apoptosis is suppressed [65,66]. It is conceivable that sublytic complement may activate NF-κB via PKC and PI3K/Akt pathways, thereby protecting cells from TNF-mediated cell death. In conclusion, our results indicate that sublytic complement induces tumour cell resistance against TNF-mediated killing by activation of NF-κB and interference with TNF-mediated caspase-8 activation and Bcl-2/Bcl-xL suppression. These data extend our current view on the impact of complement in cancer-related inflammation and underlines the importance of the microenvironment in tumour escape or even promotion. This adds to the well-known evasion strategy by over-expression of surface complement regulators which allow malignant cells to survive in a proinflammatory environment [11,67]. In this study, we excluded over-expression of surface complement regulators as a protective mechanism (not shown). A possible role of complement beyond the protection of tumour cells from cytotoxic attack has been suggested recently, based on the somewhat unexpected findings of Mariewsky et al. [68]. 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Clinical and Experimental Immunology © 2012 British Society for Immunology This article is published and distributed under the terms of the Oxford University Press, Standard Journals Publication Model (https://academic.oup.com/journals/pages/open_access/funder_policies/chorus/standard_publication_model)
TI - Sublytic complement protects prostate cancer cells from tumour necrosis factor-α-induced cell death
JO - Clinical & Experimental Immunology
DO - 10.1111/j.1365-2249.2012.04596.x
DA - 2012-07-06
UR - https://www.deepdyve.com/lp/oxford-university-press/sublytic-complement-protects-prostate-cancer-cells-from-tumour-Q73NeQVIGy
SP - 100
EP - 108
VL - 169
IS - 2
DP - DeepDyve
ER -