Abstract
Cisplatin is widely and effectively used for the treatment of various types of cancer. However, its biochemical mechanisms are still unelucidated. Previously, we reported that membrane sphingomyelin (SM) was important for FAS-mediated apoptosis through lipid raft function. In this study, we strikingly show that cisplatin combined with CH11 (anti-FAS antibody, IgM) was able to induce marked apoptosis in SM synthase-restored WR/Fas-SMS1 cells, but not in SM synthase-deficient WR/FAS-SM(−) cells. In addition, we demonstrated that membrane SM played an important role in cisplatin/CH11-induced apoptosis through the classical caspase-dependent pathway, mainly by enhancing the formation of FAS-associated signaling complexes.
The caspase-dependent intrinsic pathway is one of the main death pathways activated by specific cellular damage. In this pathway, FAS plays a predominant role by forming death-inducing signaling complex (DISC) composed of FAS, FAS-associated death domain (FADD) and caspase-8 (1, 2). Recently, there is accumulating evidence that lipid rafts are involved in Fas-induced apoptosis through translocation and clustering of FAS into lipid rafts upon stimulation (3-5). Lipid rafts are membrane microdomains enriched in cholesterol and sphingolipids such as sphingomyelin (SM), and float freely within the cellular membrane bilayer or cluster to form large ordered platforms on activation (6). In addition, membrane SM is catalyzed by acid sphingomyelinase (aSMase) to ceramide, which may induce coalescence of elementary rafts and/or reorganization of these domains. We previously reported that membrane SM was crucial in FAS-mediated apoptosis through clustering of Fas in lipid rafts, formation of death-inducing signaling complex (DISC) and initiation of the caspase-dependent cell death pathway (7).
Cisplatin (the platinum coordination complex cis-diaminedichloroplatinum II), a widely and effectively used chemotherapeutic agent in the treatment of various tumors, has been reported to induce cellular damage at several structural levels (8). Besides its primary target DNA, cisplatin has been shown to interact with some transport proteins on the cellular membrane and in the cytoplasm (9). For example, cisplatin has been shown to cluster and activate FAS in a FAS ligand-(FASL) independent manner (10), sequentially activating caspase-8, -3 and -6 (11). However, these limited biochemical function modes are not sufficient to explain cisplatin-induced cytotoxicity. Recently, it has been reported that aSMase triggers apoptosis in response to several apoptotic stimuli (12), and that cisplatin was able to activate aSMase, resulting in an increase of ceramide and induction of apoptosis (13).
We have cloned the gene encoding the SM synthase SMS1, and subsequently established SM synthase-restored cells (WR/FAS-SMS1) by transfection of SMS1 gene into SM synthase-defective WR19L/FAS cells that were transfected with the human FAS gene (WR/FAS-SM(−)) (14). In the present study using WR/FAS-SM(−) and WR/FAS-SMS1 cells, we examined Fas and cisplatin-induced apoptosis.
Materials and Methods
Cell lines. WR19L cells, a mouse T-cell lymphoma cell line, were transfected with the cDNA of the human FAS gene (WR19L/FAS cells) (15). We obtained SM-defective cells from the original WR19L/Fas cells by limiting dilution (WR/Fas-SM(−)). SMS1 gene was subcloned into the pLIB expression vector and transfected into the WR/Fas-SM(−) cells in VSV-G retroviral particles to establish SM synthase-restored cells (WR/Fas-SMS1) (14).
Antibodies and reagents. Anti-FADD (1F7, mouse IgG1), anti-caspase-3 (1F9, mouse IgG1), anti-Fas death domain (3D5, mouse IgG1), and anti-caspase-8 (5D3, mouse IgG1) monoclonal antibodies purchased from MBL International Corporation (Woburn, MA, USA), anti-β-actin antibodies obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA) were all used or a concentration of 1:1000 in the analysis of immunoprecipitation and Western blotting. Secondary antibodies, such as rabbit anti-mouse IgM and horseradish peroxidase-conjugated goat anti-mouse or anti-rabbit IgG antibodies were purchased from Zymed Laboratories (San Francisco, CA, USA) and Amersham Biosciences Inc. (Piscataway, NJ), respectively. RNase and saponin were purchased from Nacalai Tesque. A cell viability assay kit using WST-1 was purchased from Wako Co. Ltd. (Osaka, Japan). The enhanced chemiluminescence (ECL) immunodetection system was obtained from Amersham Biosciences Inc. (Piscataway, NJ, USA). Cisplatin was obtained from Bristol-Myers Squibb (Princeton, NJ, USA). The anti-FAS antibody (CH11, IgM) was purchased from MBL International Corporation.
Morphological evaluation. Cells were treated with 5 μg/ml of cisplatin and/or 50 ng/ml of CH11 for 2 hours, then the apoptotic featues were analyzed by phase-contrast microscopy. The number of dead cells was counted by using a blood cell counting dish after trypan blue staining.
Flow cytometry. Cell apoptosis was determined by flow cytometry as described previously (7). In brief, aliquots of cells (1×106/ml) were seeded in 24-well plates and cultured for 12 h. Cells were treated with cisplatin and/or anti-FAS antibody (clone: CH11) at specified concentrations for the hours indicated in the Figures. Ethanol-fixed cell suspension was centrifuged and then 50 ml of RNase solution was added. Thereafter, propidium iodide solution (450 ml, final concentration 50 mg/ml) was added to each tube. Cells were washed twice and subsequently analyzed by flow cytometry (BD Biosciences, Palo Alta, CA, USA).
Immunoprecipitation and Western blotting analysis. Immunoprecipitation and Western blotting were carried out as described previously (16). In brief, cell lysates were separated by SDS-PAGE and transferred electrophoretically onto polyvinylidene difluoride (Immobilon-P) membranes (Sigma-Aldrich, St. Louis, MO, USA). The membranes were blocked with 5% nonfat dried milk in Tris buffer saline (TBS) and then incubated with the above primary antibodies overnight at 4°C. Horseradish peroxidase-conjugated anti-rabbit or anti-mouse antibodies were used as secondary antibodies. The immunoreactive bands were visualized using the ECL protocol. Densitometry of the protein bands was performed using NIH Image (17). The quantification of bands was corrected to the density of β-actin and depicted as arbitrary units.
Cell viability assay. WST-1 assay was used to quantify cell viability following exposure to 5 μg/ml cisplatin or 50 ng/ml CH-11, or both in combination for 12 h, as described elsewhere (18). The absorbance value of untreated cells was considered 100% viability.
Statistical analysis. All data were expressed as means±standard deviation (SD). Comparisons between two values were performed by Student's t-test using Stat View statistical software (SAS Institute, Cary, NC, USA). P<0.05 was taken to indicate statistical significance.
Results
Synergistic effects of cisplatin and CH11 on cell viability in WR/Fas-SM(−) and WR/Fas-SMS1 cells. Firstly, we examined the morphological change and cell viability of WR/Fas-SM(−) and WR/Fas-SMS1 cells under culture with cisplatin (5 μg/ml) and/or CH11 (50 ng/ml). Although neither cisplatin nor CH11 treatment caused remarkable morphological changes in WR/Fas-SM(−) cells, both agents, especially in combination, showed strong cytotoxicity against WR/Fas-SMS1 cells (Figure 1A). Cell viability determined by trypan blue staining was significantly decreased in WR/Fas-SMS1 cells cultured with CH11 or with combination of cisplatin and CH11, but not in WR/Fas-SM(−) cells (Figure 1B). Similarly, growth of WR/Fas-SM(−) cells and WR/Fas-SMS1 cells, when treated with cisplatin alone (Figure 1C), however, significantly lower than those of WR/Fas-SM(−) cells, when treated with the combination of cisplatin and CH11 (Figure 1D).
Synergistic effects of cisplatin and CH11 on apoptosis in WR/Fas-SM(−) and WR/Fas-SMS1 cells. Since it has been reported that cisplatin increases membrane FAS expression (19), we examined the effects of cisplatin on Fas expression in WR/Fas-SM(−) and WR/Fas-SMS1 cells. We found that human FAS was highly and equally expressed in both cell lines and that cisplatin did not enhance FAS expression in either cell type at any concentration throughout incubation time (Figure 2A). Previously, we reported that membrane SM plays a key role in FAS-mediated apoptosis through its involvement in the efficient clustering of FAS and lipid rafts (4). Therefore, we examined whether cisplatin induced apoptosis in WR/Fas-SM(−) and WR/Fas-SMS1 cells. Cisplatin itself was not able to induce apoptosis in either cell line at any concentration examined (Figure 2B) nor at any harvest time point (Figure 2C). However, 50 ng/ml of CH11 markedly induced apoptosis in WR/Fas-SMS1 cells, but not in WR/Fas-SM(−) cells. Moreover, combination of cisplatin (5 ng/ml) with CH11 (50 ng/ml) augmented apoptosis more remarkably in WR/Fas-SMS1 cells (Figure 2D and 2E).
Association of FADD and FAS in WR/Fas-SM(−) and WR/Fas-SMS1 cells after the treatment with cisplatin and/or CH11. According to the current model of type I apoptosis, binding of either the FASL or an agonistic antibody induces aggregation of FAS followed by a conformational change in its cytoplasmic domain that results in formation of the DISC (20-22). Previously, we have reported that FAS stimulation enhanced association of FAS with FADD and caspapse-8 in WR/Fas-SMS1 cells, but not in WR/Fas-SM(−) cells (7). In this study, we examined association of FAS with FADD after treatment with cisplatin and/or CH11. As shown in Figure 3, we did not detect association of FAS with FADD in WR/Fas-SM(−) cells after treatment with cisplatin and/or CH11. A low level of association of FAS with FADD was detected in WR/Fas-SMS1 cells treated with CH11 alone. Interestingly, association of FAS with FADD was significantly induced after treatment of WR/Fas-SMS1 cells with the combination of cisplatin and CH11 (Figure 3).
Detection of active caspase-8 and caspase-3 in WR/FAS-SM(−) and WR/FAS-SMS1 cells after the treatment with cisplatin and/or CH11. Sequential activation of two distinct different groups of caspases i.e. initiator caspases (caspase-8, -9, -10) and executor caspases (caspase-3, -6, -7), are proposed in FAS-mediated apoptosis signaling (20, 21, 23). We examined the expression of active caspase-8 and caspase-3 in two cell lines. Western blot analysis using a caspase-8- and caspase-3-specific antibodies revealed that the active fragments (p23) of caspase-8 and (p17) of caspase-3 cleaved from each pro-caspase form were expressed at a higher level in WR/Fas-SMS1 cells compared to WR/Fas-SM(−) cells after the treatment with cisplatin and CH11 (Figure 4A). Furthermore, strong induction of active caspase-8 and caspase-3 was detected in WR/Fas-SMS1 cells after the combined treatment of cisplatin and CH11 (Figure 4B and 4C). However, caspase-8 and caspase-3 activity of WR/Fas-SM(−) cells remained at baseline.
Discussion
Cisplatin is a well known DNA-damaging agent and the current thinking is that binding of DNA and cisplatin is an essential first step in the cytotoxic activity of the drug. However, many cellular components that have nucleophilic sites such as DNA, RNA, proteins, membrane phospholipids, cytoskeletal microfilaments, and thiol-containing molecules react with cisplatin. Thus, cisplatin does not always induce cell death by inhibition of DNA synthesis, but through DNA damage-independent pathways. In fact, cisplatin binds to mitochondrial DNA, interacts with phospholipids and phosphatidylserine in membranes, disrupts the cytoskeleton, and affects the polymerization of actin (24). Cisplatin can induce apoptotic cell death in a number of cultured tumor cell lines, and the main death pathway activated by specific cellular damage induced by cisplatin is a caspase-dependent intrinsic pathway (2, 19). In this pathway, cisplatin firstly clusters and activates FAS receptor (10), and sequentially forms FAS/FADD/procaspase-8 complex known as DISC, catalyzes the proteolytic conversion of procaspase-8 into active caspase-8, which activates caspase-3 and other downstream effector caspases, triggering cell death (11, 25). Although we failed to find increased apoptosis in WR/Fas-SM(−) and WR/Fas-SMS1 cells treated by cisplatin alone at the dose and time points examined (Figure 2B and 2C), the combined treatment of cisplatin and CH11 induced more remarkable cell death than CH11 treatment alone did (Figure 2D and 2E). These results suggest that cisplatin still played an important role in the apoptosis of the cells in our system.
It has been widely discussed that cytotoxic drugs may act in a synergistic manner with FAS to induce tumor cell death (26, 27). Lacour et al. has reported that cisplatin induces clustering of FAS at the surface of the cell and redistribution of DISC into cholesterol- and sphingomyelin-enriched microdomains, lipid rafts, and that these events are inhibited by the inhibitor of acid sphigomyelinase and the cholesterol sequestering agent that disrupt lipid rafts (19). SM is the major component of lipid rafts on the cellular membrane, which play a central role in many cellular processes such as membrane trafficking, cell polarization, and signal transduction (28-30). The lipid rafts are important for efficient formation of receptor-associated signaling complexes, and which can produce complicated biological outcomes, including T-cell receptor (TCR)-mediated signal transduction and FAS-mediated cell death (31-33). Previously, we reported that expression of membrane SM enhances FAS-mediated apoptosis through increasing DISC formation, activation of caspases, efficient translocation of FAS into lipid rafts and subsequent FAS clustering (7). Interestingly, the combined treatment of cisplatin and CH11 had discordant effects on WR/Fas-SM(−) and WR/Fas-SMS1 cells in this study. We clearly demonstrated an inhibition of cell viability (Figure 1A and 1B), a decrease of survival of cells (Figure 1C and 1D), a significant enhancement of apoptosis (Figure 2D and 2E), an up-regulated association of FAS with FADD (Figure 3) and activation of caspase-8 and -3 (Figure 4) in WR/Fas-SMS1 cells, but not WR/Fas-SM(−) cells. Meanwhile, cisplatin is known to activate several proteins in this cascade, such as caspase-8 (34), which can be considered as a synergetic manner with FAS. Altogether, these results suggest that cisplatin may play an important role in FAS-mediated apoptosis through lipid raft function.
Acknowledgements
This work was supported by grants 13557160, 15024236, 15390313, 13877075, 15024236 and 15390313 from Japanese Ministry of Education and Science and Culture, Uehara Memorial Foundation, The Vehicle Racing Commemorative Foundation, and Kanazawa Medical University Research Foundation.
- Received October 25, 2009.
- Revision received April 8, 2010.
- Accepted April 16, 2010.
- Copyright© 2010 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved