Abstract. Cisplatin is widely and effectively used for the treatment of various types of cancer. However, its biochemical mechanisms are still unelucidated.
ANTICANCER RESEARCH 30: 2065-2072 (2010)
Cisplatin Augments FAS-mediated Apoptosis through Lipid Rafts CHENG-RI HUANG1, ZHE-XIONG JIN1, LINGLI DONG1,2, XIAO-PENG TONG1, SUN YUE1, TAKAFUMI KAWANAMI1, TOSHIOKI SAWAKI1, TOMOYUKI SAKAI1, MIYUKI MIKI1, HARUKA IWAO1, AKIO NAKAJIMA1, YASUFUMI MASAKI1, YOSHIHIKO FUKUSHIMA1, MASAO TANAKA1, YOSHIMASA FUJITA1, HIDEO NAKAJIMA3, TOSHIRO OKAZAKI4 and HISANORI UMEHARA1
Departments of 1Hematology and Immunology, and 3Medical Oncology, Kanazawa Medical University, Uchinada-machi, Kahoku-gun, Ishikawa 920-0293, Japan; 2Department of Rheumatology, Tongji Hospital, Huazhong University of Science and Technology, Wuhan, Hubei, 430030, P.R. China; 4Faculty of Medicine, Tottori University, Yonago, Tottori 683-8504, Japan
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/FasSMS1 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 deathinducing signaling complex (DISC) composed of FAS, FASassociated 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),
Correspondence to: Hisanori Umehara, MD, Ph.D., Department of Hematology and Immunology, Kanazawa Medical University, 1-1 Daigaku, Uchinada-machi, Kahoku-gun, Ishikawa 920-0293, Japan. Tel: +81 762188158, Fax: +81 762869290, e-mail: umehara@ kanazawa-med.ac.jp Key Words: Cisplatin, FAS, apoptosis, sphingomyelin, lipid rafts.
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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 cisdiaminedichloroplatinum 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/FASSMS1 cells, we examined Fas and cisplatin-induced apoptosis.
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ANTICANCER RESEARCH 30: 2065-2072 (2010) 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), anticaspase-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 antirabbit 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.
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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
