Published online before print November 7, 2005
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School of Pharmacy, Tokyo University of Pharmacy and Life Science, Hachioji, Japan
2 Correspondence: Tokyo University of Pharmacy and Life Science, 1432-1 Horinouchi, Hachioji, Tokyo 192-0392, Japan. E-mail: aramaki{at}ps.toyaku.ac.jp
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Key Words: reactive oxygen species • bcl-2 family • cationic liposome • macrophage • apoptosis
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Apoptosis plays a major role in development, in homeostasis, and in many disease processes including cancer and acquired immunodeficiency syndrome [9 ]. Unlike necrotic cell death, apoptosis is characterized by cell shrinkage, chromatin condensation, DNA fragmentation, and eventual disintegration into membrane-enclosed apoptotic bodies [10 ]. Mitochondria play a pivotal role in the regulation of apoptosis, and the physiological and biochemical alterations associated with apoptosis induce a disruption of mitochondrial transmembrane potential, permeability transition, the generation of superoxide radicals, and the release of apoptogenic factors including cytochrome c [11 12 13 ]. Recently, we have examined whether the cytotoxicity of cationic liposomes is a result of apoptosis and showed that the cationic liposomes induced apoptosis in mouse splenic macrophages [14 ], the mouse macrophage-like cell line RAW264.7 [14 ], and the immature B cell line WEHI 231 [15 ]. Cationic liposome-induced apoptosis exhibited the following features: the generation of reactive oxygen species (ROS) [14 , 16 , 17 ]; mitochondrial dysfunction and the release of cytochrome c [17 ]; and the activation of caspase-3 [17 ]. However, it remains unclear how the ROS, generated following treatment with cationic liposomes, transmit an apoptotic signal(s) to mitochondria and cause the release of cytochrome c, which has an important role in inducing apoptosis.
The mitogen-activated protein kinase (MAPK) cascade is an evolutionarily conserved phosphorylation-regulated protein kinase cascade, which is involved in controlling the fate of cells survival or cell death [18 ]. The major MAPKs are extracellular signal-regulated kinase (ERK), c-jun N-terminal kinase (JNK), and p38. ERK is generally associated with cell proliferation and growth. JNK and p38 are stress-inducible and involved in cytokine-mediated differentiation and cell death [19 , 20 ]. However, the direct effects of cationic liposomes on the MAPK signaling pathway have not been fully characterized. Caspases, a family of aspartate-cysteine proteases, are key effectors responsible for many morphological and biochemical changes in apoptosis [21 , 22 ]. Caspase-8 has been established as an important component of the FasR death-inducing signaling complex (DISC) [23 ]. Activation of caspase-8 in the DISC results in the activation of downstream caspases and cleavage of cytosolic substances such as Bid, a Bcl-2 family protein. Recently, Choi et al. [24 ] reported the involvement of p38 in the activation of caspase-8 in dopaminergic neurons.
The release of cytochrome c from mitochondria is regulated through the activation of proapoptotic Bcl-2 family members, along with the possible inactivation of antiapoptotic family members [25 ]. In mammals, multiple proapoptotic bcl-2 homology 3 (BH3) domain-only members of the Bcl-2 family are specialized in sensing partially distinct stimuli. The BH3 domain-only family is upstream activators of apoptosis, which signal to downstream, proapoptotic Bcl-2 family members such as Bax or Bak, leading to their oligomerization at the mitochondria [26 ]. Activated caspase-8 can cleave p22 Bid to generate a p15 active, truncated Bid (tBid) fragment, which is then recruited to the mitochondria [27 ]. Cleavage of Bid to tBid, which is active in mitochondria, is a feature of caspase-8-mediated apoptosis induced via death receptors, which enables amplification of the apoptotic signal through the mitochondrial release of cytochrome c [28 ].
The present study was undertaken with the objective of understanding the signaling pathways mediating apoptosis in the macrophage-like cell line RAW264.7 upon treatment with cationic liposomes composed of stearylamine. The results indicated that cationic liposome-induced apoptosis involves the ROS-mediated activation of p38 MAPK and subsequent activation of caspase-8 and cleavage of Bid. These findings revealed that the generation of ROS by cationic liposomes is critical to the translocation of Bid to mitochondria and subsequent cationic liposome-induced cell death.
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Cell culture
The mouse macrophage-like cell line RAW264.7 was purchased from the Riken Cell Bank (Ibaraki, Japan) and maintained in RPMI-1640 medium supplemented with 10% heat-inactivated fetal bovine serum, 100 units/ml penicillin, and 100 µg/ml streptomycin in a humidified atmosphere containing 5% CO2 at 37ºC. Cell culture plates were washed to remove nonadherent cells, and adherent cells were >95% viable as determined with the trypan blue dye exclusion test.
Preparation of liposomes
PC from egg yolk was obtained from Nippon Oil and Fat Co. PS from calf brain and SA were obtained from Sigma Chemical Co. Liposomes of multilamellar vesicles were prepared by vortexing [29
] and were passed though a membrane filter (0.45 µm, Iwaki Co., Tokyo) before use. Lipid compositions of liposomes were PC:cholesterol = 1:1 (PC-liposomes, neutral liposomes), PC:PS:cholesterol = 1.5:0.5:2.0 (PS-liposomes, anionic liposomes), and PC:SA:cholesterol = 1.5:0.5:2.0 (SA-liposomes, cationic liposomes).
DNA content
RAW264.7 cells (5x105/well) were treated with liposomes (0.5 µmol lipid/ml) in the presence or absence of SB203580 (p38 MAPK inhibitor) or SP600125 (JNK inhibitor) for specified periods. After treatment, the cells were collected and fixed with 70% ethanol at 4ºC for at least 4 h. The cells were then centrifuged at 2000 g for 5 min, and ethanol was thoroughly removed. The cells were resuspended in phosphate-citrate buffer and allowed to stand at room temperature for at least 30 min. After centrifugation at 2000 g for 5 min, the cell pellets were suspended in RNase A solution (100 µg/ml, Amresco Inc., Dallas, TX) and incubated at 37ºC for 30 min to deplete RNA. RNase A-treated cells were suspended in 1.0 ml PI (50 µg/ml) and subjected to flow cytometry (FACSCalibur, Becton Dickinson, San Jose, CA).
PS externalization
PS externalization was determined using TACSTM Annexin V-FITC apoptosis detection kits (R&D Systems). In brief, RAW264.7 cells (5x105) were treated with liposomes (0.5 µmol lipid/ml) in the presence or absence of a caspase-8 inhibitor, Z-IETD-FMK, for specified periods. After treatment, the cells were washed with phosphate-buffered saline (PBS), resuspended in the binding buffer (attached to the apoptosis detection kit and composed of 0.25 mg/ml FITC-Annexin V, 0.1 M HEPES, pH 7.4, 1.5 M NaCl, 0.5 M KCl, 0.1 M MgCl2, and 0.18 M CaCl2), and incubated for 15 min before being analyzed with a flow cytometer (Becton Dickinson, FACSCalibur).
Western blotting
For the determination of phosphorylated p38, JNK, and ERK, RAW264.7 cells (5x105) were treated with liposomes (0.5 µmol lipid/ml) in the presence or absence of various inhibitors for given periods. The cells were lysed with lysis buffer [10 mM Tris buffer, pH 7.2, 150 mM NaCl, 1% Triton X-100, 0.1 mM Na3VO4, 1 mM phenylmethylsulfonyl fluoride (PMSF), 5 mM EDTA, 10 mg/ml aprotinin, and 10 mg/ml leupeptin] at 4ºC for 1 h. Cell lysates (10 µg as protein) were subjected to 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), transferred onto Immobilion P membranes (Nihon Millipore, Tokyo, Japan), and analyzed using a Phospho plus p44/p42 MAPK (Thr202/Tyr204) antibody kit, Phospho plus p38 MAPK (Thr180/Tyr182) antibody kit, and Phospho plus stress-activated protein kinase/JNK (Thr183/Tyr185) antibody kit (Cell Signaling Technology, Beverly, MA), respectively.
For the determination of caspase-8, RAW264.7 cells (5x105) were treated with liposomes (0.5 µmol lipid/ml) in the presence or absence of various inhibitors for specific periods. The cells were lysed with lysis buffer (10 mM Tris buffer, pH 7.2, 150 mM NaCl, 1% Triton X-100, 0.1 mM Na3VO4, 1 mM PMSF, 5 mM EDTA, 10 µg/ml aprotinin, and 10 µg/ml leupeptin) at 4ºC for 1 h. Cell lysates were subjected to 10.5% SDS-PAGE, transferred onto Immobilion P membranes (Nihon Millipore), and analyzed using anticaspase-8 polyclonal antibody (PharMingen). For the determination of ß-actin, above, the same methods were carried out using anti-ß-actin monoclonal antibody (mAb; Sigma Chemical Co.). Band intensity was analyzed with National Institutes of Health (Bethesda, MD) Image in each case [30 ].
Translocation of Bid to mitochondria
RAW264.7 cells (5x105) were treated with liposomes (0.5 µmol lipid/ml) in the presence or absence of various inhibitors for given periods. Fractionation was then performed using the ApoAlert cell fractionation kit following the manufacturer’s instructions. The cells were collected, washed with PBS, and resuspended in the buffer solution provided with the kit on ice for 10 min. The cells were then broken with 10 passages through a 26-gauge needle, and the homogenate was centrifuged at 700 g for 10 min. The supernatant was further centrifuged at 10,300 g for 25 min. The supernatant obtained was designated as the cytosolic fraction. The mitochondrial pellet was resuspended in the buffer solution of the ApoAlert cell fractionation kit. Cytosolic and mitochondrial fractions were used for quantification of the translocation of Bid. The proteins of both fractions (10 µg as protein) were subjected to 15% SDS-PAGE, transferred onto Immobilion P membranes (Nihon Millipore), and analyzed using anti-mouse mAb (Bid, R&D Systems). The membrane was then stripped and reprobed with anti-cytochrome c oxidase 4 (cox4) antibody (Clontech) to confirm that the mitochondrial fractions were separated successfully from cytosolic fractions.
Statistical analysis
Data are given as the mean ± SD. The statistical significance of differences was determined by ANOVA with Duncan’s test for multiple comparisons. The Pvalue for significance was set at 0.05.
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Figure 1. Effects of liposomes on phosphorylation of p38 (p-p38; A), JNK (p-JNK2/1; B), and ERK (p-ERK1/2; C). RAW264.7 cells were treated with liposomes (0.5 µmol lipid/ml) for the period indicated. Following treatment, equal amounts of protein from whole cellular lysates (10 µg) were analyzed by Western blotting. The figure is representative of three experiments showing similar results.
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Figure 2. Effects of SB203580, SB202474, or SP600125 on hypodiploid DNA content in RAW264.7 cells treated with liposomes. (A) RAW264.7 cells were pretreated with 0–50 µM SB203580 or 0–50 µM SB202474 for 30 min and then treated with or without SA-liposomes (0.5 µmol lipid/ml) for 24 h. ( , SB203580 alone; •, SB203580+SA-liposomes;  , SB202474 alone;  , SB202474+SA-liposomes). Following treatment, cells were stained with PI, and hypodiploid DNA content was analyzed using flow cytometry. Data are presented as the mean ± SD from three independent experiments. (B) Cells were pretreated with 0–10 µM SP600125 for 30 min and then treated with or without liposomes (0.5 µmol lipid/ml) for 24 h. (, SP600125 alone; •, SP600125+SA-liposomes). Following treatment, cells were stained with PI, and hypodiploid DNA content was analyzed. Data are presented as the mean ± SD from three independent experiments.
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Figure 3. Effects of different charged liposomes on phosphorylation of p38. RAW264.7 cells were treated with liposomes (0.5 µmol lipid/ml) for 150 min. Following treatment, equal amounts of protein from whole cellular lysates (10 µg) were analyzed by Western blotting. The figure is representative of three experiments showing similar results. C, Control.
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Figure 4. Effects of NAC on phosphorylation of p38 in RAW264.7 cells treated with liposomes. RAW264.7 cells were pretreated with 0–50 mM NAC 0–600 µM tocopheral for 30 min and then treated with liposomes (0.5 µmol lipid/ml) for the period indicated. Following treatment, equal amounts of protein from the whole cellular lysates (10 µg) were analyzed by Western blotting. The figure is representative of three experiments showing similar results.
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Figure 5. Effects of SA-liposomes on activation of caspase-8. RAW264.7 cells were treated with liposomes (0.5 µmol lipid/ml) for the period indicated. Following treatment, equal amounts of protein from the whole cellular lysates (10 µg) were analyzed by Western blotting. ß-Actin expression was used as an internal control. The figure is representative of three experiments showing similar results.
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Figure 6. Effects of SB203580 or NAC on activation of caspase-8 in RAW264.7 cells treated with liposomes. (A) RAW264.7 cells were pretreated with 0–10 µM SB203580 for 30 min and then treated with liposomes (0.5 µmol lipid/ml) for 6 h. Following treatment, equal amounts of protein from whole cellular lysates (10 µg as protein) were analyzed by Western blotting. (B) RAW264.7 cells were pretreated with 0–50 mM NAC for 30 min and then treated with liposomes (0.5 µmol lipid/ml) for 6 h. Following treatment, equal amounts of protein from whole cellular lysates (10 µg as protein) were analyzed by Western blotting. ß-Actin expression was used as an internal control. The figure is representative of three experiments showing similar results.
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Figure 7. Effects of Z-IETD-FMK on PS externalization in RAW264.7 cells treated with liposomes. RAW264.7 cells were pretreated with 0–50 µM Z-IETD-FMK for 30 min and then treated with or without liposomes (0.5 µmol lipid/ml) for 6 h. (, Z-IETD-FMK alone; •, Z-IETD-FMK+SA-liposomes). Following treatment, cells were stained with FITC-Annexin V, and PS externalization was analyzed. Data are presented as the mean ± SD from three independent experiments.
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Figure 8. Involvement of Bid in apoptosis of RAW264.7 cells treated with liposomes. (A) RAW264.7 cells were treated with liposomes (0.5 µmol lipid/ml) for the period indicated. Following treatment, cells were fractionated into mitochondrial and cytosolic fractions, and proteins of both fractions (10 µg) were subjected to Western blotting to examine the activation of Bid. The figure is representative of three experiments showing similar results. (B) Effect of caspase-8 inhibitor on the activation of Bid was investigated. RAW264.7 cells were pretreated with 0–20 µM Z-IETD-FMK for 30 min and then treated with SA-liposomes (0.5 µmol lipid/ml) for 6 h. Following treatment, cells were fractionated into mitochondrial and cytosolic fractions, and proteins of both fractions (10 µg) were analyzed by Western blotting to estimate the involvement of caspase-8 in the activation of Bid. The figure is representative of three experiments showing similar results.
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Figure 9. The effects of SB203580 and Z-IETD-FMK on cytochrome c release from mitochondria of RAW264.7 cells treated with SA-liposomes. RAW264.7 cells were pretreated with SB203580 (10 µM) or Z-IETD-FMK (10 µM) for 30 min and then treated with SA-liposomes (0.5 µmol lipid/ml) for 6 h, and mitochondria were isolated. Mitochondria lysates were electrophoresed, blotted, and stained with anti-cytochrome c and anti-cox4 antibodies.
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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It has been reported that MAPK signaling has an important part in the response to mitogen or stress to determine cell proliferation or apoptosis, and ROS is considered to be a factor in the activation of MAPK [29 ]. We thus initially investigated the requirement for activated MAPKs in SA-liposome-induced apoptosis. Tyrosine phosphorylation of p38 and JNK began at 2 and 3 h after treatment, respectively, and was observed even at 6 h, indicating that SA-liposomes induced a sustained activation of p38 MAPK and JNK (Fig. 1) . Although both kinases were activated, only p38 contributed to the apoptosis, as suggested by the finding that SA-liposome-induced apoptosis was only inhibited in the presence of the p38-specific inhibitor SB203580 (Fig. 2) . Furthermore, NAC inhibited SA-liposome-induced activation of p38 MAPK (Fig. 3) . Apoptosis signal-regulating kinase 1 (ASK1) is a MAPK kinase kinase family member and is activated in response to ROS, tumor necrosis factor (TNF), and Fas through distinct mechanisms and relays signals to stress-activated MAPKs such as p38 and JNK [39 ]. Furthermore, ASK1 is selectively required for the TNF- and ROS-induced, sustained activation of JNK and p38 and apoptosis [29 ]. From these findings, the activation of p38 is required for the apoptosis induced by SA-liposomes, and the sustained activation of this MAPK might be regulated by ASK1. However, the reason why JNK did not contribute to the SA-liposome-induced apoptosis is still unclear.
The activation of caspases is known to be a general mechanism in the induction of apoptosis [21 , 22 ]. It has been shown that MAPK family members have a role in activating caspase cascades [40 ]. Recently, Bhattacharyya et al. [18 ] reported that p38 MAPK signaling was linked to the activation of caspase-8, which has been reported to be an important component of the Fas receptor death-inducing signaling complex (DISC), and activation of caspase-8 in the DISC results in the activation of downstream caspases and the cleavage of cytosolic substances such as Bid, a BH3 domain-only protein [23 ]. We thus examined the activation by Western blotting and found that caspase-8 was activated by SA-liposome treatment. Treatment with SB203580 was associated with inhibition of the activation. Furthermore, NAC also inhibited the activation. These findings suggested that p38 MAPK signaling plays a crucial role in the activation of caspase-8. However, inhibitors for p38 MAPK and caspase-8, especially caspase-8, failed to fully prevent the SA-liposome-induced apoptosis, suggesting an existence of other signaling mechanisms involved in the SA-liposome-induced apoptosis. Further identification of caspase-8-independent apoptosis signal(s) is also worth investigating.
Pro- and antiapoptotic bcl-2 members regulate apoptosis induced by the mitochondrial pathway. We speculated that the caspase-8-mediated cleavage of Bid leads to the release of cytochrome c from mitochondria and carried out a time-dependent analysis of caspase-8 and Bid by Western blotting in RAW264.7 cells. Bid, a proapoptotic bcl-2 family member containing only the BH3 domain, is exclusively a cytosolic protein and can be cleaved by caspase-8 [27 ]. The cleaved form, tBid, translocates to the mitochondria, where it promotes the release of cytochrome c [28 ]. Li et al. [27 ] reported that Bid is a mediator of mitochondrial damage induced by caspase-8. We observed that the intensity of the full-length Bid decreased over time following SA-liposome treatment, and the change seems to be a result of the cleavage of Bid to tBid (Fig. 8) . The cleavage of Bid was inhibited by the addition of a caspase-8 inhibitor (Fig. 8) , suggesting that caspase-8-dependent Bid cleavage in SA-liposome-induced apoptosis triggers the mitochondrial pathway.
It is generally thought that the expression of full-length Bax increases following death signaling, and Bax is then translocated into the mitochondria to release cytochrome c [26 ]. However, no increase in the expression or translocation into mitochondria of Bax was observed by Western blotting analysis (data not shown). Furthermore, cationic liposome treatment did not alter the expression of bcl-2, which is an antiapoptotic protein (data not shown).
Cytochrome c binds to Apaf-1 and forms a complex that processes and activates procaspase-9, which can cleave and activate the down-stream caspase cascade including caspase-3 and -7 [41 ]. In our previous report [17 ], the effect of SA-liposomes on the activation of caspase-3 was evaluated by measuring the conversion of procaspase-3 (32 kDa) into caspase-3 (20, 19, 17 kDa) using Western blotting, and a large processed subunit of caspase-3 was observed when RAW264.7 cells were treated with SA-liposomes but not with PC- or PS-liposomes. Furthermore, processing of caspase-3 was inhibited by addition of NAC. As the release of cytochrome c from mitochondria was inhibited by NAC, as mentioned above, the suppression of caspase-3 processing by NAC may be a result of the inhibition of cytochrome c release.
DNA is polyanionic and easily forms a complex with cationic substances via electrostatic interactions. Cationic liposomes are therefore candidates as nonviral vectors. However, cationic liposomes showed apoptosis, which may deleteriously affect application of cationic liposomes as nonviral vectors. Thus, to clarify the mechanism of apoptosis exerted by cationic liposomes may provide important information for the use of cationic liposomes as nonviral vector. Taking these findings into consideration, a mechanism of SA-liposome-induced apoptosis is speculated as follows: ROS generated by RAW264.7 cells, which were treated with SA-liposomes, activates p38 MAPK, and p38 MAPK signaling may also regulate caspase-8 activity by phosphorylating caspase-8 itself. Then, caspase-8-mediated cleavage of Bid and its translocation to the mitochondria are associated with the release of cytochrome c, leading to the formation of an apoptosome. Our finding is the first observation that the contribution of the p38 MAPK–caspase-8 pathway on apoptosis was induced by cationic liposome. We are now investigating how cationic liposomes interact with macrophages and generate ROS. To clarify the mechanism of apoptosis may lead to safe application of cationic liposomes for nonviral vectors.
Received April 8, 2005; revised July 27, 2005; accepted September 15, 2005.
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