Molecular Cancer Research 3:373-379 (2005)
© 2005 American Association for Cancer Research
DNA Damage and Cellular Stress Responses
Lipopolysaccharide Prevents Doxorubicin-Induced Apoptosis in RAW 264.7 Macrophage Cells by Inhibiting p53 Activation
Ferdaus Hassan,
Shamima Islam,
Mya Mya Mu,
Hiroyasu Ito,
Naoki Koide,
Isamu Mori,
Tomoaki Yoshida and
Takashi Yokochi
Department of Microbiology and Immunology, Aichi Medical University School of Medicine, Nagakute, Aichi, Japan
Requests for reprints: Takashi Yokochi, Department of Microbiology and Immunology, Aichi Medical University School of Medicine, Nagakute, Aichi 480-1195, Japan. Phone: 81-561-62-3311; Fax: 81-561-63-9187. E-mail: yokochi{at}aichi-med-u.ac.jp
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Abstract
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The effect of lipopolysaccharide on doxorubicin-induced cell death was studied by using mouse RAW 264.7 macrophage cells. Pretreatment with lipopolysaccharide at 10 ng/mL prevented doxorubicin-induced cell death and the inhibition was roughly dependent on the concentration of lipopolysaccharide. Posttreatment with lipopolysaccharide for 1 hour also prevented doxorubicin-induced cell death. Lipopolysaccharide inhibited DNA fragmentation and caspase-3 activation in doxorubicin-treated RAW 264.7 cells, suggesting the prevention of doxorubicin-induced apoptosis. Lipopolysaccharide did not significantly inhibit doxorubicin-induced DNA damage detected by single-cell gel electrophoresis (comet) assay. Lipopolysaccharide definitely inhibited the stabilization and nuclear translocation of p53 in doxorubicin-treated RAW 264.7 cells. Lipopolysaccharide, as well as being an inhibitor of p53, abolished doxorubicin-induced apoptosis. Therefore, p53 was suggested to play a pivotal role in the prevention of doxorubicin-induced apoptosis in RAW 264.7 cells by lipopolysaccharide.
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Introduction
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Doxorubicin, a derivative of anthracyclines, is a frequently used anticancer drug in the treatment of numerous human malignancies. Understanding the mechanisms of tumor cell–killing by anthracyclines still remains an active area of research. Anthracyclines are known for their complex cytotoxic mechanism involving (a) inhibition of enzymes such as topoisomerase II, RNA polymerase, cytochrome c oxidase, and others; (b) intercalation into DNA; (c) chelation of iron and generation of reactive oxygen species; and (d) induction of apoptosis (1). Although doxorubicin induces apoptosis in a wide range of cells (2-4), the detailed mechanism is not completely clear. Moreover, a variety of factors including proapoptotic and antiapoptotic molecules influences doxorubicin-induced apoptosis.
Bacterial lipopolysaccharide is present on the outer membranes of all Gram-negative bacteria and is known to cause systemic inflammatory response syndrome, septic shock, and disseminated i.v. coagulation (5, 6). The activation of macrophages by lipopolysaccharide leads to the production of inflammatory mediators, and causes cell damage and cell death. In fact, lipopolysaccharide exhibits a cytotoxic action on various types of cells via apoptosis (7-9). Therefore, it was possible that lipopolysaccharide augmented the cell death induced by anticancer drugs. However, there is no report concerning the effect of lipopolysaccharide on DNA-damaging anticancer drug–induced cell death. In the present report, we studied if and how lipopolysaccharide influenced doxorubicin-induced cell death by using RAW 264.7 macrophage cells. Unexpectedly, lipopolysaccharide prevented doxorubicin-induced apoptosis in RAW 264.7 macrophage cells. Here, we discuss the possible role of p53 tumor suppressor protein in the prevention of doxorubicin-induced apoptosis by lipopolysaccharide.
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Results
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Prevention of Cell Death in Doxorubicin-Treated RAW 264.7 Cells by Lipopolysaccharide
RAW 264.7 cells were pretreated with lipopolysaccharide at 10, 100, and 1,000 ng/mL for 30 minutes and incubated with doxorubicin at 5 or 10 µmol/L for 24 hours. Cell death was determined with the reduction of 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) activity 24 hours after doxorubicin treatment. Doxorubicin at 5 µmol/L significantly reduced the cell viability of RAW 264.7 cells (Fig. 1). Doxorubicin at 10 µmol/L reduced the MTT activity more markedly than doxorubicin at 5 µmol/L. Pretreatment of lipopolysaccharide definitely prevented the cell death of RAW 264.7 cells treated by doxorubicin at 5 µmol/L, and to a lesser extent, prevented it in the case of doxorubicin at 10 µmol/L. Lipopolysaccharide alone at any concentration tested did not affect the cell viability of RAW 264.7 cells. Next, the effect of pre- and posttreatment of lipopolysaccharide on doxorubicin-induced cell death was studied. RAW 264.7 cells were treated with various concentrations of lipopolysaccharide 1 hour before and after addition of the doxorubicin at 5 µmol/L. One hour of pretreatment and posttreatment with lipopolysaccharide significantly prevented cell death induced by doxorubicin at 5 µmol/L. However, 6 hours of posttreatment with lipopolysaccharide at 10 ng/mL did not prevent it. In the following experiments, the protective action of lipopolysaccharide was studied by the exposure of RAW 264.7 cells pretreated with lipopolysaccharide (100 ng/mL) for 30 minutes to doxorubicin (5 µmol/L) unless otherwise stated. In addition, lipopolysaccharide prevented doxorubicin-induced cell death in physiologic peritoneal macrophages from lipopolysaccharide-sensitive BALB/c mice, but not lipopolysaccharide-resistant C3H/HeJ mice (Fig. 2).
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FIGURE 1. Effect of lipopolysaccharide on doxorubicin-induced cell death. RAW 264.7 cells were seeded at 2 x 105/100 µL/well into a 96-well microplate and pretreated with lipopolysaccharide (100 ng/mL) for 30 minutes before the addition of doxorubicin at 5 and 10 µmol/L concentration. The MTT activity was measured 24 hours after doxorubicin treatment. Columns, means; bars, SD.
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FIGURE 2. Effect of lipopolysaccharide on doxorubicin-induced cell death in peritoneal cells from C3H/HeJ and BALB/c mice. Peritoneal cells were isolated from lipopolysaccharide-resistant C3H/HeJ and sensitive BALB/c mice by washing the peritoneal cavity and were cultured overnight in a 96-well plate. The cells were pretreated with lipopolysaccharide (100 ng/mL) for 30 minutes and cultured with doxorubicin (5 µmol/L) for 24 hours. The MTT activity was measured 24 hours after the addition of doxorubicin. Columns, means; bars, SD.
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Induction of Apoptosis in Doxorubicin-Treated RAW 264.7 Cells and Prevention by Lipopolysaccharide
In order to clarify the preventive action of lipopolysaccharide on doxorubicin-induced cell death, we studied whether lipopolysaccharide prevented necrosis or apoptosis in doxorubicin-treated RAW 264.7 cells. First, DNA fragmentation, a characteristic of apoptosis (10), was examined in agarose gel electrophoresis (Fig. 3A). RAW 264.7 cells were treated with doxorubicin and/or lipopolysaccharide for 6 hours. Doxorubicin alone induced a clear ladder pattern of fragmented DNA in RAW 264.7 cells, whereas the treatment with both doxorubicin and lipopolysaccharide did not induce it. Lipopolysaccharide alone did not induce DNA fragmentation either. Second, activation of caspase-3 was examined by immunoblotting using an anti–cleaved caspase-3 antibody (Fig. 3B). When RAW 264.7 cells were treated with doxorubicin and/or lipopolysaccharide for 3 or 6 hours, doxorubicin alone clearly caused activation of caspase-3 at 6 hours after the treatment. On the other hand, the combination of doxorubicin and lipopolysaccharide did not induce caspase-3 activation at either 3 or 6 hours. Lipopolysaccharide alone did not cause caspase-3 activation 3 or 6 hours after the treatment. Third, cleavage of poly(ADP-ribose) polymerase (PARP), a marker of apoptotic cells (11), was examined by immunoblotting using an anti-PARP antibody (Fig. 3C). RAW 264.7 cells were treated with doxorubicin and/or lipopolysaccharide for 6 hours. Doxorubicin clearly caused cleavage of PARP 6 hours after treatment, whereas lipopolysaccharide did not. No PARP cleavage was seen in RAW 264.7 cells treated for 6 hours with doxorubicin and lipopolysaccharide or lipopolysaccharide alone. The experimental result with immunoblotting was also confirmed by an immunohistochemical staining using an anti–cleaved caspase-3 antibody (data not shown). Z-VAD-FMK as a broad-spectrum caspase inhibitor and DEVD-FMK as a caspase-3-specific inhibitor prevented the reduction of the MTT activity in doxorubicin treatment. Taken together, it became clear that lipopolysaccharide prevented doxorubicin-induced apoptosis of RAW 264.7 cells via inactivation of caspase-3. Doxorubicin-induced apoptosis was also prevented by CpG DNA, but not IFN-
and tumor necrosis factor-
(Fig. 4).
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FIGURE 3. Induction of apoptosis in doxorubicin-treated RAW 264.7 cells. A. RAW 264.7 cells were seeded at 2 x 105/mL in a 35 mm plastic dish, pretreated with lipopolysaccharide (100 ng/mL) for 30 minutes, and incubated with doxorubicin (5 µmol/L) for 6 hours. Fragmented DNA was analyzed by agarose gel electrophoresis. A typical experimental result of three independent experiments is shown. Lane 1, DNA size marker; lane 2, untreated control; lane 3, lipopolysaccharide; lane 4, doxorubicin; lane 5, lipopolysaccharide and doxorubicin. B and C, RAW 264.7 cell were pretreated with lipopolysaccharide (100 ng/mL) for 30 minutes and treated with doxorubicin (5 µmol/L) for 3 and 6 hours. Cell extracts were analyzed with immunoblotting using antibody to cleaved caspase-3 (B) or PARP (C). Results are representative of at least three independent experiments.
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No Inhibition of Doxorubicin-Induced DNA Damage by Lipopolysaccharide
Doxorubicin is known to damage DNA and make the strands break (12). DNA damage could be detected quantitatively by the simple and quick comet assay with alkaline single-cell gel electrophoresis (13, 14). In the comet assay, 
80% of doxorubicin-treated cells migrated away from the head by forming a long comet-tailed shape (Fig. 5). The cells treated with lipopolysaccharide and doxorubicin also showed a similar comet shape. There was no significant difference in the frequency of comet-shaped cells between treatment with doxorubicin alone and that with doxorubicin and lipopolysaccharide. Only a few comet-shaped cells were detected in the cells treated with lipopolysaccharide alone or without lipopolysaccharide (<10%).
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FIGURE 5. Effect of lipopolysaccharide on doxorubicin-induced DNA damage. RAW 264.7 cells seeded into comet slides were pretreated with lipopolysaccharide (100 ng/mL) for 30 minutes and incubated with doxorubicin (5 µmol/L) for 3 hours. DNA damage was analyzed with the comet assay with single cell gel electrophoresis. A. Untreated control; B, lipopolysaccharide; C, doxorubicin; D, lipopolysaccharide and doxorubicin. Original magnification, x200. A typical result from three independent experiments is shown.
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Involvement of p53 in the Prevention of Doxorubicin-Induced Apoptosis by Lipopolysaccharide
From the comet assay, it became clear that DNA damage was induced in doxorubicin-treated RAW 264.7 cells. It is known that DNA damage triggers the activation of p53 (15). In order to clarify the involvement of p53 in doxorubicin-induced apoptosis, the effect of PFT-, an inhibitor of p53 (16), on doxorubicin-induced apoptosis was studied (Fig. 6). RAW 264.7 cells were pretreated with PFT- (5 µmol/L) for 1 hour and then treated with doxorubicin (5 µmol/L) for 6 hours. The activation of caspase-3 and the expression of p53 were examined by immunoblotting using anti–cleaved caspase-3 antibody and anti-p53 antibody, respectively. Doxorubicin led to high expression of p53 and activation of caspase-3 in RAW 264.7 cells, although neither the expression of p53 nor the activation of caspase-3 was detected in untreated cells. Pretreatment of PFT- significantly inhibited p53 expression and almost completely abolished the activation of caspase-3. Thus, p53 was suggested to play an important role in doxorubicin-induced apoptosis.
Based on the pivotal role of p53 on doxorubicin-induced apoptosis, the effect of lipopolysaccharide on the expression of p53 in doxorubicin-treated RAW 264.7 cells was studied (Fig. 7). p53 expression at 3 and 6 hours after lipopolysaccharide and/or doxorubicin treatment was examined by immunoblotting using anti-p53 antibody. As shown in Fig. 7A, lipopolysaccharide significantly inhibited the expression of p53 at 3 and 6 hours after doxorubicin treatment, although doxorubicin alone clearly induced the stabilization and accumulation of p53. Immunohistochemical staining with anti-p53 antibody showed that positively stained cells were detected in doxorubicin-treated cells, but not in lipopolysaccharide and doxorubicin-treated cells (data not shown). Next, the difference in p53 expression between cytoplasmic and nuclear regions was examined in cells pretreated with or without lipopolysaccharide (Fig. 7B). The p53 expression was induced 3 hours after doxorubicin treatment and continued 6 hours after the treatment. However, cytoplasmic expression of p53 at 6 hours was lower than that at 3 hours. Pretreatment with lipopolysaccharide significantly prevented the cytoplasmic expression of p53 at 3 and 6 hours after doxorubicin treatment. Nuclear expression of p53 became much stronger 6 hours after doxorubicin treatment, compared with that at 3 hours. Nuclear translocation of p53 did not occur in lipopolysaccharide-pretreated cells. A semiquantitative reverse transcription-PCR (RT-PCR) showed no differences in the mRNA level among the cells treated with doxorubicin, lipopolysaccharide, and the combination of doxorubicin and lipopolysaccharide (Fig. 7C), suggesting a posttranscriptional modification of p53 by lipopolysaccharide. Based on the analysis with doxorubicin, we examined the effect of lipopolysaccharide on p53 activation by two other DNA-damaging agents, topotecan and 5-fluorouracil (17). Lipopolysaccharide also inhibited the p53 stabilization induced by topotecan or 5-fluorouracil (Fig. 7D).
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FIGURE 7. Effect of lipopolysaccharide on doxorubicin-induced p53 stabilization. RAW 264.7 cells were pretreated with lipopolysaccharide (100 ng/mL) and treated with doxorubicin (5 µmol/L) for 1, 3, and 6 hours. The expression of p53 was detected by immunoblotting using anti-p53 antibody. A. Total protein extract at 3 and 6 hours after doxorubicin treatment. B. Cytoplasmic and nuclear protein extracts at 1, 3, and 6 hours after doxorubicin treatment. C. The expression of p53 mRNA at 3 and 6 hours after doxorubicin treatment was analyzed by RT-PCR. The housekeeping GAPDH gene was used as a control. D. Effect of lipopolysaccharide on p53 stabilization induced by 5-fluorouracil and topotecan at 6 hours. Typical results from three independent experiments are shown.
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Discussion
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In the present study, we show that lipopolysaccharide prevents doxorubicin-induced apoptosis in RAW 264.7 macrophage cells. Lipopolysaccharide significantly prevented the cell death of RAW 264.7 cells treated with doxorubicin at 5 µmol/L, whereas it did not prevent the cell death by a higher concentration (>10 µmol/L) of doxorubicin. With >10 µmol/L of doxorubicin, it was possible to induce necrosis rather than apoptosis in RAW 264.7 cells because it has been reported that a high concentration of doxorubicin causes necrosis in rat cardiac myocytes (18). This might be the reason why lipopolysaccharide could not prevent cell death with a higher concentration of doxorubicin. Thus, lipopolysaccharide might exclusively prevent doxorubicin-induced apoptosis. As far as we know, this is the first report on the preventive effect of lipopolysaccharide on the cytotoxicity of DNA-damaging anticancer drugs, such as doxorubicin, topotecan, and 5-fluorouracil.
The present study suggests that lipopolysaccharide prevents doxorubicin-induced apoptosis through inactivation of p53. p53 is reported to cause apoptosis via activation of caspase-3 (19). In fact, doxorubicin induces stabilization and nuclear translocation of p53, and subsequently, the activation of caspase-3 in RAW 264.7 cells. Based on our finding that lipopolysaccharide inhibits doxorubicin-induced p53 activation, it is strongly suggested that p53 plays a pivotal role in the prevention of doxorubicin-induced apoptosis by lipopolysaccharide. This idea is also supported by the fact that PFT-, an inhibitor of p53, prevents doxorubicin-induced apoptosis. Considering that p53 expression is closely associated with microtubules (20), it is of interest to clarify whether lipopolysaccharide inhibits doxorubicin-induced p53 activation through disorganization of microtubules.
Doxorubicin-induced apoptosis was prevented by CpG DNA as well as by lipopolysaccharide (Fig. 4). On the other hand, it was not prevented by tumor necrosis factor- and IFN-. This finding suggests the involvement of Toll-like receptors (TLR) because lipopolysaccharide and CpG DNA exhibit their biological action through TLR4 and TLR9, respectively. Furthermore, the involvement of TLR was supported by the finding that lipopolysaccharide cannot prevent doxorubicin-induced cell death in peritoneal cells from C3H/HeJ mice with TLR4 mutation (Fig. 2). TLR4 and TLR9 share MyD88-dependent nuclear factor-
B activation (21). However, we could not obtain evidence of nuclear factor-B involvement by analysis with signal inhibitors.
Lipopolysaccharide can prevent the p53 activation and stabilization induced by DNA-damaging agents, such as doxorubicin, topotecan, and 5-fluorouracil (Fig. 4D). However, lipopolysaccharide could not prevent the cytotoxic actions of other anticancer drugs, such as taxol and cisplatin. Therefore, the preventive action of lipopolysaccharide might be specific for drugs that directly damage DNA. It is unclear whether lipopolysaccharide can protect macrophages from doxorubicin-induced cell death in clinical in vivo treatment or not. It would be of interest to selectively protect particular cell types from the cytotoxicity of anticancer drugs by cell activation.
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Materials and Methods
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Materials
Doxorubicin, 5-fluorouracil, and lipopolysaccharide from Escherichia coli O55:B5 were obtained from Sigma Chemicals, St. Louis, MO. Antibodies to p53 (IC12), PARP, and cleaved caspase-3 were purchased from Cell Signaling Technology, Beverly, MA. Topotecan was obtained from Calbiochem, San Diego, CA.
Animals
BALB/c mice were purchased from SLC (Hamamatsu, Japan) and used at about 6 weeks of age. All animal experiments were approved by the Animal Care Committee and carried out under the Guide for Care and Use of Laboratory Animals, Aichi Medical University.
Cell Culture
The murine macrophage cell line, RAW 264.7, was obtained from Riken Cell Bank (Tsukuba, Japan) and was maintained in RPMI 1640 containing 5% heat-inactivated FCS (Life Technologies, Gaithersburg, MD) and antibiotics at 37°C under 5% CO2.
Cell Viability
Cell death was determined by the MTT assay (Chemicon, Temecula, CA) as described elsewhere (22). The experiment result is expressed as the mean of triplicates ± SD.
DNA Fragmentation
Doxorubicin-treated cells were washed with cold phosphate-buffed saline (pH 7.4) and lysed with hypotonic buffer containing 1% Triton X-100, 10 mmol/L Tris-HCl (pH 7.9), and 10 mmol/L EDTA for 10 minutes at room temperature. The supernatant containing DNA fragments was collected by centrifugation at 8,000 x g for 10 minutes. DNA fragments in the supernatant were extracted with phenol/chloroform, treated with RNase (2.5 µg/mL) for 30 minutes at 37°C, and run in 2% agarose gel. The gels were stained with CYBR safe DNA gel stain (Molecular Probes, Eugene, OR) and visualized under an UV transilluminator. The 1 kb DNA size marker (Invitrogen, Carlsbad, CA) was also run to determine the approximate size of fragmented DNA.
Extraction of Cytoplasmic and Nuclear Proteins
RAW 264.7 cells were seeded in a 60 mm dish at a concentration of 8 x 105/2 mL, pretreated with lipopolysaccharides (100 ng/mL) for 30 minutes and incubated with doxorubicin (5 µmol/L) for various times. To extract cytoplasmic and nuclear proteins, the cells were lysed with lysis buffer containing 0.5 mol/L Tris-HCl, 4% SDS, 0.1% 2-mercaptoethanol, 1 mmol/L phenylmethylsulfonyl fluoride, and phosphatase inhibitor cocktail II (Sigma Chemicals) for 1 hour on ice. The lysates were centrifuged at 8,000 x g for 10 minutes and the supernatant was collected as a cytoplasmic protein extraction. The pellet was dissolved in nuclear protein extraction buffer containing 20 mmol/L HEPES (pH 7.9), 23% glycerol, 420 mmol/L NaCl, 1.5 mmol/L MgCl2, 0.2 mm EDTA, 0.5 mm DTT, and 0.2 mmol/L phenylmethylsulfonyl fluoride (23), left for 2 hours on ice and centrifuged for 10 minutes at 8,000 x g. The supernatant was collected as a nuclear protein extract.
Immunoblotting
RAW 264.7 cells were pretreated with lipopolysaccharide (100 ng/mL) for 30 minutes in a 35 mm plastic dish (4 x 105 cells/mL) and then treated with doxorubicin (5 µmol/L) for various times. The immunoblotting method was described previously (24). Briefly, the cell lysates were extracted by lysis buffer containing 0.5 mol/L Tris-HCl, 4% SDS, and 2 mercaptoethanol, and boiled at 80°C for 5 minutes. The protein concentration of each sample was determined by bicinchoninic acid protein assay reagent (Pierce, Rockford, IL). An equal amount of proteins (20 µg) were analyzed by SDS-PAGE under reducing conditions and transferred to a membrane filter. The membranes were treated overnight with an appropriately diluted antibody. The immune complexes were detected with a 1:5,000 dilution of horseradish peroxidase-conjugated protein G for 1 hour and the bands were visualized with a chemiluminescent reagent (Pierce). The chemiluminescence was analyzed by a light capture system (AE6955, Atto Corp., Tokyo, Japan) with a CS analyzer.
RT-PCR
RAW 264.7 cells were pretreated with lipopolysaccharide (100 ng/mL) for 30 minutes and then treated with doxorubicin (5 µmol/L) for various times. RNA was extracted from the cells with RNeasy mini kit (Qiagen, Valencia, CA) according to the manufacturer's instructions. Semiquantitative RT-PCR was carried out by using access quick RT-PCR system (Promega, Madison, WI). The primer sequence and PCR conditions for the p53 gene was described in detail elsewhere (25). A total of 35 cycles were run for each experiment. The sample (5 µL) was taken out after 22, 25, 28, 31, and 35 cycles, and run in 2% agarose gel. The housekeeping GAPDH gene was used as a control.
Detection of DNA Damage
RAW 264.7 cells were pretreated with lipopolysaccharide (100 ng/mL) for 30 minutes and treated with doxorubicin (5 µmol/L) for 3 hours. Doxorubicin-induced DNA damage was detected by the comet assay with single-cell gel electrophoresis (26) according to the manufacturer's instruction (Trevigen, Gaithersburg, MD). In brief, 1 x 105 cells were mixed with LMA agarose and placed onto a comet slide. Slides were then immersed in lysis solution at 4°C for 10 minutes, again immersed in freshly prepared alkali solution (pH > 13.0) for 20 minutes and run in 1x Tris-borate EDTA buffer at 1 V/cm for 10 minutes. Slides were air-dried and stained with CYBR green. The digital images were taken by a fluorescence microscope (Zeiss Axiovert-200, Jena, Germany) equipped with a camera and Meta-View software.
Statistical Analysis
Statistical significance was determined by Student's t test. Experimental results are expressed as the mean of triplicates ± SD in at least three independent experiments.
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Notes
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Grant support: Japan Society for the Promotion of Science-Kakenhi (14570247).
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Received 4/19/05;
revised 6/19/05;
accepted 6/21/05.
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Abstract
Introduction
