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Bacterial redox protein azurin induce apoptosis in human osteosarcoma U2OS cells Di-Sheng Yang a,∗ , Xu-Dong Miao a , Zhao-Ming Ye a , Jie Feng a , Rong-Zheng Xu b , Xin Huang c , Fen-Fen Ge c a
Department of Orthopaedics, Institute of Orthopaedic Research, Second Affiliated Hospital, School of Medicine, Zhejiang University, Hang Zhou 310009, PR China b Institute of Cancer Research, Second Affiliated Hospital, Zhejiang University, Hang Zhou 310009, PR China c School of Medicine, Zhejiang University, Hang Zhou 310009, PR China Accepted 7 June 2005
Abstract As a low molecular weight redox protein elaborated from the pathogenic bacteria Pseudomonas aeruginosa, azurin is one of representative bacterial products applied in the treatment of tumour. We found that the growth of U2OS cells was significantly inhibited by azurin in a dose-dependent manner with the IC50 value of 114.54 ± 7.65 mg l−1 . But the growth of MG63 cells or L02 cells was almost not inhibited by azurin (P < 0.05). Moreover, when treated with azurin, U2OS cells showed typical apoptotic morphological features observed by fluorescent microscopy (AO and Hoechst 33258) and transmission electron microscopy. Typical DNA “ladder” bands were also observed. The apoptosis rate was 35.8% tested by fluorescence-activated cell sorter (Annexin-V-FITC+ /PI− ) and the cell-cycle arrested in G1 phase. But no apoptotic features were observed in control cells. The down-regulation of Bcl-2 (an inhibitor of apoptosis) were detected in U2OS cells when azurin was added for 24 h. In contrast, the level of Bax and caspase-3 were significantly up-regulated. So we concluded that azurin could selectively induce apoptosis of human osteosarcoma U2OS cells and the induction of apoptosis by azurin was closely associated with down-regulation of Bcl-2, up-regulation of Bax and activation of caspase-3. © 2005 Elsevier Ltd. All rights reserved. Keywords: Azurin; Osteosarcoma; Apoptosis; Bax; Bcl-2; Caspase-3
1. Introduction Osteosarcoma is a malignant tumour of bone that is most prevalent in adolescents and young adults. Osteosarcoma accounts for approximately 5% of the tumours in childhood and 80% of this tumour originates around the knee [1]. Although adjuvant chemotherapy is effective in improvement of patient survival and treatment of the primary tumour [2], some groups of patients who present with metastatic disease and patients with tumours that recur after treatment continue to have a poor prognosis. In addition, the frequent acquisition of drug-resistant phenotypes and the occurrence of “second malignancy” are often associated with chemotherapy, which remains as serious problems. Therefore, there is a clear need ∗
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for newer effective agents for patients with osteosarcoma, especially for patients who present with metastatic disease or develop disease recurrence. Recently, live or attenuated pathogenic bacteria or their products were used in the treatment of cancer [3–5]. A significant regression of subcutaneous tumours in mice was observed by combining anaerobic bacteria with various chemotherapeutic agents [6]. Azurin, a cupredoxin type of electron transfer and purified low molecular weight redox protein elaborated from the pathogenic bacteria Pseudomonas aeruginosa, has been reported to induce and trigger apoptosis in several human cancer cells selectively. These findings in vitro have been confirmed in nude mice bearing tumour xenograft in vivo. Furthermore, it is completely lack of toxicity. At necropsy, none of the treated mice showed any histological evidence of toxicity and all of the viscera were within normal limits
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[7–9]. However, as a potent inducer of apoptosis, the precise mechanism of azurin-induced apoptosis is still unclear.
coloured bottle at 4 ◦ C as a stock solution, The stock was diluted to the required concentration immediately before use with growth media. 3-(4,5-Dimethylthiazol-2-yl)-2,5diphenyl tetrazolium bromide (MTT), AO, Hochest33258 were obtained from Sigma Chemical Co. (St. Louis, MO, USA). Primary antibodies to caspase-3, Bcl-2, Bax and actin were from Neomarkers (Fremont, CA, USA). Western blot chemiluminescent detection system (LumiGLO System) was purchased from KPL (Gaithersburg, MD, USA). All other regents are analytical or cultured grade purity. 2.2. Cell culture
The protein p53 has an important role in pathogenesis of neoplasia [10]. The mechanism involved entails a rapid increase in p53 protein levels and the mediation of several cellular responses including G1 arrest, DNA damage repair and induction of apoptosis [11]. One of the major signaling pathways involved in apoptotic cell death includes the intracellular caspases, a family of structurally related cysteine proteases [12]. Caspase activity is responsible, either directly or indirectly, for the cleavage of cellular proteins, which are characteristically proteolyzed during apoptosis. For example, caspases-2, -3, -6, -7 and -9 can cleave poly(ADP ribose) polymerase (PARP) [13]. Bcl-2 family proteins are one of the already identified regulators of apoptosis. Bcl-2 family of homologous proteins represents a critical checkpoint within most apoptotic pathways, acting upstream of such irreversible damage to cellular constituents [14]. At least 15 Bcl-2 family members have been identified so far in mammalian cells. They function either as pro-apoptotic (Bax, Bak and Bad) or anti-apoptotic (Bcl-2 and Bcl-XL) regulators. These proteins form heterodimers of anti- and pro-apoptotic members, thereby titrating one another’s function [14]. The ratio of anti- and pro-apoptotic proteins determines in part how cells respond to apoptotic or survival signals [15]. Despite extensive analysis of anti-tumour activities of azurin, its ability to modulate osteosarcoma growth has not yet been well characterized. We used osteosarcoma cell lines to study the effect of different concentrations of azurin on cell viability and genes related to apoptosis. Our results demonstrated that azurin selectively causes growth arrest and apoptosis in U2OS cells and the growth inhibitory effects of azurin appeared to be mediated by the regulation of Bcl-2, Bax and caspase-3. Azurin-induced apoptosis and the induction may be related to p53 status of the cell lines.
The human osteosarcoma cell line U2OS and MG63 (ATCC, CRL-1427, HTB 96, Manassas, VA, USA) and the human hepatocyte L02 (a normal liver cell line, was provided by Shanghai Institute of Biochemistry and Cell Biology, Chinese Academy of Science, China) were used for this study. Cell lines U2OS and L02 are p53 wild type and MG63 is devoid of endogenous p53. The cells were cultured in RPMI1640 (U2OS and LO2 cells) and MEM (MG63 cells) culture medium (Gibco, NY) at 37 ◦ C in a humidified incubator with a 5% CO2 atmosphere. The culture medium was supplemented with 0.03% l-glutamine (Gibco) and 10% fetal bovine serum (FBS), penicillin (50 U ml−1 ) and streptomycin (50 g ml−1 ). 2.3. Azurin treatment of cells The cells were exposed to azurin at different concentrations (0–500 mg l−1 ) and for different time (0–72 h). Cells grown in media containing equivalent amount of RPMI-1640 or MEM without azurin served as control. 2.4. Cell growth assay and IC50 value by MTT method
2. Materials and methods
The cytotoxic effect of azurin on U2OS, L02 and MG63 cells were assessed by MTT assay, which is based on the reduction of MTT by the mitochondrial dehydrogenase of intact cells to a purple formazan product. The cells were suspended in 96-well plate (Coastar from Corning, NY) at a density of 2 × 104 cells per well. After 24 h, they were treated with various concentrations (0–500 mg l−1 ) of azurin for different time intervals (0–72 h). Four hours before the end of incubation, 20 l of MTT solution (5 × 103 mg l−1 ) was added to each well. The supernatant was removed and 150 l DMSO was added to each well. An ELISA reader was used to measure the absorbance at 525 nm and IC50 value was assessed by Bliss method.
2.1. Chemical reagents
2.5. Morphological studies of apoptotic cell
Azurin, purchased from Sigma (MO, USA), was dissolved in RPMI-1640 or MEM culture medium with the final concentration of 1 mg l−1 and was stored in a dark-
2.5.1. Fluorescence microscopy observation U2OS cells exposed to different concentrations of azurin for overnight were harvested by centrifugation, washed with
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PBS and stained with 0.01% AO or fixed with 1% glutaraldehyde for 1 h at room temperature. Fixed cells were washed with PBS again and stained with 200 M Hoechst 33258, then were observed under the fluorescence microscopy (Axioskop, Zeiss Company). 2.5.2. Transmission electron microscopy (TEM) As described previously [16], cells harvested as above were washed in PBS and cell pellets were fixed with 2.5% glutaraldehyde for 2 h at 4 ◦ C and then incubated with 1% osmium tetroxide for 1 h at 4 ◦ C. After dehydration in a series concentrations of ethanol and infiltration in propylene oxide, cells were embedded in Epon 812. Ultrathin sections (60 nm) were stained with uranyl acetate and lead citrate, then cell morphology was observed by TEM (Philips Tecnai 10) at 80 kV.
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analysis using CellQuest software 6.0 as described previously [18,19]. 2.8. Western blot analysis Cells harvested were lysed with cold cell lysis buffer (Cell Signaling) and protein concentration determined using the BCA protein assay (Pierce) according to the manufacturer’s instructions. Cell lysates (50 g) were loaded onto 12% SDSpolyacrylamide gels, transferred onto PVDF membranes, and subjected to Western blot analysis. Relevant proteins were visualized using primary antibodies specific for caspase-3, -actin (Santa Cruz), Bcl-2 (Oncogene) and Bax (Cell Signaling). Blots were then probed with HRP-conjugated secondary antibody (Santa Cruz). Detection was carried out by incubating membranes with an enhanced chemiluminescence reagent (ECL) kit (Amersham).
2.6. DNA fragmentation assay Cells were incubated in RPMI-1640 medium at the presence or absence of drugs for 24 h. Cells were then collected and lysed in a buffer containing 10 mM Tris–HCl, pH 7.4, 150 mM NaCl, 5 mM EDTA and 0.5% Triton X-100 for 30 min on ice. DNA of 3 × 106 cells was extracted using DNA extraction kit (Sepa Gene, Sanko) and treated with RNase (Sigma). The procedures for DNA extraction and precipitation were repeated. Approximately 10 g DNA was electrophoresed on 2% agarose gel (Seakem GTG, BMA Products) containing 0.5 g ml−1 ethidium bromide (Wako) at 1 V cm−1 for 2 h. The DNA was visualized under UV light and photographed. 2.7. Quantification of apoptosis and cell-cycle analysis with a fluorescence-activated cell sorter (FACS) Determination of apoptosis and secondary necrosis utilizes the high affinity of Annexin-V to phosphatidylserine, which is exposed on the surface of apoptotic cells [17]. U2OS cells (1 × 106 ) were trypsinized to detach any adherent cells. Cells were harvested and washed in PBS, resuspended in binding buffer (10 mmol l−1 HEPES/NaOH, 140 mmol l−1 NaCl, 2.5 mmol l−1 CaCl2 , pH 7.4), and incubated with 0.25 mg ml−1 fluorescein isothiocyanate (FITC)-conjugated Annexin-V and 10 mg ml−1 PI. The mixture was kept on ice for 5 min and the cell fluorescence was measured by two-parameter flow cytometry (FACS Calibur; Becton Dickinson). When green fluorescence (FITC) was plotted against red fluorescence (PI), three distinct cell populations could be detected in a dot–plot: viable cells (FITC− /PI− ), apoptotic cells (FITC+ /PI− ) and secondary necrotic cells (FITC+ /PI+ ). A minimum of 10,000 events was counted per sample and data reported as the percentage of apoptotic cells (AnnexinV-FITC+ /PI− ). Cell-cycle analysis was also evaluated with the FACS using a cell-cycle analysis kit (Cycle Test Plus, Becton Dickinson, San Jose, CA, USA). Cell-cycle distribution was based on 2N and 4N DNA content for DNA content
2.8.1. Subcellular fractionation Cells were lysed in isotonic mitochondrial buffer containing 1 mM phenylmethylsulfonyl fluoride, 5 mg ml−1 leupeptin, 5 mg ml−1 aprotinin and 0.7 mg ml−1 pepstatin. After homogenization with a Dounce homogenizer, cell lysates were centrifuged at 1000 × g for 10 min to discard nuclei and unbroken cells. The postnuclear supernatant was centrifuged at 10,000 × g for 15 min to pellet mitochondriaenriched heavy membrane fraction, and the resulting supernatant was further centrifuged at 100,000 × g for 30 min to obtain cytosolic fraction. The membrane fractions were resuspended in Triton X-100 lysis buffer containing protease inhibitors. Protein concentration was determined by BCA assay (Pierce Chemical, Rockford, IL, USA) and total proteins (50 mg) from each fraction were subjected to immunoblot. 2.9. Statistics analyses In this study, all data were presented as the mean ± S.E.M. of three replicates from four separate experiments. Statistical differences were calculated using the Student’s t-test and considered significant at the ** P < 0.01 or * P < 0.05 level. All the figures shown in this article were obtained from at least four independent experiments with a similar pattern.
3. Results 3.1. Effect of azurin on the growth of U2OS and MG63 cells U2OS and MG63 osteosarcoma cell and L02 normal liver cells were treated with various concentrations of azurin (25, 50, 100, 200, 400 and 800 mg l−1 ), viable cells were measured after 48 h by MTT assay. As shown in Fig. 1 inhibitory effect of azurin on U2OS cells was in a dosedependent manner. Compared with U2OS cells, MG63 cells
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3.2. Effect of azurin-induced apoptosis on morphology of U2OS cells After the exposure to azurin, U2OS cells showed typical apoptosis morphology characterized by volume reduction, chromatin condensation, nuclear fragmentation and appearance of apoptosis bodies (Fig. 2A and B). In the mean time, the apoptosis effect was observed by Hoechst 33258 (Fig. 2C and D). 3.3. Transmission electron microscopic observation
Fig. 1. Relationship between the concentration of azurin and the survival rate of U2OS cells after 48 h culture. Dose-dependent growth inhibitory effect was observed only in U2OS cells. Only a slight effect or no cytotoxic effect was observed in MG63 and in normal human liver L02 cells, respectively.
were inhibited slightly and L02 cells were in activity. Azurin also showed time-dependent inhibitory effect on U2OS cells (data not shown). The IC50 at 48 h exposure of azurin were 114.54 ± 7.6 mg l−1 by Bliss method for U2OS cells. Morphological examination of U2OS cells using light microscope showed a cytotoxic effect: floating dead cells were observed (figure not shown).
After treatment with 200 mg l−1 azurin for 24 h, the reduced cell volume and shranked cytoplasm was observed under electron microscope. But plasma membrane remained well defined. Condensed chromatin located along nuclear envelope and formed irregularly crescents shape at the nuclear edge, or became condensed and fragmented. Nuclear membrane became irregular. Vacuoles in cytoplasma could be observed (Fig. 3). 3.4. DNA fragmentation assay DNA isolated from U2OS cells cultured with azurin 100, 200 and 400 mg l−1 for 24 h showed the characteristic “ladder” pattern of apoptosis. A comparison with molecular
Fig. 2. AO and Hoechst 33258 staining of apoptotic cells. AO staining: untreated U2OS cells depicted show homogeneous staining of their nuclei (A). In contrast, apoptotic cells represented show asymmetric staining of their nuclei as a result of chromatin condensation and nuclear fragmentation (B). Hoechst 33258 staining: untreated U2OS cells depicted show homogeneous staining of their nuclei (C). Apoptotic cells represented show asymmetric staining of their nuclei as a result of chromatin condensation and nuclear fragmentation (D). Magnification: 200×.
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Fig. 3. Apoptotic features of U2OS cell by electron microscopy Compared with the untreated cells (A), after 24 h of 100 mg l−1 azurin treatment, chromosomes (dark spots) and microtubules of U2OS cells (B) were visible in the middle of the cell. Condensed chromatin and vacuoles, swollen mitochondria and blebbing were observed in the nucleus. Magnification: (A) 5600× and (B) 6400×.
weight markers indicated that the fragments were multiples of certain band approximately (Fig. 4). 3.5. Evaluation of azurin-induced apoptosis in U2OS cells by FACS analysis (Annexin-V/PI) Apoptotic U2OS cells was measured by a fluorescenceactivated cell sorter. Annexin-V staining combined with PI staining was performed in control cells and cells treated with azurin in different concentration and time and then analyzed by flow cytometry. Early apoptotic is in right quadrant of a dot–plot graph using Annexin-V-FITC versus PI. There was significant difference between azurin groups (50, 100 and 200 mg l−1 ) and untreated control group in apoptosis rate
Fig. 4. DNA fragmentation assay was examined to confirm of late apoptotic change of U2OS cells after treatment with azurin. U2OS cells were exposed to azurin at various doses for 24 h. Lane 1: untreated control cells; lane 2: 100 mg l−1 azurin-treated cells; lane 3: 200 mg l−1 azurin-treated cells; lane 4: 400 mg l−1 azurin-treated cells; M: marker.
(%) of U2OS cells at 3, 6, 12, 24, 48 and 72 h (P < 0.01) and 3 h (P < 0.05), n = 3, mean ± S.E.M. After treatment of U2OS cells with azurin 200 mg l−1 for 48 h, the apoptosis rate was 35.8 ± 3.2%. The obvious changes in cell-cycle distribution of U2OS cells treated with azurin were characterized by increase of G0/G1 phase cells and decrease of S and G2/M phase cells, suggesting that azurin led to accumulation of U2OS cells in G0/G1 phase (Figs. 5A and 6). 3.6. Expressions of azurin on Bcl-2, Bax and caspase-3 proteins Based on the apoptotic analysis by determination of cells with sub-G1 DNA contents and Annexin-V/PI analysis on cells arrest and apoptosis, we then examined expression level of cell apoptosis molecules. In this analysis, we confirmed intracellular apoptotic events biochemically, by examining the expression levels of pro-apoptotic molecules such as active caspase-3, Bax and an anti-apoptotic molecule of Bcl2. Cell lysates were prepared at various time points after treatment of azurin and used for Western blot. As shown in Fig. 7, azurin had obvious effect on the level of Bcl-2, Bax and caspase-3 proteins in U2OS cells and slight effect in MG63 cells. Expression of Bcl-2 and pro-caspase-3 protein was reduced in azurin-treated U2OS cells, while that of Bax and active caspase-3 protein were increased with the increase of azurin concentration from 50 to 200 mg l−1 , suggesting azurin down-regulated Bcl-2 protein level and up-regulated Bax and caspase-3 level in U2OS cells (Fig. 7). In the mean time, expression of Bax at different time point in cytosol and mitochondria was also detected. Bax protein translocated from cytosol to mitochondria in U2OS cells, there was no changes in MG63 cells (Fig. 8).
4. Discussion Some researches on attenuated pathogenic bacteria or their products have been actively carried out in efforts to develop
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Fig. 5. (A) Four populations of cells treated with different concentrations of azurin for different times were distributed in dot–plot: viable cells (FITC− /PI− ), apoptotic cells (FITC+ /PI− ) and secondary necrotic cells (FITC+ /PI+ ). Cells treated with 0 mg l−1 azurin for 6 h (a). Cells treated with 50 mg l−1 azurin for 24 h (b). Cells treated with 100 mg l−1 azurin for 12 h (c). Cells treated with 200 mg l−1 azurin for 24 h (d). (B) Effect of azurin on apoptosis rate of U2OS cell with different concentrations (0, 50, 100 and 200 mg l−1 ) and times (3, 6, 12, 24, 48 and 72 h). When green fluorescence (FITC) was plotted against red fluorescence (PI), three distinct cell populations could be detected in a dot–plot: viable cells (FITC− /PI− ), apoptotic cells (FITC+ /PI− ) and secondary necrotic cells (FITC+ /PI+ ). A minimum of 10,000 events was counted per sample and data reported as the percentage of apoptotic cells (Annexin-V-FITC+ /PI− ).
Fig. 6. Cell-cycle (apoptosis, G0/G1, G2/M, S) analysis of U2OS cells exposed to azurin (200 mg l−1 for 48 h). A cell-cycle analysis kit (Cycle Test Plus, Becton Dickinson, San Jose, CA, USA) was used and cell-cycle distribution was based on 2N and 4N DNA content for DNA content analysis using CellQuest software 6.0.
new treatments that can decrease side effects or increase antitumour effects. Azurin, a cupredoxin type of electron transfer agent elaborated from the bacteria P. aeruginosa, is one of representative bacterial products in the treatment of tumour. Several previous studies have demonstrated that azurin exhibited anti-tumour activities [7–9]. In this study, we analyzed effects of azurin on proliferation and apoptosis of human osteosarcoma cell line U2OS, MG63 and normal human liver cell line L02. Our cell proliferation assays showed that azurin strongly inhibited proliferation of U2OS cells at doses ranging from 50 to 800 mg l−1 (P < 0.05), whereas slight inhibitory effects were exhibited on MG63 cells and no effects on L02 cellular growth. Azurin’s inhibitory effect on cellular proliferation was in dose- and time-dependent manner and its cytotoxic effect to osteosarcoma cell was specific (Fig. 1). Based on the results of cytotoxicity of azurin, we have done further studies: morphological changes of cells (fluorescence microscopy AO and Hoschest 33258 and transmission electron microscopy) (Figs. 2A–D and 3A and B). With the Annexin-V/PI staining (FITC+ /PI− ), azurin-induced apoptotic changes were observed in U2OS cells (Figs. 5B and 6) by FACS analysis, which represents early marker of apoptosis. We also performed DNA fragmentation assay, which represents late marker of apoptosis. After the treatment of U2OS cells with different concentrations of azurin for 48 h,
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Fig. 7. The expression of Bcl-2, Bax, pro-caspase-3, -actin and caspase-3 in azurin-treated U2OS cells and MG63 cells by Western blot. (A) Cells were treated with azurin 0, 50, 100 and 200 mg l−1 for 24 h. Cell lysates were separated by 12% SDS-PAGE electrophoresis, and Bcl-2, Bax, pro-caspase-3 and caspase-3 protein bands were detected by Western blot analysis. -Actin was used as an equal loading control. (B) Densitometric quantification of Bax/Bcl-2 rate in azurin-treated U2OS cells and MG63 cells was measured by Gel-Pro Analyzer 3.1 software. There was significant difference between U2OS cells group and MG63 cells group in each dose point. (C) Caspase-3 activation in U2OS cells and MG63 cells subjected to different concentration of azurin treatment in vivo for 24 h. Densitometric quantification rate of active form of casapse-3/-actin were measured by Gel-Pro Analyzer 3.1 software. There was significant difference between U2OS cells group and MG63 cells group in each dose point for 24 h.
significant DNA fragmentations were detected (Fig. 4). In the cell-cycle analysis, significant increase of cell population in G0/G1 phase were observed at 24, 48 and 72 h (Fig. 6). Characteristic apoptosis were observed by all of the above apoptosis-related experiments, respectively. According to our apoptotic analysis and morphological observation in U2OS cells, it may be concluded that azurin could induce apoptosis in U2OS cells. Then, we investigated the mechanism of the induction. Apoptosis is a genetically programmed event that can take place by a variety of internal or external stimuli and these signals are regulated by two distinct pathways, involving either death receptor (extrinsic) or mitochondria [20,21]. In
the mitochondrial pathway, a variety of death signals triggers the release of several pro-apoptotic proteins. A number of apoptosis-related proteins such as Bcl-2 family members display both anti- and pro-apoptotic functions by forming homo- or heterodimers [22]. They are important regulators of apoptosis [23]. In these proteins, Bax is a crucial mediator and loss of this pro-apoptotic protein contributes to drug resistance in human cancers [24]. As a tumour suppressor, it mediates the p53-induced apoptosis and increases sensitivity to chemotherapy-induced apoptosis [25,26]. Meanwhile, Bcl-2 is an anti-apoptotic protein. When it is activated or prevalent, apoptosis is prohibited. Abnormal overexpression of Bcl-2 has frequently been observed in many types of
Fig. 8. Treatment of 100 mg l−1 azurin for indicated periods regulates expression level of apoptotic-related protein – Bax – in cytosol and mitochondria of U2OS cells and MG63 cells. Cells were treated with 100 mg l−1 azurin for 0, 12, 24, 48 h and lysed in isotonic mitochondrial buffer containing protease inhibitor. After homogenization, cell lysates were centrifuged at 1000 × g. The postnuclear supernatant was centrifuged at 10,000 × g and the resulting supernatant was further centrifuged at 100,000 × g to obtain cytosolic fraction. The membrane fractions were resuspended in Triton X-100 lysis buffer containing protease inhibitors. Total proteins (50 mg) from each fraction were subjected to immunoblot analysis. Compared with MG63 cells, expression level of Bax decreased in cytosol while increased in mitochondria in U2OS cells.
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human cancers. And relative expression levels of Bcl-2 to Bax were reported to determine the sensitivity to apoptosis [27–29]. However, several studies indicated that these pro- and anti-apoptotic proteins (Bcl-2 and Bax) might function independently and without formation of heterodimers [30]. We found that azurin up-regulated Bax protein and down-regulated Bcl-2 protein in U2OS cells by Western blot analysis. It means the induction of apoptosis in U2OS cells by azurin is associated with Bcl-2 family. Whether or not Bcl-2 and Bax formed the heterodimers was not investigated, which needs us to study further. We reported here that U2OS cells containing wild-type p53 were far more sensitive to azurin at all dose levels than MG63 cells, a p53-negative cell line. It could be concluded that functional p53 was required for the apoptotic pathway in U2OS cells. p53 is not only able to up-regulate transcriptionally particular pro-apoptotic genes such as Bax, Apaf-1, caspase-9 and PUMA but also able to repress some antiapoptotic genes like Bcl-2 [31,32]. A ratio of Bax to Bcl-2 – rather than anti-apoptotic Bcl-2 alone – is important in cell survival or apoptotic death in response to death stimuli [33]. In our study, the average ratio of Bax/Bcl-2 in p53containing U2OS cells treated with azurin was higher than that of p53-defective cells. It is possible that the mechanism of azurin-induced apoptosis in U2OS cells includes functional p53’s modulation of the ratio of Bax/Bcl-2. Caspases have been shown to be activated during apoptosis in many cells and play critical roles in both the initiation and execution of apoptosis [34]. Recently, it was reported that caspase-3 is essential for DNA fragmentation and the morphological change associated with apoptosis [35]. It has been postulated that activated caspase-3 cleaves the inhibitor of caspase-activated DNase (ICAD/DEF-45), releasing from the complex the caspases activated DNase (CAD/CPAN). Once the ICAD is cleaved, CAD enters the nucleus and degrades chromatin into oligonucleosomal fragments [36,37]. This, in turn, leads to apoptosis. To obtain further insight into the mechanism of azurin action, we studied the expression of caspse-3 protein. Caspase-3 activation was evident in cells treated with azurin. This experiment suggested the possibility that the mechanism of azurin-induced apoptosis in U2OS cells also involve caspase-3 activation and the following cascades of reactions. Bax translocation from the cytosol to mitochondria [38] (Fig. 8) is a classical event in the mitochondrial apoptotic pathway and has been reported to occur in many cell lines in response to various apoptotic stimuli [38,39]. It appears that azurin-treated U20S cells, within 12–48 h, signals the mitochondrial pathway to recruit and activate caspase-9 then activates caspase-3 which in turn puts into a chain reaction that results in apoptotic cell death. In summary, we have demonstrated that azurin selectively induced apoptosis in U2OS cells. And the induction of apoptosis was closely associated with down-regulation of Bcl-2, up-regulation of Bax and activation of caspase-3. Homoor heterodimerization of these pro- and anti-apoptotic pro-
teins is one of the suggested mechanisms of azurin-induced apoptosis. It is important for us to make it clear. Human tumour suppressor protein p53 plays a major role in the cellcycle, orchestrating a number of important genes involved in cell-cycle control and apoptosis. And the level of Bcl-2 expression is consistent with p53 inactivation because p53 inhibits Bcl-2 gene expression [40]. The relationship between azurin-induced apoptosis and p53 level is also needed to be done.
Acknowledgements This project is supported by the Nature Science Foundation of Zhejiang Province in China (No. Y204358) and Scientific Research Key Program of Health Bureau of Zhejiang Province in China (No. 2003ZD007).
References [1] Link MP, Eilber F. Pediatric osteosarcoma. In: Pizzo PA, Poplack DG, editors. Principles of pediatric oncology. Philadelphia: Lippincott; 1989. p. 689–711. [2] Marina NM, Pratt CB, Rao BN, Shema SJ, Meyer WH. Improved prognosis of children with osteosarcoma metastatic to the lung(s) at the time of diagnosis. Cancer 1992;70:2722–7. [3] Sinha G. Bacterial battalions join war against cancer. Nat Med 2003;9(10):1229. [4] Chakrabarty AM. Microorganisms and cancer: quest for a therapy. J Bacteriol 2003;185(9):2683–6. [5] Da Rocha AB, Lopes RM, Schwartsmann G. Natural products in anticancer therapy. Curr Opin Pharmacol 2001;1(4):364–9. [6] Tangri S, Ishioka GY, Huang X, Sidney J, Southwood S, Fikes J, et al. Structural features of peptide analogs of human histocompatibility leukocyte antigen class I epitopes that are more potent and immunogenic than wild-type peptide. J Exp Med 2001;194(6):833–46. [7] Punj V, Bhattacharyya S, Saint-Dic D, Vasu C, Cunningham EA, Graves J, et al. Bacterial cupredoxin azurin as an inducer of apoptosis and regression in human breast cancer. Oncogene 2004;23(13):2367–78. [8] Yamada T, Goto M, Punj V, Zaborina O, Kimbara K, Das Gupta TK, et al. The bacterial redox protein azurin induces apoptosis in J774 macrophages through complex formation and stabilization of the tumor suppressor protein p53. Infect Immun 2002;70(12):7054– 62. [9] Yamada T, Goto M, Punj V, Zaborina O, Chen ML, Kimbara K, et al. Bacterial redox protein azurin, tumor suppressor protein p53, and regression of cancer. Proc Natl Acad Sci USA 2002;99(22):14098–103. [10] Greenblatt MS, Bennett WP, Hollstein M, Harris CC. Mutations in the p53 tumor suppressor gene: clues to cancer etiology and molecular pathogenesis. Cancer Res 1994;54(18):4855–78. [11] Bhaumik S, Jyothi MD, Khar A. Differential modulation of nitric oxide production by curcumin in host macrophages and NK cells. FEBS Lett 2000;483(1):78–82. [12] Salvesen GS, Dixit VM. Caspases: intracellular signaling by proteolysis. Cell 1997;91(4):443–6. [13] Nagata S. Apoptosis by death factor. Cell 1997;88(3):355–65. [14] Adams JM, Cory S. The Bcl-2 protein family: arbiters of cell survival. Science 1998;281(5381):1322–6. [15] Farrow SN, Brown R. New members of the Bcl-2 family and their protein partners. Curr Opin Genet Dev 1996;6(1):45–9.
D.-S. Yang et al. / Pharmacological Research 52 (2005) 413–421 [16] Wang T, Xia D, Li N, Wang C, Chen T, Wan T, et al. Bone marrow stromal cell-derived growth inhibitor, BDGI, inhibits growth and migration of breast cancer cells via induction of cell cycle arrest and apoptosis. J Biol Chem 2005;280(6):4374–82. [17] Pongracz J, Webb P, Wang K, Deacon E, Lunn OJ, Lord JM. Spontaneous neutrophil apoptosis involves caspase 3-mediated activation of protein kinase C-delta. J Biol Chem 1999;274(52):37329–34. [18] Yamamoto S, Mogi M, Kinpara K, Ishihara Y, Ueda N, Amano K, et al. Anti-proliferative capsular-like polysaccharide antigen from Actinobacillus actinomycetemcomitans induces apoptotic cell death in mouse osteoblastic MC3T3-E1 cells. J Dent Res 1999;78(6):1230–7. [19] Ozeki N, Mogi M, Nakamura H, Togari A. Differential expression of the Fas–Fas ligand system on cytokine-induced apoptotic cell death in mouse osteoblastic cells. Arch Oral Biol 2002;47(7):511–7. [20] Reed JC. Apoptosis-regulating proteins as targets for drug discovery. Trends Mol Med 2001;7(7):314–9. [21] Wyllie AH. Apoptisis (the 1992 Frank Rose Memorial Lecture). Br J Cancer 1993;67(2):205–8. [22] Zhou M, Gu L, Yeager AM, Findley HW. Sensitivity to Fas-mediated apoptosis in pediatric acute lymphoblastic leukemia is associated with a mutant p53 phenotype and absence of Bcl-2 expression. Leukemia 1998;12(11):1756–63. [23] Zhang H, Heim J, Meyhack B. Novel BNIP1 variants and their interaction with BCL2 family members. FEBS Lett 1999;448(1):23–7. [24] Yamaguchi H, Bhalla K, Wang HG. Bax plays a pivotal role in thapsigargin-induced apoptosis of human colon cancer HCT116 cells by controlling Smac/Diablo and Omi/HtrA2 release from mitochondria. Cancer Res 2003;63(7):1483–9. [25] Schlesinger PH, Gross A, Yin XM, Yamamoto K, Saito M, Waksman G, et al. Comparison of the ion channel characteristics of proapoptotic BAX and antiapoptotic BCL-2. Proc Natl Acad Sci USA 1997;94(21):11357–62. [26] Tu Y, Xu FH, Liu J, Vescio R, Berenson J, Fady C, et al. Upregulated expression of BCL-2 in multiple myeloma cells induced by exposure to doxorubicin, etoposide, and hydrogen peroxide. Blood 1996;88(5):1805–12. [27] Strobel T, Swanson L, Korsmeyer S, Cannistra SA. BAX enhances paclitaxel-induced apoptosis through a p53-independent pathway. Proc Natl Acad Sci USA 1996;93(24):14094–9.
421
[28] Yin C, Knudson CM, Korsmeyer SJ, et al. Bax suppresses tumorigenesis and stimulates apoptosis in vivo. Nature 1997;385(6617): 637–40. [29] Itoh N, Tsujimoto Y, Nagata S. Effect of bcl-2 on Fas antigenmediated cell death. J Immunol 1993;151(2):621–7. [30] Cheng EH, Levine B, Boise LH, et al. Bax-independent inhibition of apoptosis by Bcl-XL. Nature 1996;379(6565):554–6. [31] Gross A, Jockel J, Wei MC, Korsmeyer SJ. Enforced dimerization of BAX results in its translocation, mitochondrial dysfunction and apoptosis. EMBO J 1998;17(14):3878–85. [32] Budhram-Mahadeo V, Morris PJ, Smith MD, Midgley CA, Boxer LM, Latchman DS. p53 suppresses the activation of the Bcl-2 promoter by the Brn-3a POU family transcription factor. J Biol Chem 1999;274(21):15237–44. [33] Salomons GS, Brady HJ, Verwijs-Janssen M, Van Den Berg JD, Hart AA, Van Den Berg H, et al. The Bax alpha:Bcl-2 ratio modulates the response to dexamethasone in leukaemic cells and is highly variable in childhood acute leukaemia. Int J Cancer 1997;71(6):959–65. [34] Cohen GM. Caspases: the executioners of apoptosis. Biochem J 1997;326(Pt 1):1–16. [35] Janicke RU, Sprengart ML, Wati MR, Porter AG. Caspase-3 is required for DNA fragmentation and morphological changes associated with apoptosis. J Biol Chem 1998;273(16):9357–60. [36] Pratesi G, Polizzi D, Perego P, Dal Bo L, Zunino F. Bcl-2 phosphorylation in a human breast carcinoma xenograft: a common event in response to effective DNA-damaging drugs. Biochem Pharmacol 2000;60(1):77–82. [37] Pandey S, Smith B, Walker PR, Sikorska M. Caspase-dependent and independent cell death in rat hepatoma 5123tc cells. Apoptosis 2000;5(3):265–75. [38] Gross A, Gross A, McDonnell JM, Korsmeyer SJ. BCL-2 family members and the mitochondria in apoptosis. Genes Dev 1999;13(15):1899–911. [39] Mooney LM, Al-Sakkaf KA, Brown BL, Dobson PRM. Apoptotic mechanisms in T47D and MCF-7 human breast cancer cells. Br J Cancer 2002;87(8):909–17. [40] Miyashita T, Krajewski S, Krajewska M, Wang HG, Lin HK, Liebermann DA, et al. Tumor suppressor p53 is a regulator of bcl-2 and bax gene expression in vitro and in vivo. Oncogene 1994;9(6):1799–805.
