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Achacin induces cell death in HeLa cells through two different mechanisms
ABB Archives of Biochemistry and Biophysics 422 (2004) 103–109 www.elsevier.com/locate/yabbi
Achacin induces cell death in HeLa cells through two different mechanisms Nobuyuki Kanzawa,a,* Satoko Shintani,a Kazumasa Ohta,b Seiji Kitajima,a Tatsuya Ehara,a Hiroko Kobayashi,a Harutoshi Kizaki,b and Takahide Tsuchiyaa a b
Department of Chemistry, Faculty of Science and Technology, Sophia University, Tokyo 102-8554, Japan Department of Biochemistry, Tokyo Dental College, 1-2-2 Masago, Mihama-ku, Chiba 261-8502, Japan Received 14 October 2003, and in revised form 2 December 2003
Abstract Achacin, which belongs to the L -amino acid oxidase group, oxidizes free amino acids and produces hydrogen peroxide in cell culture systems. Morphological changes in cells incubated with achacin were similar to those of cells incubated with H2 O2 . In both cases, the end result was cell death. To examine the mechanism of achacin-associated cytotoxicity, the H2 O2 scavenger catalase was added to culture media. Features typical of apoptosis, including morphological changes, DNA fragmentation, and PARP cleavage, were observed when cells were incubated with achacin in the presence of catalase. Moreover, apoptosis was inhibited by Z–VAD– fmk, a broad-spectrum caspase inhibitor. Herein, we present evidence that two pathways are involved in achacin-induced cell death. One is direct generation of H2 O2 through the L -amino acid oxidase activity of achacin. The other is the caspase-mediated apoptotic pathway that is induced by depletion of L -amino acids by achacin. Ó 2003 Elsevier Inc. All rights reserved. Keywords: Achacin; Apoptosis; L -amino acid oxidase; Hydrogen peroxide
Achacin was originally isolated as an antibacterial glycoprotein from the body surface mucus of the giant African snail Achatina fulica Ferussac [1,2]. Sequence and biochemical data revealed that achacin is a member of the amine oxidase family, of flavin enzymes [3], and we recently reported that achacin has L -amino acid oxidase (LAO, EC 1.4.3.2) activity [4]. LAO is a member of the amine oxidase family and catalyzes oxidative deamination of L -amino acids to a-keto acids, hydrogen peroxide (H2 O2 ), and ammonia (NH3 ). LAOs are present in various organisms and are thought to induce cell death under physiological conditions through generation of hydrogen peroxide [5,6]. Dolabellanin, which has sequence similarity with achacin, is known to induce cell death in cancer cells [7–9]; however, the mode of cytotoxicity is not well documented. These facts led us to investigate how achacin induces cell death in cell
culture systems, and how the mechanism of the cytotoxicity may contribute to the host defense mechanism of achacin. Reactive oxygen species (ROS)1 are produced under a variety of physiological conditions, including acute and chronic inflammation [10], and are known to cause apoptosis [11–13]. LAOs are present in fish [14] and several snake venoms [15–17] and have been reported to induce apoptosis [5,6]; however, it is unclear if this apoptosis is due directly to the effect of H2 O2 [18]. In the present study, we measured the LAO activity of achacin in a cell culture system. Achacin generated H2 O2 from free amino acids present in the culture media and induced cell death in HeLa cells; however, the mechanism of the cell death was not due solely to H2 O2 generated through the LAO activity of achacin. Herein, we show that the LAO activity of achacin depletes amino acids from culture media and that the 1
*
Corresponding author. Fax: +011-81-3-3238-3361. E-mail address: [email protected] (N. Kanzawa). 0003-9861/$ - see front matter Ó 2003 Elsevier Inc. All rights reserved. doi:10.1016/j.abb.2003.12.007
Abbreviations used: PARP, poly (ADP-ribose) polymerase; ROS, reactive oxygen species; Z–VAD–fmk, carbobenzoyl–valyl–alanyl– aspartyl–fluoromethyl ketone.
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reductions of specific amino acids are associated with H2 O2 -independent cell death.
Materials and methods Materials Carbobenzoyl–valyl–alanyl–aspartyl–fluoromethylketone (Z–VAD–fmk) was purchased from the Peptide Institute, (Osaka, Japan) and dissolved in dimethyl sulfoxide (DMSO). Catalase and all other chemicals used in this study were purchased from Wako Pure Chemicals (Tokyo, Japan), Nacalai Tesque (Kyoto, Japan), or Sigma (St. Louis, MO). Cell culture HeLa cells were provided by the Cell Resource Center for Biomedical Research (Tohoku University, Miyagi, Japan) and maintained in Minimal Essential Medium (MEM) (Gibco BRL, Gaithersburg, MD) supplemented with 10% fetal bovine serum (FBS) at 37 °C in a humidified atmosphere containing 5% CO2 . Protein preparation Achacin was prepared as described previously [19,20]. Briefly, mucus from snails (Achatina fulica Ferussac) was mixed with an equal volume of 40 mM potassium phosphate (pH 7.0) by gently stirring for 24 h. After fractionation by 50–90% (w/v) ammonium sulfate saturation, the precipitate was dissolved in 50 mM Tris–HCl (pH 8.0) and applied to a TSK-gel DEAE-Toyopearl 650 M column (Tosoh, Tokyo, Japan). Antibacterial activity of each fraction was assessed as described previously [4]. Eluates containing antibacterial activity were pooled and purified further with a TSK-gel G3000SW column (Tosoh). Purified fractions containing antibacterial activity were used in this study. Measurement of oxidase activity Xylenol orange assay for LAO was performed with an established method [21]. Levels of activity were determined by measuring absorbance of Fe3þ (from oxidation of Fe2þ by H2 O2 ) –xylenol orange complex at 550 nm. LAO activity of achacin for each amino acid (15 mM, all purchased from Kyowa Hakko Kogyo, Tokyo, Japan) was measured after 10 min of incubation at 37 °C. Test aliquots of the culture medium were removed and analyzed after the appropriate incubation times with achacin (5 lg/ml) at 37 °C. One unit of enzyme activity was defined as the oxidation of 1 lmol of amino acid per minute.
Cell viability and microscopic analysis Cell viability was assessed by trypan blue exclusion. Morphological examination was done by phase-contrast microscopy (IX70; Olympus, Tokyo, Japan). For fluorescence microscopy, detached cells were collected by centrifugation at 3000g for 10 min, fixed with 3.4% formaldehyde in phosphate-buffered saline (PBS) for 15 min, and then stained with 5 lg/ml Hoechst 33342 for 20 min. Cells were then observed with a fluorescence microscope (Axiophoto-2; Carl Zeiss, Oberkochen, Germany). Detection of nucleosomal DNA fragmentation Total DNA was extracted according to the method of Lee and Shacter [26]. Briefly, approximately 7 105 HeLa cells were cultured in 6-well plates overnight and then incubated for 48 h under the conditions described below. Cells were harvested, washed with PBS, and lysed in buffer containing 10 mM Tris–HCl (pH 7.8), 10 mM EDTA, 0.5% Triton X-100, 200 lg/ml RNase, and 200 lg/ml proteinase K. After DNA was precipitated with isopropanol, it was then resuspended in a Tris–EDTA solution. Samples were resolved by electrophoresis on 2% agarose gels and visualized by ethidium bromide staining and UV transillumination. Western blot analysis HeLa cells were sonicated in cell lysis buffer [1% Triton X-100, 10 mM Tris–HCl (pH 7.5), 150 mM NaCl, 2 mM EGTA, 50 mM of 2-mercaptoethanol, 2 mM Na2 VO4 , 10 mM NaF, 1 mM DTT, and protease inhibitor cocktail (Complete; Roche Diagnostics, Tokyo, Japan)] and lysates were resuspended in SDS sample buffer [2.3% SDS, 5% of 2-mercaptoethanol, 63 mM Tris–HCl (pH 6.7), and 5% glycerol]. Samples were separated on 10% SDS– PAGE gels [22], transferred to Immobilon membranes (Millipore, Bedford, MA), incubated in rabbit antiPARP antibody (1:1000, Santa Cruz Biotechnology, Santa Cruz, CA), followed by HRP-conjugated secondary antibody (1:5000, DAKO, Kyoto, Japan), and visualized with an enhanced chemiluminescence (ECL) kit (Amersham–Pharmacia Biotech, Little Chalfont, UK). Quantification of amino acid composition HeLa cells were cultured in MEM containing 10% FBS with achacin for 48 h. Cells were removed by centrifugation. The concentrations of amino acids in culture media before (control) and after cells were cultured with achacin were determined with an automated amino acid analyzer (Mitsubishi Kagaku BCL, Tokyo, Japan). Amino acid concentrations were reported as relative to control.
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Amino acid depletion culture
Characterization of achacin-induced cell death
HeLa cells were cultured for 48 h in the absence of free amino acids or in the presence of 29.2 g/ml L -Gln, 5.2 g/ml L -Ile, 4.8 g/ml L -Thr, 4.6 g/ml L -Val, 3.1 g/ml L cystine, 5.2 g/ml L -Tyr, and 4.2 g/ml L -His. The modified culture medium lacks Arg, Leu, Lys, Met, Phe, and Trp, which are preferred substrates of the LAO activity of achacin.
ROS are known to cause apoptosis [11–13] and our data suggested that achacin induces cell death. To study the cytotoxicity of achacin, we examined the dose-dependent effect of achacin on cell viability in the presence or absence of catalase. HeLa cells (4 104 /well, 24-well plate) incubated with 1.5 lg/ml achacin underwent cell death after 24 h of incubation (Fig. 1B) similar to cells incubated with 300 lM H2 O2 . In both cases, membrane blebbing and shrinking of the cytoplasm were observed (Fig. 2, panels B–D). Addition of 100 U/ml catalase effectively abolished the effect of H2 O2 , resulting in a level of viability similar to that of controls (Fig. 1B). However, catalase could not suppress the cytotoxicity of achacin completely. We confirmed this by observing morphological changes in cells incubated with achacin. Cells incubated with H2 O2 together with catalase were extended and no significant changes in cell shape were observed. Cells incubated with achacin displayed a fibrous shape and some had membrane blebbing. The onset of morphological changes was detected at approximately 24 h of incubation; however, chromatin
Results LAO activity of achacin To characterize the enzymatic activity of achacin, we quantified the amount of H2 O2 produced by achacin from free amino acids as substrates (Table 1). Achacin utilizes only L -type amino acids to produce H2 O2 [4]. In the present study, achacin preferentially oxidized basic and some neutral amino acids (Table 1). We further analyzed the LAO activity of achacin in cell culture media. Levels of H2 O2 peaked 1 h after the addition of achacin to the culture medium, and H2 O2 levels then decreased with increased incubation time (Fig. 1A). Maximal production of H2 O2 was approximately 300 lM for 5 lg/ml achacin after 1 h of incubation. When catalase (100 U/ml) was added to MEM and incubated with achacin, H2 O2 levels were below the limit of detection (data not shown). Table 1 Oxidation activity of achacin Amino Activitya acid Unit (lmol/min)
Unit/mg
Lys Arg His
3
2.69 10 0.22 10 2.39 103 0.10 103 0.21 103 0.07 103
0.82 0.067 0.72 0.030 0.06 0.041
Neutral Met amino acids Leu Trp Phe Cys Asn Tyr Ala Gln Gly Ile Pro Ser Thr Val
4.67 103 0.01 103 4.38 103 0.17 103 3.58 103 0.46 103 2.16 103 0.40 103 1.10 103 0.02 103 0.99 103 0.30 103 0.84 103 0.70 103 0.36 103 0.02 103 ())b ()) ()) ()) ()) ()) ())
1.42 0.005 1.33 0.052 1.08 0.014 0.65 0.011 0.33 0.006 0.30 0.001 0.25 0.020 0.11 0.007 ()) ()) ()) ()) ()) ()) ())
Acidic amino acids
()) ())
()) ())
Basic amino acids
a
Asp Glu
3
Oxidation activity of achacin (3.3 lg/ml) was determined with amino acids (15 mM) as substrate. ()) indicates that the level of activity is below the detection limit.
L -type b
Fig. 1. Effects of achacin on cell viability. Time-dependent production of H2 O2 by achacin (5 lg/ml) detected in cell culture media (A). Mean values of three experiments are shown. Error bars are the standard deviation (SD) of the mean. (B) Cell viability was examined in HeLa cells incubated with various concentrations of achacin with or without 100 U/ml catalase. HeLa cells (4 104 ) were seeded in 24-well plates, cultured overnight, and then incubated for 24 h in MEM under the indicated conditions. Cells were counted, and the mean percent viability of control from three independent experiments is presented.
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Fig. 2. Effect of achacin on cell morphology. Morphological and chromosomal changes were examined in HeLa cells incubated with achacin with or without catalase. Pre-cultured HeLa cells were incubated for either 24 h (24 h; B, F, and J) or 48 h in MEM (48 h; A, C, D, E, G, H, I, K, and L) with 5 lg/ml achacin (+achacin; B, C, F, G, J, and K), without achacin (control; A, E, and I) or 300 lM H2 O2 (+H2 O2 ; D, H, and I) in the absence (A–D) or presence (E–L) of 100 U/ml catalase (catalase). Phase contrast images of attached cells (A–H) and fluorescent images of floating cells (I–L) were analyzed as described in the Materials and methods. Bars in (H) and (L) are 80 and 20 lm, respectively.
condensation became obvious at approximately 48 h of incubation (Fig. 2, panels J and K). These observations indicate that achacin induces apoptosis, that is independent of H2 O2 -induced cell death. Achacin-induced apoptosis is mediated by the caspase pathway To clarify the involvement of caspases in achacininduced apoptosis, we examined the effects of Z–VAD– fmk on DNA fragmentation and PARP cleavage, which are the hallmark features of apoptosis. DNA fragmentation in cells incubated with achacin and catalase was suppressed by Z–VAD–fmk, similar to that in staurosporine-treated cells; this was not prevented in the absence of catalase (Fig. 3A). Z–VAD–fmk also inhibited PARP cleavage in cells incubated with achacin alone in the presence of catalase (Fig. 3B). We then analyzed the effect of Z–VAD–fmk on achacin-induced cell death. Total numbers of attached and floating cells were counted (Fig. 4A). In the absence of catalase, living cells undergo cell death. However, achacin appears to suppress cell proliferation because total cell number did not increase regardless of the presence of Z–VAD–fmk. The possible role of caspase in achacininduced apoptosis was analyzed by comparing the numbers of detached floating cells in the presence and absence of Z–VAD–fmk (Fig. 4B). Numbers of floating dead cells were decreased considerably in the presence of catalase, and a marked effect on the number of floating cells in response to Z–VAD–fmk was also observed (P < 0:01; StudentÕs t test).
Depletion of free amino acids induces apoptosis To clarify the mechanism of cytotoxicity, HeLa cells were cultured for 48 h in pre-conditioned media that had been incubated for 48 h with achacin and filtered with Centricon YM-10 (<10 kDa, Millipore) to eliminate protein factors, and a 1/10 volume of FBS was added to the media. Approximately 30% of cells relative to control were viable when cells were cultured with achacin (Fig. 5, bars on a left half). Interestingly, the proportion of surviving cells cultured in the pre-conditioned media was approximately 50% that of the control even in the absence of achacin (Fig. 5, bars on a right half). This result suggested that an induction factor of achacin-induced cell death in the presence of catalase is in culture media, but not via protein factor. Catalase should metabolize H2 O2 generated during culture of cells with achacin; however, catalase does not affect the enzyme activity of achacin. Therefore, deamination of free amino acids in culture media may occur during incubation of cells with achacin. Murakawa et al. [14] reported that LAO induces apoptosis of HL-60 cells and this is associated with depletion of L -Lys from the culture medium. We analyzed amino acid concentrations in culture media after 48 h of incubation with achacin in the presence of catalase (Fig. 6). In most cases, the level of amino acid consumption was lower in medium with achacin than in medium without achacin. This is likely due to growth arrest of achacin-treated cells. However, Arg, Trp, Lys, and Tyr showed the highest levels of depletion in media from achacin-incubated cells, suggesting that achacin-induced apoptosis is
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Fig. 4. Effect of Z–VAD–fmk on cell death. HeLa cells (7 105 /6-cm dish) were pre-cultured overnight and then incubated for 48 h in MEM alone (control) or with 5 lg/ml achacin (achacin), 5 lg/ml achacin, and 100 U/ml catalase (achacin + catalase), or 50 nM staurosporine (staurosporine) with (+) or without ()) 50 lM Z–VAD–fmk. (A) Numbers of cells attached to the dish surface (solid bar) or detached and floating (open bar) were counted, and the mean (n ¼ 3) is presented. (B) Effect of Z–VAD–fmk is depicted as the relative ratio of floating to attached cells. *P < 0:01, paired StudentÕs t test. Fig. 3. Effect of Z–VAD–fmk on achacin-induced cell death. (A) HeLa cells (7 105 /6-cm dish) were pre-cultured overnight in MEM and further incubated for 48 h in the presence of 5 lg/ml achacin (achacin), 5 lg/ml achacin, and 100 U/ml catalase (achacin + catalase), or 50 nM staurosporine (staurosporine) with (+) or without ()) 50 lM Z–VAD– fmk. Nucleosomal DNA was isolated and separated by electrophoresis on 2% agarose gels. M, 200-bp ladder. (B) Pre-cultured HeLa cells (3 105 /well of 6-well plate) were incubated in MEM (control) or in the presence of 5 lg/ml achacin (achacin), 5 lg/ml achacin, and 100 U/ ml catalase (achacin + catalase), and 50 nM staurosporine (staurosporine) with (+) or without ()) 50 lM Z–VAD–fmk. Total protein extracts were prepared from each sample and separated by electrophoresis on 10% acrylamide gels. Gels were stained with Coomassie brilliant blue (lower panel) or transferred to Immobilon membranes followed by immunodetection (upper panel) with anti-PARP antibody. Arrowhead and double arrowhead indicate PARP protein and its degradation product, respectively. M, protein molecular mass marker. Ordinate, molecular mass in kDa.
mediated by depletion of free amino acids from the culture medium. To confirm this finding, we next analyzed the effect of amino acid starvation on apoptosis. Cells were cultured in the absence of free amino acids or in the presence of restricted amino acids. Lack of Arg, Leu, Lys, Met, Phe, and Trp, which are suitable substrates for the LAO activity of achacin, induced DNA fragmentation in HeLa cells. A higher level of fragmentation was observed in medium with restricted amino acids than in medium lacking free amino acids (Fig. 7A). In addition, fibrous changes in cell shape and chromatin condensation were observed in cells cultured in both modified MEMs (Fig. 7B), and this is similar to changes observed in cells
Fig. 5. Cell viability tests in pre-conditioned media. HeLa cells (7 105 /6-cm dish) were pre-cultured for 48 h in the presence of catalase (100 U/ml) with (+) or without ()) achacin (5 lg/ml). Thereafter, the culture medium was filtered over Centricon CY-10 (<10 kDa), 1/10 volume of FBS was added, and the medium was transferred to new wells. HeLa cells (7 105 /6-cm dish) were seeded in the wells and incubated for 48 h. The average number of surviving cells from three separated experiments was compared to that of controls, which were cells cultured for 48 h with (+) or without ()) achacin (5 lg/ml) in the same conditioned media without the pre-incubation. (n), Culture medium was filtered without the pre-culturing with achacin.
incubated with achacin and catalase. These findings indicate that depletion of amino acids from culture medium induces apoptosis of HeLa cells.
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Fig. 6. Amino acid concentrations after incubation with achacin and catalase. The concentrations of amino acids in culture media after 48 h of incubation with (solid bar) or without (open bar) 5 lg/ml achacin in the presence of 100 U/ml catalase were measured. The consumption ratio is reported. Cys2 , Cystine.
Discussion Achacin was initially identified in the body surface mucus of the giant African snail and is thought to contribute to the innate immunity of the snail [4,20]. In higher vertebrates, LAOs, such as fish LAO [14,23], interleukin 4 (IL-4)-induced gene [15], and mouse milk LAO [24], have been reported to act as host defense factors. However, the mechanism by which LAOs do so has not been well documented. In the present study, we showed that achacin induces cell death of HeLa cells. Our present results provide evidence that achacin-induced cell death occurs through two different mechanisms. The first mechanism is rapid cell death due
directly to H2 O2 generated through the LAO activity of achacin. HeLa cells incubated with achacin show disturbed cell growth after 7.5 h of incubation (data not shown), and membrane blebbing is present in most cells after 24 h of incubation, similar to H2 O2 -treated cells. The second mechanism is slow cell death mediated by caspases, and this form is induced by depletion of free amino acids. Cells incubated with achacin in the presence of an H2 O2 scavenger undergo slow cell death. Sufficient levels of catalase abolish H2 O2 -mediated rapid cell death; however, chromatin condensation, DNA fragmentation, and PARP cleavage are still observed in cells after 48 h of incubation. These apoptotic phenomena are suppressed by Z–VAD–fmk, a caspase inhibitor. Our present findings clearly show that achacin induces apoptosis of HeLa cells through the independent H2 O2 induced cell death pathway. Notably, apoptosis of HeLa cells was observed after 48 h of incubation with achacin in the presence of catalase, but it was not observed with less than 24 h of incubation. Moreover, we examined cell viability in pre-conditioned media (pre-incubated with achacin followed by elimination of achacin) and found that apoptosis is induced in pre-conditioned media even in the absence of achacin. Therefore, the slow apoptotic cell death appears to exclude direct effects of achacin, such as binding to the cell membrane. Apoptosis-inducing protein (AIP), which is an LAO isolated from Anisakis-infected mackerels, is known to induce apoptosis of H2 O2 -insensitive cells. It was recently reported that depletion of L -Lys in culture medium through the deamination activity of AIP results in H2 O2 -independent apoptosis [14]. Therefore, we quantified amino acid concentrations in cell culture media. Achacin hydrolyzes
Fig. 7. Depletion of L -amino acids causes apoptotic cell death. (A) DNA fragmentation was examined in cells cultured for 48 h in MEM (control), in MEM lacking free amino acids (-free), or MEM containing selected amino acids (selected) as described in the Materials and methods. M, 200-bp ladder marker. (B) Chromatin condensation was observed in cells cultured in MEM (control; a and d), in amino acid-free medium (-free; b and e), and in selected amino acid medium (selected; c and f). (a–c) Fluorescent images of floating cells. (d–f) Phase contrast images of attached cells. Scale bars indicated under (a–c) and (d–f) are 20 and 80 lm, respectively.
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a variety of amino acids with the highest specificity for Met and Leu when each amino acid is used as substrate. However, depletion of Arg and Trp and reductions in levels of other essential amino acids were observed in culture media that contained higher concentrations of Arg (546 nmol/ml) than Leu (372.4 nmol/ml). Arg is an essential factor for cell proliferation, and restriction of Arg is thought to induce cell death of cultured HeLa cells and fibroblasts [25]. These findings suggest that depletion of essential amino acids is the cause of LAOassociated apoptosis. In the present study, we found that both H2 O2 -dependent and -independent cell deaths were induced by achacin. Achacin-induced apoptosis, which is accompanied by activation of caspases, occurs only in the presence of catalase, which is different from AIP-induced cell death. AIP activates two caspase cascades through different molecular mechanisms that are dependent on the presence or absence of an H2 O2 scavenger [14]. In both cases, cells incubated with AIP undergo apoptosis. Whether apoptosis is induced directly by H2 O2 is still controversial. Depending on the concentration of H2 O2 employed and the type of cell studied, the mode of cell death induced by H2 O2 has been reported to be apoptotic or necrotic [12,13,26], with necrotic cell death generally being reported with higher concentrations of oxidant [18]. We measured levels of H2 O2 in the media of cells cultured with achacin. The concentration of H2 O2 (approximately 300 lM) used in the present study is not sufficient to induce necrosis, and necrotic features were not observed in cells incubated with achacin in the absence of catalase. Therefore, we believe that the coordinated effects of H2 O2 and amino acid depletion cause caspase-independent cell death in the absence of an H2 O2 scavenger. Programmed cell death plays important roles in innate immunity and in cellular process necessary for embryogenesis, organ metamorphosis, and tissue homeostasis [27,28]. LAOs are widely distributed in living organisms and in several tissues [4,6,15,23,24]; however, the roles of LAOs in physiological systems are not fully understood. Our results suggest that depletion of free amino acids through the deamination activity of LAO may play an important role in several situations, where the concentrations of free amino acids used in experimental scenarios would not be expected.
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Achacin induces cell death in HeLa cells through two different mechanisms Nobuyuki Kanzawa,a,* Satoko Shintani,a Kazumasa Ohta,b Seiji Kitajima,a Tatsuya Ehara,a Hiroko Kobayashi,a Harutoshi Kizaki,b and Takahide Tsuchiyaa a b
Department of Chemistry, Faculty of Science and Technology, Sophia University, Tokyo 102-8554, Japan Department of Biochemistry, Tokyo Dental College, 1-2-2 Masago, Mihama-ku, Chiba 261-8502, Japan Received 14 October 2003, and in revised form 2 December 2003
Abstract Achacin, which belongs to the L -amino acid oxidase group, oxidizes free amino acids and produces hydrogen peroxide in cell culture systems. Morphological changes in cells incubated with achacin were similar to those of cells incubated with H2 O2 . In both cases, the end result was cell death. To examine the mechanism of achacin-associated cytotoxicity, the H2 O2 scavenger catalase was added to culture media. Features typical of apoptosis, including morphological changes, DNA fragmentation, and PARP cleavage, were observed when cells were incubated with achacin in the presence of catalase. Moreover, apoptosis was inhibited by Z–VAD– fmk, a broad-spectrum caspase inhibitor. Herein, we present evidence that two pathways are involved in achacin-induced cell death. One is direct generation of H2 O2 through the L -amino acid oxidase activity of achacin. The other is the caspase-mediated apoptotic pathway that is induced by depletion of L -amino acids by achacin. Ó 2003 Elsevier Inc. All rights reserved. Keywords: Achacin; Apoptosis; L -amino acid oxidase; Hydrogen peroxide
Achacin was originally isolated as an antibacterial glycoprotein from the body surface mucus of the giant African snail Achatina fulica Ferussac [1,2]. Sequence and biochemical data revealed that achacin is a member of the amine oxidase family, of flavin enzymes [3], and we recently reported that achacin has L -amino acid oxidase (LAO, EC 1.4.3.2) activity [4]. LAO is a member of the amine oxidase family and catalyzes oxidative deamination of L -amino acids to a-keto acids, hydrogen peroxide (H2 O2 ), and ammonia (NH3 ). LAOs are present in various organisms and are thought to induce cell death under physiological conditions through generation of hydrogen peroxide [5,6]. Dolabellanin, which has sequence similarity with achacin, is known to induce cell death in cancer cells [7–9]; however, the mode of cytotoxicity is not well documented. These facts led us to investigate how achacin induces cell death in cell
culture systems, and how the mechanism of the cytotoxicity may contribute to the host defense mechanism of achacin. Reactive oxygen species (ROS)1 are produced under a variety of physiological conditions, including acute and chronic inflammation [10], and are known to cause apoptosis [11–13]. LAOs are present in fish [14] and several snake venoms [15–17] and have been reported to induce apoptosis [5,6]; however, it is unclear if this apoptosis is due directly to the effect of H2 O2 [18]. In the present study, we measured the LAO activity of achacin in a cell culture system. Achacin generated H2 O2 from free amino acids present in the culture media and induced cell death in HeLa cells; however, the mechanism of the cell death was not due solely to H2 O2 generated through the LAO activity of achacin. Herein, we show that the LAO activity of achacin depletes amino acids from culture media and that the 1
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Corresponding author. Fax: +011-81-3-3238-3361. E-mail address: [email protected] (N. Kanzawa). 0003-9861/$ - see front matter Ó 2003 Elsevier Inc. All rights reserved. doi:10.1016/j.abb.2003.12.007
Abbreviations used: PARP, poly (ADP-ribose) polymerase; ROS, reactive oxygen species; Z–VAD–fmk, carbobenzoyl–valyl–alanyl– aspartyl–fluoromethyl ketone.
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reductions of specific amino acids are associated with H2 O2 -independent cell death.
Materials and methods Materials Carbobenzoyl–valyl–alanyl–aspartyl–fluoromethylketone (Z–VAD–fmk) was purchased from the Peptide Institute, (Osaka, Japan) and dissolved in dimethyl sulfoxide (DMSO). Catalase and all other chemicals used in this study were purchased from Wako Pure Chemicals (Tokyo, Japan), Nacalai Tesque (Kyoto, Japan), or Sigma (St. Louis, MO). Cell culture HeLa cells were provided by the Cell Resource Center for Biomedical Research (Tohoku University, Miyagi, Japan) and maintained in Minimal Essential Medium (MEM) (Gibco BRL, Gaithersburg, MD) supplemented with 10% fetal bovine serum (FBS) at 37 °C in a humidified atmosphere containing 5% CO2 . Protein preparation Achacin was prepared as described previously [19,20]. Briefly, mucus from snails (Achatina fulica Ferussac) was mixed with an equal volume of 40 mM potassium phosphate (pH 7.0) by gently stirring for 24 h. After fractionation by 50–90% (w/v) ammonium sulfate saturation, the precipitate was dissolved in 50 mM Tris–HCl (pH 8.0) and applied to a TSK-gel DEAE-Toyopearl 650 M column (Tosoh, Tokyo, Japan). Antibacterial activity of each fraction was assessed as described previously [4]. Eluates containing antibacterial activity were pooled and purified further with a TSK-gel G3000SW column (Tosoh). Purified fractions containing antibacterial activity were used in this study. Measurement of oxidase activity Xylenol orange assay for LAO was performed with an established method [21]. Levels of activity were determined by measuring absorbance of Fe3þ (from oxidation of Fe2þ by H2 O2 ) –xylenol orange complex at 550 nm. LAO activity of achacin for each amino acid (15 mM, all purchased from Kyowa Hakko Kogyo, Tokyo, Japan) was measured after 10 min of incubation at 37 °C. Test aliquots of the culture medium were removed and analyzed after the appropriate incubation times with achacin (5 lg/ml) at 37 °C. One unit of enzyme activity was defined as the oxidation of 1 lmol of amino acid per minute.
Cell viability and microscopic analysis Cell viability was assessed by trypan blue exclusion. Morphological examination was done by phase-contrast microscopy (IX70; Olympus, Tokyo, Japan). For fluorescence microscopy, detached cells were collected by centrifugation at 3000g for 10 min, fixed with 3.4% formaldehyde in phosphate-buffered saline (PBS) for 15 min, and then stained with 5 lg/ml Hoechst 33342 for 20 min. Cells were then observed with a fluorescence microscope (Axiophoto-2; Carl Zeiss, Oberkochen, Germany). Detection of nucleosomal DNA fragmentation Total DNA was extracted according to the method of Lee and Shacter [26]. Briefly, approximately 7 105 HeLa cells were cultured in 6-well plates overnight and then incubated for 48 h under the conditions described below. Cells were harvested, washed with PBS, and lysed in buffer containing 10 mM Tris–HCl (pH 7.8), 10 mM EDTA, 0.5% Triton X-100, 200 lg/ml RNase, and 200 lg/ml proteinase K. After DNA was precipitated with isopropanol, it was then resuspended in a Tris–EDTA solution. Samples were resolved by electrophoresis on 2% agarose gels and visualized by ethidium bromide staining and UV transillumination. Western blot analysis HeLa cells were sonicated in cell lysis buffer [1% Triton X-100, 10 mM Tris–HCl (pH 7.5), 150 mM NaCl, 2 mM EGTA, 50 mM of 2-mercaptoethanol, 2 mM Na2 VO4 , 10 mM NaF, 1 mM DTT, and protease inhibitor cocktail (Complete; Roche Diagnostics, Tokyo, Japan)] and lysates were resuspended in SDS sample buffer [2.3% SDS, 5% of 2-mercaptoethanol, 63 mM Tris–HCl (pH 6.7), and 5% glycerol]. Samples were separated on 10% SDS– PAGE gels [22], transferred to Immobilon membranes (Millipore, Bedford, MA), incubated in rabbit antiPARP antibody (1:1000, Santa Cruz Biotechnology, Santa Cruz, CA), followed by HRP-conjugated secondary antibody (1:5000, DAKO, Kyoto, Japan), and visualized with an enhanced chemiluminescence (ECL) kit (Amersham–Pharmacia Biotech, Little Chalfont, UK). Quantification of amino acid composition HeLa cells were cultured in MEM containing 10% FBS with achacin for 48 h. Cells were removed by centrifugation. The concentrations of amino acids in culture media before (control) and after cells were cultured with achacin were determined with an automated amino acid analyzer (Mitsubishi Kagaku BCL, Tokyo, Japan). Amino acid concentrations were reported as relative to control.
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Amino acid depletion culture
Characterization of achacin-induced cell death
HeLa cells were cultured for 48 h in the absence of free amino acids or in the presence of 29.2 g/ml L -Gln, 5.2 g/ml L -Ile, 4.8 g/ml L -Thr, 4.6 g/ml L -Val, 3.1 g/ml L cystine, 5.2 g/ml L -Tyr, and 4.2 g/ml L -His. The modified culture medium lacks Arg, Leu, Lys, Met, Phe, and Trp, which are preferred substrates of the LAO activity of achacin.
ROS are known to cause apoptosis [11–13] and our data suggested that achacin induces cell death. To study the cytotoxicity of achacin, we examined the dose-dependent effect of achacin on cell viability in the presence or absence of catalase. HeLa cells (4 104 /well, 24-well plate) incubated with 1.5 lg/ml achacin underwent cell death after 24 h of incubation (Fig. 1B) similar to cells incubated with 300 lM H2 O2 . In both cases, membrane blebbing and shrinking of the cytoplasm were observed (Fig. 2, panels B–D). Addition of 100 U/ml catalase effectively abolished the effect of H2 O2 , resulting in a level of viability similar to that of controls (Fig. 1B). However, catalase could not suppress the cytotoxicity of achacin completely. We confirmed this by observing morphological changes in cells incubated with achacin. Cells incubated with H2 O2 together with catalase were extended and no significant changes in cell shape were observed. Cells incubated with achacin displayed a fibrous shape and some had membrane blebbing. The onset of morphological changes was detected at approximately 24 h of incubation; however, chromatin
Results LAO activity of achacin To characterize the enzymatic activity of achacin, we quantified the amount of H2 O2 produced by achacin from free amino acids as substrates (Table 1). Achacin utilizes only L -type amino acids to produce H2 O2 [4]. In the present study, achacin preferentially oxidized basic and some neutral amino acids (Table 1). We further analyzed the LAO activity of achacin in cell culture media. Levels of H2 O2 peaked 1 h after the addition of achacin to the culture medium, and H2 O2 levels then decreased with increased incubation time (Fig. 1A). Maximal production of H2 O2 was approximately 300 lM for 5 lg/ml achacin after 1 h of incubation. When catalase (100 U/ml) was added to MEM and incubated with achacin, H2 O2 levels were below the limit of detection (data not shown). Table 1 Oxidation activity of achacin Amino Activitya acid Unit (lmol/min)
Unit/mg
Lys Arg His
3
2.69 10 0.22 10 2.39 103 0.10 103 0.21 103 0.07 103
0.82 0.067 0.72 0.030 0.06 0.041
Neutral Met amino acids Leu Trp Phe Cys Asn Tyr Ala Gln Gly Ile Pro Ser Thr Val
4.67 103 0.01 103 4.38 103 0.17 103 3.58 103 0.46 103 2.16 103 0.40 103 1.10 103 0.02 103 0.99 103 0.30 103 0.84 103 0.70 103 0.36 103 0.02 103 ())b ()) ()) ()) ()) ()) ())
1.42 0.005 1.33 0.052 1.08 0.014 0.65 0.011 0.33 0.006 0.30 0.001 0.25 0.020 0.11 0.007 ()) ()) ()) ()) ()) ()) ())
Acidic amino acids
()) ())
()) ())
Basic amino acids
a
Asp Glu
3
Oxidation activity of achacin (3.3 lg/ml) was determined with amino acids (15 mM) as substrate. ()) indicates that the level of activity is below the detection limit.
L -type b
Fig. 1. Effects of achacin on cell viability. Time-dependent production of H2 O2 by achacin (5 lg/ml) detected in cell culture media (A). Mean values of three experiments are shown. Error bars are the standard deviation (SD) of the mean. (B) Cell viability was examined in HeLa cells incubated with various concentrations of achacin with or without 100 U/ml catalase. HeLa cells (4 104 ) were seeded in 24-well plates, cultured overnight, and then incubated for 24 h in MEM under the indicated conditions. Cells were counted, and the mean percent viability of control from three independent experiments is presented.
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Fig. 2. Effect of achacin on cell morphology. Morphological and chromosomal changes were examined in HeLa cells incubated with achacin with or without catalase. Pre-cultured HeLa cells were incubated for either 24 h (24 h; B, F, and J) or 48 h in MEM (48 h; A, C, D, E, G, H, I, K, and L) with 5 lg/ml achacin (+achacin; B, C, F, G, J, and K), without achacin (control; A, E, and I) or 300 lM H2 O2 (+H2 O2 ; D, H, and I) in the absence (A–D) or presence (E–L) of 100 U/ml catalase (catalase). Phase contrast images of attached cells (A–H) and fluorescent images of floating cells (I–L) were analyzed as described in the Materials and methods. Bars in (H) and (L) are 80 and 20 lm, respectively.
condensation became obvious at approximately 48 h of incubation (Fig. 2, panels J and K). These observations indicate that achacin induces apoptosis, that is independent of H2 O2 -induced cell death. Achacin-induced apoptosis is mediated by the caspase pathway To clarify the involvement of caspases in achacininduced apoptosis, we examined the effects of Z–VAD– fmk on DNA fragmentation and PARP cleavage, which are the hallmark features of apoptosis. DNA fragmentation in cells incubated with achacin and catalase was suppressed by Z–VAD–fmk, similar to that in staurosporine-treated cells; this was not prevented in the absence of catalase (Fig. 3A). Z–VAD–fmk also inhibited PARP cleavage in cells incubated with achacin alone in the presence of catalase (Fig. 3B). We then analyzed the effect of Z–VAD–fmk on achacin-induced cell death. Total numbers of attached and floating cells were counted (Fig. 4A). In the absence of catalase, living cells undergo cell death. However, achacin appears to suppress cell proliferation because total cell number did not increase regardless of the presence of Z–VAD–fmk. The possible role of caspase in achacininduced apoptosis was analyzed by comparing the numbers of detached floating cells in the presence and absence of Z–VAD–fmk (Fig. 4B). Numbers of floating dead cells were decreased considerably in the presence of catalase, and a marked effect on the number of floating cells in response to Z–VAD–fmk was also observed (P < 0:01; StudentÕs t test).
Depletion of free amino acids induces apoptosis To clarify the mechanism of cytotoxicity, HeLa cells were cultured for 48 h in pre-conditioned media that had been incubated for 48 h with achacin and filtered with Centricon YM-10 (<10 kDa, Millipore) to eliminate protein factors, and a 1/10 volume of FBS was added to the media. Approximately 30% of cells relative to control were viable when cells were cultured with achacin (Fig. 5, bars on a left half). Interestingly, the proportion of surviving cells cultured in the pre-conditioned media was approximately 50% that of the control even in the absence of achacin (Fig. 5, bars on a right half). This result suggested that an induction factor of achacin-induced cell death in the presence of catalase is in culture media, but not via protein factor. Catalase should metabolize H2 O2 generated during culture of cells with achacin; however, catalase does not affect the enzyme activity of achacin. Therefore, deamination of free amino acids in culture media may occur during incubation of cells with achacin. Murakawa et al. [14] reported that LAO induces apoptosis of HL-60 cells and this is associated with depletion of L -Lys from the culture medium. We analyzed amino acid concentrations in culture media after 48 h of incubation with achacin in the presence of catalase (Fig. 6). In most cases, the level of amino acid consumption was lower in medium with achacin than in medium without achacin. This is likely due to growth arrest of achacin-treated cells. However, Arg, Trp, Lys, and Tyr showed the highest levels of depletion in media from achacin-incubated cells, suggesting that achacin-induced apoptosis is
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Fig. 4. Effect of Z–VAD–fmk on cell death. HeLa cells (7 105 /6-cm dish) were pre-cultured overnight and then incubated for 48 h in MEM alone (control) or with 5 lg/ml achacin (achacin), 5 lg/ml achacin, and 100 U/ml catalase (achacin + catalase), or 50 nM staurosporine (staurosporine) with (+) or without ()) 50 lM Z–VAD–fmk. (A) Numbers of cells attached to the dish surface (solid bar) or detached and floating (open bar) were counted, and the mean (n ¼ 3) is presented. (B) Effect of Z–VAD–fmk is depicted as the relative ratio of floating to attached cells. *P < 0:01, paired StudentÕs t test. Fig. 3. Effect of Z–VAD–fmk on achacin-induced cell death. (A) HeLa cells (7 105 /6-cm dish) were pre-cultured overnight in MEM and further incubated for 48 h in the presence of 5 lg/ml achacin (achacin), 5 lg/ml achacin, and 100 U/ml catalase (achacin + catalase), or 50 nM staurosporine (staurosporine) with (+) or without ()) 50 lM Z–VAD– fmk. Nucleosomal DNA was isolated and separated by electrophoresis on 2% agarose gels. M, 200-bp ladder. (B) Pre-cultured HeLa cells (3 105 /well of 6-well plate) were incubated in MEM (control) or in the presence of 5 lg/ml achacin (achacin), 5 lg/ml achacin, and 100 U/ ml catalase (achacin + catalase), and 50 nM staurosporine (staurosporine) with (+) or without ()) 50 lM Z–VAD–fmk. Total protein extracts were prepared from each sample and separated by electrophoresis on 10% acrylamide gels. Gels were stained with Coomassie brilliant blue (lower panel) or transferred to Immobilon membranes followed by immunodetection (upper panel) with anti-PARP antibody. Arrowhead and double arrowhead indicate PARP protein and its degradation product, respectively. M, protein molecular mass marker. Ordinate, molecular mass in kDa.
mediated by depletion of free amino acids from the culture medium. To confirm this finding, we next analyzed the effect of amino acid starvation on apoptosis. Cells were cultured in the absence of free amino acids or in the presence of restricted amino acids. Lack of Arg, Leu, Lys, Met, Phe, and Trp, which are suitable substrates for the LAO activity of achacin, induced DNA fragmentation in HeLa cells. A higher level of fragmentation was observed in medium with restricted amino acids than in medium lacking free amino acids (Fig. 7A). In addition, fibrous changes in cell shape and chromatin condensation were observed in cells cultured in both modified MEMs (Fig. 7B), and this is similar to changes observed in cells
Fig. 5. Cell viability tests in pre-conditioned media. HeLa cells (7 105 /6-cm dish) were pre-cultured for 48 h in the presence of catalase (100 U/ml) with (+) or without ()) achacin (5 lg/ml). Thereafter, the culture medium was filtered over Centricon CY-10 (<10 kDa), 1/10 volume of FBS was added, and the medium was transferred to new wells. HeLa cells (7 105 /6-cm dish) were seeded in the wells and incubated for 48 h. The average number of surviving cells from three separated experiments was compared to that of controls, which were cells cultured for 48 h with (+) or without ()) achacin (5 lg/ml) in the same conditioned media without the pre-incubation. (n), Culture medium was filtered without the pre-culturing with achacin.
incubated with achacin and catalase. These findings indicate that depletion of amino acids from culture medium induces apoptosis of HeLa cells.
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Fig. 6. Amino acid concentrations after incubation with achacin and catalase. The concentrations of amino acids in culture media after 48 h of incubation with (solid bar) or without (open bar) 5 lg/ml achacin in the presence of 100 U/ml catalase were measured. The consumption ratio is reported. Cys2 , Cystine.
Discussion Achacin was initially identified in the body surface mucus of the giant African snail and is thought to contribute to the innate immunity of the snail [4,20]. In higher vertebrates, LAOs, such as fish LAO [14,23], interleukin 4 (IL-4)-induced gene [15], and mouse milk LAO [24], have been reported to act as host defense factors. However, the mechanism by which LAOs do so has not been well documented. In the present study, we showed that achacin induces cell death of HeLa cells. Our present results provide evidence that achacin-induced cell death occurs through two different mechanisms. The first mechanism is rapid cell death due
directly to H2 O2 generated through the LAO activity of achacin. HeLa cells incubated with achacin show disturbed cell growth after 7.5 h of incubation (data not shown), and membrane blebbing is present in most cells after 24 h of incubation, similar to H2 O2 -treated cells. The second mechanism is slow cell death mediated by caspases, and this form is induced by depletion of free amino acids. Cells incubated with achacin in the presence of an H2 O2 scavenger undergo slow cell death. Sufficient levels of catalase abolish H2 O2 -mediated rapid cell death; however, chromatin condensation, DNA fragmentation, and PARP cleavage are still observed in cells after 48 h of incubation. These apoptotic phenomena are suppressed by Z–VAD–fmk, a caspase inhibitor. Our present findings clearly show that achacin induces apoptosis of HeLa cells through the independent H2 O2 induced cell death pathway. Notably, apoptosis of HeLa cells was observed after 48 h of incubation with achacin in the presence of catalase, but it was not observed with less than 24 h of incubation. Moreover, we examined cell viability in pre-conditioned media (pre-incubated with achacin followed by elimination of achacin) and found that apoptosis is induced in pre-conditioned media even in the absence of achacin. Therefore, the slow apoptotic cell death appears to exclude direct effects of achacin, such as binding to the cell membrane. Apoptosis-inducing protein (AIP), which is an LAO isolated from Anisakis-infected mackerels, is known to induce apoptosis of H2 O2 -insensitive cells. It was recently reported that depletion of L -Lys in culture medium through the deamination activity of AIP results in H2 O2 -independent apoptosis [14]. Therefore, we quantified amino acid concentrations in cell culture media. Achacin hydrolyzes
Fig. 7. Depletion of L -amino acids causes apoptotic cell death. (A) DNA fragmentation was examined in cells cultured for 48 h in MEM (control), in MEM lacking free amino acids (-free), or MEM containing selected amino acids (selected) as described in the Materials and methods. M, 200-bp ladder marker. (B) Chromatin condensation was observed in cells cultured in MEM (control; a and d), in amino acid-free medium (-free; b and e), and in selected amino acid medium (selected; c and f). (a–c) Fluorescent images of floating cells. (d–f) Phase contrast images of attached cells. Scale bars indicated under (a–c) and (d–f) are 20 and 80 lm, respectively.
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a variety of amino acids with the highest specificity for Met and Leu when each amino acid is used as substrate. However, depletion of Arg and Trp and reductions in levels of other essential amino acids were observed in culture media that contained higher concentrations of Arg (546 nmol/ml) than Leu (372.4 nmol/ml). Arg is an essential factor for cell proliferation, and restriction of Arg is thought to induce cell death of cultured HeLa cells and fibroblasts [25]. These findings suggest that depletion of essential amino acids is the cause of LAOassociated apoptosis. In the present study, we found that both H2 O2 -dependent and -independent cell deaths were induced by achacin. Achacin-induced apoptosis, which is accompanied by activation of caspases, occurs only in the presence of catalase, which is different from AIP-induced cell death. AIP activates two caspase cascades through different molecular mechanisms that are dependent on the presence or absence of an H2 O2 scavenger [14]. In both cases, cells incubated with AIP undergo apoptosis. Whether apoptosis is induced directly by H2 O2 is still controversial. Depending on the concentration of H2 O2 employed and the type of cell studied, the mode of cell death induced by H2 O2 has been reported to be apoptotic or necrotic [12,13,26], with necrotic cell death generally being reported with higher concentrations of oxidant [18]. We measured levels of H2 O2 in the media of cells cultured with achacin. The concentration of H2 O2 (approximately 300 lM) used in the present study is not sufficient to induce necrosis, and necrotic features were not observed in cells incubated with achacin in the absence of catalase. Therefore, we believe that the coordinated effects of H2 O2 and amino acid depletion cause caspase-independent cell death in the absence of an H2 O2 scavenger. Programmed cell death plays important roles in innate immunity and in cellular process necessary for embryogenesis, organ metamorphosis, and tissue homeostasis [27,28]. LAOs are widely distributed in living organisms and in several tissues [4,6,15,23,24]; however, the roles of LAOs in physiological systems are not fully understood. Our results suggest that depletion of free amino acids through the deamination activity of LAO may play an important role in several situations, where the concentrations of free amino acids used in experimental scenarios would not be expected.
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