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Recombinant arginine deiminase as a potential anti-angiogenic agent
Cancer Letters 183 (2002) 155–162 www.elsevier.com/locate/canlet
Recombinant arginine deiminase as a potential anti-angiogenic agent Karin Beloussow a, Li Wang a, Jun Wu a, David Ann b, Wei-Chiang Shen a,* a
Department of Pharmaceutical Sciences, University of Southern California School of Pharmacy, PSC 404B, 1985 Zonal Avenue, Los Angeles, CA 90089-9121, USA b Department of Molecular Pharmacology and Toxicology, University of Southern California School of Pharmacy, Los Angeles, CA 90089-9121, USA Received 17 May 2001; received in revised form 5 October 2001; accepted 9 October 2001
Abstract Arginine deiminase (ADI), isolated from Mycoplasma cell extracts, has been suggested to inhibit endothelial cell growth in vitro. However, anti-angiogenic activity by ADI has not yet been demonstrated. In this study, we investigated the in vitro effect of recombinant ADI (rADI) on the growth, migration, and tube formation of human umbilical vein endothelial (HUVE) cells. Mycoplasma arginine deiminase was cloned by PCR and the rADI was expressed in Escherichia coli. and purified to near homogeneity. The purified recombinant protein was found to have characteristics similar to those of the native enzyme: molecular weight (48 kDa) and specific enzymatic activity of converting l-arginine into citrulline (32.7 U/mg). This recombinant enzyme also exhibited an inhibitory effect on the growth of HUVE cells. The anti-angiogenic activity was demonstrated by in vitro inhibition of migration into the scratch wounded area in HUVE cell monolayers and the inhibition of microvessel tubelike formation of HUVE cells on Matrigel-coated surfaces. These results suggest that arginine deiminase is a potential inhibitor for angiogenesis, and that arginine concentrations may play an important role in regulating neovascularization. q 2002 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Arginine deiminase; Recombinant; Anti-angiogenesis; Endothelial cells
1. Introduction Arginine deiminase (ADI) is a mycoplasma enzyme that catalyzes the imine hydrolysis of arginine to produce citrulline and ammonia. ADI has previously been suggested as an inhibitor of cell proliferation [1,2]. Furthermore, in vivo anti-tumor activity of this enzyme has also been reported [3]. It was postulated that the anti-tumor action of this enzyme is mediated by a direct inhibition of tumor * Corresponding author. Tel.: 11-323-442-1902; fax: 11-323442-1390. E-mail address: [email protected] (W.-C. Shen).
cell growth via the reduction of the polyaminebiosynthesis in tumor cells [2]. However, the putative anti-tumor mechanism of ADI as a direct inhibitor of tumor cell growth has not yet been verified. Since ADI appeared to be highly inhibitory to the growth of cultured endothelial cells, it has been suggested recently that the anti-tumor activity is due to the inhibition of tumor angiogenesis [4]. Angiogenesis is a complex sequence of events leading to the formation or sprouting of capillaries from pre-existing vessels [5]. Angiogenesis occurs continuously during embryogenesis and fetal development, but is restricted in adults except during ovulation,
0304-3835/02/$ - see front matter q 2002 Elsevier Science Ireland Ltd. All rights reserved. PII: S 0304-383 5(01)00793-5
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wound healing, placenta formation, and menstruation [6]. Thus, capillary endothelial cells within most adult tissues are considered to be quiescent under normal physiological conditions. However, the growth of new capillaries can occur under many pathological conditions, including the growth of solid tumors [7] and the development of many other diseases [8]. Therefore, inhibitors of angiogenesis have long been considered as potential therapeutic agents for the treatment of various angiogenesis-related diseases in humans. Recently, promising results have been obtained from the development of anti-angiogenic agents as novel drugs in the emerging anticancer strategies targeting the neovascularization in solid tumors [9,10]. We reported previously that a 48-kDa protein with anti-angiogenic activity was purified from the culture medium of HL-60 human promyelocytic leukemic cells [11]. This protein was later identified as ADI, which possibly was a product from a mycoplasma contamination in the cell culture. In this report, mycoplasma ADI was successfully cloned and expressed in Escherichia coli and results from angiogenic studies using the recombinant ADI are presented. To our knowledge, this is the first time that it has been reported that ADI is inhibitory in experimental angionesis models. 2. Materials and methods Chemicals and reagents were obtained from Sigma Chemical Company unless otherwise stated. Primers, restriction enzymes and Taq polymerase were purchased from Gibco-BRL. The TA cloning kit was obtained from Invitrogen. The site-mutagenesis kit was a product of the Clonetech company. Ionexchange and arginine-affinity column (Q Sepharose Fast Flow,Arginine Sepharosee 4B) were obtained from Amersham Pharmacia Biotech. 2.1. Expression of mycoplasma arginine deiminase in E. coli and its preparation The method previously described by Misawa et al. [12] was used with some modification. Briefly, a 1230 base pair fragment representing the entire arginine deiminase coding sequence from Mycoplasma arginini genomic cDNA (ATCC 23838D) was isolated by PCR. The PCR product was subsequently cloned
into the pCR 2.1 vector using a TA cloning kit. Sitedirected mutagenesis was performed in order to change the five mycoplasma tryptophan codons (TGA, which is also the STOP codon of the standard genetic code) in the ADI gene to the tryptophan codon (TGG) that would not cause termination of translation in E. coli. The modified ADI gene fragment was then introduced into the high level expression vector PTTQ118. All DNA sequences in the various steps were confirmed by DNA sequence analysis on both strands. The recombinant expression plasmid (PTTQ118ADI) was transformed into E. coliDH5a cells, and subsequently grown at 378C until the A600 reached 0.6–0.8. IPTG was added and incubation was continued for an additional 3.5 h. The rADI, expressed as inclusion bodies in the cytosol, was solubilized, renatured, and subsequently purified as previously described [12]. Briefly, the E. coli cells were disrupted by sonication, centrifuged, and washed with 10 mM potassium phosphate buffer (pH 7.0) containing 4% (w/v) Triton X-100 and 1 mM EDTA. The inclusion bodies, which remained in the final pellet, were then solubilized under reducing conditions and any remaining cell debris removed by centrifugation. Renaturation of the solubilized rADI was carried out by dilution in 175 fold by volume of 10 mM potassium phosphate buffer (pH 7.0) containing 1 mM EDTA and 0.02% NaN3 for 45 h at 258C. After filtration, the renaturation mixture was applied to Q Sepharose Fast Flow column and then subsequently to an Arginine-Sepharose column at room temperature. The active rADI was eluted from the ArginineSepharose column with a linear gradient (0.2–1.0 M NaCl) in the equilibration buffer. Active fractions identified by ADI enzymatic activity were pooled and concentrated, then analyzed for purity and molecular weight as determined by 10% SDS-polyacrylamide gel electrophoresis (PAGE). Gel electrophoresis under non-reducing conditions was also performed. The single-band non-denatured gel was further analyzed by slicing one of the sample lanes into six fractions, homogenizing each slice with a Dounce homogenizer in phosphate buffered saline (PBS) pH 7.4, then gel fragments were removed by centrifugation and the supernatants containing extracted proteins sterilized by filtration. These supernatant
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fractions were further tested for enzymatic activity and cell growth inhibition in HUVE cell culture. 2.2. 2.2 Arginine deiminase (ADI) enzyme activity The ADI activity was determined by the amount of l-citrulline (l-cit) produced from l-arg as described by the method of Oginsky [13], where one unit (U) of ADI activity was defined as the amount of enzyme that converted 1 mmol of l-arg to l-cit per min under assay conditions. 2.3. Cells and cell culture Human umbilical vein endothelial (HUVE) cells were obtained from Clonetics and maintained in Clonetics’ endothelial cell growth media system, EGM-2-MV BulletKit. Cell culture reagents: Trypsin/EDTA, Trypsin Neutralizing Solution, and HEPES Buffered Saline Solution (HBSS) were also purchased from Clonetics. Matrigel was purchased from Becton-Dickinson Labware. 2.4. Dose-dependent restoration of cell proliferation assay 2
HUVE cells were seeded (2500 cells/cm ) into 24well cluster plates in 1.0 ml of appropriate medium and supplements. An aliquot of rADI solution, corresponding to 0.3 £ 10 23, 1 £ 10 23, and 3 £ 10 23 Units, was added to each ml of medium in test wells. Cell monolayers were incubated at 378C, 95% air/5% CO2, in the presence or absence of the enzyme (control). On day 4 (time 0), the medium was removed and cell monolayers were rinsed with HBSS and fresh medium replaced, thereby removing rADI, and cultured for an additional 3 days. The restoration of proliferation of the endothelial cells was observed under an inverted light microscope at 40£ and photographed on day 0, 1 and 3 post medium replacement. 2.5. Migration assay HUVE cells were seeded (2500 cells/cm 2) into 24well cluster plates and grown to confluence. The ‘scratch wound’ in the confluent monolayers was made using a razor blade, then each well was rinsed with HBSS, and fresh medium replaced with rADI (1 £ 10 23 U/ml) or without (control) [14]. The plates were incubated at 378C, 5% CO2 in air for 48 h. The
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plates were then rinsed with cold PBS, fixed in 3.7% formaldehyde, quenched with 50 mM NH4Cl, and stained with 0.2% coomassie blue. The extent of migration into the empty area was observed under an inverted light microscope at 40£ and photographed. 2.6. In vitro endothelial cell microvessel formation Confluent HUVE cell monolayers were treated with rADI (1 £ 10 23 U/ml) in 12-well cell culture plates for 48 h before being harvested and plated onto Matrigelcoated 24-well cluster plates (4 £ 10 4 cells/well) using medium that had been pre-treated with rADI (1 £ 10 23 U /ml) or without (control) for 24 h at 378C. Microvessel formation was observed using an inverted light microscope at 40£. Photographs were taken at 2.5, 5.0, and 22.0 h after plating. 3. Results 3.1. Characterization of rADI The renaturation mixture containing r-ADI was submitted to purification by arginine-sephorose column chromatography at 258C. Active fractions, assayed by ADI enzymatic activity [13], were pooled together and concentrated. From 1 L of cultured E. coli, 1 mg of active rADI was obtained. The specific activity of the renatured rADI was 32.7 U/mg protein, similar to that of Mycoplasma ADI, 37 U/mg [1]. Proteins isolated from E. coli containing the expression vector alone had no detectable ADI activity (data not shown). The concentrated rADI was then analyzed by SDSPAGE (Fig. 1), yielding a single active band with a molecular mass of 48 kDa, similar to that of Mycoplasma ADI [12]. The concentrated rADI also displayed a single band in non-denatured PAGE; this single band (Slice 3) contained the majority of ADI enzymatic activity and accordingly displayed the greatest cell growth inhibition in HUVE cell culture (Fig. 1). 3.2. Dose-dependent restoration of proliferation in rADI-treated endothelial cells in culture The reversible inhibitory effect of rADI on the
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Fig. 1. (A) SDS-PAGE analysis of pooled fractions with ADI enzymatic activity eluted from an arginine-sepharose column. Molecular weight markers left lane, purified rADI sample (48 kDa) right lane. (B) The sample lane in the non-denatured gel (shown) was sliced into six fractions as indicated, each homogenized using a Dounce homogenizer, then gel fragments removed by centrifugation and supernatants containing extracted proteins sterilized by filtration. Each supernatant fraction was tested for enzymatic activity (line graph) and cell growth inhibition (column graph) in HUVE cell culture. Error bars on column graph represent standard deviation, n ¼ 3.
growth of cultured HUVE cells is presented in Fig. 2. An arrest of HUVE cell growth was achieved after a 3-day incubation with rADI-containing medium. However, once rADI was removed by washing and replacing with fresh rADI-free medium, the growth of the endothelial cells was restored at the lower doses. At day 1 post medium replacement, both 0.3 £ 10 23 and 1 £ 10 23 U/ml, displayed considerable growth whereas 3 £ 10 23 U/ml remained similar to Time 0. On day 3 without further medium replacement, cell growth at 0.3 £ 10 23 and 1 £ 10 23 U/ml was similar to control. Therefore, 1 £ 10 23 U/ml was chosen for subsequent studies. At 3 £ 10 23 U/ml, in addition to the non-significant restoration of cell growth, there was the appearance of larger multinucleated cells, which is similar to previously reported morphology changes in cells grown in arginine deficient medium [15]. 3.3. rADI inhibits the migration of endothelial cells in culture Endothelial cell motility is an important factor in
angiogenesis. Therefore, the effect of rADI on endothelial cell migration was examined. HUVE cell migration from the scraped edge into the denuded area of the confluent endothelial cell monolayer is shown in Fig. 3. Endothelial cells of the confluent monolayers treated with 1 £ 10 23 U rADI /ml, migrated much less than control into the empty space. 3.4. rADI inhibits the formation of microvascular structures in HUVE cells Endothelial differentiation was determined by the commonly used Matrigel assay [16]. As shown in Fig. 4, rADI-treated HUVE cells, unlike the control HUVE cells, were unable to complete the microvascular structure formation over the 24 h time period. The cells in untreated control wells differentiated in morphology, elongating to form networks of capillary-like tubes and loss of area covered by the endothelial cell monolayer, whereas the treated wells had decreased tube formation resulting in sporadic and incomplete networks. Cell number in triplicate wells, during and at the conclusion of the
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Fig. 2. Dose-dependent restoration of cell proliferation in rADI-treated endothelial cells.HUVE cells were seeded (2500 cells/cm 2) into 24-well cluster plates and treated with rADI (0.3 £ 10 23, 1 £ 10 23, and 3 £ 10 23 U/ml) for 3 days. On day 4 (time 0), cells were rinsed with HBSS and the medium replaced, thereby removing rADI, then cultured for an additional 3 days. The restoration of proliferation of the endothelial cells was observed under an inverted light microscope at 40£. Control cells (without rADI treatment) at Time 0 in comparison to rADI-treated cells at Time 0, day 1 and 3 post medium replacement are shown.
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Fig. 3. Effect of rADI (1 £ 10 23 U/ml) on endothelial migration. Confluent monolayers were scratched with a razor blade as described in the Section 2.5. The cells were then incubated for 48 h, fixed, stained and photographed using an inverted light microscope at 40£. Arrows indicate original wound edge.
of ADI can arrest the cell cycle in the G1 phase and only at high concentrations of the enzyme, i.e., .200 ng/ml, could apoptosis be induced [4]. Therefore in the case of ADI, the reduction in endothelial cell number seen in proliferation assays may be attributed, in part, to the induction of apoptosis due to modulation of the cell cycle by ADI, but it remains to be determined whether this effect contributes to the observed anti-tumor activity of ADI in vivo. In a recent investigation, it was shown that a cytostatic protein purified from ASC170 sertoli cell-conditioned medium had ADI enzymatic activity and was homologous to mycoplasma ADI [17]. This protein could actually inhibit taxolinduced apoptosis in DU145 cancer cells. It was
experiment, for rADI-treated wells remained consistent to that of control wells (seeding at 4 £ 10 4 cells per well, ending with 30,417 ^ 1488 and 28,743 ^ 3997 cells per well, control and rADI-treated, respectively). This indicates the lack of tube formation was not due to a difference in cell number.
4. Discussion In this report, purified recombinant ADI was used to investigate the effects of this enzyme on angiogenesis. Our results revealed that rADI, at a concentration that is non-cytotoxic, can inhibit migration (Fig. 3) as well as capillary vessel formation in cultured HUVE cells (Fig. 4). Our data in cultured endothelial HUVE cells also demonstrated that by removing this enzyme from the medium cell growth was restored, indicating that the cells were viable and capable of a proliferative response (Fig. 2). This would suggest that a reversible cytostatic effect, rather than cytotoxicity, is responsible for the cell growth inhibition, an observation that is consistent with what others have reported [4]. Together these observations, the inhibition of endothelial motility and the reversible inhibitory effect on endothelial cell growth, would indicate that ADI is more likely an anti-angiogenic rather than only an anti-proliferative agent. ADI has been shown to have an inhibitory effect on tumor cells in vitro [1,2]. This effect is thought to be cytotoxic, due to depletion of extracellular l-arg. However, in endothelial cells, the apparent cytostatic effect coincides with the report that low concentrations
Fig. 4. The effect of rADI on microvessel formation of HUVE cells grown on Matrigel-coated surfaces. HUVE cells were pretreated with 1 £ 10 23 U/ml of rADI for 48 h before being harvested and plated onto a Matrigel-coated 24-well cluster plate at 4 £ 10 4 cells/ well. The microvessel formation was observed using an inverted light microscope at 40£. Photographs comparing control (without rADI) to rADI pre-treated were taken at 2.5, 5.0, and 22.0 h after plating.
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suggested that the ADI-induced arginine depletion inhibited the required protein synthesis of apoptotic cell death, thereby resulting in protection of the cancer cells. It has also been reported that ADI is more potent than arginase in inhibiting leukemic cell growth in vitro [18]. The mechanism was speculated to be related to ADI’s ability to deplete l-arg thereby arresting the cell cycle and eventually leading to apoptotic cell death. This does not necessarily preclude possible anti-angiogenic properties of ADI. It has been speculated that angiogenesis not only has a critical role in the growth of solid tumors but also liquid tumors, such as leukemia [19]. The expansion of the endothelium, angiogenesis, and solid tumor growth are entwined by a balance of proliferative and apoptotic signals in the tumor microenvironment. If a similar model exists in the growth of liquid tumors it would then be highly desirable for an anti-cancer agent to have a combination of effects, i.e. direct toxicity and anti-angiogenic activity. Endothelial cell apoptosis has an important regulatory role in the process of angiogenesis. Angiogenic stimulatory factors have apoptotic protective effects in endothelial cells via various survival promoting pathways that block apoptosis execution and ultimately promoting endothelial cell survival. In contrast, it has also been shown that negative regulators of angiogenesis not only effect endothelial cell proliferation, migration, and remodeling, but also concomitantly stimulate endothelial apoptosis either directly or indirectly [20]. Excessive apoptosis could limit angiogenesis by counteracting endothelial cell proliferation and lead to tumor regression. However it is likely that angiogenesis is regulated by a balance between nitric oxide (NO) and polyamine synthesis pathways which could be determined by extracellular arginine levels. The regulation of arginine metabolism, such as the production of NO and polyamines, has recently been suggested to play an important role in tumor angiogenesis [21]. Interestingly, it has been implied that the healing of chronic gastric ulcerations in rats by intragastric administration of high doses of arginine is due to the promotion of angiogenesis [22]. On the other hand, the depletion of extracellular arginine for the reduction of angiogenesis has not yet been considered as a potential regulatory therapy for angiogenesisrelated diseases.
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The mechanism of the observed anti-angiogenic activity of ADI is still not clear at the present time. The protection of the cells by the substrate, arginine, indicates that the cell growth inhibition is not due to products of the enzymatic reaction, citrulline and ammonium, or products from other subsequent reactions [1]. Therefore, we assume that the anti-angiogenic activity is primarily due to the depletion of arginine from the culture medium. However, since complete reversal was not achieved in endothelial cells, perhaps alternate mechanisms are also involved, as has been suggested [4]. Whether the anti-tumor effect of ADI as reported in experimental animals is due to an inhibition of tumorassociated angiogenesis or an inhibition of tumor cell proliferation remains to be determined.
Acknowledgements This study was supported by a grant, 6IB-0045, from the California Breast Cancer Research Program.
References [1] H. Takaku, M. Matsumoto, S. Misawa, K. Miyazaki, Antitumor activity of arginine deiminase from mycoplasma arginini and its growth-inhibitory mechanism, Jap. J. Cancer Res. 86 (1995) 840–846. [2] K. Miyazaki, H. Takadu, M. Umeda, T. Fujita, W. Huang, T. Kimra, J. Yamashita, T. Horio, Potent growth inhibition of human tumor cells in culture by arginine deiminase purified from a culture medium of mycoplasma-infected cell line, Cancer Res. 50 (1990) 4522–4527. [3] H. Takaku, M. Takase, S. Abe, H. Hayashi, K. Miyazaki, In vivo anti-tumor activity of arginine deiminase purified from mycoplasma arginini, Int. J. Cancer 51 (1992) 244–249. [4] H. Gong, F. Zolzer, G. von Recklinghausen, J. Rossler, S. Breit, W. Havers, T. Fotsis, L. Schweigerer, Arginine deiminase inhibits cell proliferation by arresting cell cycle and inducing apoptosis, Biochem. Biophys. Res. Commun. 261 (1999) 10–14. [5] J. Folkman, Y. Shing, Angiogenesis, J. Biol. Chem. 267 (1992) 10931–10934. [6] W. Auerbach, R. Auerbach, Angiogenesis inhibition: a review, Pharmacol. Ther. 63 (1994) 265–311. [7] J. Folkman, Tumor angiogenesis: therapeutic implications, New England J. Med. 285 (1971) 1182–1186. [8] J. Folkman, Angiogenesis in cancer, vascular, rheumatoid and other disease, Nature Med. 1 (1) (1995) 27–31. [9] G. Gastl, T. Hermann, M. Steurer, J. Zmija, E. Gunsilius, C.
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Chun, B.G. Chun, B.H. Min, Cytoprotective effect of arginine deiminase on taxol-induced apoptosis in DU145 human prostate cancer cells, Molecules Cells 10 (3) (2000) 331–337. H. Gong, F. Zolzer, G. von Recklinghausen, W. Havers, L. Schweigerer, Arginine deiminase inhibits proliferation of human leukemia cells more potently than asparaginase by inducing cell cycle arrest and apoptosis, Leukemia 14 (2000) 826–829. G.J. Roboz, S. Dias, G. Lam, W.J. Lane, S.L. Soignet, R.P. Warrell Jr., S. Rafii, Arsenic trioxide induces dose- and timedependant apoptosis of endothelium and may exert an antileukemic effect via inhibition of angiogenesis, Blood 96 (4) (2000) 1525–1530. S. Dimmeler, A.M. Zeiher, Endothelial cell apoptosis in angiogenesis and vessel regression, Circulation Res. 87 (6) (2000) 434–439. M. Joshi, L. Fuller, G. Batchelor, l-arginine metabolites regulate DNA synthesis and nitric oxide synthase activity in cultured human dermal microvascular endothelial cells. Potential positive and negative regulators of angiogenesis derived from l-arginine, Free Radical Biol. Med. 22 (1997) 573–578. T. Brzozowski, S.J. Konturek, D. Drozdowicz, A. Dembiriski, J. Stachura, Healing of chronic gastric ulcerations by l-arginine, Digestion 56 (1995) 463–471.
Recombinant arginine deiminase as a potential anti-angiogenic agent Karin Beloussow a, Li Wang a, Jun Wu a, David Ann b, Wei-Chiang Shen a,* a
Department of Pharmaceutical Sciences, University of Southern California School of Pharmacy, PSC 404B, 1985 Zonal Avenue, Los Angeles, CA 90089-9121, USA b Department of Molecular Pharmacology and Toxicology, University of Southern California School of Pharmacy, Los Angeles, CA 90089-9121, USA Received 17 May 2001; received in revised form 5 October 2001; accepted 9 October 2001
Abstract Arginine deiminase (ADI), isolated from Mycoplasma cell extracts, has been suggested to inhibit endothelial cell growth in vitro. However, anti-angiogenic activity by ADI has not yet been demonstrated. In this study, we investigated the in vitro effect of recombinant ADI (rADI) on the growth, migration, and tube formation of human umbilical vein endothelial (HUVE) cells. Mycoplasma arginine deiminase was cloned by PCR and the rADI was expressed in Escherichia coli. and purified to near homogeneity. The purified recombinant protein was found to have characteristics similar to those of the native enzyme: molecular weight (48 kDa) and specific enzymatic activity of converting l-arginine into citrulline (32.7 U/mg). This recombinant enzyme also exhibited an inhibitory effect on the growth of HUVE cells. The anti-angiogenic activity was demonstrated by in vitro inhibition of migration into the scratch wounded area in HUVE cell monolayers and the inhibition of microvessel tubelike formation of HUVE cells on Matrigel-coated surfaces. These results suggest that arginine deiminase is a potential inhibitor for angiogenesis, and that arginine concentrations may play an important role in regulating neovascularization. q 2002 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Arginine deiminase; Recombinant; Anti-angiogenesis; Endothelial cells
1. Introduction Arginine deiminase (ADI) is a mycoplasma enzyme that catalyzes the imine hydrolysis of arginine to produce citrulline and ammonia. ADI has previously been suggested as an inhibitor of cell proliferation [1,2]. Furthermore, in vivo anti-tumor activity of this enzyme has also been reported [3]. It was postulated that the anti-tumor action of this enzyme is mediated by a direct inhibition of tumor * Corresponding author. Tel.: 11-323-442-1902; fax: 11-323442-1390. E-mail address: [email protected] (W.-C. Shen).
cell growth via the reduction of the polyaminebiosynthesis in tumor cells [2]. However, the putative anti-tumor mechanism of ADI as a direct inhibitor of tumor cell growth has not yet been verified. Since ADI appeared to be highly inhibitory to the growth of cultured endothelial cells, it has been suggested recently that the anti-tumor activity is due to the inhibition of tumor angiogenesis [4]. Angiogenesis is a complex sequence of events leading to the formation or sprouting of capillaries from pre-existing vessels [5]. Angiogenesis occurs continuously during embryogenesis and fetal development, but is restricted in adults except during ovulation,
0304-3835/02/$ - see front matter q 2002 Elsevier Science Ireland Ltd. All rights reserved. PII: S 0304-383 5(01)00793-5
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wound healing, placenta formation, and menstruation [6]. Thus, capillary endothelial cells within most adult tissues are considered to be quiescent under normal physiological conditions. However, the growth of new capillaries can occur under many pathological conditions, including the growth of solid tumors [7] and the development of many other diseases [8]. Therefore, inhibitors of angiogenesis have long been considered as potential therapeutic agents for the treatment of various angiogenesis-related diseases in humans. Recently, promising results have been obtained from the development of anti-angiogenic agents as novel drugs in the emerging anticancer strategies targeting the neovascularization in solid tumors [9,10]. We reported previously that a 48-kDa protein with anti-angiogenic activity was purified from the culture medium of HL-60 human promyelocytic leukemic cells [11]. This protein was later identified as ADI, which possibly was a product from a mycoplasma contamination in the cell culture. In this report, mycoplasma ADI was successfully cloned and expressed in Escherichia coli and results from angiogenic studies using the recombinant ADI are presented. To our knowledge, this is the first time that it has been reported that ADI is inhibitory in experimental angionesis models. 2. Materials and methods Chemicals and reagents were obtained from Sigma Chemical Company unless otherwise stated. Primers, restriction enzymes and Taq polymerase were purchased from Gibco-BRL. The TA cloning kit was obtained from Invitrogen. The site-mutagenesis kit was a product of the Clonetech company. Ionexchange and arginine-affinity column (Q Sepharose Fast Flow,Arginine Sepharosee 4B) were obtained from Amersham Pharmacia Biotech. 2.1. Expression of mycoplasma arginine deiminase in E. coli and its preparation The method previously described by Misawa et al. [12] was used with some modification. Briefly, a 1230 base pair fragment representing the entire arginine deiminase coding sequence from Mycoplasma arginini genomic cDNA (ATCC 23838D) was isolated by PCR. The PCR product was subsequently cloned
into the pCR 2.1 vector using a TA cloning kit. Sitedirected mutagenesis was performed in order to change the five mycoplasma tryptophan codons (TGA, which is also the STOP codon of the standard genetic code) in the ADI gene to the tryptophan codon (TGG) that would not cause termination of translation in E. coli. The modified ADI gene fragment was then introduced into the high level expression vector PTTQ118. All DNA sequences in the various steps were confirmed by DNA sequence analysis on both strands. The recombinant expression plasmid (PTTQ118ADI) was transformed into E. coliDH5a cells, and subsequently grown at 378C until the A600 reached 0.6–0.8. IPTG was added and incubation was continued for an additional 3.5 h. The rADI, expressed as inclusion bodies in the cytosol, was solubilized, renatured, and subsequently purified as previously described [12]. Briefly, the E. coli cells were disrupted by sonication, centrifuged, and washed with 10 mM potassium phosphate buffer (pH 7.0) containing 4% (w/v) Triton X-100 and 1 mM EDTA. The inclusion bodies, which remained in the final pellet, were then solubilized under reducing conditions and any remaining cell debris removed by centrifugation. Renaturation of the solubilized rADI was carried out by dilution in 175 fold by volume of 10 mM potassium phosphate buffer (pH 7.0) containing 1 mM EDTA and 0.02% NaN3 for 45 h at 258C. After filtration, the renaturation mixture was applied to Q Sepharose Fast Flow column and then subsequently to an Arginine-Sepharose column at room temperature. The active rADI was eluted from the ArginineSepharose column with a linear gradient (0.2–1.0 M NaCl) in the equilibration buffer. Active fractions identified by ADI enzymatic activity were pooled and concentrated, then analyzed for purity and molecular weight as determined by 10% SDS-polyacrylamide gel electrophoresis (PAGE). Gel electrophoresis under non-reducing conditions was also performed. The single-band non-denatured gel was further analyzed by slicing one of the sample lanes into six fractions, homogenizing each slice with a Dounce homogenizer in phosphate buffered saline (PBS) pH 7.4, then gel fragments were removed by centrifugation and the supernatants containing extracted proteins sterilized by filtration. These supernatant
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fractions were further tested for enzymatic activity and cell growth inhibition in HUVE cell culture. 2.2. 2.2 Arginine deiminase (ADI) enzyme activity The ADI activity was determined by the amount of l-citrulline (l-cit) produced from l-arg as described by the method of Oginsky [13], where one unit (U) of ADI activity was defined as the amount of enzyme that converted 1 mmol of l-arg to l-cit per min under assay conditions. 2.3. Cells and cell culture Human umbilical vein endothelial (HUVE) cells were obtained from Clonetics and maintained in Clonetics’ endothelial cell growth media system, EGM-2-MV BulletKit. Cell culture reagents: Trypsin/EDTA, Trypsin Neutralizing Solution, and HEPES Buffered Saline Solution (HBSS) were also purchased from Clonetics. Matrigel was purchased from Becton-Dickinson Labware. 2.4. Dose-dependent restoration of cell proliferation assay 2
HUVE cells were seeded (2500 cells/cm ) into 24well cluster plates in 1.0 ml of appropriate medium and supplements. An aliquot of rADI solution, corresponding to 0.3 £ 10 23, 1 £ 10 23, and 3 £ 10 23 Units, was added to each ml of medium in test wells. Cell monolayers were incubated at 378C, 95% air/5% CO2, in the presence or absence of the enzyme (control). On day 4 (time 0), the medium was removed and cell monolayers were rinsed with HBSS and fresh medium replaced, thereby removing rADI, and cultured for an additional 3 days. The restoration of proliferation of the endothelial cells was observed under an inverted light microscope at 40£ and photographed on day 0, 1 and 3 post medium replacement. 2.5. Migration assay HUVE cells were seeded (2500 cells/cm 2) into 24well cluster plates and grown to confluence. The ‘scratch wound’ in the confluent monolayers was made using a razor blade, then each well was rinsed with HBSS, and fresh medium replaced with rADI (1 £ 10 23 U/ml) or without (control) [14]. The plates were incubated at 378C, 5% CO2 in air for 48 h. The
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plates were then rinsed with cold PBS, fixed in 3.7% formaldehyde, quenched with 50 mM NH4Cl, and stained with 0.2% coomassie blue. The extent of migration into the empty area was observed under an inverted light microscope at 40£ and photographed. 2.6. In vitro endothelial cell microvessel formation Confluent HUVE cell monolayers were treated with rADI (1 £ 10 23 U/ml) in 12-well cell culture plates for 48 h before being harvested and plated onto Matrigelcoated 24-well cluster plates (4 £ 10 4 cells/well) using medium that had been pre-treated with rADI (1 £ 10 23 U /ml) or without (control) for 24 h at 378C. Microvessel formation was observed using an inverted light microscope at 40£. Photographs were taken at 2.5, 5.0, and 22.0 h after plating. 3. Results 3.1. Characterization of rADI The renaturation mixture containing r-ADI was submitted to purification by arginine-sephorose column chromatography at 258C. Active fractions, assayed by ADI enzymatic activity [13], were pooled together and concentrated. From 1 L of cultured E. coli, 1 mg of active rADI was obtained. The specific activity of the renatured rADI was 32.7 U/mg protein, similar to that of Mycoplasma ADI, 37 U/mg [1]. Proteins isolated from E. coli containing the expression vector alone had no detectable ADI activity (data not shown). The concentrated rADI was then analyzed by SDSPAGE (Fig. 1), yielding a single active band with a molecular mass of 48 kDa, similar to that of Mycoplasma ADI [12]. The concentrated rADI also displayed a single band in non-denatured PAGE; this single band (Slice 3) contained the majority of ADI enzymatic activity and accordingly displayed the greatest cell growth inhibition in HUVE cell culture (Fig. 1). 3.2. Dose-dependent restoration of proliferation in rADI-treated endothelial cells in culture The reversible inhibitory effect of rADI on the
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Fig. 1. (A) SDS-PAGE analysis of pooled fractions with ADI enzymatic activity eluted from an arginine-sepharose column. Molecular weight markers left lane, purified rADI sample (48 kDa) right lane. (B) The sample lane in the non-denatured gel (shown) was sliced into six fractions as indicated, each homogenized using a Dounce homogenizer, then gel fragments removed by centrifugation and supernatants containing extracted proteins sterilized by filtration. Each supernatant fraction was tested for enzymatic activity (line graph) and cell growth inhibition (column graph) in HUVE cell culture. Error bars on column graph represent standard deviation, n ¼ 3.
growth of cultured HUVE cells is presented in Fig. 2. An arrest of HUVE cell growth was achieved after a 3-day incubation with rADI-containing medium. However, once rADI was removed by washing and replacing with fresh rADI-free medium, the growth of the endothelial cells was restored at the lower doses. At day 1 post medium replacement, both 0.3 £ 10 23 and 1 £ 10 23 U/ml, displayed considerable growth whereas 3 £ 10 23 U/ml remained similar to Time 0. On day 3 without further medium replacement, cell growth at 0.3 £ 10 23 and 1 £ 10 23 U/ml was similar to control. Therefore, 1 £ 10 23 U/ml was chosen for subsequent studies. At 3 £ 10 23 U/ml, in addition to the non-significant restoration of cell growth, there was the appearance of larger multinucleated cells, which is similar to previously reported morphology changes in cells grown in arginine deficient medium [15]. 3.3. rADI inhibits the migration of endothelial cells in culture Endothelial cell motility is an important factor in
angiogenesis. Therefore, the effect of rADI on endothelial cell migration was examined. HUVE cell migration from the scraped edge into the denuded area of the confluent endothelial cell monolayer is shown in Fig. 3. Endothelial cells of the confluent monolayers treated with 1 £ 10 23 U rADI /ml, migrated much less than control into the empty space. 3.4. rADI inhibits the formation of microvascular structures in HUVE cells Endothelial differentiation was determined by the commonly used Matrigel assay [16]. As shown in Fig. 4, rADI-treated HUVE cells, unlike the control HUVE cells, were unable to complete the microvascular structure formation over the 24 h time period. The cells in untreated control wells differentiated in morphology, elongating to form networks of capillary-like tubes and loss of area covered by the endothelial cell monolayer, whereas the treated wells had decreased tube formation resulting in sporadic and incomplete networks. Cell number in triplicate wells, during and at the conclusion of the
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Fig. 2. Dose-dependent restoration of cell proliferation in rADI-treated endothelial cells.HUVE cells were seeded (2500 cells/cm 2) into 24-well cluster plates and treated with rADI (0.3 £ 10 23, 1 £ 10 23, and 3 £ 10 23 U/ml) for 3 days. On day 4 (time 0), cells were rinsed with HBSS and the medium replaced, thereby removing rADI, then cultured for an additional 3 days. The restoration of proliferation of the endothelial cells was observed under an inverted light microscope at 40£. Control cells (without rADI treatment) at Time 0 in comparison to rADI-treated cells at Time 0, day 1 and 3 post medium replacement are shown.
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Fig. 3. Effect of rADI (1 £ 10 23 U/ml) on endothelial migration. Confluent monolayers were scratched with a razor blade as described in the Section 2.5. The cells were then incubated for 48 h, fixed, stained and photographed using an inverted light microscope at 40£. Arrows indicate original wound edge.
of ADI can arrest the cell cycle in the G1 phase and only at high concentrations of the enzyme, i.e., .200 ng/ml, could apoptosis be induced [4]. Therefore in the case of ADI, the reduction in endothelial cell number seen in proliferation assays may be attributed, in part, to the induction of apoptosis due to modulation of the cell cycle by ADI, but it remains to be determined whether this effect contributes to the observed anti-tumor activity of ADI in vivo. In a recent investigation, it was shown that a cytostatic protein purified from ASC170 sertoli cell-conditioned medium had ADI enzymatic activity and was homologous to mycoplasma ADI [17]. This protein could actually inhibit taxolinduced apoptosis in DU145 cancer cells. It was
experiment, for rADI-treated wells remained consistent to that of control wells (seeding at 4 £ 10 4 cells per well, ending with 30,417 ^ 1488 and 28,743 ^ 3997 cells per well, control and rADI-treated, respectively). This indicates the lack of tube formation was not due to a difference in cell number.
4. Discussion In this report, purified recombinant ADI was used to investigate the effects of this enzyme on angiogenesis. Our results revealed that rADI, at a concentration that is non-cytotoxic, can inhibit migration (Fig. 3) as well as capillary vessel formation in cultured HUVE cells (Fig. 4). Our data in cultured endothelial HUVE cells also demonstrated that by removing this enzyme from the medium cell growth was restored, indicating that the cells were viable and capable of a proliferative response (Fig. 2). This would suggest that a reversible cytostatic effect, rather than cytotoxicity, is responsible for the cell growth inhibition, an observation that is consistent with what others have reported [4]. Together these observations, the inhibition of endothelial motility and the reversible inhibitory effect on endothelial cell growth, would indicate that ADI is more likely an anti-angiogenic rather than only an anti-proliferative agent. ADI has been shown to have an inhibitory effect on tumor cells in vitro [1,2]. This effect is thought to be cytotoxic, due to depletion of extracellular l-arg. However, in endothelial cells, the apparent cytostatic effect coincides with the report that low concentrations
Fig. 4. The effect of rADI on microvessel formation of HUVE cells grown on Matrigel-coated surfaces. HUVE cells were pretreated with 1 £ 10 23 U/ml of rADI for 48 h before being harvested and plated onto a Matrigel-coated 24-well cluster plate at 4 £ 10 4 cells/ well. The microvessel formation was observed using an inverted light microscope at 40£. Photographs comparing control (without rADI) to rADI pre-treated were taken at 2.5, 5.0, and 22.0 h after plating.
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suggested that the ADI-induced arginine depletion inhibited the required protein synthesis of apoptotic cell death, thereby resulting in protection of the cancer cells. It has also been reported that ADI is more potent than arginase in inhibiting leukemic cell growth in vitro [18]. The mechanism was speculated to be related to ADI’s ability to deplete l-arg thereby arresting the cell cycle and eventually leading to apoptotic cell death. This does not necessarily preclude possible anti-angiogenic properties of ADI. It has been speculated that angiogenesis not only has a critical role in the growth of solid tumors but also liquid tumors, such as leukemia [19]. The expansion of the endothelium, angiogenesis, and solid tumor growth are entwined by a balance of proliferative and apoptotic signals in the tumor microenvironment. If a similar model exists in the growth of liquid tumors it would then be highly desirable for an anti-cancer agent to have a combination of effects, i.e. direct toxicity and anti-angiogenic activity. Endothelial cell apoptosis has an important regulatory role in the process of angiogenesis. Angiogenic stimulatory factors have apoptotic protective effects in endothelial cells via various survival promoting pathways that block apoptosis execution and ultimately promoting endothelial cell survival. In contrast, it has also been shown that negative regulators of angiogenesis not only effect endothelial cell proliferation, migration, and remodeling, but also concomitantly stimulate endothelial apoptosis either directly or indirectly [20]. Excessive apoptosis could limit angiogenesis by counteracting endothelial cell proliferation and lead to tumor regression. However it is likely that angiogenesis is regulated by a balance between nitric oxide (NO) and polyamine synthesis pathways which could be determined by extracellular arginine levels. The regulation of arginine metabolism, such as the production of NO and polyamines, has recently been suggested to play an important role in tumor angiogenesis [21]. Interestingly, it has been implied that the healing of chronic gastric ulcerations in rats by intragastric administration of high doses of arginine is due to the promotion of angiogenesis [22]. On the other hand, the depletion of extracellular arginine for the reduction of angiogenesis has not yet been considered as a potential regulatory therapy for angiogenesisrelated diseases.
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The mechanism of the observed anti-angiogenic activity of ADI is still not clear at the present time. The protection of the cells by the substrate, arginine, indicates that the cell growth inhibition is not due to products of the enzymatic reaction, citrulline and ammonium, or products from other subsequent reactions [1]. Therefore, we assume that the anti-angiogenic activity is primarily due to the depletion of arginine from the culture medium. However, since complete reversal was not achieved in endothelial cells, perhaps alternate mechanisms are also involved, as has been suggested [4]. Whether the anti-tumor effect of ADI as reported in experimental animals is due to an inhibition of tumorassociated angiogenesis or an inhibition of tumor cell proliferation remains to be determined.
Acknowledgements This study was supported by a grant, 6IB-0045, from the California Breast Cancer Research Program.
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