Noriko Yasuda

Takuma Sasaki

Yukari Shimo

Yukio Kimura

安田 紀子

Noriko YASUDA

Mukogawa Women’s University Repository / 武庫川女子大学リポジトリ

Bu/. Mukogawa Women's Univ. Nat. Sciリ 42，63 -66(1994) 武庫川女子大紀要(自然科学)In Vitro Antitumor Activity of Carboxypeptidase G3 from Pseudomonas sp. M田27Noriko Yasuda1， Yukari Shimo1*， Yukio Kimura and Takuma SasakF 1) Faculty of Pharmaceutical Sciences， Mukogawa Women's University， Nishinomiya， 663， ]，伊an2) Cancer Research Institute， Kanazawa University， Kanazawa 920， ]apan Carboxypeptidase 03 from Pseudomonas sp. M・27hydrolyzed predominately N-acyl glutamic acid. We found that carboxypeptidase 03， capable of liberating the carboxyl-terminal gluta-mate from folic acid analogue， showed in vitro antitumor activities against human carcinoma KB and PC-3 cells among ten kinds of human carcinoma cell lines. It can be expected to be useful as an anticancer agent against limited cell lines. Introduction In 1948， Ferber et al. 1) demonstrated the in・duction of brief remissions in children with acute leukemia using the folate analogue aminopterin. Since then， methotrexate has been widely used as a folic acid antagonist in the chemotherapy of cancer， notably leukemias and choriocarcinoma. Its therapeutic action is based on inhibition of di-hydrofolate reductase. However， resistance to methotrexate has been associated with increased levels of the target enzyme， dihydrofolate reductase， and with impaired cellular uptake of the drug. In a search for new antifolate chemotherapeutic agents， several enzymes which hydrolyze the pep-tide linkage of the C-terminal glutamate residue of folic acid， carboxypeptidase 0，2) 013) and 024) have been found. The action of folic acid-de-grading enzyme led to investigation of its useful-ness as an antineoplastic agent， and its antitumor activity was demonstrated -against several murine tumors in vitro5) and in vivo.。Previously during purification of polymyxin ac-ylase from Pseudomonas sp. M・27strain which produces it， we found a new enzyme， carbox-*present address : Osaka Medicine Co.，Ltd. Osaka 540， Japan ypeptidase 03， that hydrolyzed only N-fatty acyl glutamic acid or aspartic acid， but not polymyxins.7) The enzyme resembles carboxypeptidase 01 or O2 in its glutamate-releasing activity. However， itacts not only on the L-from but also on the D-form of acidic amino acids and shows affinity for the long-chain fatty acyl group but not the benzoyl group. In this paper， we describe the in vitro antitumor activity of carboxypeptidase 03 against murine and human tumor cels and its substrate specificity for folic acid and its analogues. Materials and Methods Preparation of carboxypeptidase G3 Carboxypeptidase 03 was purified to homogeneity from an acetone輔driedcel powder of Pseudomonas sp. M-27 by the procedure described in our preceding paper. 7) This enzyme had a single peak with a molecular weight of 60，000 on a calibrated Superdex column and consisted of four subunits of identical molecular weights (Mr:15， 000). Its iso-electric point was estimated to be 7. 2. The enzyme hydrolyzed predominately acidic peptides and N-acyl amino acids with glutamic acid or aspartic acid in the C-terminus. The observed Vmax and Km for N-octanoyl・DL-glutamic acid were 33μmollmin/mg and 3.5mM， respectively. This ? ???(Yasuda' Shimo・Kimura'Sasaki) enzyme was not strongly affected by thiol enzyme inhibitors (p・chloromercuribenzoate，iodoacetic acid) or serine protease inhibitors (diisopropylphosphoro fluoridate， phenylmethane sulfonylfluoride)， but was inhibited by metal chelators. Enzyme assay Carboxypeptidase or acyl amino acid acylase activity was assayed from the nin-hydrin color spot of liberated amino acid on the TLC plate as follows: the reaction mixture (10μ1) containing 1μmol of folic acid and its analogue， lmM ZnClz and the enzyme solution (25μ1) in 20mM Tris buffer (pH8.0) was incubated at 370C for several hours. An 1・μ1portion of the reaction mixture was developed by TLC on a silica gel plate Csolvent system upper layer of n・BuOH-AcOH-H20 (4:1:3) containing 1/20 pyridine by volumeJ. The amount of amino acid liberated was measured by the ninhydrin method with Chro-moscan 200/201 (Joyce-Loebl， England). During enzyme purification， this assay method using N二octanoyl-DL-glutamic acid was used. The amount of liberated amino acid in the mixture was mea-sured colorimetrically by the ninhydrin method. In vitro antitumor activity of carboxypeptidase G3 The terazolium幽basedsemiautomated colorimetric assay (MTT assay) developed by Carmichael et al.8) was modified and used for the in vitro assay. Briefly， 2000 cels in 180μ1 of RPMI-1640 medium (Nissui Pharmaceutical Co.， Osaka， Japan) sup-plemented with 10同 heat-inactivatedfetal bovine serum (Gibco Lab.， Grand Island， NY， USA)， penicillin G (100 units/ml) and streptomycin (100μg/ml) were seeded in a 96-well flat bottom microtest plate (InterMed， Roskilde， Denmark) and 20μ1 each of drug solutions of graded con-centrations was simultaneously added to each well triplicate samples. The plate was incubated for 3 d at 370C in a humidified atmosphere of 5町CO2・MTT reagent [3・(4，5-dimethylthiazol-2・yl)-2， 5-diphenyltetrazolium bromideJ (sigma Chemical Co.， St Louis， MO. USA) was prepared at a concentration of 2mg/ml in Dulbecco's phosphate buffered saline without ca1cium and magnesium and strored in a refrigerator. On the third day， MTT reagent (25μ1) was added to each well. After another 4-h incubation at 370C， the microplate was centrifuged at 1500 rpm for 10 min and the medium was removed by aspiration. To solubilize the resulting MTT-formazan， 0.2ml of dimethylsulfoxide was added to each well followed thorough mixing with a mechanical plate mixer. Absorbance at 540 nm (OD540) was measured with an ImmunoReader NJ・2000 (InterMed Japan， Tokyo， Japan). The percentage of cel1 growth inhibition was ca1culated by the following formula 070 of cel growth inhibition (1-T/C) x 100， where C is the mean OD540 of the control group and T is that of the treated group. ICso value was derermined graphically from the dose-response curve with at least 3 drug concentration points. Results and Discussion Table 1 shows the activity of carboxypeptidase G3 for folic acid and its analogues. Folic acid was exclusively hydrolyzed by this enzyme， but for methotrexate the glutamate-releasing activity was very weak as compared with those of carbox-ypeptidase G1 (725μmol/min/mg) and G2 (215μmol/min/mg). To investigate the antitumor activity of carboxypeptidase G3， murine leukemia cels and a variety of human neoplastic cels were used. As shown in Table 2， appreciable antitumor effects were observed for human carcinoma KB and PC-3 cels specifically， but the other cels were very slightly affected. Earlier， in a search for new antifolate chemotherapeutic agents， McCullough and Chabner3，9) isolated and purified an enzyme from Pseudomonas stutzeri， carboxypeptidase G1， which hydrolyzed the peptide linkage of the C-terminal glutamate residue of folic acid analogue. They observed that carboxypeptidase G1 inhibited the growth of L5178Y and L1210 murine leukemia and indicated that the antineoplastic activity was a consequence of folic acid depletion.5，6) Chabner et al. 10) reported that carboxypeptidase G1 has no selective inhibitory effect on thymidylate synthesis， as does methotrexate， but probably depresses DNA synthesis through its effects on purine synthesis or amino acid metabolism. Valerino et al.lI) reported -64-In Vitro Antitumor Activity of Carboxypeptidase G3 from Pseudomonas sp. M-27 Table 1. Substrate Specificity of Carboxypeptidase G3 for Folic Acid and Its Analogues. Substrate Activity (μmol/min/mg) Folic acid Dihydrofolic acid Methotrexate Aminopterin Leucovorin 5・Methyltetrahydrofolicacid 1.4 0.6 0.4 0.2 0.02 0.04 The enzymatic reaction conditions are described in the text. Table 2. Inhibitory Effects of Carboxypeptidase G3 on the Growth of Various Murine and Hunian Tumor Cell Lines in vitro. Cellline Origin ICso(μg/ml) L5178Y Murine leukemia >25 KB Human epidermoid carcinoma 15 NPC-TY861 Human epipharynx squamous carcinoma >25 PC-3 Human lung adenocarcinoma 7.4 PC-8 Human lung adenocarcinoma >25 PC-13 Human lung large-cell carcinoma >25 QGδ6 Human lung squamous cel carcinoma >25 KATO-ill Human stomach adenocarcinoma >25 ST-KM Human stomach adenocarcinoma >25 NUGC-4 Human stomach adenocarcinoma >25 OST Human osteosarcoma >25 that carboxypeptidase G from Pseudomonas can be Walker carcinoma in rats: Carboxypeptidase G3 used in rescue therapy following high-dose has similar properties such as carboxyl-terminal methotrexate regimes， because of its ability to glutamate-releasing activity and requirement of hydrolyze the glutamate moiety from methotrexate Zn2ヘbutthe optical specificity of the enzyme is to form 4・amino-.N1O-methylpteroate，which is a markedly different from that of the known enzyme， much les active inhibitor of dihydrofolate reduc- G， G1 and G2， that is， carboxypeptidase C3 acts tase. Sherwood et al.4) found that carboxypepti- not only on the L-form but also on the o-form of dase G2 from Peudomonas sp. RS-16 had similar glutamic acid. Carboxypeptidase G3， capable of Km values to G1 for folate (4.0μM) and meth- liberating the carboxyl-terminal glutamate from otrexate (8.0μM)， but les affinity for leucovorin folic acid analogue， inhibited very slightly the (120μM). This may be of significance if the en- growth of muriJ;le leukemia， but showed remark-勾meis used in rescue resimes together with leu- able antitumor activities against human carcinoma covorin in cases where high-dose methotrexate KB and PC・3cels among ten kinds of human therapy leads to toxicity. Carboxypeptidase O2 also carcinoma cel lines. The mechanism of the an-exhibited antitumor activity in its own right against titumor activity of carboxypeptidase G3 seems to ???(Yasuda. Shimo' Kimura . Sasaki) be based on the degradation of folic acid. Ac-cordingly， carboxypeptidase G3 can be expected to be useful as an anticancer agent against limited cel lines， which can regulate the level of folic acid in vivo， and be also used in rescue therapy following high-dose methotrexate regimes. Thus carboxypep-tidase G3 is promising as an enhancer of antitumor activity when used with other active reagents， and also may be of significance for chemotherapy to target specific human neoplastic cels， such as KB and PC・3.Acknowledgment We thank Professor Judy Noguchi for her advice in preparing the manuscript. References 1) S. Farber， L. K. Diamond， R. D. Marcer， R. F. Sylvester， and J.A. Wolff， New Engl. J. Med.， 238， 787-793(1948). 2) C. C. Levy and P. Goldman， J. Biol. Chem.， 242， 2933-2938(1967). 3) J. L.恥1cCullough，B. A. Chabner and J. R. Ber-tino， J. Biol. Chem.， 246， 7207-7213 (1971). 4) R. F. Sherwood， R. G. Melton， S. M. Alwan and P. Hughes， Eur. J. Biochem.， 148， 447-453 (1985) . 5) J. R. Bertino， P. O'Brien and J. L. McCullough， Science， 172， 161-162(1971). 6) B. A. Chabner， J， L， McCullough and J. R. Ber-tino， Proc. Amer. Ass. Cancer Res.， 12， 88(1971) • 7) N. Yasuda， M. Kaneko and Y. Kimura， Biosci. Biotech， Biochem.， 56， 1536-1540(1992). 8) J. Carmichael， W. G. DeGraff， A. F. Gazdar， J. D. Minna and J. B. Mitchell， Cancer Res，. 47， 936-942 (1987). 9) B.A.Chabner and J.R.Bertino， Biochem. Biophys. Acta.， 276， 234-240(1972). 10) B. A. Chabner， P. L. Chello and J. R. Bertino， Cancer Res.， 32， 2114-2119(1972). 11) D. M. Valerino， D. G. Johns， D. S. Zaharko and V. T. Oliverio， Biochem Pharmαcol.， 21， 821-831 (1972) . -66-

In Vitro Antitumor Activity of Carboxypeptidase G3 from Pseudomonas sp. M-27

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In Vitro Antitumor Activity of Carboxypeptidase G3 from Pseudomonas sp. M-27

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