Purification of Thiol Protease Inhibitor from Human Lung Cancer Tissue*)

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1 Hiroshima Jornal of Medical Sciences Vol. 33, No., 1..,, December, 19 HJM 33-ll 1 Prification of Thiol Protease nhibitor from Hman Lng Cancer Tisse*) Tsneo OKUMCH, Masayki NSHK, Smiyoshi T AKASUG, Motoi YAMANE and Haro EZAK The nd Department of Srgery, Hiroshima University School of Medicine, 1--3, Kasmi, Minami-k, Hiroshima 73, Japan (Received September 5, L9) Key words: Thiol protease inhibitor, Hman lng cancer ABSTRACT t was confirmed that the thiol protease inhibitory activities in hman lng cancer extracts were significantly higher than normal lng tisse extracts. Frthermore the thiol protease inhibitor was extracted from hman lng cancer tisse and prified by papain-sepharose and Sephadex G-1 colmn chromatographies. The prified inhibitor inhibited papain and ficin, and its moleclar weight determined by sodim dodecyl slfate-polyacrylamide gel electrophoresis was abot 13,. NTRODUCTON The mechanism of the process by which malignant tmors invade and metastasize is complex, and not yet flly elcidated. t has been reported that proteolytic. enzymes sch as plasminogen activator 3 l (one of serine proteases), cathepsin B l (one of thiol proteases) and collagenase 7 l (one of metallo proteases) might be associated with invasiveness and metastasis of malignant tmors. Althogh an enormos amont of literatre has been accmlated on the association of proteolytic enzymes and malignant tmors, there have been few reports abot the association of tmors and protease inhibitors 1 1 a l. n a previos paper l, the athors reported that hman lng cancer tisse contained a rinary trypsin inhibitor-like inhibitor, and proposed that the protease inhibitor might play an important role as an inhibitor of the promoting or metastatic factor in lng cancer. Only few reports were fond abot the association of thiol protease inhibitors and tmors5 17l, sch as Giraldi et al. 9 l reported that the bovine spleen netral proteinase inhibitor (SNP-1), which inhibited thiol proteinases cathepsin B and H, redced the formation of spontaneos plmonary metastases in mice bearing Lewis lng carcinoma. We thoght it worthwhile to stdy abot the presence: of tliol protease inhibitor in hman lng cancer tisse. n the present stdy we extracted and prified a thiol protease inhibitor from hman lng cancer tisse. MATERALS AND METHODS Assay of thiol proteases and protein: The caseinolytic activity of the sample was determined by the method of Lowry et al. oi as reported previosly 1 i, sing % casein sbstrate (caseinolysis method). The amidolytic activity of the sample was determined with chromogenic sbstrates S-51 (H-D-Val-Le-Lys-pNA, Kabi Diagnostica) and S-3 (H-D-Pro-Phe Arg-pNA, Kabi Diagnostica) by the method of Bang and Mattler 3 l (Testzym method). The inhibitory activities of the sample on thiol proteases were determined from the residal caseinolytic and amidolytic activities of the *>![, -ggtez, Mitifff, 11rm, U.llffiJra : r JtrM#il.il (/) :r:t ' i:i :r t 1 :,; tj - (J)fr!J '1

2 ' T. Okmichi et al. proteases after incbation with a mixtre of protease and varios amonts of inhibitors for predetermined pri9d at. 37 C. The concentration of the thiol prote'ases were adjsted to 5 µg/ml with. 1 M phosphate bffer, ph 7. containing 75 mm cysteine in caseinolysis method and to 1 µg/ml with. 5 M Tris -HClbffer, ph 7. containing 75 mm cysteine in Testzym method and. 1 ml of the protease was sed. One nit (U) of thiol protease inhibitory activity was defined as the amont inhibiting 1 µg of the protease. Protein concentration was determined by the method of Lowry et al. > sing bovine albmin (Merck) as the standard. Extraction of thiol protease inhibitor from lng cancer tisse and normal lng tisse: Hman lng tisse samples were obtained from both sites of cancer tisse and apparently healthy parts of 1 lng cancer patients at the time of srgical operations. Ten patients were diagnosed histologically as adenocarcinoma, 7 were as sqamos cell carcinoma and 1 was as large cell carcinoma. For patients were diagnosed as Stage (Stage by p-tnm 31 >), were as Stage, were as Stage and were as Stage V. The extracts of tisses with M KSCN soltion were prepared according to the procedre 9f Astrp et al. > with the modification as reported by s previosly >. Papain-Sepharose affinity chromatography: Papain-Sepharose gel was prepared by the method of Catrecasas > ; Sepharose B (Pharmacia) was activated by treatment with CNBr, and copled with papain. The resltant papain-sepharose gel was inactivated by KSCN and acetic acid and washed with. M trisodim phosphate containing 3M. KSCN, ph 1. 1 and. 1 M phosphate bffer,. 1 M NaCl, ph.. A colmn (. 7 x 1 cm) was packed with the inactivated papain Sepharose gel and eqilibrated with. 1 M phosphate bffer,. 1 M NaCl, ph. (starting bffer). The pooled lng cancer extract was applied to the colmn at a flow rate of ml/hr and collected fractions of 1 ml. Unadsorbed material was elted with the starting bffer, non specifically adsorbed proteins were elted with the starting bffer containing 3M KCl and washed with the starting bffer. The adsorbed inhibitor was elted with. M trisodim phosphate,. 1 M NaCl, ph Finally, the colmn was washed with. M acetate bffer, 3M KSCN, ph. and the starting bffer. This method of affinity chromatography was accorded to.the procedre of Jarvinen 13 >. The cllected fractions containing 1.nhibitor from papain-sepharose affinity chromatography were concentrated on celllose tbing (Visking) and applied to a colmn of Sephadex G-1. Gel filtration: A colmn (. x cm) of Sephadex G-1 (Pharmacia) was eqilibrated with. 1 M phosphate bffer,. 1 M NaCl, ph.. The separation was performed at C and the efflent was collected in fractions of 3 ml at a flow rate of 15 ml/hr. The moleclar weight of the inhibitor was estimated according to Andrews 1 > by sing moleclar weight standards (Pharmacia). Sodim dodecyl slfate- polyacrylamide gel electrophoresis (SDS-PAGE): SDS-PAGE was carried ot by the method of Weber and Osborn 3 > sing 7. 5% gel with 1% SDS and M rea. After electrophoresis, the gel was stained with Coomassie brilliant ble. Moleclar weight was determined from a simltaneos rn of moleclar weight standards. RESULTS Measrement of thiol protease inhibitory activities in tisse extracts: Thiol protease inhibitory activities in tisse extracts from 1 lng cancer patients were determined sing ficin (Sigma) and papain (Sigma) as thiol protease. As shown in Fig. 1, ficin inhibitory activities were measred by both Testzym (S-51) and caseinolysis (casein) method. The inhibitory activities measred by S-51 in lng cancer extracts were obviosly higher than in normal lng tisse extracts, and the inhibitory activities by casein in lng cancer extracts were also significantly higher than in normal lng tisse extracts (Fig. 1-A). Althogh, the inhibitory activities of individal patients were widely different, almost all the lng cancer extract had higher inhibitory activity compared with the normal lng tisse extract of the same patient. On the other hand, there were no significant difference between adenocarcinoma and sqamos cell carcinoma in the inhibitory activities

3 TP from Hman Lng Cancer 3 A B 1 1 S-51 CASEN CA NOR CA NOR l A B 1 1 S-3 CA NOR CASEN CA NOR Ad Sq c 1 Ad Sq Ad Sq d ll c 1 Ad Sq V V Fig. 1. Ficin inhibitory activities in tisse extracts from lng cancer patients. Ficin inhibitory activities (FA) were determined by Testzym (S-5L) and caseinolysis (casein) method, and expressed by U/mg protein (M±SD). A: FA in lng cancer extract (CA) and normal lng tisse extract (NOR). B: The comparison of FA in lng cancer extract by histologic types (Ad= adenocarcinoma, Sq= sqamos cell carcinoma) C: The comparison of FA in lng cancer extracts by p-tnm Stage. sing both sbstrates (Fig. 1-B). Frthermore, the differencies of the inhibitory activities in lng cancer extracts from varios stages classified by p-tnm Stage were not fond by both methods (Fig. 1-C). Fig. shows the papain inhibitory activities measred by Testzym (S-3) and caseino]ysis (casein) method. The inhibitory activities in lng cancer extracts measred by both methods were significantly higher than in normal lng tisse extracts (Fig. -A). There were no significant difference of inhi- Fig.. Papain inhibitory activities of tisse extracts from lng cancer patients. Papain inhibitory activities (PA) were determined by Testzym (S-3) and caseinolysis (casein) method, and expressed by U/mg protein (M±SD). A, B, C were the same as described in Fig. l. bitory activities between adenocarcinoma and sqamos cell carcinoma, nor among varios lng cancer stages sing both methods (Figs. -B, C). Prification of thiol protease inhibitor from hman lng cancer extract: As shown Figs. 1-A and -A, the thiol protease inhibitory activities in lng cancer extracts were significantly higher than in normal 1 ng tisse extracts. Then we tried to prify the thiol protease inhibitor from pooled lng cancer extract. The pooled lng cancer extracts were collected from lng cancer patients who were diagnosed histologicaly as adenocarcinoma, and applied to the papain-sepharose colmn. The eltion pattern of the papain-sepharose colmn is shown

4 T. Okmichi et al. in Figs. 3 and. Papain inhibitory activities in each fraction was determined by caseinolysis method. Nonspecifically adsorbed proteins were elted with elent. and the papain inhibitory activities in these fractions were apparently high, however, almost of the increased activities were de to the elent itself. So the inhibitory activities of these fractions after dialysis against. 1 M phosphate bffer,. 1 M NaCl, ph. were almost disappeared. The adsorbed inhibitor was elted with elent (. M trisodim phosphate,.1 M NaCl, ph 1.1) (Fig. 3). Papain inhibitory activity in each fraction determined by Testzym method was almost the same as caseinolysis method. Ficin inhibitory activity in each fraction was determined by Testzym method (Fig. ). The eltion pattern of the inhibitory activities were almost the same as the papain inhibitory activities. Thogh the inhibitory activities were strongly inflenced by the elent, the tre inhibitory activities were existed in the fraction which were elted with elent (Fig. ). Ficin inhibitory activity in each fraction determined by caseinolysis method was almost the same as Testzym method. The inhibitor elted with the elent was collected (fractions Nos ), dialyzed against. 1 M phosphate bffer,. 1 M NaCl, ph. and concentrated. The concentrated inhibitor was applied to Sephadex G-1 colmn. The eltion pattern of the protein and thiol protease inhibitory activity are shown in Fig. 5. The peaks of papain and ficin inhibitory activities were coincided (moleclar weight abot 13, ) and were apparently different from the peaks of the proteins. n this way, the thiol protease inhibitor from lng cancer tysse was prified and its moleclar weight was abot 13,. A typical prification procedre is smmarized in Table 1. The recovery rates of papain Sepharose affinity chromatography and Sephadex G-1 gel filtration were 5. % and. %, respectively. The prified inhibitor from Sephdex G-1 gel filtration gave a single broad and faint s. ""- >..o ell c :.a 1. '@. ell <l <l Fraction nmber N Cl Q. ell!... (!).5 ]!... ' A \ t! --ATA-A-.O..C Fig. 3. Papain inhibitory activity on papain-sepharose affinity chromatography of pooled lng cancer extract. Pooled lng cancer extract (adenocarcinoma) was applied to papain-sepharose affinity chromatography. Papain inhibitory activity in each fraction (LO ml) was determined by caseinolysis method, L- were elents (1=.1 M phosphate bffer,,lm NaCl, ph., =.lm phosphatebffer,3mkcl,ph.,3=.l, =, M trisodim phosphate,.1 M NaCl, ph L. L, 5=, M acetate bffer, 3M KSCN, ph., =1).

5 TP from Hman Lng Cancer 5. > '.P ro c.s 1. ;.5 ].s 1 t '1?1,1 1' 1:,, 11 11, b! \ q p-o (1 'o q, / ''O 5 +,, f v v-o,o-o-o-q--=-...--' Fraction nmber.5 N Q.5.s Cl) -< p... Fig.. Ficin inhibitory activity on papain-sepharose affnity chromatography of pooled lng cancer extract. Sample was pooled lng cancer extract (adenocarcinoma). Ficin inhibitory activity was determined by Testzym method. 1- were elents which were the same as described in Fig... AB c D J J f f ] ]... '--. N o A >. >..._. :;: :e i:: tl '.P ro ro ro r >. c.., -< Cl).s. / 1. :.a i:: A,, i::,, F..s.s f,,\ ro,,,,. -< ro p... ' p... : 1 11 ' ' 1, 1. f, ', J -{l 3 5 Fraction nmber Fig. 5. Sephadex G-1 chromatogj.'.aphy of the inhibitor in pooled lng cancer extract from papain-sepharose affinity chromatography. Sample was pooled lng cancer extract (adenocarcinoma). Papain and ficin inhibitory activities were determined by Testzym method. A-D were moleclar weight standards (A=Albmin 7,, B=Ovalbmin 3,, C=Chymotrypsinogen A 5,, D=Ribonclease A 13, 7).

6 T. Okmichi et al. Table 1. Prification procedre of TP from pooled lng cancer extract Prification step Volme ml Protein mg/ml Ficin inhibitory activity Specific Recovery U /ml Total(U) activity % Extract Papain-Sepharose..51 Sephadex G A Fig.. SDS-polyacrylamide gel electrophoresis of the inhibitor in pooled lng cancer extract (adenocarcinoma ). 1 =Moleclar weight standard (A=Ferritin,, B=Catalase 3,, C=Phosphorylase b 9,, D=Albmin 7,, E=Ovalbmin 3,, F= Chymotrypsinogen A 5,, G=Soy bean trypsin inhibitor, 1, H=Cytochrome C 1, 5). =TP from the papain-sepharose affinity chromatography of pooled lng cancer extract (adenocarcinoma ). 3 = Prifid TP in pooled lng cancer extract (adenocarc:noma) by papain Sepharose affinity chromatography and Sephadex G-1 gel filtration. ( [ ) =broad and faint band by TP. band, and was estimated as moleclar weight of abot 13, by SDS-P AGE (Fig. ). The thiol protease inhibitor from the pooled lng cancer extract which was collected from lng sqamos cell carcinoma patients, was also prified throgh papain-sepharose affinity chromatography and Sephadex G-1 gel filtration. The prified thiol protease inhibitor from sqamos cell carcinoma had a moleclar weight of abot 13, same as the inhibitor from adenocarcinoma (data is not shown). DSCUSSON n the present stdy, we first scceeded in prification of a thiol protease inhibitor from hman lng cancer tisse. There has been no reports abot thiol protease inhibitor in hman lng cancer tisse. As shown in Figs. 1 and, it was confirmed that the thiol protease inhibitory activities in lng cancer extracts were significantly higher than in normal lng tisse extracts. Moreover, the athors cold prify a thiol protease inhibitor (TP) from hman lng cancer tisse throgh papain-sepharose affinity chromatography and Sephadex G-1 gel filtration (Figs. 3,, 5). The prified TP from hman lng cancer tisse gave a single band on SDS-polyacrylamide gel electrophoresis and was estimated as moleclar weight of abot 13, (Fig. ). This prified inhibitor inhibited some thiol proteases sch as ficin and papain. The frther inhibitory spectra of the TP are now nder investigation in or laboratory. Recently, many investgators have reported the interaction among protease inhibitors and varios diseases, and prified some inhibitors from tisse extracts. Blackwood et al. > reported that in hman gynecological tmors the trypsin inhibitor concentration was lower in advanced tmors and metastases than in the

7 TP from Hman Lng Cancer 7 less malignant tmors or control tisses, and they sggested that the inhibitor might be consmed by proteases. However, in a previos stdy > we confirmed that trypsin inhibitory activity in lng cancer tisse was obviosly higher than in normal lng tisse. On the other hand, since Finkenstaedt > fond a inhibitor which cold inhibit cathepsin B in spernatant of rat liver homogenate, many stdies have been reported concerning TPis in varios tisses, plasma and rine. Jarvinen et al. 15 > reported that the hman epidermal SH-protease inhibior was demonstratable in sqamos cell carcinoma of the bronchs, bt not in plmonary adenocarcinoma or small-cell anaplastic carcinoma of the bronchs. However, we confirmed the TP activities in both adenocarcinoma and sqamos cell carcinoma tisse extracts and cold prify the inhibitor from both histologic types. The moleclar weight of the TP from hman lng cancer tisse is similar to the TP from Arths lesions of rabbit skin 33 l, rat liver11, 1 >, bovine nasal cartilage l, rat lng and hog kidney 19 l and hman spleen 1 >. t was of particlar interest that the moleclar weight of TP from hman lng cancer tisse was obviosly smaller than hman plasma TPis (9, -17, ) 5,7l, and rinary TPis (, 5-7. ) 913 >. The reason for these differences in moleclar weights among TPis of tisses, plasma and rine is still nclear. Althogh, the reglatory mechanism of thiol protease activities by the inhibitor has been reported 1 >, varios problems concerning the pathophysiological significance of TP represent sbject for ftre stdy. The athors speclate that TP from hman lng cancer tisse may play a protective role against tmor cell, inhibiting the degradation of the extracelllar matrix by thiol proteases. ACKNOWLEDGEMENT This research was fnded in part by Grantin-Aid for Scientific Research, Project 513, of the Japanese Ministry of Edcation. REFERENCES L. Andrews, P The gel-filtration behavior or proteins related to their moleclar weights over a wide range. Biochem. J. 9 : Astrp, T. and Albrechtsen,. K Estimation of the plasminogen activator and the trypsin inhibitor in animal and hman tisses. Scand. J. Clin. & Lab. nvest. 9 : Bang, N. V. and Mattler, L. E. L97. Sensitivity and specificity of plasma serine protease chromogenic sbstrates. Haemostasis 7 : 9-1. Blackwood, C. E., Mandl,. and Long, M. E Proteolytic enzymes and their inhibitors in hman gynecological tmors. Am. J. Obst. & Gynec. 91 : Borek, C., Miller, R., Pain, C. and Troll, W Conditions for inhibiting and enhancing effects of the protease inhibitor antipain on X ray-indced neoplastic transformation in hamster and mose cells. Proc. Natl. Acad. Sci. USA 7 : Catrecasas, P Protein prification by affinity chromatography. Derivatizations of agarose and polyacrylamide beads. J. Biol. Chem. 5 : Dresden, M. H., Heilman, S. A. and Schmidt, J. D Collagenolytic enzymes in hman neoplasms. Cancer Res. 3 : Finkenstaedt, J. T ntracelllar distribtion of proteolytic enzymes in rat liver tisse, Proc. Soc. Exp, Biol. Med. 95 : Giraldi, T., Sava, G., Kopitar, M., Brizin, J. and Trk V. 19. Netral proteinase inhibitors and antimetastatic effects in mice. Er. J. Cancer 1 : 9-5. LO. Harris, C. C., Primack, A. and Cohen, M. H Elevated alpha 1-antitrypsin serm levels in lng cancer patients. Cancer 3 : -1. l l. Hirado M., wata, D., Niinobe, M. and Fjii, S. 19 L Prification and properties of thiol pro tease inhibitor from rat liver cytosol. Biochim. Biophys. Acta 9 : Hozmi, M., Ogawa, M., Sgimra, T., Takechi, T. and Umezawa, H nhibition of tmorigenesis in mose skin by lepeptin, a protease inhibitor from actinomycetes, Cancer Res. 3 : Jarvinen, M Prification and properties of two protease inhibitors from rat skin inhibiting papain and other SH-proteases, Acta Chem. Scand. B3 : !, Jarvinen, M. and Rinne. A. 19. Hman spleen cysteine proteinase inhibitor: Prification, fractionation into isoelectric variants and some properties of the variants. Biochim. Biophys. Acta 7: L5. Jarvinen, M., Rinne, A. and Rasanen, A protein reminiscent of epidermal SH-protease inhibitor occrs in sqamos carcinoma of the lng. Acta histochem, Bd. 3, S Katnma, N. and Kominami, E Strctres and fnctions of lysosomal thiol proteinases and thier endogeneos inhibitor. Crr. Top. Cell

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