Extraction and Isolation of the Anti2tumor Protein Components from Earthworm ( Eisenia fetida a ndrei) and the Anti2tumor Activity

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1 ISSN CN ΠQ Chinese Journal of Biochemistry and Molecular Biology 19 (3) : ,,,,,, (, ) 3, ( ) ; Fe Zn Cu Se, % ( n = 5), MTT SRB ( HCT2116 SY5Y K562 MGc803 HeLa) (HEK293 COS27), 50 % mgπl ;,100 5 min, aprotinin PMSF, (S180), 28 mgπkg 36 mgπkg, % %, ( ) (7615 %),,,, Q51, Q55 Extraction and Isolation of the Anti2tumor Protein Components from Earthworm ( Eisenia fetida a ndrei) and the Anti2tumor Activity XIE Jiang2bi, HE Wei2guo, WENG Ning, GUO Zhen2quan, YU Mei2min, LIU Ning, RU Bing2gen 3 ( College of Life Sciences, Peking University, Beijing , China) Abstract A group of anti2tumor protein components ( Eisenia fetida extract, EE) was extracted and isolated from earthworm Eisenia fetida andrei by acetone sedimentation and gel filtration The protein content of EE was % ( n = 5) EE was also rich in trace elements, such as Zn, Cu, Fe and Se In vitro the concentrations of EE required for 50 % growth inhibition of different human tumor cell strains ( HCT2116, SY5Y, K562, MGc803 and HeLa) were between 60 and 110 mgπl And the inhibition of normal cell strains, HEK293 and COS27, by EE were much weaker than those of most human tumor cell strains tested However, the cell2killing activity of EE was completely diminished after boiling for 5 min By fibrin plate assay EE was found to be fibrinolytic as well as plasminogen2activating The cell2killing activity of EE in vitro could be inhibited by serine protease inhibitors, aprotinin and phenylmethyl sulfonyl fluoride ( PMSF) EE (28 mgπkg and 36 mgπkg, i p ) in vivo were found to prolong the life span of ascites tumor (S180)2bearing mice by % and %, respectively, and even to improve their physical conditions Meanwhile, EE was found to be low toxic as compared with the cyclophosphamide treatment (7615 %) Key words Eisenia fetida andrei, anti2tumor protein components, extraction and isolation, fibrinolytic activity, assay of anti2tumor activity,, : , : Tel : (010) , Fax : (010) , E2mail : edu cn ( lumbnofebrine),, , Received :December 2,2002 ;Accepted :March 6, Corresponding author Tel : (010) , Fax : (010) E2mail : edu cn Fe Mn Cu Zn Se (lumbnitin) (tennestrolumbrolysin)

2 ,, (4 h, 4 ) ; ( [1 ], g, 30 min, 4 ), 3 4 B ( - 30 ), [2 ] ; ( - 20 ) (2 h) ;,, ( g, 30 min, 0 ),, [3 ], ( - 30 ),,, ( - 30 ) (, ( ) ),, ml (0115 molπl NaCl in HCT ml, 600 ml B ( MGc803 HeLa ), Hi Prep TM 26Π10, HEK293 COS27 S180, 114 SY5Y 1 ( SDS, K562 2 ),, 5 % (, %, 20 ma,, n = 40, (20 2) g, Grade, certificate number 005) (BSA), ( Eisenia fetida andrei) Lowry, Bio2Rad DC Hi Prep TM 26Π10, Hi Prep TM 26Π60 Sephacryl S2100 KTA prime ICP2MS (4 500, Agilent) Pharmacia, (sulforhodamine B, SRB) ( 32( 4, 52dimethyl thiazol222yl)22,52diphenyl tetrazolium bromide, MTT) (BSA, > 9919 %) Sigma, (DC Protein Assay) Bio2Rad, RPMI1640 Gibco, (0115 molπl NaCl, 0102 molπl Tris2HCl, ph 810), ( aprotinin ) ( phenylmethyl sulfonyl molπl Tris2HCl, ph 810), ( g, 5 min), Hi Prep TM 26Π 60 Sephacryl S mlπmin, R mg, 3 % HNO 3 10 ml, ( 0115 g), 3 ml A (0102 molπl Tris2HCl, ph 810),37 ; 0115 g, 20 ml B,58 30 min ;, fluride, PMSF) Boehringer Mannheim, (10 BP 100 l A), ( = 510, 3 cm) Nunc, ( = 510 cm) ;,80 15, 20 min,4,,, 200 l 113 ( l A), 3, 4 (0102 molπl PB, ph 810), l

3 3 : 361, 37, 4 10 h, MTT aprotinin PMSF (HCT2116 SY5Y MGc803 HeLa HEK293 COS2, 150 l Tris (10 mmolπl, ph 1015) 515 nm 650 nm ( A 515nm - A 650nm ), 2 ( A %) Fig11 Fig 2 ( PAGE Fig 1, 3 80 mgπl) A B C, B ( ) 30 min min, HCT2116, 7) 1,215 gπl, RPMI1640 Aprotinin 58, ( 5 % ) (10 4 ; 10 5 Πml) ( RPMI 1640 (ph ) ) ; 96, 100 l, ( 5 % CO % 37 ), (30 min, 4 ), 24 h ( ) 100 l PMSF RPMI1640 ( ),, PMSF ( = 0122 m), (100 mmolπl ), (100 lπ ) (100 l ) (30 min, 4 ) (100 l ) 4 6 MTT h, l MTT (1 gπl ) S180 4 h, 150 l (10 6 ), 4 (DMSO), (formazan) ( 10 ) 1,, ( Spectra Rainbow, Tecan Austria) 012 ml ; nm 650 nm (, (20 mgπkg) A ; 3 4, A 650 ), :, 28 mgπkg 36 A % = [ ( A 0 - A) ΠA 0 ] 100 % mgπkg A A 0 ( = 0122 m) 2 d SRB K562, MTT, d, 20 d, SRB MTT, 96,, T % = [ ( T - C) ΠC] 100 % TCA( 100 gπl,4 ) ( T C 1 h, 4 ) ;, (days) ; 100 l SRB (4 gπl 1 % 119 ), 10 min 1 %, ( gx s), t 211 (HCT2116 ), ( HCT2116), MTT C (Fig 2),

4 ;B C,, ; F I Fig 1 Gel filtration chromatography of Eisenia fetida acetone powder The inhibition of cell growth ( HCT2116 tested) and the fibrinolytic activity of different parts separated were calculated by A % 100 and diameter of fibrin lysis (mm), respectively Fig 2 SDS2PAGE of different peaks isolated from acetone powder by gel filtration 1 : Protein standard molecular weight marker ; 2 : Eisenia fetida acetone powder ; 3 11 : Peak A I isolated from Eisenia fetida acetone powder Eisenia fetida tumor cell strain, HCT (A) The cytotoxicity 1) of EE which had been pre2incubated under % different temperatures for 30 min ( n = 5), Zn 5414 gπl Cu 4319 gπl Fe 1910 gπl Ni 1198 gπl Mn 1100 gπl Se 0170 gπl Co 0116 gπl Mo 0108 gπl, ;, Fe Cu Zn, Se, [4 ], Data are gx s ( n = 3) [5 ] 213 Table 1 (A) (80 mgπl, 24 h), ;100 5 min,,,, Table 1 (B) (24 h, 37 ), 80 mgπl, HCT2116, Table 1 In vitro cytotoxicity of E fetida extract ( EE) to human tπ A % 2) ) in 24 hours experiment, calculated by A % ; 2) MTT assay

5 3 : 363 (B) The cytotoxicity 1) of EE with different concentrations ( EE)Πmg L - 1 Data are gx s ( n = 3) A % 2) ) in 24 hours experiment,calculated by A % ; 2) MTT assay 3 A B C, B ( ) ;,, 215 Fig 4,, (aprotinin), aprotinin, 80 mgπl ( 50 mgπl) ; PMSF( ) (0 2 mmolπl) 214, Fig 3, a b Fig 3 Fibrin plate assay of different peaks isolated from Eisenia fetida acetone powder by gel filtration (a) Negative plate without plasminogen ; (b) Positive plate with plasminogen (a) and (b) were tested under the same conditions (0 1 g, 37, 4 hours) Obviously, only Peak A, B and C were fibrinolytic and plasminogen2activating Fig 4 Inhibition of antitumor activity in vitro of Eisenia fetida extract ( EE) by two serine protease inhibitors (A) Aprotinin inhibitor ; Control ; EE(50 mgπl) (B) PMSF inhibitor ; Control ; EE(60 mgπl) Inhibitors had been mixed with EE separately and later added to HCT2116 cells At last, the inhibition was analyzed with MTT assay after 24 h incubation X2coordinate denoted the concentration of the inhibitor tested Y2coordinate denoted the inhibition of cell growth calculated by A % 100

6 Table 2, mgπl, (HCT2116 MGc803 HeLa K562), ( HEK293 COS27) ( ), ;,, 50 % mgπl ;,,K562, Table 2 In vitro cytotoxicity 1) of E fetida extract ( EE) to different human tumor cell strains and several normal cell strains Tested material EE Cisplatin Concentration for 50 % cytotoxicityπmg L - 1 HCT2116 2) MGc803 2) SY5Y 2) HeLa 2) K562 3) HEK293 2) COS27 2) Data are gx s ( n = 3) 1) in 24 hours experiment, calculated by A % ; 2) MTT assay ; 3) SRB assay 217 Table 3 (36 mgπkg) ;,,, (135 3 % S180, %) (7615 %) Table 3 Effect of E fetida extract ( EE) on ascites tumor reduction in mice (kunming strain, male) Tested material DoseΠmg kg - 1 (i p ) No of animals with tumor No of days survived Increase in life span 1) Control Normal saline 10Π Cyclophosphamide 20 10Π ) 7615 EE 28 10Π ) EE 36 10Π ) Data are gx s ( n = 10) 1) calculated by T % 100 2),3),4) 3),4) 4) P < 0 01 v control P < 0 01 v cyclophosphamide P > 0 05 v EE (28 mgπkg) S180, ;,,,, S180 9 d,,, ( %) (13513 %), ( P > 0105),, 3,, ( ), ( ),, ;, ( K562 HCT2116 HeLa MGc803),, 2,

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