ISSN 100727626 CN 1123870ΠQ 2003 6 Chinese Journal of Biochemistry and Molecular Biology 19 (3) :359 366,,,,,, (, 100871) 3, ( ) ; Fe Zn Cu Se, 60143 2136 % ( n = 5), MTT SRB ( HCT2116 SY5Y K562 MGc803 HeLa) (HEK293 COS27), 50 % 60 110 mgπl ;,100 5 min, aprotinin PMSF, (S180), 28 mgπkg 36 mgπkg, 13513 % 12315 %, ( ) (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 100871, 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 60 43 2136 % ( 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 13513 % and 12315 %, 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,, :2002212202, :2003203206 3 Tel : (010) 62751842, Fax : (010) 62751842, E2mail : Rulab @pku edu cn ( lumbnofebrine),,1973 11, Received :December 2,2002 ;Accepted :March 6,2003 3 Corresponding author Tel : (010) 62751842, Fax : (010) 62751842 E2mail : Rulab @pku edu cn Fe Mn Cu Zn Se (lumbnitin) (tennestrolumbrolysin)
360 19 80 90,, (4 h, 4 ) ; (10 000 [1 ], g, 30 min, 4 ), 3 4 B ( - 30 ), [2 ] ; ( - 20 ) (2 h) ;,, (12 000 g, 30 min, 0 ),, [3 ], ( - 30 ),,, ( - 30 ) (, ( ) ),, - 20 11312 10 ml (0115 molπl NaCl in 0102 1 111 HCT2116 8 ml, 600 ml B ( MGc803 HeLa ), Hi Prep TM 26Π10, HEK293 COS27 S180, 114 SY5Y 1 ( SDS, K562 2 ),, 5 % (, 4 6 15 %, 20 ma,, n = 40, (20 2) g, Grade, certificate number 005) 115 112 (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), (10 000 g, 5 min), Hi Prep TM 26Π 60 Sephacryl S2100 018 mlπmin, R2250 1 mg, 3 % HNO 3 10 ml, 116 2 ( 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 (12 200 l 11311 A), 3, 4 (0102 molπl PB, ph 810), 10 15 l
3 : 361, 37, 4 10 h, 37 117 11711 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 %) 11713 Fig11 Fig 2 ( PAGE Fig 1, 3 80 mgπl) 4 20 37 56 A B C, B ( ) 30 min 100 5 min, 11714 HCT2116, 7) 1,215 gπl, RPMI1640 Aprotinin 58, ( 5 % ) (10 4 ; 10 5 Πml) ( RPMI 1640 (ph 712 714) ) ; 96, 100 l, ( 5 % CO 2 100 % 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 96 24 h, 118 50 l MTT (1 gπl ) S180 4 h, 150 l (10 6 ), 4 (DMSO), (formazan) ( 10 ) 1,, ( Spectra Rainbow, Tecan Austria) 012 ml ; 2 550 nm 650 nm (, (20 mgπkg) A 550 - ; 3 4, A 650 ), :, 28 mgπkg 36 A % = [ ( A 0 - A) ΠA 0 ] 100 % mgπkg A A 0 ( = 0122 m) 2 d 11712 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),
362 19 ;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, HCT2116 212 (A) The cytotoxicity 1) of EE which had been pre2incubated under 60143 2136 % 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) 100 4 54 014 20 49 111 37 40 116 56 29 016 100-15 019 1) in 24 hours experiment, calculated by A % ; 2) MTT assay
3 : 363 (B) The cytotoxicity 1) of EE with different concentrations ( EE)Πmg L - 1 Data are gx s ( n = 3) A % 2) 100 20 13 0 5 40 12 0 8 50 23 0 7 60 32 0 6 70 37 1 3 80 50 0 8 90 47 0 8 100 35 0 5 110 29 0 9 120 18 0 6 130 30 1 0 140 24 1 7 170 29 0 4 1) 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
364 19 216 Table 2, 120 150 mgπl, (HCT2116 MGc803 HeLa K562), ( HEK293 COS27) ( ), ;,, 50 % 60 110 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) 79 3 6 90 2 5 111 3 2 80 3 5 60 2 4 154 1 8 120 3 6 26 1 5 33 2 0 29 2 1 28 1 2 10 1 5 20 2 3 10 1 3 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, 12315 %) (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 10Π10 17 118 Cyclophosphamide 20 10Π10 30 210 2) 7615 EE 28 10Π10 40 215 3) 13513 EE 36 10Π10 38 312 4) 12315 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,,, ( 12315 %) (13513 %), ( P > 0105),, 3,, ( ), ( ),, ;, ( K562 HCT2116 HeLa MGc803),, 2,
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