(M u ltidrug resistance, M DR )

V o l. 26 N o. 8 2 0 0 5 8 CH EM ICAL JOU RNAL O F CH IN ESE UN IV ERS IT IES 1446 1450 1, 1, 2 (1., 132013; 2., 130023) V CR Bel27402 M DR,.,, V CR. Bel27402, V CR, V CR G2, P2170, M DR 1gmRNA, Topo gαmrna. ; ; ; ; R 735 A 025120790 (2005) 0821450205 (M u ltidrug resistance, M DR ).,,. M DR,,. (P2 ) ; ;, P53 Bcl22; DNA [1 3 ]. M DR,,. T su ruo [4 ],, (Calcium channel b locker, CCB ),., CCB [5 ]. Bel27402,,. 1 1. 1 H ew lett 1100 ; Cou lter Ep ics XL ; M odel 550 (B IO 2RAD ) ; 9700 PCR ; Kodak (EDA S). Bel27402 ( ) ; (BCDC), (V er2 apam il, V R P, ) ; (V incristine, V CR., ) ; P2gp JSB 21 ( ) ; AM V (GibcogBRL ) ; T ripure (GibcogBRL ) ; T aq ( ) ; ( ). 1. 2 1. 2. 1 M T T 0125%, 10% R PM I 1640 3 10 3 gml, 96, : 2004210229. : ( : 200026) ( : 200104). : (1962 ),,,,. E2m ail: gaixiaodong@ yahoo. com. cn

N o. 8 : 200 ΛL,, 6 : (1) BCDC. 5 ΛL BCDC 300, 320, 340, 350, 360, 380, 390, 400 460 ΛggmL ; (2) BCDC+ V CR. BCDC 300, 320, 340, 350, 360, 380, 390, 400 460 ΛggmL, 5 ΛL V CR ( 0102 ΛggmL ) ; (3) V R P+ V CR. V R P 5 ΛL ( 5 ΛggmL ), 5 ΛL V CR ( 0102 ΛggmL ) ; (4), ; (5) V CR. 5 ΛL V CR ( 0102 ΛggmL ) ; (6),. 37 5% CO 2 4 d, 50 ΛL M T T (1 m ggml ), 4 h,, 150 ΛL DM SO, 10 m in, OD 570 nm. : (% ) = OD 570 - OD 570 OD 570 - OD 570 = 1. 2. 2 [ 6 ], CD I= A B g(a B ) CD I, A B, A B, CD I< 1,. 1. 2. 3 Bel27402 V CR [7 ] Bel27402 25 ml, 5 10 5 gml, 24 h, 3 : 110 ΛggmL ) ; (1) Bel27402 + V CR ( (2) Bel27402 + V CR ( 110 ΛggmL ) + V R P ( 5 Λgg ml ) ; (3) Bel27402 + V CR ( 110 ΛggmL ) + BCDC ( 390 ΛggmL ). 24 h, 900 rgm in 10 m in, 019% N ac l 3, 15 ml g, 900 rgm in 7 m in,, 114 ml DDW, - 20 3, 900 rgm in 30 m in,, 4,. Zo rbax2od S, 0102 mo lgl K 2H PO 4 (ph = 616) 2 ( 20 80), 110 ml g m in, 50, 267 nm. 1. 2. 4 Bel27402 10 5 gml, 80% 90%, : (1)Bel27402 ; ( 2)Bel27402 + V CR ( 0102 ΛggmL ) + V R P ( 5 Λggm l) ; (3) Bel27402 + BCDC ( 390 ΛggmL ) ; (4) Bel27402 + BCDC ( 390 ΛggmL ) + V CR ( 0102 ΛggmL ). 3,, 900 rgm in 5 m in,, 5 10 6 gml, PBS 2, 5 ml 70%,, 4 24 h, 1 000 rgm in 5 m in,, PBS 2, PBS 1 ml, RN ase, 50 ΛggmL, 37 1 h, (P I), 50 ΛggmL, 4, 30 m in, 200,. 1. 2. 5 P2gp S2P P2 (P2gp ). 1. 2. 6 R T 2PCR M DR Bel27402 10% R PM I1640, 37 5% CO 2, 3 10 4 gml. Bel27402 ; Bel27402 + 390 ΛggmL BCDB, 24, 48, 72 96 h M DR. T rizo l RNA, R T 2PCR, cdna - 20. PCR : M DR 1, M R P, GST Π, Topog Α Β2actin 1. : 94 3 m in, 94 30 s, 57 45 s, 72 1 m in, 72 10 m in 30, 115%., M DR 1gΒ2actin, M R PgΒ2actin, GST ΠgΒ2actin Topog gβ2actin, M DR 1, M R P, GST Π Topog ΑmRNA. 1447

V o l. 26 Gene M DR1 Table 1 The sequence of pr imers and the length of multiplication products(bp) M ultip lication M ultip lication Sequence of p rim ers Gene Sequence of p rim ers p roduct (bp) p roduct (bp) 5 CCGCT GT TCGT T TCCT T TA GGT 3 136 GST Π 5 CCGT GGTCTA T T TCCCA GT TCG3 466 5 TCT TCT T T GCTCCTCCA T T GCG3 5 CA GCAA GTCCA GCA GGT T GTA G3 Topog Α 5 GGGT T TA CGA T GAA GA T GT T GGC3 114 Β2A ctin 5 AA GA GA GGCA TCCT GA CCCT 3 218 M RP 5 GGGTCCCT T T GT T T GT T GTCCG 3 5 A T GCTCA CT T TCT GGCT GGTA G3 460 5 A CCT T GGAA CTCTCT T TCGGCT 3 2 2. 1 Bel-7402 5 TA CA T GGCT GGGGT GT T GAA 3 300 390 ΛggmL,,, P > 0105; 400 ΛggmL,, P < 0105. 390 ΛggmL Bel27402 V CR,, P < 0101, Bel27402, 390 ΛggmL ( 2)., CD I 0189, 390 ΛggmL, 718, V R P 1516. A dded arug Cell Contro l V CR V RP+ V CR Table 2 D ose-dependen t exper imen t of BCDC reversal on Bel-7402 Cell L ine 3 01667 01037 01683 01007 01582 01015 b lg(λg ml - 1 ) 0 390 400 460 BCDC 01667 01051 01549 01039 c 01438 01038 b BCDC+ V CR 01608 01012 d 01549 01050 d 01296 01033 d a. T he date are the m ean values SD, n= 6; b. P < 0101 as compared w ith the contro l cell; c. P < 0105, as compared w ith the con2 tro l cell; d. P < 0101 as compared w ith the cells treated by V CR. 2. 2 Bel-7402 VCR, V CR, V CR, V CR 21957 m in,., ngg10 ΛL. 390 ΛggmL (131287 01015) nggm 2 (121716 01088) nggm 2, P < 0101, (131506 01007) nggm 2. V CR V CR. 2. 3 Bel-7402, (390 ΛggmL ),, V CR G2 30187% 37143% (P < 0101), V R P+ V CR G2 (42177% ), ( 3). Bel27402 M DR V CR, V CR,, G2 [8 ]. 10%,, Bel27402 V CR. Group Table 3 The effect of BCDC treatmen t on Cell Cycle a Cell Cycle (% ) G0G1 S G2M Bel27402 60160 0170 10103 0171 29137 1101 Bel27402+ V RP+ V CR 51180 1166 5147 1125 42177 0165 b Bel27402+ 390 ΛggmL BCDC 58113 1127 11100 0192 30187 0135 Bel27402+ 390 ΛggmL DCDC+ V CR 56140 1140 6153 0164 37143 0190 b a. T he cell cycle w as analyzed after 3 days of expo sure to the compounds as described in the experim ental m ethods. A ll data w ere the m ean value SD in trip licate. b. P < 0101 as compared w ith contro l cell group. 1448

N o. 8 : 2. 4 P-gp P2gp [ 1 (A ) ],, Bel27402 P2gp [ 1 (B ) ]. M DR M DR 1 P2170. P2170,, [9, 10 ]. P2170,., F ig. 1 Expression of P-gp in Bel-7402 cells (A ) Bel27402 cells. S2P m ethod 400; (B) Bel27402 cells after BCDC treatm ent fo r 3 d. S2P m ethod 400. 2. 5 4. M DR 1, GST Π Topog Α Bel27402, M R P 30. M DR 1 mrna, ; Topog Α, ; GST Π ( 2). Table 4 The expression changes of mrna related to MD R in Bel-7402 cell treated with DCDC 3 Group T im egd DR 1 GST Π Topog Α Bel27402 1 0181 0103 1160 0114 2123 0105 2 0180 0107 1157 0105 2117 0109 3 0181 0104 1173 0112 2117 0105 4 0184 0101 1173 0109 2110 0108 Bel27402+ BCDC 390 ΛggmL 1 0175 0102 1143 0105 2110 0108 2 0165 0106 1140 0136 2183 0105 a 3 0145 0101 a 1153 0112 3177 0112 a 4 0165 0101 a 1183 0112 3103 0147 3 M ean SD, n= 3; a. P < 0101 as compared w ith contro l cell group at sam e tim e. M DR 1 P2gp,. M R P ABC, M R P [11 ]. GST, GST Π. [12 ], F ig. 2 The level of MD R1, GSTΠ and Topog Α gene transcr ipt in Bel-7402 and Bel-7402 cells treated by 390 ΛggmL BCDC (lane 1, 2, 3, 4, 5, 6 respectively) DNA DNA,. Topog,, DNA 2Topog. Topog, [3 ].., Bel27402 M R P, M DR 1gmRNA P2gp, Topog ΑmRNA, 1449

V o l. 26 Topog Α. 3, Bel27402 M DR, Bel27402 M DR 1gmRNA, P2, Topog Α mrna, Bel2 7402 V CR V CR, V CR G2M.,,. [ 1 ] Endico tt J. A., L ing V.. A nn. Rev. B iochem. [J ], 1989, 58: 137_ 171 [ 2 ] T etu B., B risson J., P lante V. et a l.. M od. Patho l. [J ], 1998, 11 (9) : 823_ 830 [ 3 ] Pomm ier Y., L eteurte F., Fesen M. R. et a l.. Cancer Invesṫ [J ], 1994, 12: 530 542 [ 4 ] T suruo T., L ida H., T sukago sh i S. et a l.. Cancer Res. [J ], 1981, 41: 1967_ 1972 [ 5 ] GAO X ian2jun ( ). Journal of Integrated T raditional Ch inese and W estern M edicine ( ) [J ], 1990, 10 (7) : 447_ 448 [ 6 ] SU J ian ( ), ZH EN Yong2Su ( ), Q I Chang2Q ing ( ) et al.. A cta Pharm aceutica Sinica ( ) [J ], 1994, 29: 656_ 661 [ 7 ] L I D an2rong ( ), TU W en2sheng ( ), L I L i( ) et al.. Ch inese Journal of Ch rom atography ( ) [J ], 1998, 16 (1) : 50_ 51 [ 8 ] L in H. L., L iu T. Y., W u C. W. et a l.. B r. J. Cancer[J ], 1999, 8 (13) : 416_ 422 [ 9 ] Scam bia G., Ranelletti F. O., Benedetti Panici P. et a l.. Cancer Chemo theṙ Pharmoco l. [J ], 1994, 34: 459_ 464 [ 10 ] L in H. L., L iu T. Y., L uiw. Y. et a l.. Cancer[J ], 1999, 85 (9) : 1937_ 1942 [ 11 ] Jedlitschky G., Kepp ler D.. V itam Ho rm [J ], 2002, 64: 153_ 184 [ 12 ] T ew K. D.. Cancer Res. [J ], 1994, 54: 4313_ 4320 Effect of Bupleurun Ch inese DC (BCDC) on the MD R Reversal of Hepatocellular Carc inoma and Related M echan ism GA I X iao2dong 13, ZEN G Chang2Q ian 1, HON G M in 2 (1. L aboratory of L if e S cience and B iolog ical T echnology, B eihua U niversity, J ilin 132013, Ch ina; 2. M ed icine P rotein and P roteom ics laboratory, J ilin U niversity, Chang chun 130023, Ch ina) Abstract T he reversal effect and m echan ism s of BCDC w ere evaluated by u sing Bel27402 cell line w ith innate resistance in th is study. T he iden tification of new and less tox ic reversal agen t, BCDC, w ou ld open a new perspectives in cancer treatm en ṫ T he resu lts show that BCDC has an effect on M DR reversal of Bel27402 cell line and a synergetic effect w ith V CR. T he reversal m echan ism s w ere due to supp ress M DR 1 tran scrip tion and P2gp exp ression, as a resu lt, to leave TopogΑmRNA level, to increase in tracellu lar V CR concen tration of Bo l27402 cell, to enhance cell G2M phase arrest induced by V CR. Keywords H epatocellu lar carcinom a; M u ltidrug resistance (M DR ) ; M DR reversal; V incristine (V CR ) ; Bup leu run Ch inese DC (Ed. : D, I) 1450