------------------------------------------------------------------------------------------I -----------------------------------------------------------------------------------------II ------------------------------------------------------------------------------------------1 ----------------------------------------------------------------------------------------37 Eb --------------------------37 1. MTT SRB Eb ----------------38 2., Eb DDP Lewis ---53 3. MTT SRB Eb -------------54 4. Eb ---------------------------------------------------------------56 5. -------------------------------------------------------------------------------------62 Eb ADM DDP TAXOL --------------------------64 1. ----------------------------------------------------------------------------65 2. -------------------------------------------------------------65 3. ----------------------------------------------------------------------65 4. -------------------------------------------------------------------------------------84 Eb Eb ---------86 1. ------------------------------------------------------------------------------------86 1-1 FCM Eb DNA --86 1-2 Eb ---------88 1-3 Annexin-V --------------------------------------------------88 1-4 -------------89 2. ---------------------------------------------------------------------------89 3. Eb -----------------------------------------100
3-1 Hoechst 33342 Eb --100 3-2 Annexin-V FCM Eb --103 4. ----------------------------------------------------------------------------------104 Eb Eb GSH GSH-PX ----------------------------------------106 1. -------------------------------------------------------------------------------106 2.Eb GSH ---------------------------107 3.Eb GSH-PX ----------------------109 -----------------------------------------------------------------------------------------111 --------------------------------------------------------------------------------113 -----------------------------------------------------------------------------------------------114 -----------------------------------------------------------------------------------------116
Eb Eb Ebselen 01118666.6 Eb Eb 0.05µmol/L 0.5µmol/L Eb DDP 5.0µmol/L 50µmol/L 100µmol/L Eb DDP P<0.01 Eb Eb DDP ADM A Taxol T Eb DDP ADM A Taxol T FCM Confocus Eb Eb G0/G1 S Eb Eb GSH GSH-PX 0.05µmol/L 0.5µmol/L 1.0µmol/L Eb GSH GSH-PX Eb Eb A/DDP/T
The investigation of anti-tumor activity of a novel organic selenium compound (Eb) and the related mechanism Abstract Eb (Patent No.: 01118666.6), developed by our research group, is a novel organic selenium compound, as one of the derivatives of Ebselen. It showed a positive anti-tumor activity and became a potential agent with the ability of anticancer on the basis of the screening study in anti-inflammation, anti-tumor and cellular immunity. Here, we studied the growth inhibition of Eb to nine human tumor cells and two human normal cells in vitro for the first time. The results showed that Eb has a strong inhibition to those cells, but the effect to the tumor cells is stronger than that to the normal cells. It was also found that the effect of Eb in tumor cells was the same as or slightly weaker than DDP when the concentration was 0.05µmol/L or 0.5µmol/L, but the effect was much more powerful than that of DDP when the concentration was 5.0µmol/L, 50µmol/L, or 100µmol/L, and there was significant difference between them (P<0.01). The results suggested that the novel organic selenium compound (Eb) was a potential anti-tumor agent. That was also supported by the experiments on the level of animals. We also examined additive or synergistic effect of Eb in combination with standard anti-cancer drugs, cisplatin (DDP), adriamycin (ADM A), and taxol(t). Furthermore, we discovered that Eb was able to induce apoptosis of tumor cells by using some techniques, such as flow cytometry (FCM), confocus and agarose electrophoresis separation. We observed that Eb effected the G0/G1 and S phase in cell cycle of the apoptosis tumor cells, and that different concentrations had different effects to the preapoptosis, midapoptosis or postapoptosis formation. We also discuss the relationship between concentrations of Eb and the GSH level and GSH-PX activity in tumor cells and in liver and kidney of animals with tumors. The results showed that Eb can up-regulate the GSH level and GSH-PX activity on the level of 0.05µmol/L, 0.5µmol/L and 1.0µmol/L. Key words novel organic Selenium compound Eb Combination of Eb and DDP/ADM/T Anti-tumor activity Anti-tumor mechanism
1817 Berlius alkali disease blind staggers Nelson 1943 50 Clagtan Nelson DAB Schwarz Foltz 1957 V E 1973 Rotruck Hoekstra GPX [1] WHO 1978 35 [2] 17 (Selenocysteine, Sec) (Selenomethionine,Se Met) Sec UGA
[3] 1. 2 0 60, [4] Na 2 SeO 3 20 200 1970 Na 2 SeO 3 [5] 1. 2. T 2 3.
[6,7] [8] Na 2 SeO 3 [9,10] 1 C 2 3 4 5 [11 12] M Ig M G Ig G [6] [13 14] HIV 20 Gladyshev HIV T c-gpx ph-gpx TR 15-kDa [15] [16] [6] [6]
1. 2. 3. 4. [6] [16 17] [18] ph-gpx ph-gpx MCS [19] Simonoff Wang Lifen Schlieuger Adnan [20] E C [21] 1 2 camp MAP 3 S6 4 Berg [22] Lizuka [23 ] mrna, Zhu [24] Osamu [21], Na 2 SeO 4 Na 2 SeO 3,,,,,
,,,, E Vit E [25], Vit E Vit E, Vit E Vit E,,, 2.,, Wenberg 0.1 2ppm 3 10ppm 10ppm [26], 10,30 50 [27], 2.1 1856 Nebraska K.W.Franke alkali disease blind staggers 60 [28 29] [30 31]
PH As Ag Cu [32] [4] 0.02mg/L, 1.02mg/L ( ) 22 g/d ( ) 50 g/d ( ) 550 g/d 400 g/d 200-250 [33 34] 2.2, 317,,,,, 0.2 g/g 3 4 g/g 16 g/g [35,36] L- D- [37],
, [38] 2.3, Seko [39], Hsien Ganther 1 [40] (SeO3 2- ) (GSH) H 2 Se (O 2- ) 1, [41] Hu [41] 1991 Yan [42],, L- H 2 O 2, H 2 O 2 Kitahara [43,44] DNA, HO (ESR) H 2 Se O 2 HO,Seko,, GSH-PX O - 2, GSH-PX Se 2-, DNA,
, Se 2- RseH Spallholz [45], :1), 2) (Selenocytamine) RSeH,, 3) (Selenothers), 4) (Selenol), 5),,, H 2 O 2 ROOH,, [46] S- - - -,,,, [46],, [46],,,, S- (SAM) Hoffman [47], SAM S- (SAH), SAM,
50% [47] SAM,,, SAM,, (GSH-PX), GSH-PX, [48] GSH-PX, 1mg/Kg,, Behne [49] 2 g/g, - (IDI) [50], ( Kg 5mg) GSH-PX, GSH-PX, IDI, (PHGPX), Whanger [51] SD ( 0.2 1.0 2.0 4.0 g/g) 9, 1 g/g, GSH-PX Masaaki [52] DDY 7.5mg/Kg,72h (AM)-N- (EM)-N- P450, (AN) 7- (EC)-O- NADPH C NADPH b5 1 2 g/g 12, 4 8 g/g, AM-N- AN P450, AM-N- AN EC-O- P450 - - - ( -ALAD)
[53] Barbosa [54] -ALAD, Se-Se Se-C,,, [7],, (10 20 g/g) AST ALT A V A SD 2.05 4.05 g/g, 36% 49% [46] V A, Chen [47] 1 2.5 5 10 15 g/g,,, 15mg/L,,,,, 3. 92% 92% 81% 90% 95% 60% [48 49]
[50] [51] 82 95 26 [52] 2 8 HPLC FLD [53] 20 50 [54] 35 3 Ganther 87 40 20 8 40 6 [54]
[6] 1 1 > > > > > > > > > > > 15mg > > > > > > > 0.030±0.004mg/kg 0.0430±0.012mg/kg 0.121 0.14 mg/kg 0.25 mg/kg [55] 15kDa 2/3 34kDa [56] GPX P 50 [57 58] P [59] W