LowdoseCOX-2inhibitor(useetodolac)andarsenictrioxide

1、Low dose COX-2 inhibitor ( use etodolac ) and arsenic trioxide have additive or synergistic effect onHep3B. HepG2lower sensitivity to AS2O3 學生學生 : : 清大生科三年級清大生科三年級 黃玫璇黃玫璇 指導教授指導教授 : : 成大生化所成大生化所 賴明德老師賴明德老師 vichaels 2002 summer in NCKU賴明德教授 ( Dr.M.D. Lai )從事分子生物學、腫廇分生學、致癌基因之研究實驗室的研究主要在了解致癌基因及腫瘤抑制基因如何

2、交互作用而使細胞產生癌化現象。目前的研究集中在致癌基因neu、Ras及腫瘤抑制基因P53 的交互作用及在細胞內的作用機轉。 Human hepatoma-derived cell line = Hep3B (Huh-7, ML-15a, HepG2) Normal human hepatic cell line =Chang liverTarget Cell Line# Hep 3BType: Human hepatoblastomaTissue: liver; hepatoblastoma The line lacks the expression of both p53 and retin

3、oblastoma tumor suppressor genes (deletions).Characteristics of The Cell Line# Chang liver Type: HeLa derivative Tissue: HeLa contaminant This line was originally thought to be derived from normal liver tissue, but was subsequently found, based on isoenzyme analysis, HeLa marker chromosomes, and DNA

4、 fingerprinting, to have been established via HeLa cell contamination.# COX-1constitutively expressed in vitually all tissues underbasal conditions# COX-2over expression in different cancers ex：Colon cancer, Lung cancer, Hepatocellular carcinoma. inducible by the oncogenes ras, src and other cytokin

5、es Stimulates cellular division, angiogenesis, metastasis Inhibits apoptosisIntroduction of COX-1 & COX-2# Etodolac Selective COX-2 inhibitor =inhibit COX-2 tenfold more than COX-1# AS2O3 A novel anticancer agent for the treatment of solid cancer * Low concentration induces cell growth inhibitio

6、n and apoptosis Medication* 1. Hematologic malignancies 2. Solid cancer (1)carcinoma (2)sarcoma (3)others: ex: germ cell tumorsArachidonic acid-PGG2-PGH2 (H2O2-2H2O)- PGI2, PG2, PGD2 , PGE2 (in blood vessel, kidney, spleen, heart)COX-1 COX-2COX-1 COX-2Glutathione peroxidase# Glutathione(GSH) detoxic

7、ation in mammalian cells = ex : H2O2-2H2O effect on arsenical-induced cell injury =Cancer cells that were intrinsically sensitive to AS2O3 contained lower level of GSH. = induced PGI2 biosysthesis ( cytoprotective effect )# NSAIDs inhibit the PGI2 biosysthesis induced by GSH in AS2O3-induced cell in

8、jury & by COX-1 and COX-2Cell Culture = Standard Curve MTT assay = Combine assayTechniqueseeding (24 well)：37,1216hourremove medium,add 200l MTT working solution/well： 37,4hourremove liquid,add 600lDMSO/well(pipette up and down to depart cells from dish.)：37,10mintransfer 150l/well to the 96 wel

9、l platemeasure OD at 540nmCell Culture MTT：3-(4,5-cimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide 1. Principle： MTT is a pale yellow substrate that is cleaved by living cells to yield a dark blue MTT formazan. (This process requires active mitochondria.)MTT Assay 2. Step: cell seeding 1 104 c

10、ell number/well(24well)： 37,1216hour remove medium, add different concentration etodolac 1ml/well： 37,4872hour remove liquid, add 200l MTT working solution/well：37,4hour remove liquid, add 600l DMSO/well (pipette up and down to depart cells from dish.)：37,10min transfer 150l/well to the 96 well plat

11、e measure OD at 540nm killing curve combination assayUse A only : kill a% target cellUse B only : kill b% target cellCombine A and B : kill target cell more than (a+b)% = A and B have additive or synergistic effect to the target cell.Combination AssayCell Culture Standard Curve ( OD at 540nm v.s cel

12、l number/ml ) cell line: Hep3BAim 1st 1104 5104 10104 15104 (cell number/ml)# Target cell : Hep3B00.511.522.5OD. 540nm1104 2.5104 5104 10104 15104 (cell number/ml)# Target cell : Hep3B00.511.522.5OD(540nm)1104 2.5104 5104 10104 15104 (cell number/ml)# Target cell : Hep3B (cell number/ml)*Standard Cu

13、rve ( OD at 540nm v.s cell number/ml )# Target cell : Hep3BMTT assay cell killing curve( etodolac only / ATO only ) * chang liver, Hep3Bcombination assay * chang liverAim 2nd# Target cell : Hep3B 0 0.0625 0.125 0.25 0.50 1.0 Etodolac con.( mM )0OD(540nm)Hep3BETodolac (mM)0.00.51.01

14、.52.02.53.03.5Cell Number %02040608010012000.8OD(540nm)# Target cell : chang liver 0 0.0625 0.125 0.25 0.50 1.0 2.0 3.0 Etodolac con.( mM )*提高藥物劑量，做出毒殺曲線chang liver cell lineetodolac (mM)0.00.51.01.52.02.53.03.5Cell Number %02040608010012000.8OD(540nm) 0 0.5 1.0 2.0 4.0 5.0 8.0 10.

15、0 AS2O3 con. ( M )# Target cell : chang liverCol 2 vs Col 1 AS2O3 (M)024681012Cell Number %020406080100120# Etodolac 0, 0.0625, 0.125, 0.25, 0.5, 1.0, 2.0, 3.0 ( mM )# ATO 0, 0.5, 1.0, 2.0, 4.0, 5.0, 8.0, 10.0 (M ) *反應時間48, 72 hour *OD at 540 nmCombination Assay * : no etodolac & AS2O3 A : etodo

16、lac only B : AS2O3 only C.D.E : combine etodolac & AS2O3 Etodolac(mM) 0.125 0.25 0.5 0.125 0.125 0.125 0.25 0.25 0.25 0.5 0.5 0.5 AS2O3(M) 0.5 2 5 0.5 2 5 0.5 2 5 0.5 2 5 chang liverCell Number %02040608010012048 hr72 hr *Low dose etodolac and arsenic trioxide have no synergistic effect to chang

17、 liver. * More experiment must be done to test whether The result is verifiable.Result If the result is verrifiable, we can expect its application in clinical medicine since low dose etodolac and ATO have no synergistic effect to chang liver (normal human hepatic cell line) but have higher killing a

18、bility to human hepatoma-derived cell line,for example, Huh-7 and ML15a, than use them alone. (according to the data of 洪瑞祥學長in the M.D. lai lab.of NCKU) If it is verifiable, then unfreeze the same group of the chang liver used before. Observe its morphology to check whether the chang liver cell line is contaminat