Transcript Low dose COX-2 inhibitor ( use additive or synergistic effect on Hep3B.

Low dose COX-2 inhibitor ( use
etodolac ) and arsenic trioxide have
additive or synergistic effect on
Hep3B.
＊
HepG2＝＞lower sensitivity to
AS2O3
學生 : 清大生科三年級
指導教授 : 成大生化所
黃玫璇
賴明德老師
vichael’s 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。
Target Cell Line
Human hepatoma-derived cell line
=> Hep3B
(Huh-7, ML-15a, HepG2)
Normal human hepatic cell line
=>Chang liver
Characteristics of The Cell Line
# Hep 3B
Type: Human hepatoblastoma
Tissue: liver; hepatoblastoma
＊The line lacks the expression of both p53
and retinoblastoma tumor suppressor
genes (deletions).
# 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 fingerprinting,
to have been established via HeLa cell
contamination.
Introduction of COX-1 & COX-2
# COX-1
constitutively expressed in vitually all tissues under
basal conditions
# COX-2
over expression in different cancers
ex：Colon cancer, Lung cancer,
Hepatocellular carcinoma.
inducible by the oncogenes ras, src and other
cytokines
＊ Stimulates cellular division, angiogenesis,
metastasis
＊ Inhibits apoptosis
Medication
# 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 inhibition and
1. Hematologic malignancies
apoptosis
2. Solid cancer
(1)carcinoma
(2)sarcoma
(3)others: ex: germ cell tumors
COX-1
COX-1
COX-2
COX-2
Glutathione
peroxidase
Arachidonic acid-------->PGG2--------->PGH2
(H2O2------------>2H2O)
-- >PGI2, PG2α, PGD2 , PGE2…
(in blood vessel, kidney, spleen, heart…)
# Glutathione(GSH)
detoxication 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 injury & by COX-1 and COX-2
Technique
Cell Culture => Standard Curve
MTT assay => Combine assay
Cell Culture
seeding (24 well)：37℃,12~16hour
－－＞remove medium,add 200μl MTT working
solution/well： 37℃,4hour
－－＞remove liquid,add
600μlDMSO/well(pipette up and down
to depart cells from dish.)：37℃,10min
－－＞transfer 150μl/well to the 96 well plate
－－＞measure OD at 540nm
MTT Assay
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.)
2. Step:
cell seeding 1×104 cell number/well(24well)：
37℃,12~16hour
－－＞remove medium, add different
concentration etodolac 1ml/well：
37℃,48~72hour
－－＞remove liquid, add 200μl MTT working
solution/well：37℃,4hour
－－＞remove liquid, add 600μl DMSO/well
(pipette up and down to depart cells from
dish.)：37℃,10min
－－＞transfer 150μl/well to the 96 well plate
－－＞measure OD at 540nm
－－＞killing curve
－－＞combination assay
Combination Assay
Use A only : kill a% target cell
Use B only : kill b% target cell
Combine A and B : kill target cell more than (a+b)%
=> A and B have additive or synergistic effect
to the target cell.
Aim 1st
Cell Culture ＝＞Standard Curve
( OD at 540nm v.s cell number/ml )
＊ cell line: Hep3B
# Target cell : Hep3B
OD. 540nm
2.5
2
1.5
1
0.5
0
1×104
5×104
(cell
10×104
number/ml)
15×104
# Target cell : Hep3B
2.5
OD 540nm
2
1.5
1
0.5
0
1×104
2.5×104 5×104 10×104 15×104
(cell
number/ml)
# Target cell : Hep3B
OD(540nm)
2.5
2
1.5
1
0.5
0
1×104
2.5×104 5×104 10×104 15×104
(cell
number/ml)
# Target cell : Hep3B
2.5
OD 540nm
2
1.5
1
0.5
y = 0.4928x - 0.2556
R2 = 0.9849
0
(cell number/ml)
*Standard Curve ( OD at 540nm v.s cell number/ml )
Aim 2nd
MTT assay
＝＞cell killing curve( etodolac only / ATO only )
* chang liver, Hep3B
＝＞combination assay
* chang liver
# Target cell : Hep3B
0.6
OD(540nm)
0.5
0.4
0.3
0.2
0.1
0
0
0.0625
0.125
0.25
Etodolac con.( mM )
0.50
1.0
120
Hep3B
Cell Number %
100
80
60
40
20
0
0.0
0.5
1.0
1.5
2.0
ETodolac (mM)
2.5
3.0
3.5
# Target cell : chang liver
OD(540nm)
0.8
0.6
0.4
0.2
0
0
0.0625 0.125 0.25 0.50 1.0
Etodolac con.( mM )
*提高藥物劑量，做出毒殺曲線
2.0
3.0
chang liver cell line
120
Cell Number %
100
80
60
40
20
0
0.0
0.5
1.0
1.5
2.0
etodolac (mM)
2.5
3.0
3.5
# Target cell : chang liver
OD(540nm)
0.8
0.6
0.4
0.2
0
0
0.5
1.0
2.0
4.0
AS2O3 con. (μ M )
5.0
8.0
10.0
120
Cell Number %
100
80
Col 2 vs Col 1
60
40
20
0
0
2
4
6
AS2O3 (M)
8
10
12
Combination Assay
# 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 (μM )
*反應時間48, 72 hour
*OD at 540 nm
chang liver
120
*
100
<- A ->
48 hr
72 hr
<- B ->
Cell Number %
<- C ->
<- D -> <- E ->
80
60
40
20
0
Etodolac(mM)
AS2O3(μM)
– 0.125 0.25 0.5
–
–
–
–
–
–
– 0.125 0.125 0.125 0.25 0.25 0.25 0.5 0.5
0.5
0.5
2
5
5
0.5
2
5
0.5
2
5
0.5
* : no etodolac & AS2O3
A : etodolac only
B : AS2O3 only
C.D.E : combine etodolac & AS2O3
2
Result
Low dose etodolac and arsenic
trioxide have no synergistic effect
to chang liver.
*
More experiment must be done to test
whether The result is verifiable.
Discussion
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 ability to human hepatomaderived 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 contaminated.
~~~ THE END ~~~
Thanks for Dr. M.D. Lai , and all
enthusiastic persons that had given me
assistance.
2002 vichael’s summer
附註資料
NSAIDs(nonsteroidal anti-inflammatory
drugs) =>inhibit COX-1 and COX-2
inhibit COX-1 ＝> NSAIDs-induced gastropathy
selecitive COX-2 inhibitor ＝> less side-effect