Characterization of the combinatioCharacterization of the combinational effects of DNA hypomethylatinnal effects of DNA hypomethylating agents and histone deacetylase g agents and histone deacetylase inhibitors in human acute myeloid inhibitors in human acute myeloid

leukemialeukemia 研究生： 林秀盆研究生： 林秀盆指導教授指導教授 : : 劉興璟 博士劉興璟 博士 林建煌 教授林建煌 教授

IntroductionIntroduction

HDACIs

HDAC(Histone Deacetylase ),HAT(Histone Acetyltransferase)

(Cancer cell:July 2003)

• A).Compare the effects of sodiA).Compare the effects of sodium butyrate, phenylbutyrate & um butyrate, phenylbutyrate & suberoylanilide hydroxamic acsuberoylanilide hydroxamic acid(SAHA) on apoptosis, cell cycid(SAHA) on apoptosis, cell cycle arrest, cell proliferation and le arrest, cell proliferation and differentiation in HL-60 differentiation in HL-60 . .

MATERIALS AND MATERIALS AND METHODSMETHODS

HL-60 AML FAB M2

1)1) Histone Deacetylase InhibitorsHistone Deacetylase Inhibitors -Sod -Sodium Butyrate,Phenylbutyrate & SAium Butyrate,Phenylbutyrate & SAHA Induce Death of HL-60 Cells. HA Induce Death of HL-60 Cells.

Fig.1)Sodium Butyrate,Phenylbutyrate, & SAHA Induce Death of HL-60 Cells in a dose- and time-dependent manner.

• HDACIs apicidin and CBHA induced ceHDACIs apicidin and CBHA induced cell death via activation of the death recell death via activation of the death receptor pathway ptor pathway

(Bernhard et al., 1(Bernhard et al., 1999)999)

• SAHA and sodium butyrate, show no rSAHA and sodium butyrate, show no requirement for the death receptor patequirement for the death receptor pathwayhway

(Ruefli,A.A.,2001)(Ruefli,A.A.,2001)

HL-60 untreat PB=1mM SB=1mM SAHA=1uM SAHA=2uM

CD95 0.19±.01% 4.2±.21% 0.2±.01% 0.21±.02% 0.3±.02%

Table.1

Phenylbutyrate induced cell death mPhenylbutyrate induced cell death may be via activation of the death receay be via activation of the death receptor pathway , SAHA and sodium butptor pathway , SAHA and sodium butyrateyrate may be not.

• HDACIs effect Cell Cycle ArrestHDACIs effect Cell Cycle Arrest

(Richon et al.,2000)(Richon et al.,2000)

2)Can2)Can Sodium Butyrate,PhenylbutyratSodium Butyrate,Phenylbutyrate & SAHA effect Cell Cycle Arrest ?e & SAHA effect Cell Cycle Arrest ?

Fig.2) Sodium Butyrate,Phenylbutyrate, & SAHA effect Cell Cycle Arrest.

• HDACIs can Antiproliferative activity in cancer cells.

(Jaboin et al., 2002)(Jaboin et al., 2002)

3)Can Sodium butyrate,Sodium butyrate,

Phenylbutyrate & SAHAPhenylbutyrate & SAHA effect

cells proliferation?

Day0untreated SB=2mM

Fig.3) CFSE stain -proliferation

HL-60-CFSE meanDay0 245.11±5.24

Day4

untreated 42.56±0.56

PB=2mM 67.63±0.83

PB=3mM 84.28±0.92

SB=2mM 104.33±5.48

SB=3mM 124.71±0.19

SAHA=1uM 131.81±4.56

SAHA=2uM 190.61±3.80

Table 1.

• Sodium Butyrate,Phenylbutyrate & Sodium Butyrate,Phenylbutyrate & SAHA SAHA can Antiproliferative activity in a dose- and time-dependent manner.

• HDACIs Induce Cancer cells DifferentiaHDACIs Induce Cancer cells Differentiationtion..

(Ferrara et al.,2002;Gottlicher et al.,2001)(Ferrara et al.,2002;Gottlicher et al.,2001)

• 4) 4) )Can)Can Sodium Butyrate,Phenylbutyrate & Sodium Butyrate,Phenylbutyrate & SAHA Induce HL-60 Differentiation ?SAHA Induce HL-60 Differentiation ?

SAHA=2uM

PB=2mM

Fig.4a)NBT(nitroblue tetrazolium) reduction test

untreated

SB=2mM

untreated macrophage vitD3=0.1uM

SAHA=2uM SB=2mM PB=2mM

Fig.4b) α-Naphthyl Acetate Esterase stain

untreat PB=2mM SB=2mM SAHA=2uM vitD3=100nMRA=2.5um

NBT ± + + ++ + +NAE ± ++ + + + ±

HL-60 CD11b CD13 CD14 CD95

no drug

0.4±0.1% 0.5±0.1% 0.2±0.1% 0.19±0.01%

PB=1mM

2.4±0.32% 1.65±0.7% 2.3±0.1% 4.2±0.21%

SB=1mM

2.5±0.05% 4.2±0.5% 2.3±0.1% 0.2±0.1%

SAHA=1uM

5.1±0.3% 3.1±0.3% 1.2±0.1% 0.21±0.02%

SAHA=2uM

14±0.5% 8.0±0.8% 2.3±0.1% 0.3±0.02%

Sodium Butyrate,Phenylbutyrate & SAHA Can Induce HL-60

Monocytes

HL-60 PB SB SAHA

cell death

cell cycle arrest

antiproliferation

cell differentiation

CD95

• 5-azacytidine and histone deacetylase inhibitors are synergistic with demethylation for reexpressing the silenced genes

(Jones and Baylin, 2002)

• 5AZA in combination with HDAC

inhibitors produces a synergistic antineoplastic effect against leukemia cells.

(Shaker et al., 2003).

• B).Hypothesis:B).Hypothesis: Synergy of DNA methylation inhibitoSynergy of DNA methylation inhibito

r(Zebularine) and HDACI (SAHA) in thr(Zebularine) and HDACI (SAHA) in the re-expression of genes silenced in e re-expression of genes silenced in Leukemia.Leukemia.

DNAMethyltransferases and inhibitorinhibitors

(Cheng JC, et al.,2003)

• E-cadherin gene is a common target for E-cadherin gene is a common target for hypermethylation in hematologic malighypermethylation in hematologic malignancies. (nancies. (Melki et al.,Melki et al.,Blood. 2000)Blood. 2000)

Zeb

SAHA

(Maralice Conacci-Sorrell., J. Clin. Invest.2002)

HDAC & suberoylanilide hydroxamic acid(SAHA)

The HDACIs SAHA, sodium butyrate, The HDACIs SAHA, sodium butyrate, and trichostatin A have alland trichostatin A have all been showbeen shown to induce n to induce apoptosisapoptosis of CEM cells of CEM cells

(Ruefli,A.A.,20(Ruefli,A.A.,2001)01)

untreated SB=1mM

PB=1mM SAHA=1uM

• Hypermethylation of E-cadherin in lHypermethylation of E-cadherin in leukemiaeukemia

• ((Melki,John R., BLOOD, 15 MAY 2000)Melki,John R., BLOOD, 15 MAY 2000)

•