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Characterization of the combinational effects of DNA hypomethylating agents and histone deacetylase inhibitors in human acute myeloid leukemia 研究生: 林秀盆.

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Presentation on theme: "Characterization of the combinational effects of DNA hypomethylating agents and histone deacetylase inhibitors in human acute myeloid leukemia 研究生: 林秀盆."— Presentation transcript:

1 Characterization of the combinational effects of DNA hypomethylating agents and histone deacetylase inhibitors in human acute myeloid leukemia 研究生: 林秀盆 指導教授 : 劉興璟 博士 林建煌 教授 林建煌 教授

2 Introduction

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5 HDACIs

6 HDAC(Histone Deacetylase ), HAT(Histone Acetyltransferase)

7 (Cancer cell:July 2003)

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

10 MATERIALS AND METHODS

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12 HL-60 AML FAB M2

13 1)Histone Deacetylase Inhibitors -Sodium Butyrate,Phenylbutyrate & SAHA Induce Death of HL-60 Cells.

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

15 HDACIs apicidin and CBHA induced cell death via activation of the death receptor pathway (Bernhard et al., 1999)HDACIs apicidin and CBHA induced cell death via activation of the death receptor pathway (Bernhard et al., 1999) SAHA and sodium butyrate, show no requirement for the death receptor pathway (Ruefli,A.A.,2001)SAHA and sodium butyrate, show no requirement for the death receptor pathway (Ruefli,A.A.,2001)

16 HL-60untreatPB=1mMSB=1mMSAHA=1uMSAHA=2uM CD950.19±.01%4.2±.21%0.2±.01%0.21±.02%0.3±.02% Table.1 Phenylbutyrate induced cell death may be via activation of the death receptor pathway, SAHA and sodium butyrate Phenylbutyrate induced cell death may be via activation of the death receptor pathway, SAHA and sodium butyrate may be not.

17 HDACIs effect Cell Cycle Arrest HDACIs effect Cell Cycle Arrest (Richon et al.,2000) (Richon et al.,2000)

18 2)Can Sodium Butyrate,Phenylbutyrate & SAHA effect Cell Cycle Arrest ?

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

20 HDACIs can Antiproliferative activity in cancer cells. (Jaboin et al., 2002)

21 Sodium butyrate, 3)Can Sodium butyrate, Phenylbutyrate & SAHA Phenylbutyrate & SAHA effect cells proliferation?

22 Day0 untreated SB=2mM Fig.3) CFSE stain - proliferation Table 1.

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

24 HDACIs Induce Cancer cells Differentiation.HDACIs Induce Cancer cells Differentiation. (Ferrara et al.,2002;Gottlicher et al.,2001) (Ferrara et al.,2002;Gottlicher et al.,2001)

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

26 SAHA=2uM PB=2mM Fig.4a)NBT(nitroblue tetrazolium) reduction test untreated SB=2mM

27 untreatedmacrophage vitD3=0.1uM SAHA=2uMSB=2mMPB=2mM Fig.4b) α-Naphthyl Acetate Esterase stain

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29 HL-60CD11bCD13CD14CD95 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%

30 Sodium Butyrate,Phenylbutyrate & SAHA Can Induce HL-60 Monocytes

31 HL-60PBSBSAHA cell death cell cycle arrest antiproliferation cell differentiation CD95

32 5-azacytidine and histone deacetylase inhibitors are synergistic with demethylation for reexpressing the silenced genes (Jones and Baylin, 2002)

33 5AZA in combination with HDAC inhibitors produces a synergistic antineoplastic effect against leukemia cells. (Shaker et al., 2003).

34 B).Hypothesis:B).Hypothesis: Synergy of DNA methylation inhibitor(Zebularine) and HDACI (SAHA) in the re-expression of genes silenced in Leukemia. Synergy of DNA methylation inhibitor(Zebularine) and HDACI (SAHA) in the re-expression of genes silenced in Leukemia.

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36 inhibitor DNAMethyltransferases and inhibitors (Cheng JC, et al.,2003)

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

38 Zeb SAHA

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

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46 HDAC & suberoylanilide hydroxamic acid(SAHA)

47  The HDACIs SAHA, sodium butyrate, and trichostatin A have all been shown to induce apoptosis of CEM cells (Ruefli,A.A.,2001)

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49 untreatedSB=1mM PB=1mMSAHA=1uM

50 Hypermethylation of E-cadherin in leukemiaHypermethylation of E-cadherin in leukemia ( Melki,John R., BLOOD, 15 MAY 2000) ( Melki,John R., BLOOD, 15 MAY 2000)

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