組蛋白去乙醯酶抑制劑抑制肺癌細胞生長之機制探討

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(1)捌、附圖. Fig. 1. Histone acetylation (A) The core histones have histone-fold domains, which interact with other histones and with DNA in the nucleosome, and N-terminal tails, which extend outside of the nucleosome. The N-terminal tails of the core histones (e.g., H3) are modified by the addition of acetyl groups (Ac) to the side chains of specific lysine residues. (B) Transcriptional activators and repressors are associated with coactivators and corepressors, which have histone acetyltransferase (HAT) and histone deacetylase (HDAC) activities, respectively. Histone acetylation is characteristic of actively transcribed chromatin and may weaken the binding of histones to DNA or alter their interactions with other proteins. [Adapted from Ref.(53)]. 60.

(2) Fig. 2. Proposed mechanism of action of histone deacetylase inhibitors. With inhibition of histone deacetylases (HDACs) by HDAC inhibitors such as suberoylanilide hydroxamic acid (SAHA), histones are acetylated, and the DNA that is tightly wrapped around a deacetylated histone core relaxes. It has been proposed that there are specific sites in the promoter region of a subset of genes (eg, SP1 sites) that recruit the transcription factor complex (TFC) with HDAC and that the accumulation of acetylated histones in nucleosomes leads to increased transcription of this subset of genes (eg, CDKN1A, which encodes p21WAF1), which, in turn, leads to downstream effects that result in cell-growth arrest, differentiation, and/or apoptotic cell death. [Adapted and modified from Ref. (54)]. 61.

(3) Fig. 3. Representative structures and localities of classes I and II histone deacetylases (HDACs). References in this figure can be viewed in the online issue (26), which is available at www.interscience.wiley.com 62.

(4) Fig. 4. Classification of HDAC inhibitors. [Taken from Ref. (26)] 63.

(5) Fig. 5. Summary of the HDAC inhibitors in clinical studies [Taken from Ref. (22)]. 64.

(6) Fig. 6. Structural basis for HDAC inhibitors binding toHDAC8 active site. (A) Chemical structures of TSA, SAHA, HTPB and HDAC-44. (B) Molecular docking of SAHA, TSA, HTPB and HTPB into the active-site pocket of HDAC8. 65.

(7) Fig. 7. Dose-dependent effects of CL1-1 cells treated with HTPB, SAHA, and HDAC-44 for 6hr histones H3 and H4 as well as p53 acetylation. (A) Western blot analysis of the CL1-1 cells treated with HTPB, SAHA, or HDAC-44 at indicated concentration for 6 hours. (B) signals from (A) were quantitated by Multi Gauge program and were used to calculated the acetylation status. Ac-H3 and Ac-H4 normalized against that of β-actin or GAPDH, and Ac-p53 normalized against that of p53.. 66.

(8) Fig. 8. Time-dependent effects of CL1-1, A549, and H1299 lung cancer cells treated with HDAC-44, HTPB, and SAHA on expression level of HDAC6, HDAC1, and p53, and acetylation level of p53, histone H3 and histone H4. (A) Western blot analysis with immunoblotted for target protein. (B) Quantitative measurement of relative expression level of Ac-H3, Ac-H4, and Ac-p53 in CL1-1 cells. Ac-H3 and Ac-H4 normalized against that of β-actin or GAPDH, and Ac-p53 normalized against that of p53. 67.

(9) Fig. 9. HDAC-44, HTPB, and SAHA induced p21 and TIMP3 mRNA expressions (A) and p21 and Bcl2 protein expressions (B) in CL1-1, A549 and H1299 cells. Cells were treated with 2.5 µM HDAC-44, 5 µM HTPB, or 5 µM SAHA for indicated time. p21 and TIMP3 transcription level increased apparently by HDAC-44 and HTBP. p21 protein level increased apparently by all HDAC inhibitors, whereas increase of Bcl2 protein was only observed in H1299. 68.

(10) Fig. 10. Real-time RT-PCR of HDAC-44, HTPB, and SAHA induced p21 mRNA expressions. Real-time RT-PCR comparison of induction of p21 mRNA expression between A549 cells treated with 2.5 µM HDAC-44, 5 µM HTPB, or 5 µM SAHA for the indicated times. p21 transcription level increased most apparently by HDAC-44. p21 was normalized against that of β-actin.. 69.

(11) Fig. 11. The cytotoxicity of HDAC-44, HTPB, and SAHA in one normal and three lung cancer cell lines. Cells were treated with HDAC-44, HTPB, or SAHA at the indicated concentrations in 10% FBS-containing DMEM for 48 hours, and cell viability was assessed by trypan Blue exclusion assay. The dose-dependent effects of (A) HDAC-44, (B) HTPB and (C) SAHA on cell viability in IMR90 normal lung cells and CL1-1, A549, and H1299 lung cancer cells. Points, mean; bars, ±SD (n>3). (D) The IC50 cytotoxicity calculated under HDAC-44, HTPB, and SAHA treatments for 48hr of three cancer cell lines.. 70.

(12) Fig. 12. The cytotoxicity of HDAC-44 combination treatment with cisplatin increased the cytotoxicity in lung cancer cells. The cells were exposed to low-dose cisplatin alone for 4 hours, HDAC-44 alone for 48 hours, or pretreatment with low-dose HDAC-44 for 48 hours before low-dose cisplatin treatment for 4 hours. Cell viability was assessed by trypan blue exclusion assay. CL1-1 treated with 4.4 μM cisplatin or 0.3μM HDAC-44. A549 treated with 1.6 μM cisplatin or 0.2 μM HDAC-44. Control cells received DMSO solvent. Points, mean; bars, ±SD (n=3).. 71.

(13) Fig. 13. The effects of HDAC inhibitors on cell cycle distribution in (A) A549 or (B) H1299 cells for 48 hours. Cells treated with different HDAC inhibitors at the indicated concentrations in DMEM with 10% FBS for 48 hours and stained with PI for nuclear staining. Cell cycle distributions were determined on flow cytometry and analyzed by ModFit program. Control cells received DMSO. Numbers in each panel show mean percentage of each phase distributions. A549 and H1299 under HDAC-44, HTPB, or SAHA treatments for 48 hours arrested in G2/M phase and increased in sub-G1 phase as an indication of apoptosis.. 72.

(14) Fig. 14. Time-dependent effects of HDAC-44 on cell cycle distribution in (A) A549 or (B) H1299 cells at 2.5 μM. Cells treated with 2.5 μM HDAC-44 in DMEM with 10% FBS for indicated time. Cell cycle distributions were determined as described in Fig. 9. A549 and H1299 under 2.5 μM HDAC-44 treatments for 24 hours arrested in G2/M phase and Sub-G1 appeared at 48 hours. Increased in sub-G1 phase as an indication of apoptosis fragment.. 73.

(15) Fig. 15. HDAC inhibitors induced lung cancer cell apoptosis and down-regulated anti-apoptotic Bcl-2 protein. (A) The DNA ladder assay. A549 cells treated with DMSO, 5 μM HDAC-44, 10 μM HTPB, or 10 μM SAHA for 48hr. The data showed that the apoptosis specific characteristics of DNA fragmentation was induced after different HDAC inhibitors treatments. (B) The Western blot of anti-apoptosis marker Bcl-2. Under 2.5 μM HDAC-44, 5 μM HTPB, or 5 μM SAHA treatment for indicated time, Bcl-2 down-regulated in both A549 and H1299 lung cancer cell lines. 74.

(16) Fig. 16. HDAC-44 induced cytoskeleton abnormality, apoptosis, 75.

(17) G2/M arrest and/or cytokinesis inhibition in H1299, A549, and CL1-1 lung cancer cells. Cells were analyzed for α–tubulin staining after DMSO or 2.5μM HDAC-44 treatment for 48 hours. Cells were stained by FITC conjugated anti-α-tubulin antibody (shown in green, left panel) and DAPI nuclear stain (shown in blue, meddle panel). The α–tubulin and DAPI staining were merged in right panel. The red arrows indicate cells in the G2/M arrest and/or cytokinesis inhibition. The blue arrows refer to the apoptosis cells. The green arrow shows cytoskeleton abnormality.. 76.

(18) Fig. 17. ChIP-on-chip data of HDAC-44 induced histone acetylation in 52 common genes locus of A549, CL1-1, and H1299 lung cancer cells (A), and the pathways involved by these genes are shown in (B). Cells were treated with 2.5 µM HDAC-44 for 6hrs, and soluble chromatin was immunoprecipitated with anti-acetyl-Histone H3. The DNA isolated from the immunoprecipitated chromatin was used by ChIP-on chip analysis. 77.

(19) 玖、附表 Table 1. Representative Nonhistone Substrates of HDACs [Taken from ref. (26)]. 78.

(20) Table 2. List of antibodies used in the present study (Western blot) antibody. Final conc.. Source. Anti-HDAC6, CT. 1:4000. Upstate, USA, catalog #07-732. Anti-HDAC1. 1:2000. Upstate, USA, catalog #06-720. Acetylated-p53. 1:500. Cell Signaling Technology, USA, catalog #2525. Total p53. 1:1000. Santa Cruz , USA, catalog, catalog #SC-126. α-tubulin. 1:200. Upstate, USA, catalog #05829. β-actin. 1:5000. Novus Biologicals, USA, catalog #NB 600-501. GAPDH. 1:4000. Novus Biologicals, USA, catalog #NB 600-502. Bcl2. 1:2000. Chemicon, USA, catalog #AB1720. P21 Waf1/Cip1 (DCS60). 1:500. Cell Signaling Technology, USA, catalog #2946. Anti-acetyl-Histone H3. 1:4000. Upstate , USA, catalog #06-599. Anti-acetyl-Histone H4. 1:500. Upstate, USA, catalog #06-598. 79.

(21) Table 3. List of PCR primer sequences used in the present study Gene. primer. 5’Æ3’ sequences. p21. Forward. TCACCGAGACACCACTGGAG. (RT-PCR). Reverse. TGGAGTGGTAGAAATCTGTC. β-actin-L. Forward. ACACTGTGCCCATCTACGAGG. (RT-PCR). Reverse. AGGGGCCGGACTCGTCATACT. TIMP3. Forward. CCAAGGTGGTGGGAAAGAAGC. Reverse. AAGTCACAAAGCAAGGCAGGTA. (RT-PCR) oligo JW102. GCGGTGACCCGGGAGATCTGAATTC. oligo JW103. GAATTCAGATC. 80. PCR size Tm (bp) (oC) 281. 55. 618. 61. 344. 65.

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