1. Volume 143, Issue 1, Pages 99-109.e10 (July 2012)
Pan-Histone Deacetylase Inhibitor Panobinostat Sensitizes Gastric Cancer Cells to Anthracyclines via Induction of CITED2 
Ivonne Regel, Lisa Merkl, Teresa Friedrich, Elke Burgermeister, Wolfgang Zimmermann, Henrik Einwächter, Ken Herrmann, Rupert Langer, Christoph Röcken, Ralf Hofheinz, Roland Schmid, Matthias P. Ebert 
Gastroenterology 
Volume 143, Issue 1, Pages e10 (July 2012)
DOI: /j.gastro
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2. Figure 1
Expression of HDAC2 in gastric cancer tissue, cell lines, and mouse model. (A) HDAC2 mRNA in human gastric cancer cell lines. HDAC2 gene expression normalized to housekeeping gene shows a significant up-regulation of HDAC2 mRNA (n = 3) compared with normal tissue (NT). (B) HDAC2 in gastric cancer cell lines as analyzed by Western blotting. (C and D) HDAC2 protein expression in stomach tumors (TU) and corresponding NT (n = 19; 6 representative samples are shown in panel D as determined by Western blot analysis. (C) Band intensity was normalized to protein concentration and is displayed ± standard error (n = 19). (E) Immunohistochemical analysis reveals nuclear staining of HDAC2 in mucosa of C57BL/6 wild-type (WT) mice and tumor tissue of CEA/Tag mice. Magnification, ×200. *P < .05.
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3. Figure 2
LBH589-dependent HDAC inhibition in human gastric cancer cell lines. (A) MTT assay. IC50 of LBH589 was measured in gastric cancer cell lines. Cell survival (at 0–5000 nmol/L of LBH589) was compared with vehicle-treated cells; normalized to 100% ± standard error (n = 4). (B) Colony formation assay of AGS cells treated with LBH589 (0–100 nmol/L) for 48 hours. Colony density ± standard error (n = 5). (C) Nuclear extracts of AGS and MKN-45 cells were incubated with LBH589 (0–100 nmol/L), and HDAC enzyme activity ± standard deviation was calculated in nmol/min/mL (n = 2). (D) Western blot analysis of AGS and MKN45 cells. Time- (0–24 h) and dose-dependent (0–200 nmol/L) increase of acetylated histones after LBH589 treatment. *P < .05.
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4. Figure 3
LBH589 up-regulates CITED2. (A) Promoter analysis identified hypoxia response elements (HREs) in doxorubicin-resistance genes. HIF-1α binds to HRE, and CITED2 inhibits transactivation of the coactivator protein 300, and thereby inhibits transcription of resistance genes. (B) RT-qPCR of mRNA isolated from AGS cells treated with LBH589 (0–100 nmol/L) for 24 hours, CITED2 mRNA expression in fold-changes ± standard deviation (n = 2) to vehicle-treated control. (C) Overexpression of CITED2 after LBH589 treatment was confirmed by Western blot in AGS and MKN45 cells. (D) Western blot analysis of CITED2 in pCITED2 and pcDNA3 EV–transfected AGS cells. *P < .05.
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5. Figure 4
High expression of CITED2 prevents chemoresistance in gastric cancer cells. (A) Luciferase assay of AGS and MKN-45 cells co-transfected with reporter plasmid of FOXM1 promoter or reporter plasmid of HMGB2 promoter and pCITED2 or EV, respectively. Luciferase activity values were determined after 24 hours, normalized to protein content, and displayed as fold-change ± standard error (n = 3). (B) RT-qPCR detecting mRNAs of resistance genes as fold-change ± standard deviation (n = 2) after transfection of pCITED2. (C) Cell survival analysis of pCITED2 and pcDNA3 EV–transfected AGS cells upon 0.1 μmol/L doxorubicin or 0.1 μmol/L epirubicin treatment for 48 hours with MTT assay as 100% ± standard error (n = 3). (D) CITED2 staining of gastric cancer treated with epirubicin; staining intensity ± standard error and response status (4 complete responders [CR]; 6 partial responders [PR]; 10 nonresponders [NR]). (E) Representative pictures from CITED2 staining of gastric cancer tissue array; and comparison of staining intensity ± standard error with grade of differentiation (G1: n = 12; G2: n = 16; G3: n = 12). Scale bar: 50 μm. *P ≤ .05.
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6. Figure 5
CITED2 is regulated by HDAC1/HDAC2. (A) RT-qPCR detection of CITED2 mRNA of AGS cells transfected with 10 nmol/L siRNA (against HDAC1 or HDAC2) for 24 hours. Gene expression was normalized to the housekeeping gene and calculated as fold-change ± standard error (n = 3) compared with control scramble siRNA (siSCR). (B) Quantification of CITED2, HDAC1, HDAC2, and AcH4K12 protein by Western blot in AGS cells transfected with 10 nmol/L siRNA (scramble, HDAC1, HDAC2) for 72 hours. (C) ChIP of AGS cells treated with 50 nmol/L LBH589 for 24 hours vs vehicle-treated cells and AGS cells transfected with HDAC1 and HDAC2 siRNA for 72 hours vs siSCR-transfected cells. Antibody against HDAC2 was used to detect epigenetic regulation of CITED2 promoter. Promoter binding was measured with RT-qPCR; Ct values were normalized to input DNA and calculated as fold-change ± standard error (n = 3). *P < .05.
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7. Figure 6
Combination therapy in vitro. Combination treatment of (A) AGS and (B) MKN45 cells with 50 or 100 nmol/L LBH589 for 12 hours and 0.1, 0.5, or 1 μmol/L doxorubicin or epirubicin for 48 hours vs single treatment. Cell survival was measured with the MTT assay and compared with normalized vehicle-treated cells as 100% ± standard error (n = 3). *P < .05.
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8. Figure 7
Combination therapy in vivo. CEA/Tag mice were treated with Epi as monotherapy or with LBH/Epi as combination therapy compared with vehicle-treated controls (n = 4–6 per group). (A) Representative H&E staining of measured main tumor area (delineated by red line). Tumor area was measured in mm2 (n = 2 areas/mouse). (B) Representative Ki67 staining of each therapy group, calculation of positive nuclei in percentages ± standard error compared with total nuclei. (C) RT-qPCR of CITED2 in C57BL/6 mice (n = 3) and CEA/Tag mice (n = 5). Ct values were normalized to the housekeeping gene and calculated as fold-change ± standard error. (D) RT-qPCR of CITED2 mRNA in therapy groups (control, Epi, LBH/Epi). Gene expression was normalized to the housekeeping gene and calculated as fold-change ± standard error. Scale bar, 100 μm. *P ≤ .05.
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9. Supplementary Figure 1
HDAC2 expression in gastric cancer. Immunostaining was performed to determine the expression of HDAC2 in (A and B) diffuse- and (C and D) intestinal-type gastric cancer. (B) A weak nuclear immunostaining was found in 10%–50% of the tumor cells (HDAC2-immunoreactivity score, 2; negative according to Weichert et al3 in this diffuse-type gastric cancer). (D) To the contrary, a strong nuclear expression of HDAC2 was found in >80% of the tumor cells (HDAC2-immunoreactivity score, 12; positive according to Weichert et al3 of this intestinal-type gastric cancer). (A and C) H&E stain. (B and C) Anti-HDAC2 antibody, haemalaun counterstain. Magnification, ×400.
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10. Supplementary Figure 2
LBH589 down-regulates expression of anthracycline-resistance genes. (A) Gene set enrichment analysis (GSEA) of cDNA microarray data of AGS cells treated with 100 nmol/L LBH589 for 24 hours compared with vehicle-treated controls. Doxorubicin-resistance genes are down-regulated with LBH589 treatment (fold-change, 0.98–0.02). #Promoter analysis was performed. Fold-change of CITED2 in the cDNA microarray analysis. (B) RT-qPCR of FOXM1, HMGB2, and TYMS in AGS cells treated with 50 nmol/L LBH589 for 24 hours compared with vehicle-treated controls. Gene expression was normalized to housekeeping gene and calculated as fold-change ± standard error (n = 3). *P ≤ .05.
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11. Supplementary Figure 3
Anthracycline response depends on CITED2 expression. (A) RT-qPCR of CITED2 mRNA expression of GC cell lines (AGS, MKN-7, MKN-45, SNU-1, SNU-5, NCI-N87, KATO-III) compared with normal tissue (NT). Gene expression was normalized to housekeeping gene and calculated as fold-change ± standard error (n = 3). (B) GC cell lines were treated with 5 μmol/L doxorubicin or epirubicin for 48 hours. Cell survival was measured with the MTT assay and compared with normalized vehicle-treated cells as 100% ± standard error (n = 3).
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12. Supplementary Figure 4
Flow cytometry analysis of combination treatment of epirubicin and LBH589. Flow cytometry analysis of annexin (A) and propidium iodide (PI)-stained AGS cells with epirubicin and LBH589 single and combination treatment. A, PI, and A + PI–positive cells are summarized as apoptotic rate in percentage ± standard error (n = 3). Representative data are shown. *P ≤ .05.
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13. Supplementary Figure 5
Analysis of cleaved-caspase 3 in CEA/Tag mice. Immunohistochemical analysis was performed and representative pictures of cleaved-caspase 3 expression for each treatment group (control, Epi, and LBH/Epi) are presented, number of positive nuclei in percentage ± standard error compared with total nuclei. Scale bar, 100 μm.
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14. Supplementary Figure 6
Macroscopic assessment of tumor response in CEA/Tag mice after therapy. From each therapy group (control, Epi, and LBH/Epi), macroscopic pictures are presented and the size of the tumor area at the pylorus was measured and shown in pixels ×104. *P ≤ .05.
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15. Supplementary Figure 7
Analysis of potential drug toxicity in epirubicin and LBH589-treated mice. (A) Cardiotoxicity. Representative pictures of H&E staining and Elastica van Gieson (EvG) staining of hearts from each treatment group. Scale bar, 100 μm. (B) Weight loss analysis. Normalized weight curves of each therapy group from 41 to 82 days of age. *P ≤ .05.
Gastroenterology  , e10DOI: ( /j.gastro )
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