Effect of AN-7 on the activity and expression of cellular HDACs classes I and II.

<p>(A) The cells were exposed to AN-7 for 3 h in the presence of the HDAC fluorogenic substrate. The average % of HDAC activity as a function of 0–100 µM concentration of AN-7 of a representative experiment conducted in quadruplicates is shown. (B) The average IC₅₀±SE of AN-7 for each of the cell types is calculated from three independent experiments. (C) Lysates of cells were loaded (35 µg protein/well) on 10% SDS gels and were subjected to Western blot analysis. HDAC1, HDAC2, HDAC5 or actin were detected with the appropriate antibodies. (D) HDAC activity of three experiments (in triplicate) conducted with 4T1 (cancer) and H9C2 (non-cancer) cells treated with AN-7 (50 µM) as a single agent for a total of 3 h, 1 h prior to the addition of the fluorogenic substrate and 2 h in its presence; Dox (200 nM) as a single agent treatment, or with the combination AN-7+Dox where 50 µM AN-7 was added 1 h prior to the addition of Dox and the fluorogenic substrate and then incubated for an additional 2 h. (E) The viability of the cells treated as described in (D) was determined by a Hoechst assay after 23 or 24 h for cells treated with Dox. (F) Cells were treated with AN-7 (3 or 24 h), Dox (2 or 23 h) or AN-7 (3 or 24 h)+Dox (2 or 23 h), as described (D). The lysates of the cells were subjected to Western blot analysis as described (C).</p

Effect of AN-7, DOX and their combinations on angiogenesis.

<p>Sections of tumors and hearts were stained for (A) lo-FGF (DAB); (B) VEGF (Nova Red) and counter stained with hematoxylin. Bar = 200 µm, the 4-fold magnified picture in the insert was taken from the area indicated by the square. (C) Lysates samples (as described above) were resolved on 7.5% SDS gels and stained for TSP-1(left). Mean±SE of the ratio of TSP-1/actin expression is shown (right), ^#p<0.05, as specified, vs. untreated control; * p<0.05, as specified, vs. Dox; ^&p<0.05, AN-7+Dox vs. AN-7.</p

Effect of Dox, AN-7, and AN-7+Dox in the 4T1 murine mammary carcinoma model.

<p>(A) In the preliminary experiment, female Balb/c mice carrying 75–180 mm³ subcutaneous 4T1 tumors were randomly divided into three groups (10 mice/group) and were injected ip once a week with either vehicle, 3 or 5 mg/kg Dox. Tumor volume was measured twice a week up to day 14 when the experiment was terminated (after 2 Dox treatments). Mean±SE of tumor volume was: *p<0.05, untreated vs. the other groups; #p<0.05, Dox 5 mg/kg vs. Dox 3 mg/kg. (B) Mean±SE of tumor weight and the ratio of heart-weight to body-weight (HW/BW, g/g), measured at the experiment termination point were: *p<0.01, Dox 5 mg/kg vs. all other groups. (C) In the pivotal experiment, the Percent Failure Free mice bearing 75–180 mm³ subcutaneous 4T1 tumors, was assessed by a Kaplan-Mier graph. The mice were assigned to the following treatments: saline vehicle ip (n = 7) or po (n = 7); ip Dox, 5 mg/kg once/week (n = 14); po AN-7, 50 mg/kg 3 times/week (n = 14); po AN-7+ip Dox (n = 14). The arrows on the x-axis indicate the point in time of the second and the third Dox treatment. (D) Mean±SE of heart-weight to body-weight ratios (HW/BW, g/g) at the above experiment end-point was ^#p<0.01, Dox 5 mg/kg vs. all other groups. (E) Tumor weight at the termination point (mean±SE) was: Dox 5 (total) represents the tumor weight of all the mice treated with 5 mg/kg Dox (n = 14), as measured at their individual end-points; Dox 5 (>21) represents the tumor weight of the mice (n = 5) treated with 5 mg/kg Dox and survived beyond day 21 of treatment. *p<0.02 all groups vs. untreated control; ^#p<0.05 AN-7+Dox vs. AN-7 or Dox 5 (total) or p<0.01 Dox 5 (>21). (F) Mean±SE of lung lesions for the various treatment groups is shown. *p<0.02, untreated vs. all treatment groups; ^#p<0.05, AN-7+Dox vs. AN-7 or vs. Dox.</p

Effect of AN-7, DOX and AN-7+Dox on HDAC1 and HDAC2 expression in tumor or heart.

<p>(A) Extracts of tumors from 8 mice and hearts from 6 mice per treatment group were loaded (20 µg protein/well) on 10% SDS gels, and subjected to Western blot analysis. HDAC1, HDAC2 or actin were detected with the appropriate antibodies. (B) Expression of HDAC1, HDAC2 or actin in tumors and hearts was analyzed by Odyssey 2.1. The intensity ratio of HDACs to actin (mean±SE) is presented. *p<0.05, tumors of untreated or Dox-treated vs. AN-7 or AN-7+Dox; ^#p<0.007, HDAC2 expression in the hearts of Dox treated vs. all other treatment groups.</p

Effect of AN-7, DOX and their combinations on markers of proliferation and fibrosis.

<p>Sections of tumors and hearts were stained (DAB) for: (A) Ki-67; (B) c-Kit and both were counter stained with hematoxylin. The arrowhead marked the c-Kit positively staind cells. Bar = 200 µm, the 4-fold magnified picture in the insert was taken from the area indicated by the square. (C) Lysates samples, as described above, were resolved on 10% SDS gel and stained for c-Myc (left). The Mean±SE ratio of c-Myc/actin expression is shown (right). ^#p<0.05, as specified, vs. untreated control; *p<0.05, as specified, vs Dox. (D) Picrosirius red and fast-green for the visualization of interstitial fibrosis. Bar = 200 µm, the 2-fold magnified picture in the insert was taken from the area indicated by the square.</p

Effect of SAHA and AN-7 on the viability of MF/SS cell lines, SPBL and NPBL.

<p>Viability curves based on the MTT assay of MyLa cells, Hut78 cells, (a,b), and SPBL (n = 3) (c,d) compared to NPBL (n = 8) following treatment with SAHA (a,c) and AN-7 (b,d) for 72 h. Also shown are the IC₅₀ and SI values of SAHA and AN-7 in MF/SS cell lines and SPBL and NPBL based on viability curves a-d, and their p values (e).</p

Apoptosis induction of AN-7 and SAHA in SPBL.

<p>PBL from 2 SS patients were plated at a concentration of 0.5x10⁶ cells/mL, and were then treated with SAHA 4 μM or AN-7 200 μM for 48 h. The cells were then stained with annexin V and PI. FACS plots are shown with percent of cells in each quadruplet, and the percent of cells in apoptotic cells (early + late apoptosis) are shown also in column.</p

Toxic and apoptotic effect of SAHA and AN-7 on MF/SS cell lines as a function of exposure time.

<p>Viability curves based on trypan blue staining of MyLa and Hut78 cells following short or long exposure to SAHA (a, c) or AN-7 (b, d). IC₅₀ values of short and long exposure to SAHA and AN-7 in MF/SS cell lines based on viability curves a-d (e). Apoptosis curves based on FACS analysis of annexin V and PI staining (f-i). Percent of apoptotic MyLa cells (early + late apoptosis) after short or long exposure to SAHA (f) or AN-7 (g), and apoptotic Hut78 cells after short or continuous exposure to SAHA (h) or AN-7 (i).</p

Effect of SAHA and AN-7 on specific protein expression and modification in MF/SS cell lines.

<p>Immunoblot of apoptotic and proapoptotic proteins in MyLa and Hut78 cells treated with SAHA 10 μM or AN-7 300 μM for the indicated periods (a). Basal HDAC1 protein expression in NPBL and MF/SS cell lines (b) and in MF/SS cell lines treated with SAHA 10 μM or AN-7 300 μM (c). Acetylated H3 in the nuclear lysate of MF/SS cell lines treated with and the same concentrations of SAHA and AN-7 for the indicated periods (d).</p