Histone modification alterations in response to GE treatment.

<p>A and B, Histone modification patterns in the promoters of <i>p16</i> (A) and <i>p21</i> (B) were analyzed by ChIP assay in breast precancerous SH cells (left panel) and completed transformed breast cancer SHR cells (right panel). Histone modification enrichment was calculated from the corresponding DNA fragments amplified by ChIP-PCR. The cells were treated with 40 µM of GE as described previously and analyzed by ChIP assays using chromatin markers including acetyl-H3, acetyl-H4, trimethyl-H3K4, trimethyl-H3K9, trimethyl-H3K27 and mouse IgG control in the promoter regions of <i>p16</i> and <i>p21</i>. C. Changes of binding abilities of <i>BMI1</i> and <i>c-MYC</i> in the <i>p16</i> promoter were determined by ChIP assay as described previously. Inputs came from the total DNA and served as the same ChIP-PCR conditions. DNA enrichment was calculated as the ratio of each bound sample divided by corresponding input. Data are in triplicate from three independent experiments. Columns, mean; Bars, SD; *, <i>P</i><0.05, * * <i>P</i><0.01, significantly different from control.</p

GE treatment results in apoptosis in transformed breast cells.

<p>A. Cell apoptosis of breast precancerous SH cells and transformed breast cancer SHR cells were detected by using an Annexin V and propidium iodide (PI) staining system following FACS-based flow cytometry assay. Cells were treated with 40 µM of GE for 3 days and harvested for apoptosis analysis. Apoptotic cells are shown in the upper right (as late apoptotic cells) or lower right (as early apoptotic cells) quadrants of the FACS histogram. B, Histogram of the apoptosis rate in SH and SHR cells in response to GE treatment. The relative apoptotic rate is the percentage of early plus late apoptotic cells normalized to levels of untreated samples. The graphs shown are representative of similar results obtained from three independent experiments. Columns, mean; Bars, SD; *, <i>P</i><0.05, significantly different from control; £, <i>P</i><0.05, significantly different from SHR cells.</p

GE inhibited tumor growth in mouse SHR xenografts.

<p>a. Prevention rate  =  tumor-free mice number/total mice number; b. Tumor volume (TV)  =  (length × width²) ×0.532; c. Relative Tumor volume (RTV)  =  (TV on sacrificing day)/(TV on day 1 of injection); d. Inhibition rate on tumor growth (IR)  =  {1 – (mean RTV of the treatment group)/(mean RTV of the control group)} 100.</p

Breast tumor growth in a mouse xenograft model by dietary GE treatment.

<p>Female athymic nude mice were injected with transformed breast cancer SHR cells. GE or control diets were provided from two weeks prior to injection and continued throughout the study. A, Tumor volume during the experiment. B. Tumor weights when xenograft tumors were harvested at the termination of the experiment. Tumor volumes were calculated by using the formula: volume (mm³)  =  (length × width²) ×0.523, and represented as mean ± SD (mm³) for each group. Tumor weight is the wet weight of the tumor per mouse in each group and is reported as mean ± SD (g); C. PCNA expression in SHR tumor xenogratfs. Immunohistochemical analysis was performed in tumor samples to detect PCNA-positive cells for proliferation index. D. Graphic representation of expression. Immunohistochemical data in terms of percentage of positive cells are presented as mean ± SD from each group. PCNA-positive cells were counted in 5 different areas of the sections, and data are summarized in terms of percent positive cells from all tumor samples. Representative photograph from one field of each experimental group. E. Expression changes of tumor suppressor genes, <i>p16</i> and <i>p21</i> in mice SHR xenograft tumors; F. Expression changes of tumor promoting genes, <i>BMI1</i> and <i>c-MYC</i> in mice SHR xenograft tumors. Symbols and columns, mean; Bars, SD; * <i>p</i><0.01, **, <i>p</i><0.001 significantly different from control group.</p

GE suppressed cellular growth in transformed breast cells.

<p>A, Schematic presentation of malignant transformation of the breast tumorigenesis cellular model. Normal HMECs stably transfected with <i>SV40, hTERT</i> and <i>h-RAS</i> to generate early transformed SH cells (precancerous cells) and completely transformed SHR cells (breast cancer cells). B, C and D, Cellular viability in response to various concentrations of GE treatment in HMECs (B), precancerous SH cells (C) and breast cancer SHR cells (D). Cells were plated in 96-well plates in triplicate and exposed to various concentrations of GE for up to 3 days. Cellular viability was measured by MTT assay. Data are in triplicate from three independent experiments and calibrated to levels in untreated samples. Columns, mean; Bars, SD; *, <i>P</i><0.05, * * <i>P</i><0.01, significantly different from control; £, <i>P</i><0.05, significantly different from SHR cells with the corresponding treatment.</p

Epigenetic enzymatic activity changes in response to GE treatment.

<p>A, HDACs enzymatic activity; B, HMT (trimethyl-H3K4) activity; C. HMT (trimethyl-H3K9) activity; D. HMT (trimethyl-H3K27) activity. Nuclear proteins of SH and SHR cells were extracted after the treatment as described above. The enzymatic activity assays were performed according to the manufacturer's protocols. The values of enzymatic activities came from the means of three independent experiments. Columns, mean; Bars, SD. **, <i>P</i><0.01, significantly different from control.</p

GE treatment induced differential expression of tumor-related genes.

<p>A and B mRNA expression changes of <i>p16, p21, BMI1</i> and <i>c-MYC</i> genes in breast precancerous SH cells (A) and completed transformed breast cancer SHR cells (B). Left panel, expression changes of tumor suppressor genes, <i>p16</i> and <i>p21</i>; right panel, expression changes of tumor promoting genes, <i>BMI1</i> and <i>c-MYC</i>. The cells were treated with various concentrations of GE for 3 days as described before. Control cells were grown in parallel with the treated cells but received vehicle DMSO. Quantitative real-time PCR was performed to measure relative transcription. C, The protein levels of p16, p21, BMI1 and c-MYC genes were determined by western-blot analysis. Actin antibody was used to ensure equal loading. Representative photograph from an experiment was repeated three times. D. Histogram of quantification of the protein levels. Data are in triplicate from three independent experiments and were normalized to <i>GAPDH</i> or Actin and calibrated to levels in untreated samples. Columns, mean; Bars, SD; *, <i>P</i><0.01, * * <i>P</i><0.001, significantly different from control.</p

Bacterial isolates with zone of hydrolysis on the agar broth containing 0.4% congo red and 0.5% konjac powder.

<p>Bacterial isolates with zone of hydrolysis on the agar broth containing 0.4% congo red and 0.5% konjac powder.</p

SDS-PAGE analysis.

<p>Lane M: protein MW markers; Lanes 1–2: IPTG induced <i>E. coli</i> pET30a-MANB; Lanes 3–5: Negative control; Lane 6: the purified MANB.</p

Biochemical properties of MANB.

<p><b>A.</b> Effect of pH on the MANB activity. <b>B.</b> The pH stability of MANB. <b>C.</b> Effect of temperature on the MANB activity. <b>D.</b> The temperature stability of MANB. <b>E.</b> The enzymatic thermostability. The maximum value was considered to be 100%. The values presented correspond to the mean values of at least three replicates.</p