Inhibition of HDAC6 activity with pharmacological inhibitors enhances cisplatin-induced DNA damage in A549 and H292 cells. A

<p>, A549 cells were treated with vehicle control, cisplatin alone, vorinostat alone, or cisplatin and vorinostat in combination for 24 hours, then anti-γH2AX, anti-acetylated tubulin, anti-PARP1, anti-HDAC6, and anti-β-actin Western blotting analyses were performed. <b>B</b>, H292 cells were treated with vehicle control, cisplatin alone, vorinostat alone or cisplatin and vorinostat in combination for 24 hours, then anti-γH2AX, anti-acetylated tubulin, anti-PARP1, anti-HDAC6, and anti-β-actin Western blotting analyses were performed. The arrowhead indicated the cleaved PARP1 band. <b>C</b>, A549 cells were treated with vehicle control, cisplatin alone, tubastatin A alone or cisplatin and tubastatin A in combination for 24 hours, then anti-γH2AX, anti-acetylated tubulin, anti-PARP1, anti-HDAC6 and anti-β-actin Western blotting analyses were performed. <b>D</b>, H292 cells were treated with vehicle control, cisplatin alone, tubastatin A alone or cisplatin and tubastatin A in combination for 24 hours, and anti-γH2AX, anti-acetylated tubulin, anti-PARP1, anti-HDAC6 and anti-β-actin Western blotting analyses were performed. The arrowhead indicated the cleaved PARP1 band.</p

Knockdown of HDAC6 inhibits tumor growth in a mouse xenograft model. A and B

<p>, H292-pRS or H292-HD6KD cells were injected into 5- to 6-week-old athymic nude female mice (Nu/Nu mice) as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0044265#s2" target="_blank">Methods</a>. After 6 weeks, tumors were taken from the mice (<b>A</b>) and weighed (<b>B</b>). <i>Columns, mean from 12 mice/group.. </i><b>C</b>, The size of H292-pRS and H292-HD6KD xenografts were calculated weekly as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0044265#s2" target="_blank">Methods</a>. <b>D</b>, A tissue microarray of H292-pRS and H292-HD6KD xenografts was constructed and stained with anti-cleaved caspase 3 antibodies as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0044265#s2" target="_blank">Methods</a>. <b>E</b>, The intensity of cleaved caspase 3 staining was measured by pixels as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0044265#s2" target="_blank">Methods</a>. <i>Columns, mean from 12 xenografts/group.. </i><b>F</b>, A tissue microarray of H292-pRS and H292-HD6KD xenografts as shown in <b>D</b> was stained with anti-Ki67 antibodies as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0044265#s2" target="_blank">Methods</a>. <b>G</b>, The intensity of anti-Ki67 staining was measured by pixels as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0044265#s2" target="_blank">Methods</a>. <i>Columns, mean from 12 xenografts/group.</i></p

Knockdown of HDAC6 increases phosphorylation of DNA damage signaling proteins upon cisplatin treatment in H292 cells.

<p><b>A</b>, H292-pRS and H292-HD6KD cells were treated with vehicle (0 µM cisplatin) or cisplatin for 24 hours, and anti-phosphorylated ATR (S428), total ATR, phosphorylated Chk1 (S296), total Chk1, phosphorylated p53 (S15), total p53 and β-actin Western blotting analyses were performed. <b>B</b>, The above cells were treated with 5 µM cisplatin for the indicated hours and Western blotting analyses with indicated antibodies were carried out.</p

HDAC6 level is associated with cisplatin sensitivity in NSCLC cell lines. A

<p>, H292 cells were transfected with scramble shRNA or shRNA against HDAC6 to generate H292-pRS or H292-HD6KD stable clone, respectively. HDAC6 expression in control (H292-pRS) or HDAC6 knockdown (H292-HD6KD) cells was detected by anti-HDAC6 Western blotting analysis (upper panel). Anti-β-actin Western blotting was performed to ensure equal loading of protein (lower panel). <b>B</b>, Knockdown of HDAC6 sensitized H292 cells to cisplatin treatment. MTT assays were performed using H292-pRS or H292-HD6KD cells exposed to vehicle control (0 µM cisplatin) or indicated concentrations of cisplatin for 3 days. <b>C</b>, Knockdown of HDAC6 did not sensitize H292 cells to paclitaxel treatment. MTT assays were performed using H292-pRS or H292-HD6KD cells treated with vehicle control (0 nM paclitaxel) or indicated concentrations of paclitaxel for 3 days. <b>D</b>, HDAC6 was efficiently depleted in A549 cells. HDAC6 expression in control (A549-Sup) or HDAC6 knockdown cells (A549-HD6KD) was detected by anti-HDAC6 Western blotting analysis (upper panel). The anti-β-actin Western blotting analysis was also performed to ensure equal loading (lower panel). <b>E</b>, Knockdown of HDAC6 sensitized A549 cells to cisplatin treatment. MTT assays were performed using A549-Sup or A549-HD6KD cells treated with vehicle control or indicated concentrations of cisplatin for 3 days. <b>F</b>, Knockdown of HDAC6 did not sensitize A549 cells to paclitaxel treatment. MTT assays were performed using A549-Sup or A549-HD6KD cells treated with vehicle control or indicated concentrations of paclitaxel for 3 days. <b>G</b>, Colony formation assays were carried out with H292-pRS and H292-HD6KD cells for 8 days with vehicle control (0 µM cisplatin) or indicated concentrations of cisplatin. <b>H</b>, The quantitative results were obtained by calculating colonies from <b>G</b>. <i>Columns, mean from three or six duplicates; bars, SEM (*, P<0.05; **, P<0.01; ***, P<0.001)</i>. <b>I</b>, HDAC6 levels positively correlate with cisplatin IC₅₀ in a panel of NSCLC cell lines. HDAC6 protein levels were obtained by Western blotting analyses of 15 NSCLC lines (ADLC, A549, EPLC, H23, H292, H358, H441, H460, H522, H661, H820, H1650, H1975, H2122, and H2172) and quantified by Quantity One software. Cisplatin IC₅₀ was calculated from MTT assays after treatment of these cells for 3 days. The correlation between HDAC6 protein level and cisplatin IC₅₀ was analyzed by SAS software. A dot plot was used to illustrate the positive correlation between HDAC6 level and cisplatin IC₅₀. Spearman coefficient was 0.64875 and <i>p</i>-value was 0.0089.</p

Knockdown of HDAC6 exacerbates cisplatin-induced crosslinking and accumulates more γH2AX foci in H292 cells.

<p><b>A</b>, H292-pRS or H292-HD6KD cells were treated with vehicle (0 µM cisplatin) or cisplatin for 24 hours, then irradiated with 10 Gy of gamma radiation to introduce random single-strand breaks. Comet assays were performed as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0044265#s2" target="_blank">Methods</a>. <b>B</b>, Uncrosslinked DNA were scored by High Capacity Sides Analysis System (HCSA) software (Coats Associates, Inc.). <i>Columns, mean from 40 single cells; bars, SE (***, P<0.001).. </i><b>C</b>, Immunofluorescence staining of γH2AX foci in H292-pRS or H292-HD6KD cells treated with vehicle (0 µM cisplatin) or cisplatin as indicated. <b>D</b>, Quantitative representation of γH2AX foci in above cells. <i>Columns, mean from 600 cells.. </i><b>E</b>, H292-pRS and H292-HD6KD cells were treated with the indicated concentrations of cisplatin for 24 hours, and anti-γH2AX and anti-β-actin Western blotting analyses were then performed. <b>F</b>, The above cells were treated with vehicle or 5 µM cisplatin for the indicated days and anti-γH2AX and anti-β-actin Western blotting analyses were performed.</p

Biomarkers ranked according to weights generated by Huberized Hinge Support Vector Machine (HHSVM) for classification of benigns and cases.

<p>The weight was estimated for each marker using HHSVM to indicate its relevance in the classification. Higher weight indicates more importance of the variable (biomarker) in the classification. Abbreviations used:lysophosphatidic acid (LPA), lysophosphatidylcoline (LPC) and plasmenylphosphoethanolamine (PPE).</p

Estimated error rates for the support vector machine models for classifying benigns and cases.

<p>. One set of models was fitted with cross-validation (CV) and the other without.</p

Histopathology of epithelial ovarian cancer cases.

<p>Histopathology of epithelial ovarian cancer cases.</p

The weights generated by Huberized Hinge Support Vector Machine (HHSVM) to indicate the relevance of the biomarkers for classification of benigns and cases for the samples with either low- or high- CA125 levels (CA125<35 or CA125≥35) in the second step of the development of the two-step models.

<p>These estimated weights were used to prioritize the markers to enter the model (see the text for more details).</p

Sensitivity, specificity and error rates of the selected two-step models.

<p>The “4 marker set” includes 16∶0, 18∶1 PPE, 15∶0 LPC, 18∶2 LPA, and 18∶0, 22∶6 PPE. The “2 marker set” includes 16∶0 18∶1 PPE and 14∶0 LPC.</p