Heterogeneous in vitro effects of doxorubicin on gene expression in primary human liposarcoma cultures

<p>Abstract</p> <p>Background</p> <p>Doxorubicin is considered one of the most potent established chemotherapeutics in the treatment of liposarcoma; however, the response rates usually below 30%, are still disappointing. This study was performed to identify gene expression changes in liposarcoma after doxorubicin treatment.</p> <p>Methods</p> <p>Cells of 19 primary human liposarcoma were harvested intraoperatively and brought into cell culture. Cells were incubated with doxorubicin for 24 h, RNA was isolated and differential gene expression was analysed by the microarray technique.</p> <p>Results</p> <p>A variety of genes involved in apoptosis were up and down regulated in different samples revealing a heterogeneous expression pattern of the 19 primary tumor cell cultures in response to doxorubicin treatment. However, more than 50% of the samples showed up-regulation of pro-apoptotic genes such as <it>TRAIL Receptor2</it>, <it>CDKN1A</it>, <it>GADD45A</it>, <it>FAS</it>, <it>CD40</it>, <it>PAWR</it>, <it>NFKBIA</it>, <it>IER3</it>, <it>PSEN1</it>, <it>RIPK2</it>, and <it>CD44</it>. The anti-apoptotic genes <it>TNFAIP3</it>, <it>PEA15</it>, <it>Bcl2A1</it>, <it>NGFB</it>, and <it>BIRC3 </it>were also up-regulated. The pro-apoptotic <it>CD14</it>, <it>TIA1</it>, and <it>ITGB2 </it>were down-regulated in more than 50% of the tumor cultures after treatment with doxorubicin, as was the antiapoptotic <it>YWHAH</it>.</p> <p>Conclusion</p> <p>Despite a correlation of the number of differentially regulated genes to the tumor grading and to a lesser extent histological subtype, the expression patterns varied strongly; however, especially among high grade tumors the responses of selected apoptosis genes were similar. The predescribed low clinical response rates of low grade liposarcoma to doxorubicin correspond to our results with only little changes on gene expression level and also divergent findings concerning the up- and down-regulation of single genes in the different sarcoma samples.</p

The JNK Inhibitor XG-102 Protects against TNBS-Induced Colitis

The c-Jun N-terminal kinase (JNK)-inhibiting peptide D-JNKI-1, syn. XG-102 was tested for its therapeutic potential in acute inflammatory bowel disease (IBD) in mice. Rectal instillation of the chemical irritant trinitrobenzene sulfonic acid (TNBS) provoked a dramatic acute inflammation in the colon of 7–9 weeks old mice. Coincident subcutaneous application of 100 µg/kg XG-102 significantly reduced the loss of body weight, rectal bleeding and diarrhoea. After 72 h, the end of the study, the colon was removed and immuno-histochemically analysed. XG-102 significantly reduced (i) pathological changes such as ulceration or crypt deformation, (ii) immune cell pathology such as infiltration and presence of CD3- and CD68-positive cells, (iii) the production of tumor necrosis factor (TNF)-α in colon tissue cultures from TNBS-treated mice, (iv) expression of Bim, Bax, FasL, p53, and activation of caspase 3, (v) complexation of JNK2 and Bim, and (vi) expression and activation of the JNK substrate and transcription factor c-Jun. A single application of subcutaneous XG-102 was at least as effective or even better depending on the outcome parameter as the daily oral application of sulfasalazine used for treatment of IBD

JNK2-Bim co-precipitation.

<p>JNK1 and JNK2 immunoprecipitates (IP) from colon tissue homogenates were analyzed by Western blotting with an anti-Bim antibody. Pounceau staining demonstrated equal loading (data not shown).</p

Representative CD3 and CD68 immunofluorescence.

<p>Representative CD3 (left) and CD68 (right) immunofluorescence of the distal colon from normal mice, trinitrobenzene sulfonic acid (TNBS) administration and treatment with sc. 100 µg/kg XG-102.</p

Hematoxylin and eosin (H&E) scores.

<p>Hematoxylin and eosin (H&E) scores from distal (A) and medial (B) colon. For the tissue damage score (hatched bars), the scores of ulcer, crypts and submucosa were summed-up for each individual animal, and the mean±SEM was calculated for each group. The mean±SEM of the infiltration score (grey bar) is separately shown. ***, ** = p<0.001 and p<0.01 for all groups compared with TNBS group.</p

c-Jun and phospho-c-Jun.

<p>Western blot analysis of c-Jun and phospho-c-Jun in colon homogenates from untreated controls (co) and 12 h or 24 h following trinitrobenzene sulfonic acid (TNBS) administration without or with XG-102 (100 µg/kg sc.). These blots are representative of 3 independent experiments.</p

Tissue damage scores.

<p>Tissue damage scores.</p

Apoptosis.

<p>Western blot analysis of (A) caspase-3 and cleaved caspase-3, (B) Bax and Bim, and (C) FasL and p53 from colon extracts of untreated controls (co) 12 h, 24 h and 72 h following trinitrobenzene sulfonic acid (TNBS) administration without or with XG-102 (100 µg/kg sc.). These blots are representative of 3 independent experiments.</p

Production of TNF-α.

<p>TNF-α release (pg/ml) into the supernatant of organic colon culture from normal mice, following trinitrobenzene sulfonic acid (TNBS) only, and treatment with sc. 100 µg/kg XG-102. ** = p<0.01 for all groups compared with TNBS group.</p