24 colour multifish karyotype showing the complexity of the genomic rearrangements with rearranged chromosomes in most pairs and several unidentified marker chromosomes (bottom left)

<p><b>Copyright information:</b></p><p>Taken from "Biological characterization of two xenografts derived from human CUPs (carcinomas of unknown primary)"</p><p>http://www.biomedcentral.com/1471-2407/7/225</p><p>BMC Cancer 2007;7():225-225.</p><p>Published online 18 Dec 2007</p><p>PMCID:PMC2241840.</p><p></p> Of particular interest are the translocation of chromosome 21 (in green) to the distal chromosome 4 (in grey) and the loss of chromosomes 9. In this cell, there were 2 der(3)t(3, 15) instead of one in most other cells which were analyzed

Microsatellite tracking assay linking DNA from patient peripheral blood mononuclear cells (PBMC), tumor surgical specimen (Surg

<p><b>Copyright information:</b></p><p>Taken from "Biological characterization of two xenografts derived from human CUPs (carcinomas of unknown primary)"</p><p>http://www.biomedcentral.com/1471-2407/7/225</p><p>BMC Cancer 2007;7():225-225.</p><p>Published online 18 Dec 2007</p><p>PMCID:PMC2241840.</p><p></p> Spec.) and xenograft to the Capi3 cell line

Treatment of Nasopharyngeal Carcinoma Cells with the Histone-Deacetylase Inhibitor Abexinostat: Cooperative Effects with Cis-platin and Radiotherapy on Patient-Derived Xenografts

<div><p>EBV-related nasopharyngeal carcinomas (NPCs) still raise serious therapeutic problems. The therapeutic potential of the histone-deacetylase (HDAC) inhibitor Abexinostat was investigated using 5 preclinical NPC models including 2 patient-derived xenografts (C15 and C17). The cytotoxicity of Abexinostat used either alone or in combination with cis-platin or irradiation was assessed <i>in vitro</i> by MTT and clonogenic assays using 2 EBV-negative (CNE1 and HONE1) and 3 EBV-positive NPC models (C15, C17 and C666-1). Subsequently, the 3 EBV-positive models were used under the form of xenografts to assess the impact of systemic treatments by Abexinostat or combinations of Abexinostat with cis-platin or irradiation. Several cell proteins known to be affected by HDAC inhibitors and the small viral non-coding RNA EBER1 were investigated in the treated tumors. Synergistic cytotoxic effects of Abexinostat combined with cis-platin or irradiation were demonstrated <i>in vitro</i> for each NPC model. When using xenografts, Abexinostat by itself (12.5 mg/kg, BID, 4 days a week for 3 weeks) had significant anti-tumor effects against C17. Cooperative effects with cis-platin (2 mg/kg, IP, at days 3, 10 and 17) and irradiation (1Gy) were observed for the C15 and C17 xenografts. Simultaneously two types of biological alterations were induced in the tumor tissue, especially in the C17 model: a depletion of the DNA-repair protein RAD51 and a stronger <i>in situ</i> detection of the small viral RNA EBER1. Overall, these results support implementation of phase I/II clinical trials of Abexinostat for the treatment of NPC. A depletion of RAD51 is likely to contribute to the cooperation of Abexinostat with DNA damaging agents. Reduction of RAD51 combined to enhanced detection of EBER 1 might be helpful for early assessment of tumor response.</p></div

Protein modifications induced by Abexinostat in EBV-positive NPC cells treated <i>in vitro</i>.

<p>These experiments were done on C666-1 cells routinely propagated <i>in vitro</i> (C666-1) and on cells resulting from collagenase dispersion of C15 and C17 patient-derived xenografts (C15 and C17 cells <i>ex vivo</i>). Treatment with Abexinostat at various concentrations (0, 50, 125, 400 and 1000 nM) was applied continuously for two days (D1 and D2). Cell proteins were then extracted and separated on PAGE gels (20 µg/lane). Parallel Western blots were done for acetylated α-tubulin, α-tubulin, PARP1, RAD51, RAD23B and actin. Protein abundance in each lane was evaluated by densitometry. For loading normalization, all proteins were referred to actin except acetylated α-tubulin which was referred to total α-tubulin. Protein abundance in lanes corresponding to untreated samples was arbitrarily set at 1. Surprisingly PARP cleavage is observed for C17 cells treated with 400 nM but not 1000 nM Abexinostat. Because C17 cells were very sensitive to Abexinostat, we suspect that when treated with a high concentration of the compound they died by a non-apoptotic mechanism possibly related to necrosis.</p

Short term culture assays on NPC cells treated <i>in vitro</i> with Abexinostat plus external irradiation.

<p>The best combinations of treatment dosages are represented graphically. EBV-negative (HONE1 and CNE1) and EBV–positive NPC cells (C15 cells <i>ex vivo</i>, C17 cells <i>ex vivo</i> and C666-1 cells) were treated for 48 hours with combinations involving various concentrations of Abexinostat (25, 50, 75 and 150 nM) and doses of irradiation (1, 2 and 4 Gy 24 h after cell plating). Cell viability was determined in short term assays based on the reduction of MTT (CNE1, HONE1) or WST (C15 <i>ex vivo</i>, C17 <i>ex vivo</i> and C666-1). For each NPC model, the result obtained with the most successful combination of treatment dosages is depicted as a color disk whose size is proportional to the EOBA index (mentioned as a two-digit number). The X and Y coordinates of the disk stand for the corresponding concentration of Abexinostat and dose of irradiation, respectively. Note that for HONE1 and CNE1, the effects of the combined treatment were only additive. The highest levels of synergy were recorded for C15 and C17 cells <i>ex vivo</i>.</p

Cooperative cytotoxic effects of Abexinostat combined with CDDP or external irradiation on xenografted NPC tumors.

<p>Treatment was started 12 days following subcutaneous graft of tumor fragments (350 mg per mouse). Groups of 10 mice were used for each therapeutic condition (vehicle−Abexinostat−vehicle + CDDP or irradiation−Abexinostat + CDDP or irradiation). Abexinostat was given from D1 to D4, D8 to D11 and D15 to D17 (two intra-peritoneal (IP) injections of 12.5 mg/kg per day with an interval of 6 to 8 hours). CDDP was given IP at a dose of 2 mg/kg at D3, D10 and D17 (upper panel). Selective irradiation of the tumor volume was done at D3, D10 and D17 (lower panel). Caliper tumor measurements and weighing of the animals were done at least 3 days a week. Mice were sacrificed at day 18 following the onset of the treatment. Error bars are based on SEM. <b>A - Cooperative tumor growth reduction of C666-1 NPC xenografts when treated with Abexinostat combined with CDDP but not with irradiation.</b> Tumor growth reduction is statistically significant at day 18 for the combination of Abexinostat with CDDP by comparison with tumors treated with the vehicle, Abexinostat alone or CDDP alone (upper graph). The combination of Abexinostat with irradiation has no statistically significant effects using this NPC model (lower graph). <b>B - Cooperative tumor growth reduction of C15 NPC xenografts treated with Abexinostat combined with CDDP or irradiation.</b> Tumor growth reductions are statistically significant for the combinations of Abexinostat with CDDP (upper graph) or with irradiation (lower graph) by comparison with tumors treated with the vehicle, Abexinostat, CDDP or irradiation used as single agents. <b>C - Cooperative tumor growth reduction of C17 NPC xenografts treated with Abexinostat combined with CDDP or irradiation.</b> Tumor growth reductions are statistically significant for the combinations of Abexinostat with CDDP (upper graph) or with irradiation (lower graph) by comparison with tumors treated with the vehicle, Abexinostat, CDDP or irradiation used as a single agent.</p

Short term culture assays on NPC cells treated <i>in vitro</i> with Abexinostat plus CDDP.

<p>The best combinations of drug dosages are represented graphically. EBV-negative (HONE1 and CNE1) and EBV–positive NPC cells (C15 cells <i>ex vivo</i>, C17 cells <i>ex vivo</i> and C666-1 cells) were treated for 48 h with combinations involving various concentrations of Abexinostat (25, 50, 75 and 150 nM) and CDDP (0.5, 1 and 2 µM). Cell viability was determined in short term assays based on the reduction of MTT (CNE1, HONE1) or WST (C15 <i>ex vivo</i>, C17 <i>ex vivo</i> and C666-1) using triplicates for each experimental conditions. For each NPC model, the result of the most successful combination of drug dosages is depicted as a color disk whose size is proportional to the EOBA index. This index mentioned as a two-digit number reflects the level of synergy for the combined treatment: values greater than 10 are indicative of a synergy between the 2 compounds (see the Materials and Methods section for additional explanations). The X and Y coordinates of the disk stand for the corresponding concentrations of Abexinostat and CDDP respectively. For CNE1 and C666-1, the effects of the combined treatment were only additive. The highest levels of synergy were recorded for C17-cells <i>ex vivo</i>, HONE1 and C15-cells <i>ex vivo</i>.</p

Sensitivity of NPC cells treated <i>in vitro</i> with Abexinostat or CDDP used as single agents.

<p>Sensitivity of NPC cells treated <i>in vitro</i> with Abexinostat or CDDP used as single agents.</p

RAD51 protein and mRNA in NPC cells treated with Abexinostat combined with irradiation.

<p>A – RAD51 protein concentrations in NPC cells treated <i>in vitro.</i> Experiments were performed on cells resulting from collagenase dispersion of the C17 patient-derived xenograft (C17 cells <i>ex vivo</i>) and on C666-1 cells routinely grown <i>in vitro</i> (C666-1). Treatment with Abexinostat (200 nM in culture medium) was applied continuously for two days (D1 and D2). Irradiation was given early on D2. Protein extracts were prepared from untreated cells (Ctrl), cells treated with Abexinostat alone (Abex.), irradiation alone (1Gy) (IR) and combination of both (Abex.+IR). Protein extracts (20 µg/lane) were subjected to PAGE gel separation. Western blotting was performed for detection of RAD51 and actin. RAD51 protein abundance was evaluated by densitometry with normalization on the actin bands detected on the same blotted membrane. It was arbitrarily set at one in lanes corresponding to untreated samples (Ctrl). In C17 and C666-1 cells, RAD51 concentration was decreased to almost the same extent by Abexinostat or Abexinostat plus irradiation. These data are representative of three similar experiments. B – RAD51 protein concentrations in xenografted NPC cells treated <i>in vivo</i>. Mice bearing C17-PDX tumors or xenografted C666-1 cells (C666-1 xeno) were treated during two days (D1 and D2) with vehicle alone, Abexinostat (25 mg/kg BID), vehicle + selective tumor irradiation (2Gy early on D2) or Abexinostat + irradiation. Tumors were collected at D3 and split in fragments used either for protein or RNA extraction (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0091325#pone-0091325-g004" target="_blank">Figure 4C</a>). Tumor protein extracts were subjected to Westen blot and densitometry as reported for <i>in vitro</i> cell extracts in the legend of <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0091325#pone-0091325-g004" target="_blank">Figure 4A</a>. For the C17-PDX, we could observe a reduction in the tumor concentration of RAD51 under treatment by Abexinostat. This reduction was even more marked when Abexinostat was combined to irradiation. In contrast, the depletion of RAD51 was modest for C666-1 xeno. These data are representative of three similar experiments. C – RAD51 mRNA concentrations in xenografted NPC cells treated <i>in vivo</i>. Total RNA was extracted from fragments of xenografted tumors subjected to various treatments in order to investigate the abundance of the RAD51 messenger RNA by real time RT-PCR. Examples of amplification curves are given for RAD51 and GAPDH (glyceraldehyde-3-phosphate dehydrogenase) mRNA which was used as an internal reference. The relative concentrations of RAD51 mRNA obtained for each type of treatment were calculated by the 2^−ΔΔCt method. RNAs from the tumors treated with the vehicle only (vhc) were used as external references or calibrators. Values given in the histogram are means of two distinct experiments. The impact of Abexinostat on RAD51 mRNA was modest and not consistent for C666-1 and C17. For example, it was decreased in the C666-1 xenograft treated with Abexinostat only whereas it was mildly increased in the corresponding C17-PDX. In the C17-PDX treated with Abexinostat combined to irradiation the depletion of the RAD51 mRNA was modest in comparison with the protein depletion in the same tumor fragments (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0091325#pone-0091325-g005" target="_blank">Figure 5B</a>).</p

Clonogenic assays on NPC cells treated <i>in vitro</i> with Abexinostat combined with external irradiation.

<p>The experiments corresponding to the most successful combinations of treatment dosages are presented for HONE1, CNE1 and C666-1. Cells were plated as explained in the Materials and Methods section. Treatment by Abexinostat was started 24 hours following cell plating and irradiation was done 24 h after the onset of Abexinostat. About 3 weeks later cell clones were numbered for calculation of the percentages of growth inhibition (which are mentioned under the picture of each well). For C666-1 cells treated with Abexinostat by itself (50 nM), clonal growth was slightly enhanced instead of being inhibited. The EOBA which reflects the level of synergy for the combined treatment is mentioned for each NPC model. The panel of each model is representative of 3 similar experiments.</p