Down-regulation of XIAP by AEG35156 in paediatric tumour cells induces apoptosis and sensitises cells to cytotoxic agents.

Resistance to conventional chemotherapy is a major problem in several paediatric tumours. One explanation for this is that tumour cells are unable to engage apoptosis after cytotoxic drug-induced damage. Inhibitor of apoptosis proteins (IAPs) function by inhibiting both effector (9) and initiator (3 and 7) caspases. Repression of the widely expressed X-linked IAP (XIAP) by RNAi sensitises adult tumour cells to cytotoxics in vitro. Antisense oligonucleotide (ASO)-induced down-regulation of XIAP is effective at inducing cell death and delaying the growth of adult tumour cells as xenografts and these agents are currently in phase II clinical trials. The importance of XIAP in paediatric tumours has not been characterised but high expression correlates with poor survival in childhood AML. We have used the novel XIAP ASO (AEG35156) to evaluate the effects of down-regulation of XIAP in paediatric tumour cells. Here, we show that AEG35156 can down-regulate XIAP in a number of paediatric cell lines including models of osteosarcoma, rhabdomyosarcoma and Ewing's sarcoma. Cell death assays demonstrated a higher proportion of dead cells after XIAP down-regulation by ASO and these cells displayed increased levels of cleaved caspase-3 and cleaved PARP, showing cell death was due to apoptosis. In long-term clonogenic assays, XIAP ASO sensitised 791T osteosarcoma cells to doxorubicin, etoposide and vincristine. The work presented here suggests that AEG35156, as a monotherapy or in combination with cytotoxic agents, may be of benefit in the treatment of paediatric tumours.

Outcome of gram negative infection in immunocompromised children.

BACKGROUND: Gram negative infection is an important cause of mortality in patients receiving chemotherapy for malignant disease or as conditioning for stem cell transplantation. The risk of infection is thought to be greatest in those patients who are neutropenic and it is routine for these patients to receive broad spectrum antibiotic therapy when febrile. This study evaluates the outcome of gram negative infection in a mixed haematology/oncology population in a single institution. PROCEDURE: All episodes of laboratory proven gram negative infection in patients receiving chemotherapy between January 2004 and March 2006 were included. A retrospective case-note based analysis was carried out to identify diagnosis, treatment, neutrophil count at time of infection, antibiotic therapy and clinical outcome. RESULTS: One hundred six episodes of gram negative infection in 72 patients were identified. Sixty-seven percent of these were in patients receiving chemotherapy for malignant disease, 33% were in patients undergoing stem cell transplantation. Overall ICU admission rate was 13% and mortality was 6%. Coliforms, Pseudomonas and Klebsiella were the commonest isolates, and ICU admission and mortality rates were higher with Pseudomonas and Klebsiella infection than coliforms. After stem cell transplantation most episodes of gram negative infection occurred in non-neutropenic patients, while the reverse was true after chemotherapy. CONCLUSION: Gram negative infection remains a significant cause of both ICU admission and mortality. Gram negative infection is largely confined to neutropenic periods after chemotherapy but not after stem cell transplantation. The isolation of Pseudomonas or Klebsiella confers a higher chance of both ICU admission and death.

Chronic hypoxia promotes hypoxia-inducible factor-1alpha-dependent resistance to etoposide and vincristine in neuroblastoma cells.

Hypoxia is widespread in solid tumors as a consequence of poorly structured tumor-derived neovasculature. Direct measurement of low oxygen levels in a range of adult tumor types has correlated tumor hypoxia with advanced stage, poor response to chemotherapy and radiotherapy, and poor prognosis. Little is known about the importance of hypoxia in pediatric tumors; therefore, we evaluated the effects of hypoxia on the response of the neuroblastoma cell lines SH-EP1 and SH-SY5Y to the clinically relevant drugs, vincristine, etoposide, and cisplatin. Short periods of hypoxia (1% O2) of up to 16 hours had no effect on drug-induced apoptosis or clonogenic survival. Prolonged hypoxia of 1 to 7 days leads to reduction in vincristine- and etoposide-induced apoptosis in SH-SY5Y and SH-EP1 cells, and this was reflected in increased clonogenic survival under these conditions. Neither short-term nor prolonged hypoxia had any effect on the clonogenic response to cisplatin in SH-SY5Y cells. Hypoxia-inducible factor-1 (HIF-1) alpha was stabilized in these cell lines within 2 hours of hypoxia but was no longer detectable beyond 48 hours of hypoxia. Up-regulation of carbonic anhydrase IX showed HIF-1alpha to be transcriptionally active. Down-regulation of HIF-1alpha by short hairpin RNA interference and the small-molecule 3-(5'-hydroxymethyl-2'-furyl)-1-benzylindazole reduced hypoxia-induced drug resistance. These results suggest that prolonged hypoxia leads to resistance to clinically relevant drugs in neuroblastoma and that therapies aimed at inhibiting HIF-1alpha function may be useful in overcoming drug resistance in this tumor.