A29
Use of nuclear export inhibitors to enhance responses to the anti-tumour agent TRAIL
Natalie Wellsby, Darroll Barrott, Neil Cross
Sheffield Hallam University, Sheffield, UK
Background
Tumour Necrosis Factor-Related Apoptosis-Inducing Ligand (TRAIL) preferentially induces apoptosis in transformed cells via activation of Death Receptor 4/5 (DR4/5). Agonists of DR4/5 are currently in clinical trials, however TRAIL-insensitivity, whether acquired or pre-existing, remains a problem. Leptomycin is a potent inhibitor of CRM1/Exportin-1, which shuttles proteins with nuclear export signals from the nucleus to the cytosol. Increased nuclear export of tumour suppressor nuclear proteins has been associated with tumour progression and therapy resistance, which may be reversed by Nuclear Export Inhibitors (NEIs). In this study, we investigated whether NEIs could enhance TRAIL sensitivity and overcome TRAIL-resistance.
Method
Breast cancer cell lines MDA231 and T47D, and PC3 prostate cancer cells were challenged with 0-20ng/ml Leptomycin in the presence or absence of TRAIL (0-50ng/ml) or DR5 agonist antibody (0-1000ng/ml). Apoptosis was determined by DAPI staining and assessment of nuclear morphology and in addition, cell cycle analysis performed.
Results
TRAIL (5ng/ml) significantly induced apoptosis in PC3 and MDA231 cells, however <10% of cells were apoptotic. T47D was unresponsive to 50ng/ml TRAIL or 1000ng/ml DR5 agonist antibody. Leptomycin alone (0-20nM) induced low levels of apoptosis (<5%). Combination of 5ng/ml TRAIL with 2-20nM TRAIL synergistically and potently enhanced TRAIL responses in PC3 and MDA231. In PC3 cells, Leptomycin (20nM) induced G1 arrest, and in combination with TRAIL, increased the sub-G1 fraction. Leptomycin (5nM) also synergistically enhanced TRAIL (50ng/ml) responses in the TRAIL-resistant cell line T47D.
Conclusion
Leptomycin enhances TRAIL responses both in TRAIL-sensitive and TRAIL-insensitive cell lines. The recent studies have suggested that induction of apoptosis by Leptomycin alone may be dependent on wild-type p53. Here we demonstrate that enhancement of TRAIL responses by Leptomycin occurs cell lines which are p53-null (PC3) or with mutant p53 (MDA231, T47D). NEIs may therefore be useful for enhancing the responses to anti-tumour agents, including DR4/5 agonists.
