P-282 Impact of death-associated protein-3 (DAP3) and DAP3 binding cell death enhancer 1 (DELE1) on drug sensitivity in colorectal cancer cells

Abstract

Death association protein-3 (DAP3) is an important growth/death regulator of cells, has been found to have important clinical value in various cancer. DELE1 (DAP3 Binding Cell Death Enhancer 1) has been reported to be a partner of DAP3 and plays a key part in mitochondrial metabolism and mitochondria stress signalling (1). DELE1 also appears to regulate the apoptotic signalling in cells including cancer cells. However, the role played by the two molecules markedly vary depending on cell and tumour types with only a very limited tumour types have been investigated. High expressions of DAP3 and DELE1 have been found to strongly correlated with poor overall survival, disease free survival and distant metastasis free survival in colorectal cancer (2). The present study was to assess the impact of DAP3 and DELE1 on the drug sensitivity of colorectal cancers in vitro. Human colorectal cancer cell lines, RKO and HT115 were used. RKO and HT115 cells were transfected with the lentivirus particles that contained an anti-DAP3 shRNA to create the DAP3 knockdown cell model, designated as RKODAP3kd and HT115DAP3kd, respectively. A DELE1 siRNA was also applied to these cells in order to silence the expression of DELE1 (the new submodels designated as RKODELE1kd and HT115DELE1kd). The DAP3/DELE1 Dual knockdown cell models were established by using the anti-DAP3 shRNA and DELE1 siRNA combination transfection and are referred here as RKODAP3kd/DELE1kd and HT115DAP3kd/DELE1kd. The knockdown of the respective gene transcripts was confirmed by quantitative PCR analysis and respective proteins by Western blot method. Cytotoxicity tests were performed on these sub-models using 5-fluorouracil (5-FU), Docetaxel (DTX) and Methotrexate (MTX) with different concentrations. Additionally, the mitochondrial response following genetic knockdowns were also evaluated. Knockdown DAP3 and knockdown of DELE1 sensitised the RKO cells to 5-FU, MTX and DTX with a IC50 at for control cells at respectively at 14.2mM, 1.6mM, 57.1nM and for RKODAP3kd at 10.4mM, 1.2mM and 51.0nM. Although knocking down DELE1 had less impact than the DAP3 knockdown, DAP3/DELE1 double knockdown however had more profound effects compared with single knockdowns and with wild type controls (IC50 for 5-FU, MTX and DTX in RKODAP3kd/DELE1kd cells being respectively at 7.0mM, 0.6mM and 1.5nM). In the metabolic analyses including metabolism of glucose, lactate, NAD(P)H and nitrogen dioxide (NO2), we did not detect a significant change following DAP3 knockdown, DELE1 knockdown or the double knockdown, arguing an mitochondria pathway independent regulation of the drug sensitivity by the two molecules. DAP3 and DELE1, important growth/death regulators and prognostic factors of in colorectal cancer, have a role in the chemosensitivity in colorectal cancer cells. The findings suggest that molecules are potential targets in therapeutic intervention in colorectal cancer.