Engineering diphteria toxin in pancreatic cancer cells: a promising tool for therapy.

Introduction. Diphteria toxin is a promising tool for cancer cell killing. It causes cell death by an apoptosis-mediated pathway catalysing ADP-ribosylation of elongating factor 2. Our aim was to test DT catalytic subunit (fragment A 1-193 aa residues) (DTA) efficacy in killing 5 different pancreatic cancer cell lines obtained from primary (BxPC3, PANC1, PSN1 and MIAPaCa2) or metastatic (CAPAN1) tumors. Methods. DTA was subcloned in an eukariotic expression vector (pRc) under the control of a constitutive promoter (RSV). Chemical transfection (Lipofectamine 2000) efficiency was evaluated by FACS analysis, using a FITColigonucleotide as tracer. Transcription efficiency was evaluated by FACS analysis and western blotting using a reporter gene (GFP) cloned in pRc. Study design was. 1. transfection of 250.000 cells for 6 h with 4 ug DNA; 2. after 24 h cells were seeded in quadruplicate in a 96-well plate; 3. cell growth was evaluated daily for 3 days by the cell viability XTT test. For each cell line two controls were run in parallel: lipofectamine only treated cells and cells transfected with the empty DNA vector (pRc). Results. More than 50% of cells were efficiently transfected. In four cell lines GFP transcription was recorded: CAPAN-1 did not translate the inserted gene. In agreement this cell line was resistant to DTA gene transfer. A complete growth inhibition was achieved after DTA gene transfer in BxPC3, PANC1, PSN1 and MIAPaCa2 in comparison with the two corresponding control cells (MANOVA: within and between subjects = p<0.0001 for all cell lines). Conclusions. DTA expression is lethal for pancreatic cancer cells and this supports the potential use of this toxin for pancreatic cancer gene therapy. The lack of gene translation found in CAPAN-1 cells, a metastatic cell line, might be consequent to the absence of specific transcription factors recognizing the RSV promoter.