Abstract
BackgroundTumor Necrosis Factor (TNF)-Related Apoptosis-Inducing Ligand (TRAIL) selectively induces apoptosis in cancer cells but not in normal cells. Despite this promising feature, TRAIL resistance observed in cancer cells seriously challenged the use of TRAIL as a death ligand in gene therapy. The current dispute concerns whether or not TRAIL receptor expression pattern is the primary determinant of TRAIL sensitivity in cancer cells. This study investigates TRAIL receptor expression pattern and its connection to TRAIL resistance in breast cancer cells. In addition, a DcR2 siRNA approach and a complementary gene therapy modality involving IKK inhibition (AdIKKβKA) were also tested to verify if these approaches could sensitize MCF7 breast cancer cells to adenovirus delivery of TRAIL (Ad5hTRAIL).MethodsTRAIL sensitivity assays were conducted using Molecular Probe's Live/Dead Cellular Viability/Cytotoxicity Kit following the infection of breast cancer cells with Ad5hTRAIL. The molecular mechanism of TRAIL induced cell death under the setting of IKK inhibition was revealed by Annexin V binding. Novel quantitative Real Time RT-PCR and flow cytometry analysis were performed to disclose TRAIL receptor composition in breast cancer cells.ResultsMCF7 but not MDA-MB-231 breast cancer cells displayed strong resistance to adenovirus delivery of TRAIL. Only the combinatorial use of Ad5hTRAIL and AdIKKβKA infection sensitized MCF7 breast cancer cells to TRAIL induced cell death. Moreover, novel quantitative Real Time RT-PCR assays suggested that while the level of TRAIL Decoy Receptor-4 (TRAIL-R4) expression was the highest in MCF7 cells, it was the lowest TRAIL receptor expressed in MDA-MB-231 cells. In addition, conventional flow cytometry analysis demonstrated that TRAIL resistant MCF7 cells exhibited substantial levels of TRAIL-R4 expression but not TRAIL decoy receptor-3 (TRAIL-R3) on surface. On the contrary, TRAIL sensitive MDA-MB-231 cells displayed very low levels of surface TRAIL-R4 expression. Furthermore, a DcR2 siRNA approach lowered TRAIL-R4 expression on surface and this sensitized MCF7 cells to TRAIL.ConclusionThe expression of TRAIL-R4 decoy receptor appeared to be well correlated with TRAIL resistance encountered in breast cancer cells. Both adenovirus mediated IKKβKA expression and a DcR2 siRNA approach sensitized MCF7 breast cancer cells to TRAIL.
Highlights
Tumor Necrosis Factor (TNF)-Related Apoptosis-Inducing Ligand (TRAIL) selectively induces apoptosis in cancer cells but not in normal cells
MCF7 breast carcinoma cells were efficiently transduced with recombinant adenoviruses In order to find out the efficacy of transduction of breast cancer cells by first generation adenoviral vectors, MCF7 cells were infected with increasing Multiplicity of Infection (MOI) of adenovirus encoding Enhanced Green Fluorescent Protein (AdEGFP)
While an multiplicity of infection (MOI) of 5000 DNA particles/cell was sufficient to transduce more than 90 % of the cells, nearly 100 % of the cells were transduced with AdEGFP at an MOI of 10,000 DNA particles/ cell
Summary
Tumor Necrosis Factor (TNF)-Related Apoptosis-Inducing Ligand (TRAIL) selectively induces apoptosis in cancer cells but not in normal cells Despite this promising feature, TRAIL resistance observed in cancer cells seriously challenged the use of TRAIL as a death ligand in gene therapy. There is a 40 % recurrence rate after ten years following the diagnosis and removal of primary tumor in patients with breast cancer [7]. For all these reasons, novel treatment methods are needed for the treatment of patients with breast cancer
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