Surface TRAIL decoy receptor-4 expression is correlated with TRAIL resistance in MCF7 breast cancer cells.
BACKGROUND
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. 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 (AdIKKbetaKA) were also tested to verify if these approaches could sensitize MCF7 breast cancer cells to adenovirus delivery of TRAIL (Ad5hTRAIL).


METHODS
TRAIL 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.


RESULTS
MCF7 but not MDA-MB-231 breast cancer cells displayed strong resistance to adenovirus delivery of TRAIL. Only the combinatorial use of Ad5hTRAIL and AdIKKbetaKA 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.


CONCLUSIONS
The expression of TRAIL-R4 decoy receptor appeared to be well correlated with TRAIL resistance encountered in breast cancer cells. Both adenovirus mediated IKKbetaKA expression and a DcR2 siRNA approach sensitized MCF7 breast cancer cells to TRAIL.
Sanlioglu
Ahter D
AD
The Human Gene Therapy Unit, Akdeniz University, Faculty of Medicine, Antalya, Turkey. ahter@akdeniz.edu.tr
Dirice
Ercument
E
Aydin
Cigdem
C
Erin
Nuray
N
Koksoy
Sadi
S
Sanlioglu
Salih
S
D000256:Adenoviridae / Q000235:genetics / Q000378:metabolism; D017304:Annexin A5 / Q000737:chemistry; D017209:Apoptosis; D001943:Breast Neoplasms / Q000378:metabolism / Q000628:therapy*; D016923:Cell Death; D045744:Cell Line, Tumor; D002470:Cell Survival; D019008:Drug Resistance, Neoplasm; D005434:Flow Cytometry; D058851:GPI-Linked Proteins; D015972:Gene Expression Regulation, Neoplastic; D015316:Genetic Therapy / Q000379:methods*; D006801:Humans; D016328:NF-kappa B / Q000378:metabolism; D034741:RNA, Small Interfering / Q000378:metabolism; D018124:Receptors, Tumor Necrosis Factor / Q000096:biosynthesis*; D053320:Receptors, Tumor Necrosis Factor, Member 10c; D020133:Reverse Transcriptase Polymerase Chain Reaction; D015533:Transcriptional Activation; D053319:Tumor Necrosis Factor Decoy Receptors
D017304:Annexin A5; D058851:GPI-Linked Proteins; D016328:NF-kappa B; D034741:RNA, Small Interfering; D018124:Receptors, Tumor Necrosis Factor; D053320:Receptors, Tumor Necrosis Factor, Member 10c; C108661:TNFRSF10C protein, human; C451501:TNFRSF10D protein, human; D053319:Tumor Necrosis Factor Decoy Receptors