Journal de recherche sur les tumeurs et rapports

Abstrait

Synthesis and characterization of X-aptamers against growth hormone eleasing hormone (1-29) peptide and investigate their apoptotic effect on PC3, HT29 and MIA PaCa-2 cells

Zeynep-Elif Apaydın

Growth Hormone Releasing Hormone (GHRH), 44 amino acid containing hypothalamic hormone, retained its biological activity by first 29 amino acids

1. GHRH (NH2 1-29) peptide antagonists inhibits growth of prostate, breast, ovarian, renal, gastric, pancreatic cancer in vitro and in vivo

2. Aptamers, single-strand RNA or DNA oligonucleotides are capable of binding against target molecules with high affinity

3. Our aim in this study is to synthesize and select aptamers against GHRH (1-29) peptide and demonstrate synthesized aptamers’ inhibitive effect on PC3, HT29 and MIA PaCa-2 cells. Aptamers against GHRH (NH2 1-29) peptides were synthesized by X-aptamer selection kit after biotinylating of target protein. Binding affinity (Kd) of GHRH (NH2 1-29) X-aptamers were determined by dot-blot method. Binding of aptamers against GHRH and its receptor and blocking of GHRH signaling was determined by GH, GHRH-R immunofluorescence assay. Dose- and time-dependent effect of X-aptamers on cell viability, mitochondrial membrane potential, apoptotic effects on PC3, HT29, MIA PaCa-2 cells were determined by MTT cell viability assay, DiOC6, DAPI, PI staining, and Annexin V/PI.

FACS flow analysis, respectively. Binding affinity of two of five putative GHRH (1-29) X-aptamers were by 1.8-fold, TKY.T2.08 and TKY.T2.09 X-aptamers have significant suppression on GH, GHRHR expression without any alteration in intracellular Ca+2 and cAMP levels in HT29, MIA PaCa-2 cells. 500 nM TKY.T2.08/TKY.T2.09 X-aptamer decreased cell viability loss by 16/22 %, 41/20%, 22/10% in PC3, HT29 and MIA PaCa-2 cells for 72 h, respectively. TKY.T2.08/ TKY.T2.09 Xaptamer induced subG1 population accumulation by 5.4/4.5%, 3.7/4.7%, 24/14.7% in PC3, HT29 and MIA PaCa-2 cells for 72 h, respectively. In conclusion, two selected GHRH (1-29) X-aptamer triggered cell viability loss and induced apoptotic cell death in PC3, HT29, MIA PaCa-2 cells via suppressing the expression of GH and GHRH-R. The project was funded by TUBİTAK-1001 National Scientific Research