Value Added Abstract
Zeynep-Elif Apaydın
Abstract
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 TUBÄ°TAK-1001 National Scientific Research Biography: Zeynep Elif Apaydın was born in 1993 in Samsun, Turkey. When she was a student in high school, she decided to study molecular biology and genetics. She has started to study in T.C. Ä°stanbul Kültür University and moved to Ä°stanbul in 2012. She currently pursues a master's degree at Ä°stanbul Kültür University and continues her studies on cancer research. She has given 2 poster presentations in international congresses related to her research. She is planning to continue her work on academical field after graduation from master's programme Speaker Publications: M. R. Ehlers, “Recombinant human GHRH (1-44) NH 2: Clinical utility and therapeutic development program,” Endocrine, vol. 14, no. 1, pp. 137–141, 2001, doi: 10.1385/ENDO: 14:1:137. F. G. Rick et al., “Antagonists of growth hormone-releasing hormone inhibit growth of androgen-independent prostate cancer through inactivation of ERK and Akt kinases,” Proc. Natl. Acad. Sci. U. S. A., vol. 109, no. 5, pp. 1655–1660, 2012, doi: 10.1073/pnas.1120588109. C. Walss-Bass et al., “X-Aptamer Technology Identifies C4A and ApoB in Blood as Potential Markers for Schizophrenia,” Mol. Neuropsychiatry, vol. 5, no. 1, pp. 52–59, 2019, doi: 10.1159/000492331. 17th International Conference on Cancer; Barcelona, Spain - June 15-16, 2020. Abstract Citation: Zeynep-Elif Apaydın, Synthesis and characterization of X-aptamers against Growth hormone releasing hormone (1-29) peptide and investigate their apoptotic effect on PC3, HT29 and MIA PaCa-2 cells, Cancer Research 2020, 17th International Conference on Cancer; Barcelona, Spain - June 15-16, 2020. (https://cancer-research.cancersummit.org/abstract/2020/investigation-of-the-circadian-clock-genes-in-human-melanoma-cells)