“The role ER stress in X-aptamers against Growth Hormone Releasing Hormone (GHRH)-mediated apoptotic cell death in HT29 and MiaPaca”

Marmara University, Medical Faculty,

Medical Genetics Department

Thursday, 27 2022

13:30

Abstract

Cancer is a complex disease in which cells undergo malignant transformation via various genomic and proteomic alterations, leading to uncontrollable cellular growth and proliferation [1]. Cancer cells are known to produce growth factors that induce their proliferation, and thus cell division is continually stimulated in these cells [2]. Growth Hormone Releasing Hormone (GHRH) is a hypothalamic neuropeptide that stimulates the pituitary gland for the production and secretion of Growth Hormone (GH) [3]. Instead of the neuroendocrine function, the peripheral expression of GHRH and its receptor was evident in various surgical samples of the prostate, breast, ovarian, and endometrial cancers [4]. The expression and secretion of GHRH in non-pituitary cell types imply the effect of GHRH on the regulation of cell proliferation, differentiation, and carcinogenesis [5]. GHRH peptide antagonist triggered apoptotic cell death via inhibiting GHRH signaling in prostate, endometrial, colon, lung cancer in vitro, and in vivo [6], [7]. Aptamers are single-strand nucleic acid molecules (DNA or RNA) that can bind to target molecules such as proteins, peptides, carbohydrates, bacteria, viruses, and also cancer cells for detection and diagnosis [7]. VEGF aptamer (Macugen) is used for the treatment of macular degeneration [8]. Recently, new generation aptamer synthesis has been performed by using a magnetic bead-dependent modified ssDNA library for target binding and amplification of candidate sequences termed as X-aptamer technology [9]. Our aim is to synthesize, select X-aptamers against GHRH NH2 (1-44) and NH2 (1-29) peptides, and also illustrate the binding affinity of selected putative X-aptamers against its target molecule with regarding their anti-carcinogenic effect on colon and pancreatic cancer cell lines.  Aptamers against GHRH NH2 1-44 and NH2 1-29 peptide were synthesized and binding affinity (Kd) of 24 putative X-aptamers was determined by the dot-blot method, co-immunofluoresences staining and, SPR analysis. Serum stability of 4 X-aptamers were 90-120 h, respectively. The dose-dependent binding of 3 of 24 putative X-aptamers on GHRHR in MIA PaCa-2 was approved by co-IF assay results. Moreover, SPR analysis indicated the Kd (47.5, 12.1 and 40 nM) levels of 3 putative X-aptamers, respectively. Our results illustrates the synthesis of 24 putative X-aptamers against GHRH peptide and 3 X-aptamers has apoptotic effect on MIAPaca-2 pancreatic and HT29 colon cancer cell lines.