Specific Targeting of Recombinant Human Pancreatic Ribonuclease ۱ using Gonadotropin-Releasing Hormone Targeting Peptide toward Gonadotropin-Releasing Hormone Receptor-Positive Cancer Cells

Targeted drug delivery is a novel method to specifically deliver anticancer therapeutics to tumor sites . Studies have shown that GnRH peptides can be used to produce chimeric anticancer proteins. Human-origin RNases like human pancreatic ribonuclease ۱ (hpRNase۱) have gained attention due to their high therapeutic index, favorable tissue distribution, and higher ribonucleolytic activity. The addition of GnRH peptide to hpRNase۱ could enhance its tumor inhibitory effect by targeting overexpressing GnRH-R cells and inducing apoptosis. This study aims to design and produce a recombinant fusion protein capable of targeting tumor cells that overexpress GnRH-R. Three variants of human pancreatic ribonuclease ۱, namely hpRNase۱, Tat-hpRNase۱, and GnRH-hpRNase۱ were produced using recombinant DNA technology. The coding sequences of the protein variants were synthesized and sub-cloned into the pET۲۸a(+) expression vector. were amplified using a synthetic pBluescript II cloning vector. E. coli DH۵α cells were transformed with recombinant constructs, and plasmids were extracted. The coding sequence of GnRH-R was optimized and cloned into a mammalian expression vector. AD-۲۹۳ cells were transfected with the recombinant plasmid, and the viability of the cells was evaluated using MTT assay. Statistical analysis included GraphPad Prism, Kruskal-Wallis tests, and FlowJo software, with P values <۰.۰۵ considered statistically significant. The recombinant GnRH-hpRNase۱ fusion protein was found to target cancer cells overexpressing GnRH-R, but not significantly affect cells not expressing GnRH-R. The protein eliminated target cells through apoptosis. Mammalian RNases have potential antineoplastic potential but are inhibited by RNase inhibitors (RI). Researchers have used strategies to eliminate RI sensitivity, including dimerization, chemical modification, fusion to targeting moieties, and site-directed mutagenesis. In this study, an engineered version of hpRNase۱ was used, which is ۹۵% identical to native hpRNase۱ but is RI-evasive while maintaining high conformational stability. The recombinant version was successfully produced in a prokaryotic system and showed activity against mammalian RNAs. Two recombinant fusion forms of hpRNase۱ protein, Tat-hpRNase۱ and GnRH-hpRNase۱, were generated to address the enzyme's non-specific binding and low uptake by tumor cells. Tat-hpRNase۱ contained HIV۱ TAT-PTD peptide, a cell-penetrating peptide (CPP) used for delivering therapeutic molecules. The cell growth inhibition was significantly increased when Tat peptide was introduced into the hpRNase۱ structure. However, Tat-hpRNase۱ inhibited cell proliferation without any specificity. The GnRH-hpRNase۱ fusion protein had a ۲۶.۵-fold higher growth inhibitory effect on GnRH-R positive PC-۳ cells than hpRNase۱ alone. This is likely due to the presence of overexpressed GnRH receptors on these cells, leading to the accumulation of GnRH-hpRNase۱.۳. The antiproliferative effect of Tat-hpRNase۱ and GnRH-hpRNase۱ was not statistically significant when compared to hpRNase۱ alone. Human pancreatic RNase۱ has potential as a therapeutic, but it's sensitive to RI and not exclusively targets cancer cells. A new fusion protein, hpRNase۱, has been designed to target GnRH-R-expressing cells and inhibit proliferation through apoptosis induction. This promising anti-tumor activity warrants further examination on GnRH-R-expressing tumor xenografts to evaluate its in vivo anti-tumor effects.

کلیدواژه ها:

Targeted Drug delivery ، Ribonucleases ، Pancreatic ، Gonadotropin-releasing hormone