Recombinant Engineered Human Pancreatic RNase1 Efficiently Targets and Eliminates Prostate Cancerous Cells

Authors
Omid Mikaili
Zahra Kiadehi
Vida Khalatbari Limaki
Reza Goudarzi
Amin Ramezani
Nasrollah Erfani
Amir Maleksabet
10.61186/ibj.25th-11th-IACRTIMSS
Abstract
Introduction: Targeted drug delivery has opened up a novel window for the specific delivery of anticancer therapeutics directly to tumor sites. Gonadotropin-releasing hormone (GnRH) is a decapeptide that has received attention for its potential use in targeted drug delivery due to its targeting properties. It exhibits a high affinity for its receptor and is not immunogenic in humans. Human pancreatic ribonuclease 1 (hpRNase1) has demonstrated anticancer properties when fused with targeting moieties such as growth hormones, antibodies, and their derivatives. The present study aimed to attach a GnRH-targeting peptide to the N-terminus of hpRNase1 to enhance its specificity for cells expressing the GnRH receptor (GnRH-R).

Methods and Materials: The coding gene was designed, synthesized, and cloned in the pET28a expression vector to produce the recombinant enzyme and subsequently expressed in Escherichia coli BL21 (DE3) bacteria. After induction of expression, the identity of the resulting protein was confirmed by SDS-PAGE and Western blot. Next, the recombinant protein was purified by affinity chromatography, and its cytotoxic effects on cancer cells expressing the GnRH-R were evaluated.

Results: The GnRH-hpRNase1 chimeric protein significantly inhibited the proliferation of PC-3 (p = 0.021), LNCaP (p = 0.034), and AD-Gn (p = 0.041) cells, while the growth of negative cells (AD-293) was not significantly affected (p = 0.081). GnRH-hpRNase1 decreased the IC50 values more than non-fused hpRNase1 by approximately 26.5-fold (p = 0.036) for PC-3 cells and exerted its growth inhibitory effects through apoptosis induction.

Conclusion and Discussion: Ribonucleases, particularly human pancreatic RNase1, have shown intriguing features for developing new therapeutics. However, they suffer from two main shortcomings: (1) being RI sensitive and (2) acting poorly specific to cancer cells. We showed that the engineered GnRH-hpRNase1 can specifically target the GnRH receptor-expressing cells and inhibit their proliferation through inducing apoptosis. Owing to its promising anti-tumor activity, the fusion enzyme can be further examined on GnRH-R-expressing tumor xenografts to evaluate its anti-tumor effects in vivo.



Keywords
Drug delivery systems
Gonadotropin-Releasing Hormone
Pancreatic ribonuclease 1