Abstract
rAj-HRP30 is a recombinant peptide derived from the wild-type rAj-HRP of Apostichopus japonicus through a gene-shortening mutation. It has a high histidine content (53.3% in its primary structure) and a molecular weight of 3.919 kDa, classifying it as a histidine-rich peptide. The literature reports indicate that human histidine-rich peptides exhibit antitumor activity. Previous research by our group demonstrated similar properties in rAj-HRP, the precursor of rAj-HRP30. Therefore, this study used Panc01 (human) and Panc02 (mouse) cells-highly malignant models with limited targeted therapies-to investigate the antitumor activity and mechanisms of rAj-HRP30 and evaluate its potential for pancreatic cancer treatment. This study designed a gene-shortening strategy for rAj-HRP and artificially synthesized the gene sequence of rAj-HRP30. The cDNA sequence of rAj-HRP30 was cloned into the pET23b vector, and the recombinant plasmid pET23b-HRP30 was transformed into E. coli BL21 for expression. Following IPTG induction, the recombinant peptide was purified using nickel ion affinity chromatography, yielding rAj-HRP30 with a purity exceeding 95%. rAj-HRP30 markedly inhibited the adhesion, migration, and invasion of Panc01 and Panc02 cells. It also disrupted cellular morphology and cytoskeletal structure while inducing apoptosis. These effects were dose-dependent. After confirming the in vitro anticancer activity of rAj-HRP30, this study employed Panc02 cells as a model to investigate its inhibitory mechanisms using Western blot analysis. The results revealed that rAj-HRP30 reduced FGFR1 expression in Panc02 cells and inhibited the downstream FYN and FAK signaling pathways, subsequently blocking the PI3K/AKT signaling and apoptosis pathways. In the apoptotic pathway, rAj-HRP30 was able to downregulate the expression of Bcl-2, Caspase-9, Caspase-3, Caspase-7, and PARP1 and upregulate the expression of Bax, cleaved Caspase-9, cleaved Caspase-3, cleaved Caspase-7, and cleaved-PARP1 to induce apoptosis in Panc02 cells. Furthermore, rAj-HRP30 also downregulated the expression of MMP2 and MMP9, thereby inhibiting the migration and invasion of Panc02 cells. Conclusion: rAj-HRP30 exhibits significant inhibitory effects on pancreatic ductal adenocarcinoma Panc01 and Panc02 cells in vitro. Its mechanism involves FGFR1-related signaling and apoptosis pathways. rAj-HRP30 shows promise as a therapeutic agent targeting FGFR for pancreatic cancer.