Song Y et al. (2025), Inhibition Effects and Mechanism Study of rAj-H...

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Int J Mol Sci 2025 Feb 11;264:. doi: 10.3390/ijms26041485.

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Inhibition Effects and Mechanism Study of rAj-HRP30, a Recombinant Histidine-Rich Peptide from Apostichopus japonicus, on the Viability of Pancreatic Ductal Adenocarcinoma Cells Panc01 and Panc02.

Song Y , Gao S , Jiang J , Zhang Y , Zhang J , Wang X , Lv L , Zhou Z , Wang J .

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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.

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	Figure 1. (a) rAj-HRP30 is a truncated peptide derived from the wild-type rAj-HRP, with the amino acid sequence of rAj-HRP30 highlighted in red underline. (b) The artificially synthesized cDNA sequence of rAj-HRP and its deduced amino acid sequence, * represents the termination codon. (c) Phylogenetic tree analysis of rAj-HRP30 (with rAj-HRP30 highlighted in yellow).
	Figure 2. (a) Schematic representation of the pET23b vector containing the rAj-HRP30 gene and the restriction sites identified through double digestion. Arrow 1 represents the insertion direction of the multiple cloning sites (MCS); Arrow 2 represents the copy direction of f1 origin; Arrow 3 represents the transcription direction of the resistance gene Amp; Arrow 4 represents the copy direction of ori. (b) Restriction enzyme digestion analysis of the pET23b-HRP30 plasmid: approximately 200–1000 ng of plasmid DNA was digested at 37 °C for 30–60 min and analyzed on a 1% agarose gel. Lane M: DNA size marker (KB ladder); Lane 1: undigested pET23b-HRP30 recombinant plasmid; Lane 2: pET23b-HRP30 recombinant plasmid digested with Bgl I and Xho I (This data is from Kingsley Biotechnology (Nanjing, China)). (c) Sequencing results of the pET23b-HRP30 recombinant plasmid: The green marker represents the DNA sequence of pET23b plasmid. The yellow markers represent the Nde I and Xho I restriction enzyme sites. The red marker represents the cDNA sequence of HRP30.
	Figure 3. The tricine SDS-PAGE results of rAj-HRP30 purification. Lane 1: marker; Lane 2: purified rAj-HRP30; Lane 3: purified rAj-HRP30.
	Figure 4. The effect of rAj-HRP30 on Panc01 and Panc02 cell proliferation was determined using the CCK-8 method (n = 3). The control group was treated with a blank medium, and the experimental group was treated with rAj-HRP30. Significant difference between the blank group and each experimental group ( p < 0.01, and * p < 0.001).
	Figure 5. (a) Effect of rAj-HRP30 on the morphology of Panc01 cells (OLYMPUS digital imaging microscope, Tokyo, Japan, 200×; scale: 2 μm). (b) Effect of rAj-HRP30 on the morphology of Panc02 cells (OLYMPUS digital imaging microscope, Tokyo, Japan, 200×; scale: 2 μm). (c) The effects of rAj-HRP30 on the cytoskeleton and nucleus of Panc01 were determined using an FITC-ghost cyclic peptide assay (Zeiss laser scanning confocal microscope, Oberkochen, Baden-Wurttemberg, Germany, 200×; scale: 2 μm). (d) The effects of rAj-HRP30 on the cytoskeleton and nucleus of Panc02 were determined using an FITC-ghost cyclic peptide assay (Zeiss laser scanning confocal microscope, Oberkochen, Baden-Wurttemberg, Germany,200×; scale: 2 μm). (e) Effect of rAj-HRP30 on changes in relative fluorescence intensity of the Panc01 cytoskeleton (n = 3). The control group was treated with a blank medium, and the experimental group was treated with rAj-HRP30. ns indicates no statistical significance; indicates significant difference between the blank group and each experimental group ( p < 0.01). (f) Effect of rAj-HRP30 on changes in relative fluorescence intensity of the Panc02 cytoskeleton (n = 3). The control group was treated with a blank medium, and the experimental group was treated with rAj-HRP30. ns indicates no statistical significance; , indicates significant difference between the blank group and each experimental group ( p < 0.01 and **** p < 0.0001).
	Figure 6. (a) The effect of rAj-HRP30 on the adhesion of Panc01 cells was determined (n = 3); significant differences between the experimental group treated with rAj-HRP30 and the control group treated with empty medium are indicated by * p < 0.05, p < 0.01, and ** p < 0.0001. (b) The effect of rAj-HRP30 on the adhesion of Panc02 cells was determined (n = 3); significant differences between the experimental group treated with rAj-HRP30 and the control group treated with empty medium are indicated by * p < 0.05, p < 0.01. (c) The degree of Panc01 cell migration under different administered concentrations (OLYMPUS digital imaging microscope, scale: 200 μm). (d) The amount of Panc01 cell migration under different administered concentrations (n = 3). The experimental group was treated with bFGF- or rAj-HRP30, and the control group was treated with bFGF+ without medication. Significant differences are expressed by p < 0.0001. (e) The degree of Panc02 cell migration under different administered concentrations (OLYMPUS digital imaging microscope, scale: 200 μm). (f) The amount of Panc02 cell migration under different administered concentrations (n = 3). The experimental group was treated with bFGF- or rAj-HRP30, and the control group was treated with bFGF+ without medication. Significant differences are expressed by p < 0.01, * p < 0.001, and ** p < 0.0001.
	Figure 7. (a) Representative results of Panc01 cell invasion in each group (OLYMPUS digital imaging microscope, scale: 200 μm). (b) Statistics on the number of Panc01 cells invaded in each group (n = 3); significant differences between the experimental group treated with bFGF- or rAj-HRP30 and the control group treated with bFGF are indicated by p < 0.01 and p < 0.0001. (c) Representative results of Panc02 cell invasion in each group (OLYMPUS digital imaging microscope, scale: 200 μm). (d) Statistics on the number of Panc02 cells invaded in each group (n = 3); significant differences between the experimental group treated with bFGF- or rAj-HRP30 and the control group treated with bFGF are expressed by ** p < 0.0001. (e) Bar graph illustrating the Western blot results for MMP2 and MMP9 expression in Panc02 cells treated with rAj-HRP30. (f) Statistical analysis of the relative optical densities of MMP2 and MMP9 (target proteins) normalized to GAPDH levels (n = 3). The control group was treated with a blank medium, and the experimental groups were treated with rAj-HRP30. Significant differences between the blank group and the experimental groups are expressed by * p < 0.05, p < 0.01, * p < 0.001.
	Figure 8. (a) The apoptotic effect of rAj-HRP30 on Panc01 cells was determined using the Hoechst method (Zeiss laser scanning confocal microscope, 630×; scale: 10 μm). (b) The apoptotic effect of rAj-HRP30 on Panc02 cells was determined using the Hoechst method (Zeiss laser scanning confocal microscope, 630×; scale: 10 μm). (c) The apoptotic effect of rAj-HRP30 on Panc01 cells was determined using the TUNEL method (Zeiss laser scanning confocal microscope, 630×; scale: 10 μm). (d) The apoptotic effect of rAj-HRP30 on Panc02 cells was determined using the TUNEL method (Zeiss laser scanning confocal microscope, 630×; scale: 10 μm).
	Figure 9. (a) Bar graph illustrating the Western blot results for Bax and Bcl-2 expression in Panc02 cells treated with rAj-HRP30. (b) Statistical analysis of the relative optical densities of Bax (target proteins) normalized to GAPDH levels. (c) Statistical analysis of the relative optical densities of Bcl-2 (target proteins) normalized to GAPDH levels. (d) Statistical analysis of the relative optical densities of Bcl-2/Bax (target proteins) normalized to GAPDH levels. (e) Bar graph illustrating the Western blot results for Caspase-9 and cleaved Caspase-9 expression in Panc02 cells treated with rAj-HRP30. (f) Bar graph illustrating the Western blot results for Caspase-7 and cleaved Caspase-7 expression in Panc02 cells treated with rAj-HRP30. (g) Bar graph illustrating the Western blot results for Caspase-3 and cleaved Caspase-3 expression in Panc02 cells treated with rAj-HRP30. (h) Bar graph illustrating the Western blot results for PARP1 and cleaved PARP1 expression in Panc02 cells treated with rAj-HRP30. (i) Statistical analysis of the relative optical densities of Caspase-9 and cleaved Caspase-9 (target proteins) normalized to GAPDH levels. (j) Statistical analysis of the relative optical densities of Caspase-7 and cleaved Caspase-7 (target proteins) normalized to GAPDH levels. (k) Statistical analysis of the relative optical densities of Caspase-3 and cleaved Caspase-3 (target proteins) normalized to GAPDH levels. (l) Statistical analysis of the relative optical densities of PARP1 and cleaved PARP1 (target proteins) normalized to GAPDH levels. (n = 3); the control group was treated with a blank medium, and the experimental groups were treated with rAj-HRP30. Significant differences between the blank group and the experimental groups are expressed by * p < 0.05, p < 0.01, * p < 0.001, and **** p < 0.0001.
	Figure 10. (a) Bar graph illustrating the Western blot results for FGRF1, FYN, and FAK expression in Panc02 cells treated with rAj-HRP30. (b) Statistical analysis of the relative optical densities of FGRF1 (target proteins) normalized to GAPDH levels (n = 3); the control group was treated with a blank medium, and the experimental groups were treated with rAj-HRP30. Significant differences between the blank group and the experimental groups are expressed by **** p < 0.0001. (c) Statistical analysis of the relative optical densities of FAK (target proteins) normalized to GAPDH levels (n = 3); the control group was treated with a blank medium, and the experimental groups were treated with rAj-HRP30. Significant differences between the blank group and the experimental groups are expressed by * p < 0.05, p < 0.01, and * p < 0.001. (d) Statistical analysis of the relative optical densities of FYN (target proteins) normalized to GAPDH levels (n = 3); the control group was treated with a blank medium, and the experimental groups were treated with rAj-HRP30. Significant differences between the blank group and the experimental groups are expressed by * p < 0.05, * p < 0.001, and p < 0.0001. (e) Bar graph illustrating the Western blot results for PI3K, p-PI3K, AKT, and p-AKT expression in Panc02 cells treated with rAj-HRP30. (f) Statistical analysis of the relative optical densities of PI3K and p-PI3K (target proteins) normalized to GAPDH levels (n = 3); the control group was treated with a blank medium, and the experimental groups were treated with rAj-HRP30. Significant differences between the blank group and the experimental groups are expressed by p < 0.01 and ** p < 0.0001. (g) Statistical analysis of the relative optical densities of AKT and p-AKT (target proteins) normalized to GAPDH levels (n = 3); the control group was treated with a blank medium, and the experimental groups were treated with rAj-HRP30. Significant differences between the blank group and the experimental groups are expressed by p < 0.01, * p < 0.001, and ** p < 0.0001.

	Figure 1. (a) rAj-HRP30 is a truncated peptide derived from the wild-type rAj-HRP, with the amino acid sequence of rAj-HRP30 highlighted in red underline. (b) The artificially synthesized cDNA sequence of rAj-HRP and its deduced amino acid sequence, * represents the termination codon. (c) Phylogenetic tree analysis of rAj-HRP30 (with rAj-HRP30 highlighted in yellow).
	Figure 2. (a) Schematic representation of the pET23b vector containing the rAj-HRP30 gene and the restriction sites identified through double digestion. Arrow 1 represents the insertion direction of the multiple cloning sites (MCS); Arrow 2 represents the copy direction of f1 origin; Arrow 3 represents the transcription direction of the resistance gene Amp; Arrow 4 represents the copy direction of ori. (b) Restriction enzyme digestion analysis of the pET23b-HRP30 plasmid: approximately 200–1000 ng of plasmid DNA was digested at 37 °C for 30–60 min and analyzed on a 1% agarose gel. Lane M: DNA size marker (KB ladder); Lane 1: undigested pET23b-HRP30 recombinant plasmid; Lane 2: pET23b-HRP30 recombinant plasmid digested with Bgl I and Xho I (This data is from Kingsley Biotechnology (Nanjing, China)). (c) Sequencing results of the pET23b-HRP30 recombinant plasmid: The green marker represents the DNA sequence of pET23b plasmid. The yellow markers represent the Nde I and Xho I restriction enzyme sites. The red marker represents the cDNA sequence of HRP30.
	Figure 3. The tricine SDS-PAGE results of rAj-HRP30 purification. Lane 1: marker; Lane 2: purified rAj-HRP30; Lane 3: purified rAj-HRP30.
	Figure 4. The effect of rAj-HRP30 on Panc01 and Panc02 cell proliferation was determined using the CCK-8 method (n = 3). The control group was treated with a blank medium, and the experimental group was treated with rAj-HRP30. Significant difference between the blank group and each experimental group ( p < 0.01, and * p < 0.001).
	Figure 5. (a) Effect of rAj-HRP30 on the morphology of Panc01 cells (OLYMPUS digital imaging microscope, Tokyo, Japan, 200×; scale: 2 μm). (b) Effect of rAj-HRP30 on the morphology of Panc02 cells (OLYMPUS digital imaging microscope, Tokyo, Japan, 200×; scale: 2 μm). (c) The effects of rAj-HRP30 on the cytoskeleton and nucleus of Panc01 were determined using an FITC-ghost cyclic peptide assay (Zeiss laser scanning confocal microscope, Oberkochen, Baden-Wurttemberg, Germany, 200×; scale: 2 μm). (d) The effects of rAj-HRP30 on the cytoskeleton and nucleus of Panc02 were determined using an FITC-ghost cyclic peptide assay (Zeiss laser scanning confocal microscope, Oberkochen, Baden-Wurttemberg, Germany,200×; scale: 2 μm). (e) Effect of rAj-HRP30 on changes in relative fluorescence intensity of the Panc01 cytoskeleton (n = 3). The control group was treated with a blank medium, and the experimental group was treated with rAj-HRP30. ns indicates no statistical significance; indicates significant difference between the blank group and each experimental group ( p < 0.01). (f) Effect of rAj-HRP30 on changes in relative fluorescence intensity of the Panc02 cytoskeleton (n = 3). The control group was treated with a blank medium, and the experimental group was treated with rAj-HRP30. ns indicates no statistical significance; , indicates significant difference between the blank group and each experimental group ( p < 0.01 and **** p < 0.0001).
	Figure 6. (a) The effect of rAj-HRP30 on the adhesion of Panc01 cells was determined (n = 3); significant differences between the experimental group treated with rAj-HRP30 and the control group treated with empty medium are indicated by * p < 0.05, p < 0.01, and ** p < 0.0001. (b) The effect of rAj-HRP30 on the adhesion of Panc02 cells was determined (n = 3); significant differences between the experimental group treated with rAj-HRP30 and the control group treated with empty medium are indicated by * p < 0.05, p < 0.01. (c) The degree of Panc01 cell migration under different administered concentrations (OLYMPUS digital imaging microscope, scale: 200 μm). (d) The amount of Panc01 cell migration under different administered concentrations (n = 3). The experimental group was treated with bFGF- or rAj-HRP30, and the control group was treated with bFGF+ without medication. Significant differences are expressed by p < 0.0001. (e) The degree of Panc02 cell migration under different administered concentrations (OLYMPUS digital imaging microscope, scale: 200 μm). (f) The amount of Panc02 cell migration under different administered concentrations (n = 3). The experimental group was treated with bFGF- or rAj-HRP30, and the control group was treated with bFGF+ without medication. Significant differences are expressed by p < 0.01, * p < 0.001, and ** p < 0.0001.
	Figure 7. (a) Representative results of Panc01 cell invasion in each group (OLYMPUS digital imaging microscope, scale: 200 μm). (b) Statistics on the number of Panc01 cells invaded in each group (n = 3); significant differences between the experimental group treated with bFGF- or rAj-HRP30 and the control group treated with bFGF are indicated by p < 0.01 and p < 0.0001. (c) Representative results of Panc02 cell invasion in each group (OLYMPUS digital imaging microscope, scale: 200 μm). (d) Statistics on the number of Panc02 cells invaded in each group (n = 3); significant differences between the experimental group treated with bFGF- or rAj-HRP30 and the control group treated with bFGF are expressed by ** p < 0.0001. (e) Bar graph illustrating the Western blot results for MMP2 and MMP9 expression in Panc02 cells treated with rAj-HRP30. (f) Statistical analysis of the relative optical densities of MMP2 and MMP9 (target proteins) normalized to GAPDH levels (n = 3). The control group was treated with a blank medium, and the experimental groups were treated with rAj-HRP30. Significant differences between the blank group and the experimental groups are expressed by * p < 0.05, p < 0.01, * p < 0.001.
	Figure 8. (a) The apoptotic effect of rAj-HRP30 on Panc01 cells was determined using the Hoechst method (Zeiss laser scanning confocal microscope, 630×; scale: 10 μm). (b) The apoptotic effect of rAj-HRP30 on Panc02 cells was determined using the Hoechst method (Zeiss laser scanning confocal microscope, 630×; scale: 10 μm). (c) The apoptotic effect of rAj-HRP30 on Panc01 cells was determined using the TUNEL method (Zeiss laser scanning confocal microscope, 630×; scale: 10 μm). (d) The apoptotic effect of rAj-HRP30 on Panc02 cells was determined using the TUNEL method (Zeiss laser scanning confocal microscope, 630×; scale: 10 μm).
	Figure 9. (a) Bar graph illustrating the Western blot results for Bax and Bcl-2 expression in Panc02 cells treated with rAj-HRP30. (b) Statistical analysis of the relative optical densities of Bax (target proteins) normalized to GAPDH levels. (c) Statistical analysis of the relative optical densities of Bcl-2 (target proteins) normalized to GAPDH levels. (d) Statistical analysis of the relative optical densities of Bcl-2/Bax (target proteins) normalized to GAPDH levels. (e) Bar graph illustrating the Western blot results for Caspase-9 and cleaved Caspase-9 expression in Panc02 cells treated with rAj-HRP30. (f) Bar graph illustrating the Western blot results for Caspase-7 and cleaved Caspase-7 expression in Panc02 cells treated with rAj-HRP30. (g) Bar graph illustrating the Western blot results for Caspase-3 and cleaved Caspase-3 expression in Panc02 cells treated with rAj-HRP30. (h) Bar graph illustrating the Western blot results for PARP1 and cleaved PARP1 expression in Panc02 cells treated with rAj-HRP30. (i) Statistical analysis of the relative optical densities of Caspase-9 and cleaved Caspase-9 (target proteins) normalized to GAPDH levels. (j) Statistical analysis of the relative optical densities of Caspase-7 and cleaved Caspase-7 (target proteins) normalized to GAPDH levels. (k) Statistical analysis of the relative optical densities of Caspase-3 and cleaved Caspase-3 (target proteins) normalized to GAPDH levels. (l) Statistical analysis of the relative optical densities of PARP1 and cleaved PARP1 (target proteins) normalized to GAPDH levels. (n = 3); the control group was treated with a blank medium, and the experimental groups were treated with rAj-HRP30. Significant differences between the blank group and the experimental groups are expressed by * p < 0.05, p < 0.01, * p < 0.001, and **** p < 0.0001.
	Figure 10. (a) Bar graph illustrating the Western blot results for FGRF1, FYN, and FAK expression in Panc02 cells treated with rAj-HRP30. (b) Statistical analysis of the relative optical densities of FGRF1 (target proteins) normalized to GAPDH levels (n = 3); the control group was treated with a blank medium, and the experimental groups were treated with rAj-HRP30. Significant differences between the blank group and the experimental groups are expressed by **** p < 0.0001. (c) Statistical analysis of the relative optical densities of FAK (target proteins) normalized to GAPDH levels (n = 3); the control group was treated with a blank medium, and the experimental groups were treated with rAj-HRP30. Significant differences between the blank group and the experimental groups are expressed by * p < 0.05, p < 0.01, and * p < 0.001. (d) Statistical analysis of the relative optical densities of FYN (target proteins) normalized to GAPDH levels (n = 3); the control group was treated with a blank medium, and the experimental groups were treated with rAj-HRP30. Significant differences between the blank group and the experimental groups are expressed by * p < 0.05, * p < 0.001, and p < 0.0001. (e) Bar graph illustrating the Western blot results for PI3K, p-PI3K, AKT, and p-AKT expression in Panc02 cells treated with rAj-HRP30. (f) Statistical analysis of the relative optical densities of PI3K and p-PI3K (target proteins) normalized to GAPDH levels (n = 3); the control group was treated with a blank medium, and the experimental groups were treated with rAj-HRP30. Significant differences between the blank group and the experimental groups are expressed by p < 0.01 and ** p < 0.0001. (g) Statistical analysis of the relative optical densities of AKT and p-AKT (target proteins) normalized to GAPDH levels (n = 3); the control group was treated with a blank medium, and the experimental groups were treated with rAj-HRP30. Significant differences between the blank group and the experimental groups are expressed by p < 0.01, * p < 0.001, and ** p < 0.0001.