Abstract

   Human umbilical cord serum is full of molecules that play vital roles
   in foetal development. This study aimed to explore the effects of
   RR-171, a novel peptide derived from umbilical cord serum, on
   pancreatic cancer cells and to elucidate its mechanisms. The
   anti-pancreatic cancer properties of RR-171 were detected by a cell
   counting kit-8, colony formation, flow cytometry, LDH release and EdU
   incorporation assays. RNA sequencing and gene enrichment analysis were
   applied to identify the differentially expressed genes and enriched
   pathways. Western blotting analysis was used to detect the expression
   of proteins. A subcutaneous xenograft model was used to examine the
   effect of RR-171 on pancreatic cancer cells in vivo. The results
   demonstrated that RR-171 inhibited the viability, proliferation and
   colony formation of pancreatic cancer cells in a dose-dependent manner.
   Gene enrichment analysis revealed that RR-171 inhibits the Wnt
   signaling pathway. Moreover, RR-171 significantly induced apoptosis and
   pyroptosis in pancreatic cancer cells in a dose-dependent manner.
   Z-VAD-FMK partly reversed the proapoptotic effect of RR-171, and VX-765
   partly reversed the pro-pyroptotic effect of RR-171. Finally, RR-171
   inhibited the growth of pancreatic cancer cells in a subcutaneous
   xenograft mice model and suppressed the expression of Ki-67 and PCNA in
   tumors. In conclusion, RR-171 induces apoptosis and pyroptosis through
   multiple pathways and inhibits pancreatic cancer growth, suggesting
   that RR-171 might be a potential agent for the treatment of pancreatic
   cancer.

Supplementary Information

   The online version contains supplementary material available at
   10.1038/s41598-025-96465-x.

   Keywords: Umbilical cord serum, Peptide, RR-171, Apoptosis, Pyroptosis,
   Pancreatic cancer

   Subject terms: Cancer, Drug discovery, Medical research, Molecular
   medicine

Introduction

   Pancreatic cancer is the seventh major cause of cancer-related death
   and ranks 12th among 36 cancers in terms of morbidity^[36]1,[37]2. The
   common risk factors for pancreatic cancer consist of smoking, chronic
   pancreatitis, diabetes, obesity, and other occupational chemical
   exposures^[38]3,[39]4. Surgery is still the cornerstone of treatment
   for pancreatic cancer, but comprehensive treatments, including
   radiotherapy and chemotherapy, also play vital roles in the fight
   against this disease^[40]5,[41]6. Although, advances have been made in
   the treatment of pancreatic cancer in recent years. Owing to the lack
   of prompt screening and diagnostic methods, most patients are already
   in an advanced stage when they are diagnosed, the success of these
   treatments is relatively low, and the overall 5-year survival ratio is
   not more than 10%^[42]7. Moreover, pancreatic cancer is an
   immunologically “cold” cancer characterized by a lack of infiltration
   of effector T cells, prominent myeloid inflammation and a low tumor
   mutational burden, which thwarts immunotherapy^[43]8,[44]9. Thus, novel
   agents for the treatment of pancreatic cancer are urgently needed.

   Peptides are tiny molecules with less than fifty amino acids in length
   and are often derived from natural or synthetic products^[45]10.
   Peptides can be used as pharmaceuticals for various diseases, including
   asthma, stroke, diabetes, heart disease and wound healing^[46]11. In
   terms of anticancer treatment, peptides have also been exploited for
   various therapeutic and diagnostic strategies^[47]12. For example, the
   peptides leuprolide and goserelin are clinically used to treat prostate
   cancer^[48]13. In addition, peptides can be applied as tumor-targeting
   carriers in peptide-drug conjugates through the combination of
   cytotoxic drugs with peptide ligands that can target tumor-specific
   biomarkers or cancer cell-membrane receptors, which can lessen side
   effects and improve the specificity of cytotoxic drugs^[49]14,[50]15.
   The peptide-drug conjugate ^177Lu-dotatate has been approved for the
   treatment of gastroenteropancreatic neuroendocrine tumors^[51]16. The
   above results suggest that peptides might be promising choices for
   pancreatic cancer therapy.

   Human umbilical cord blood, which is characterized by the presence of
   various stem cells, proteins, cytokines and lipids, plays a vital role
   in foetal development^[52]17. Up to now, human umbilical cord blood has
   been widely applied in clinical settings by utilizing hematopoietic
   stem/progenitor cells, for example, Fanconi anemia, tissue
   regeneration, and malignant hematological diseases^[53]18. In recent
   years, umbilical cord blood serum (UCBS) has attracted increasing
   attention because it contains abundant cytokines and molecules that
   play important roles in cell proliferation, differentiation and immune
   regulation^[54]19. However, studies on the role of UCBS components in
   the oncology field are rare. A previous study reported that UCBS
   inhibits the viability, proliferation, and migration of human prostate
   cancer cell lines^[55]20, which represents a novel direction in the
   oncology therapy field. Thus, anticancer components might be found in
   UCBS, which might stimulate the discovery of new agents for pancreatic
   cancer treatment. RR-171, a novel peptide with seventeen amino acids
   (peptide sequence: RRRRLVAGVLVLLRRRR), was identified and modified in
   our laboratory from UCBS though proteomics biotechnology method. The
   biological function segment is the sequence LVAGVLVLL, and the four
   arginines at both ends are added to increase its solubility. The
   molecular weight of RR-171 is 2145.68 g/mol, the isoelectric point of
   RR-171 is 12.4, and net charge is 8^[56]21. The random coil of RR-171
   accounts for 35.7%, the antiparallel structure accounts for 33.9%, and
   beta-turn accounts for 20.1%^[57]21. In our previous study, we reported
   that RR-171 exhibits anticancer properties^[58]21. However, the
   specific role of RR-171 in pancreatic cancer has not been investigated.
   In the current study, we investigated the antitumor effect of RR-171
   peptide on human pancreatic cancer cells both in vitro and in vivo and
   then further explored the underlying mechanisms of this effect in
   pancreatic cancer cells.

Materials and methods

Cell culture and reagents

   The hTERT-immortalized human normal pancreatic epithelial cell line
   (HPNE) and pancreatic cancer cell lines (Panc-1, Bxpc-3, and Capan-2)
   were purchased from ATCC. HPNE and Panc-1 cells were cultured in
   Dulbecco’s modified Eagle’s medium (DMEM, Gibco, Carlsbad, USA)
   containing 10% fetal bovine serum (FBS) (Gibco) and 1%
   penicillin/streptomycin (Gibco). Bxpc-3 and Capan-2 were cultured in
   RPMI 1640 medium (Gibco) containing 10% FBS (Gibco) and 1%
   penicillin/streptomycin (Gibco). These cells were cultured at 37℃ in a
   5% CO[2] humidified incubator. Z-VAD-FMK and VX-765 were purchased from
   Selleck Chemicals (Selleck, Houston, USA). Both reagents were dissolved
   in DMSO (Sigma Aldrich), and diluted to 100 mM and stored in a − 80℃
   freezer. The peptide RR-171 was synthesized by Glbiochem (Shanghai,
   China).

Cell viability assay

   The cells were seeded in 96-well plates (4500 cells/well). After cell
   adhesion, the medium was replaced with fresh medium or medium
   containing DMSO or different concentrations of RR-171 for 24 h. Then,
   the cells were incubated with medium containing 10% Cell Counting Kit-8
   reagent (CCK-8; Beyotime Biotechnology, China) for 2 h at 37℃. The OD
   value was measured with a microplate reader (BioTek Epoch, USA) at
   450 nm. The IC[50] values were counted by nonlinear regression analysis
   using GraphPad Prism 8 software.

Colony formation assay

   The cells were seeded in 6-well plates (800 cells/well). After the
   cells adhesion steadily, the medium was replaced with fresh medium or
   different concentrations of RR-171 for 24 h, after which the medium was
   replaced with fresh medium for 14 days. These cells were washed with
   PBS and then fixed with 4% paraformaldehyde (Beyotime Biotechnology,
   China). After fixation, the cells were washed once with PBS and then
   stained with crystal violet (Beyotime Biotechnology, China) for 30 min.

EdU incorporation assay

   The cells were seeded in 96-well plates (2500 cells/well). After cell
   adhesion, fresh medium or different concentrations of RR-171 were added
   to Bxpc-3 and Capan-2 cells for 48 h. The BeyoClick™ EdU Cell
   Proliferation Kit with Alexa Fluor 488 (Beyotime Biotechnology, China)
   was used according to the manufacturer’s protocol. The cells were
   observed and imaged with a fluorescence microscope after EdU staining.

RNA sequencing and gene enrichment analysis

   Capan-2 cells were incubated with 20 µM RR-171 for 24 h and then lysed
   in Trizol reagent (Thermo Fisher Scientific, USA). The total RNA
   quantity and purity were assessed using NEBNext Ultra TM RNA Library
   Prep Kit (NEB, E7490) and AMPure XP system. After the library was
   constructed, we performed PE150 sequencing on an Illumina Novaseq 6000
   according to the manufacturer’s protocol. We applied the DEGseq
   algorithm to filter the differentially expressed genes (DEGs) after
   significance analysis and FDR analysis under the criteria
   (|log2FC| > 1, P value < 0.05). Gene set enrichment analysis (GSEA) was
   performed using GSEA software (Broad Institute, Cambridge, MA). The R
   package ClusterProfiler was used to identify the significant GO (Gene
   Ontology) categories and KEGG (Kyoto Encyclopedia of Genes and Genomes)
   pathways^[59]22–[60]24. A network was built on the basis of the
   significant pathways (P value < 0.05). Volcano plots and bubble plots
   were drawn by the R software on the basis of the differential gene and
   pathway analyses.

Flow cytometry analysis of apoptosis

   Capan-2 and Bxpc-3 cells were seeded in 6-well (3 × 10^5 cells/well)
   plates, and then the peptide RR-171 was added to the wells at
   concentrations of 0, 5, 10, and 20 µM for 24 h. After that, the cells
   were digested and washed twice with PBS and then resuspended in Annexin
   V binding Buffer. Next, the cell suspension were stained with 10 µl
   Propidium Iodide and 5 µl FITC Annexin V (Biolegend, San Diego, CA) or
   5 µl APC Annexin V (eBioscience, San Diego, CA) reagents at room
   temperature (25℃) for 15 min in the dark. Subsequently, 400 µl Annexin
   V binding Buffer was added to each tube. Finally, the stained samples
   were detected by using a flow cytometer (MA900, Sony, Japan). The
   results were analyzed by using FlowJo 10.

Scanning electron microscopy

   Capan-2 cells were seeded in 24-wells plates (5 × 10^4 cells/well).
   After the cells adhesion, RR-171, or fresh medium for the control
   group, was added to the wells and incubated for 24 h. Then, the cells
   were washed with PBS, fixed in PBS containing 2.5% glutaraldehyde for
   2 h and washed with PBS for three times for 15 min. Next, the cells
   were dehydrated with increasing concentrations of ethanol (from 30%,
   40%, 50%, 60%, 70%, 80%, 90–100%). The cells were then sprayed with an
   ion sputter. The cells were subsequently observed via an S-3400 N
   scanning electron microscope (Hitachi, Tokyo, Japan).

Protein analysis by Western blotting

   Protein extraction was performed using RIPA lysis buffer and
   phenylmethylsulfonyl fluoride. Western blot analysis of Capan-2 and
   Bxpc-3 cells treated with different concentrations of RR-171 was
   performed as previously described^[61]25. After protein extraction,
   protein quantification was performed via a BCA protein quantification
   kit (BOSTER, Wuhan, China). The protein samples were separated via
   SDS-PAGE and transferred to polyvinylidene fluoride membranes. Next,
   the membranes were blocked with QuickBlock™ Blocking Buffer (Beyotime
   Biotechnology, China) for 15 min and then incubated with primary
   antibody at 4 °C overnight. The membranes were subsequently washed with
   TBST (10 min, 3 times) and incubated with secondary HRP-conjugated
   antibodies for 2 h at room temperature. The membranes were subsequently
   washed with TBST (10 min, 3 times) and developed with a
   chemiluminescence kit (Thermo Fisher Scientific Inc. IL, USA). Finally,
   the protein signals were detected using the ChemiDoc MP Imaging System
   (Bio-Rad, Hercules, CA, USA). Antibodies against Bax and Bcl-2 were
   purchased from BOSTER Biological Technology. Antibodies against
   Caspase-1, Caspase-3, Caspase-7, Caspase-9, Cleaved-PARP, IL-1β, IL-18,
   GSDMD, NLRP-3 and HRP-conjugated antibodies were purchased from Cell
   Signaling Technology. Antibodies against Wnt-1, β-actin, β-catenin and
   GSK3β were purchased from Proteintech.

Lactate dehydrogenase (LDH) release analysis

   A lactate dehydrogenase release assay kit (c0017, Beyotime, China) was
   used to detect the percentage of LDH released by the cells. The cells
   were seeded in 96 well plates (5000–6000 cells/well); after the cells
   were adhesion, different agent treatments were performed. After
   treatment, the 96-well plates were centrifugated (400g, 5 min), the
   supernatant was collected, and the experiment was subsequently
   performed based on the manufacturer’s instructions. The percentage of
   LDH release was calculated as follows: LDH release = (LDH sample/LDH
   maximum) × 100%.

Subcutaneous xenograft model

   The animal experiments were reviewed and approved by the Animal Ethics
   Committee of Air Force Medical University. The animal study was
   designed in accordance with the ARRIVE guidelines. Female nude mice
   (BALB/c nude, 5–6 weeks old) were obtained from GemPharmatech (Jiangsu,
   China) and housed at the Air Force Medical University Animal Experiment
   Center. Capan-2 cells (6 × 10^6) were injected subcutaneously into the
   right flanks of the nude mice. Five days later, the mice were randomly
   divided into two groups (n = 6). The experimental group was
   intraperitoneally injected with RR-171 (20 mg/kg) every 3 days for
   three weeks. The control group was intraperitoneally injected with
   100 µl of normal saline solution. Tumor length (L) and width (W) and
   mouse weight were measured every third day. The tumor volume was
   calculated as follows: tumor volume (V) = (L×W^2)/2. Finally, at the
   end of the experiment, the nude mice were euthanized by sodium
   pentobarbital overdose by intraperitoneal injection, after which the
   lungs, livers, spleens, kidneys, and hearts were excised, and the
   tumors were removed and weighed. All methods were carried out in
   accordance with relevant guidelines and regulations.

Hematoxylin-eosin (HE) and immunohistochemical staining

   Hematoxylin-eosin (HE) staining was performed using a Hematoxylin and
   eosin staining kit (c0105M, Beyotime, China). The experiment was
   performed according to the manufacturer’s instructions. The tumor
   tissues were fixed with 4% paraformaldehyde, embedded in paraffin and
   sectioned. Immunohistochemical staining was performed as described
   previously^[62]26. The immunohistochemical staining results were
   analyzed by Image j 1.53 software. The average density was calculated
   via the following formula: average density = IOD/area.

Statistical analysis

   All experimental data were statistically analyzed by using GraphPad
   Prism 8.0 software. The data are expressed as the mean ± standard
   deviation (SD). An unpaired t test (between two groups) or one-way
   ANOVA (multiple groups) was used for comparisons. P value less than
   0.05 was identified as statistically significant.

Results

RR-171 inhibits pancreatic cancer cell viability and proliferation

   At the beginning, we treated Panc-1, Capan-2, Bxpc-3 and HPNE cells
   with RR-171 and conducted CCK-8 experiments. The results revealed that
   RR-171 obviously inhibited pancreatic cancer cell lines in a
   dose-dependent manner. The IC[50] values of RR-171 in Capan-2, Bxpc-3
   and Panc-1 cells at 24 h were 24.70 µM, 15.71 µM and 75.68 µM,
   respectively (Fig. [63]1A). While the inhibitory effect of RR-171 on
   HPNE cells was attenuated, the IC[50] value was 144.50 µM
   (Supplementary Fig. [64]1). On the basis of these results, we selected
   Capan-2 and Bxpc-3 cells for further study. The results of plate
   cloning experiments indicated that the number of clones formed by
   Capan-2 and Bxpc-3 cells dramatically decreased with increasing RR-171
   concentrations (Fig. [65]1B). The results of the EdU staining assay
   also showed that RR-171 distinctly suppressed the proliferation of
   Capan-2 and Bxpc-3 cells in a dose-dependent manner (Fig. [66]1C).
   These results demonstrated that RR-171 inhibited pancreatic cancer cell
   viability and proliferation. However, the inhibitory effect of RR-171
   on normal pancreatic cells was limited.

Fig. 1.

   [67]Fig. 1
   [68]Open in a new tab

   RR-171 suppresses pancreatic cancer cell viability and proliferation in
   vitro. (A) Capan-2, Bxpc-3, and Panc-1 cell viability was determined
   using a CCK-8 analysis after treatment with different concentrations of
   RR-171. *P < 0.05, **P < 0.01, and ***P < 0.001 compared with the 0 µM
   RR-171 treatment group. (B) Colony formation assay to identify the
   effect of RR-171 on pancreatic cancer cell proliferation. *P < 0.05,
   **P < 0.01, and ***P < 0.001 compared with the 0 µM RR-171 treatment
   group. (C) The proliferation of Capan-2 and Bxpc-3 cells was determined
   using an EdU assay after treatment with increasing concentrations of
   RR-171. The nuclei were stained with Hoechst (blue), and the
   proliferating cells were stained with EdU (green) (200×).

Differential gene expression and pathway enrichment analysis of pancreatic
cancer cells after RR-171 treatment

   Capan-2 cells were treated with RR-171 for 24 h, after which RNA was
   extracted for RNA sequencing. Compared with those in the control group,
   there were 1616 downregulated genes and 556 upregulated genes in the
   RR-171-treated group (|log 2 fold change(FC)| > 1, false discovery rate
   (FDR) < 0.05) (Fig. [69]2A). Gene Ontology (GO) and Kyoto Encyclopedia
   of Genes and Genomes (KEGG) pathway enrichment analyses and gene set
   enrichment analysis (GSEA) of the genes in Capan-2 cells after RR-171
   treatment were performed. The DEGs were enriched mainly in the negative
   regulation of the cell population, apoptosis, cell death and other
   signaling pathways (Fig. [70]2B–D). Through further GO and pathway
   network analysis, we found that the Wnt signaling and MAPK pathway were
   focused (Fig. [71]2E). Combined with the GSEA results, Wnt signaling
   attracted our attention. Thus, we performed western blot analysis for
   key proteins related to canonical Wnt signaling pathway. The results
   showed that Wnt-1, GSK3β and β-catenin were decreased in a
   dose-dependent manner after RR-171 treatment (Fig. [72]2F). These
   results suggest that RR-171 can inhibit the canonical Wnt signaling
   pathway in pancreatic cancer cells, which might be one of mechanism by
   which RR-171 inhibits pancreatic cancer cells. Moreover, the results of
   the Go and KEGG analyses suggested further exploration of the other
   effects of RR-171 on pancreatic cancer cells.

Fig. 2.

   [73]Fig. 2
   [74]Open in a new tab

   Differential gene expression and pathway enrichment analysis of
   pancreatic cancer cells treated with RR-171. (A) Capan-2 cells were
   treated with RR-171 for 24 h for RNA sequencing, and a volcano plot of
   differential expression was generated. [Downregulated genes are in
   blue, upregulated genes are in red, nonregulated genes are in gray
   (|log 2 fold change (FC)| > 1, false discovery rate (FDR) < 0.05)].
   Volcano plots were drawn by the R software via differential gene
   analysis. (B) GSEA of the differentially expressed genes using GSEA
   software. (C) GO pathway analysis of differentially expressed genes.
   (D) KEGG pathway analysis of differentially expressed genes.
   ClusterProfiler package was applied to identify the significant GO and
   KEGG categories, and FDR was used to correct the p-values via R
   software. (E) Pathway network analysis of the KEGG pathways [the
   network was built on the basis of the relationships among KEGG pathways
   that are significant terms (P value < 0.05)]. (F) Western blot analysis
   of cell lysates treated with different concentrations of RR-171 for
   24 h. Detection of Wnt signaling pathway-related proteins. Relative
   expression of protein = protein/β-actin band intensity.

RR-171 induces caspase-dependent apoptosis in pancreatic cancer cells

   After incubation with RR-171, the cells were stained with PI and
   Annexin-V. Flow cytometry results indicated that RR-171 distinctively
   increased the percentage of apoptotic Capan-2 and Bxpc-3 cells in a
   dose-dependent manner (Fig. [75]3A). The western blot results
   demonstrated that, as the RR-171 concentration increased in Capan-2 and
   Bxpc-3 cells, the expression of the antiapoptotic protein Bcl-2
   decreased, whereas the expression of the proapoptotic protein Bax
   increased in a dose-dependent manner (Fig. [76]3B).Moreover, we
   examined apoptosis-associated proteins in the caspase signaling
   pathway. We found that the levels of caspase-3, caspase-7, and
   caspase-9 decreased in a dose-dependent manner, whereas the level of
   cleaved PARP increased (Fig. [77]3C), which suggested that RR-171
   activated the apoptosis-associated caspase signaling pathway to induce
   the apoptosis of pancreatic cancer cells. Next, we treated Capan-2 and
   Bxpc-3 cells with the pan-caspase inhibitor Z-VAD-FMK alone or in
   combination with RR-171 for 24 h. Flow cytometry demonstrated that
   Z-VAD-FMK treatment significantly reduced the ratio of apoptosis in
   both cell lines (Fig. [78]3D). Taken together, these results suggest
   that RR-171 induces caspase-dependent apoptosis in pancreatic cancer
   cells.

Fig. 3.

   [79]Fig. 3
   [80]Open in a new tab

   RR-171 induces apoptosis in pancreatic cancer cells. (A) After Capan-2
   and Bxpc-3 cells were treated with RR-171 for 24 h, the ratio of
   apoptotic cells was detected by flow cytometry using PI/Annexin V-FITC
   staining. *P < 0.05 and ***P < 0.001compared with the 0 µM RR-171
   treatment group. (B,C) Western blotting detection of proteins related
   to apoptosis in Capan-2 and Bxpc-3 cells treated with 0, 5, 10, and 20
   µM RR-171 for 24 h. (D) Capan-2 and Bxpc-3 cells were treated with
   RR-171 and Z-VAD-FMK or DMSO, and the apoptosis ratio was detected by
   flow cytometry using PI/Annexin V-APC double staining. **P < 0.01 and
   ***P < 0.001 compared with the control group.

RR-171 induces pyroptosis in pancreatic cancer cells

   The differential gene expression enrichment analysis results suggest
   that cell death might play a part in the effect of RR-171 on pancreatic
   cancer cells. Thus, we explored the morphology of Capan-2 cells treated
   with RR-171. Scanning electron microscopy (SEM) observed that the
   connections among cells were reduced with 5 µM RR-171 treatment,
   balloon-like membrane protrusions formed with10 µM RR-171 treatment,
   and cell debris was induced with 20 µM RR-171 treatment (Fig. [81]4A).
   We then observed that pores formed in the cell membrane, which might
   have caused cell swelling and rupture (Fig. [82]4B). Additionally, LDH
   release from Capan-2 and Bxpc-3 cells was significantly increased after
   RR-171 treatment (Fig. [83]4C; P < 0.01). These results suggest that
   pyroptosis might be induced by RR-171. Thus, we detected
   pyroptosis-associated proteins. The western blotting results
   demonstrated that RR-171 treatment led to elevated levels of caspase-1,
   NLRP-3, GSDMD, IL-1β and IL-18 in a dose-dependent manner in both cell
   lines (Fig. [84]4E, F). Next, we applied the pan-caspase inhibitor
   Z-VAD-FMK to explore the effect of RR-171 combined with Z-VAD-FMK. We
   found that Z-VAD-FMK did not inhibit the increase in LDH release in
   Capan-2 cells and partly inhibited the increase in LDH release in
   Bxpc-3 cells (Fig. [85]4D). We then utilized VX-765, a pyroptosis
   inhibitor, for RR-171 treatment of pancreatic cancer cells. The results
   showed that VX-765 significantly inhibited the increase in LDH release
   induced by RR-171 in both cell lines (Fig. [86]4G; P < 0.05). Western
   blot analysis also showed that VX-765 decreased the elevated protein
   levels of IL-18, Il-1β, Caspase-1, and NLRP-3 (Fig. [87]4H, I).

Fig. 4.

   [88]Fig. 4
   [89]Open in a new tab

   RR-171 induces pyroptosis in pancreatic cancer cells. (A,B) Capan-2
   cells were treated with different concentrations of RR-171, and
   morphological changes were observed via SEM (red arrows, morphological
   changes). (C) After Capan-2 and Bxpc-3 cells were treated with RR-171
   for 24 h, cytotoxicity was detected by lactate dehydrogenase (LDH)
   release into the cell culture medium. ***P < 0.001 compared with the
   control group. (D) Capan-2 and Bxpc-3 cells were treated with Z-VAD-FMK
   and RR-171 for 24 h. Cytotoxicity was detected by LDH release assay.
   *P < 0.05; ***P < 0.001; ns means nonsignificant. (E,F) Western
   blotting detection of proteins of pyroptosis markers in Capan-2 and
   Bxpc-3 cells treated with 0, 5, 10, 20 µM RR-171 for 24 h. (G) Capan-2
   and Bxpc-3 cells were treated with VX-765 and RR-171 for 24 h.
   Cytotoxicity was detected by LDH release assay. *P < 0.05; **P < 0.01;
   and ***P < 0.001. (H,I) Western blotting detection of proteins of
   pyroptosis markers in Capan-2 and Bxpc-3 cells treated with VX-765
   alone or in combination with RR-171 for 24 h.

RR-171 suppresses tumor growth in Capan-2 cell xenograft mice

   To identify whether RR-171 has anti-pancreatic cancer tumor properties
   in vivo, we established Capan-2 cell subcutaneous xenografts in nude
   mice. The tumor volume was significantly reduced beginning on Day 17 in
   the xenograft model injected intraperitoneally with RR-171
   (Fig. [90]5A–C). At the end of the animal experiment, the tumor weight
   was also significantly reduced (Fig. [91]5D). There was no significant
   difference in the body weight of the nude mice between the two groups
   (Fig. [92]5D). To explore the potential toxicity effects of RR-171 on
   nude mice, we obtained lung, heart, liver, spleen, and kidney samples
   from the nude mice at the end of the experiment. HE staining results
   indicated that RR-171 had no significant effects on the above five
   parenchymal organs (Supplementary Fig. [93]2). Moreover, the
   immunohistochemistry results showed that RR-171 significantly decreased
   the expression levels of Ki-67 and PCNA (Fig. [94]5E). These results
   demonstrated that RR-171 can inhibit pancreatic cancer cell tumor
   growth in vivo.

Fig. 5.

   [95]Fig. 5
   [96]Open in a new tab

   RR-171 inhibits tumor growth in pancreatic cancer cell xenograft mice.
   (A,B) Images of the mice and subcutaneous xenograft tumors formed in
   the nude mice model. (C) Tumor volumes were measured and counted every
   three days. **P < 0.01; ***P < 0.001; the arrow indicates the beginning
   of treatment. (D) Tumor weight and body weight at the end of the
   experiment. **P < 0.01. (E) Immunohistochemistry results of Ki-67 and
   PCNA expression in the tumors (200×). *P < 0.05 and **P < 0.01.

Discussion

   Pancreatic cancer remains a troubling illness to treat^[97]27. Surgery
   continues to be the only chance for curing pancreatic cancer, and it
   has become a relatively safe treatment in experienced medical
   centers^[98]28. However, surgery alone is not sufficient because most
   affected patients present with advanced stage disease and miss the
   chance for surgery, and high-rate of relapse after curative surgery
   without addition therapy^[99]29. Thus, comprehensive treatment
   strategies, including chemotherapy, radiotherapy, targeted therapy, and
   immunotherapy, have been developed in recent decades^[100]30. Although
   the overall 5-year survival of patients with pancreatic cancer has
   improved over the preceding years, the overall prognosis of patients
   with pancreatic cancer remains unsatisfactory, and the treatment for
   patients with pancreatic cancer is still a formidable
   conundrum^[101]29,[102]31. Identifying potential agents for the
   treatment of pancreatic cancer has important implications. In this
   study, we explored the peptide RR-171 as a potential drug for the
   treatment of pancreatic cancer.

   Umbilical cord blood has been developed as a therapy for numerous
   applications, including stem cell transplantation, immunotherapy, and
   regenerative medicine^[103]18,[104]32. In recent years, umbilical cord
   blood derived molecules or cells have been explored for the treatment
   of malignancies^[105]19. For example, Chen et al. reported that the use
   of umbilical cord blood-derived natural killer cells alone or in
   combination with Bevacizumab can be a novel treatment for patients with
   colorectal cancer^[106]33. Raquel et al. found that umbilical cord
   blood serum can significantly inhibit the viability, proliferation and
   migration of human prostate cancer cell lines^[107]34, but they did not
   identify the specific constituents that play anticancer roles. In our
   previous study, we reported that the peptide RR-171 derived from
   umbilical cord blood serum has anti-pancreatic cancer
   properties^[108]21. The peptide RR-171 has 17 amino acids, the core
   function part of which is the medial nine amino acids, and the terminal
   eight arginines are added to increase the solubility of the peptide. In
   present study, RR-171 inhibited the viability and proliferation of
   pancreatic cancer cell lines. However, normal pancreatic ductal
   epithelial cells were less sensitive to the cellular cytotoxicity of
   RR-171. We speculated that, just as normal epithelial cells are less
   sensitive to chemotherapy drugs, RR-171 might also be less toxic to
   relatively poorly proliferating cells. We subsequently explored the
   mechanisms by which RR-171 inhibits pancreatic cancer cell lines. This
   study revealed that RR-171 can inhibit the canonical Wnt signaling
   pathway. Increasing evidence indicates that Wnt signaling pathway
   activation plays a vital role in all aspects of human tumor development
   and progression, including growth, stemness, angiogenesis, resistance,
   and immune evasion^[109]35. Wnt signaling pathway-associated inhibitors
   have also been developed for preclinical and clinical trials in the
   treatment of malignancies^[110]36. A previous study showed consistent
   activation of core signaling pathways, including the Wnt signaling
   pathway, in pancreatic cancer^[111]37. Further studies have also
   demonstrated that epithelial Wnt signaling is crucial for pancreatic
   cancer progression^[112]38. In brief, RR-171 might exert a tumor
   suppressive role in pancreatic cancer through the inhibition of the
   canonical Wnt signaling pathway. Interestingly, in our previous study,
   we found that RR-171 can activate the NF-KappaB pathway in
   hepatocellular carcinoma^[113]21. The reason for this difference might
   be that the two pathways play different key roles in the two tumors.
   Moreover, the RNA sequencing results suggested that RR-171 treatment
   might induce apoptosis in pancreatic cancer cell lines. We revealed
   that RR-171 can significantly induce apoptosis in both pancreatic
   cancer cell lines by activating caspase pathway signaling. This result
   is similar to our previous finding that RR-171 can promote the
   apoptosis of hepatocellular carcinoma cells. In addition, through SEM
   scanning, we found that pores formed on the surface of pancreatic
   cancer cell lines and that LDH release increased after treatment with
   RR-171. These results suggest that RR-171 might induce pyroptosis in
   pancreatic cancer cell lines. We examined markers of pyroptosis,
   including NLRP-3, GSDMD, IL-1β, IL-18 and Caspase-1, which were
   increased after treatment with RR-171. We then applied the pyroptosis
   inhibitor VX765, which reversed the increase in LDH release. These
   results demonstrated that RR-171-induced pyroptosis in pancreatic
   cancer cells might occur through triggering the Caspase-1/NLRP-3/GSDMD
   pathway. In our previous study, pores were observed in hepatocellular
   carcinoma cells after RR-171 treatment^[114]21, while deeply studies
   have not investigated this phenomenon. In conclusion, RR-171 can induce
   apoptosis and pyroptosis in pancreatic cancer cells. Next, to identify
   the anticancer functions of RR-171 in vivo, we established a
   subcutaneous xenograft model to examine the function of RR-171 in vivo.
   The results showed that the volume and weight of tumors were
   significantly suppressed by RR-171 treatment. The immunohistological
   results revealed that RR-171 inhibited the expression of Ki-67 and
   PCNA, which was consistent with the tumor volume. In addition, the HE
   results demonstrated that the parenchymal organ toxicity of RR-171 was
   not apparent in vivo, which suggests that RR-171 might be a relatively
   safe agent in nude mice. We further revealed that RR-171 can inhibit
   the growth of pancreatic cancer in vivo, which is consistent with in
   vitro results. In our previous study, we demonstrated that RR-171 might
   interact with α-tubulin and inhibit tubulin polymerization in
   hepatocellular carcinoma cells^[115]21. Another study showed that
   anti-tubulin agents can arrest cell division and facilitate one of the
   apoptosis pathways and that modified tubulin inhibitors have the
   ability to improve the outcomes of patients with pancreatic cancer
   through overcoming chemoresistance in these patients^[116]39,[117]40.
   These results suggest that the specific target of RR-171 in pancreatic
   cancer cells might also be associated with tubulin polymerization,
   which needs further study. Eventually, the bioactivity of RR-171 was
   not high enough, and the IC[50] reached only micromolar concentrations.
   Modifications might be explored to improve the bioactivity of RR-171 in
   our future studies. Moreover, the effects of combined treatment with
   RR-171 and radiotherapy or chemotherapy might be evaluated in our
   future studies, and modifications must be explored in the future to
   improve its bioactivity.

   Overall, this study revealed that a new peptide, RR-171, induces
   apoptosis and pyroptosis through multiple pathways and inhibits
   pancreatic cancer growth. RR-171 might exert anti-pancreatic cancer
   effects by suppressing the Wnt signaling pathway. Our study provides
   new directions and strategies for the development of new agents for the
   treatment of pancreatic cancer.

Electronic supplementary material

   Below is the link to the electronic supplementary material.
   [118]Supplementary Material 1^ (2MB, pdf)

Acknowledgements