Trypsin-protease activated receptor-2 signaling contributes to pancreatic cancer pain

Abstract

Pain treatment is a critical aspect of pancreatic cancer patient clinical care. This study investigated the role of trypsin-protease activated receptor-2 (PAR-2) in pancreatic cancer pain. Pancreatic tissue samples were collected from pancreatic cancer (n=22) and control patients (n=22). Immunofluorescence analyses confirmed colocalization of PAR-2 and neuronal markers in pancreatic cancer tissues. Trypsin levels and protease activities were higher in pancreatic cancer tissue specimens than in the controls. Supernatants from cultured human pancreatic cancer tissues (PC supernatants) induced substance P and calcitonin gene-related peptide release in dorsal root ganglia (DRG) neurons, and FS-NH₂, a selective PAR-2 antagonist, inhibited this effect. A BALB/c nude mouse orthotopic tumor model was used to confirm the role of PAR-2 signaling in pancreatic cancer visceral pain, and male Sprague-Dawley rats were used to assess ambulatory pain. FS-NH₂ treatment decreased hunch scores, mechanical hyperalgesia, and visceromotor reflex responses in tumor-bearing mice. In rats, subcutaneous injection of PC supernatant induced pain behavior, which was alleviated by treatment with FS-NH₂ or FUT-175, a broad-spectrum serine protease inhibitor. Our findings suggest that trypsin-PAR-2 signaling contributes to pancreatic cancer pain in vivo. Treatment strategies targeting PAR-2 or its downstream signaling molecules might effectively relieve pancreatic cancer pain.

Keywords: pain; pancreatic cancer; pancreatic cancer pain model; protease activated receptor-2; trypsin.

Figure 1. Histology of pancreatic tissue specimens and immunofluorescence assay for PAR-2 and PGP 9.5 in human pancreatic tissue specimens

Hematoxylin and eosin stained section of pancreatic tissues from control patients (A) and patients with pancreatic cancer (B). Scale bar = 50μm. (C) To visualize neuronal axons, tissues were immunostained with a monoclonal antibody to PGP-9.5. The colocalization of PAR-2 and PGP 9.5 was significant increased in specimen from pancreatic pancreatic cancer tissue. White arrow indicated the PAR-2 and PGP 9.5 co-expressing cells. Scale bar = 50μm.

Figure 2. Trypsin(ogen) concentration and protease activity released from pancreatic cancer and control specimens

(A) Trypsin (ogen) released from the specimens of pancreatic cancer tissue was significantly higher than that released from the non-tumor specimens (1.38±0.12 vs 0.97±0.18 μg/ml). (B) Trypsin activity determined by the substrate BAEE. Protease activity released from specimens of pancreatic cancer patients was 2 to 3 fold higher than the activity in control specimens (9.32±1.17 vs 3.50±1.28 BAEE U/ml). PC: supernatant cultured from pancreatic cancer tissues. NC: supernatant cultured from normal pancreatic tissues. *P<0.05; n = 22 in each group.

Figure 3. Intraplantar administration with supernatants cultured from pancreatic cancer tissues (PC supernatant, 25ul) induced ambulatory pain, thermal and mechanical hyperalgesia

(A) Rats receiving injection of PC supernatant exhibited higher ambulatory score in 4h after injection. Addition of FUT-175 (50 μg/ml) into the PC supernatants immediately before injection reduced ambulatory pain in rats at each time point during observation. (B) Rats receiving injection of PC supernatant exhibited decreased PWL in response to thermal stimulus. (C) Rats receiving injection of PC supernatants exhibited decreased PWT in response to mechanical stimulus. Addition of FUT-175 reversed the PWL and PWT induced by PC supernatants. PC: supernatants cultured from pancreatic cancer tissues. NC: supernatants cultured from normal pancreatic tissues. F: a broad-spectrum serine protease inhibitor, FUT-175. S: sham control. *P<0.05 compared with normal group; **P<0.01compared with normal group; # P<0.05 compared with tumor group; ## P<0.01 compared with tumor group. n=6 in each group.

Figure 4. PAR-2 antagonist reversed the ambulatory pain, thermal and mechanical hyperalgesia induced by PC supernatant

(A) Addition of PAR-2 antagonist FS-NH₂ (100 μM) into the PC supernatant (25μl) immediately before injection reduced ambulatory pain in rats at each time point during observation. (B) FS-NH₂ increased the PWL in rats received PC supernatants injection in response to thermal stimulus. (C) FS-NH₂ increased the PWT in rats received PC supernatants injection in response to mechanical stimulus. PC: supernatants cultured from pancreatic cancer tissues; FS: FSLLRY-NH₂, PAR-2 antagonist peptide; LR: LRGILS-NH_2, control peptide. *P<0.05, **P<0.01 compared with control peptide group; n=6 in each group.

Figure 5. PC supernatant induced DRG neurons releasing of SP and CGRP in a protease-dependent way

After incubation for 2h, the releasing of SP (A) and CGRP (B) form cultured DRG neurons were higher in PC supernatant group than that in NC supernatant group. Similarly, the mRNA levels for SP (C) and CGRP (D) were significantly increased in DRG neurons treated with PC supernatants. Co-incubation with protease inhibitor FUT-175 (50μg/ml) and PC supernatant reduced this neuropeptide release effect in cultured DRG neurons. Ctr: unstimulated DRG. PC: supernatants cultured from pancreatic cancer tissues. NC: supernatants cultured from normal pancreatic tissues. F: a broad-spectrum serine protease inhibitor, FUT-175.*P<0.05 n=6 in each group.

Figure 6. PAR-2 antagonist reversed PC supernatant induced releasing of SP and CGRP in cultured DRG neurons

(A and B) FS-NH₂ (100μM), a selective antagonist peptide of PAR-2, caused a marked decrease in SP and CGRP releasing in the medium of cultured DRG neurons in contrast to the control peptide LR-NH₂ (100μM). (C and D) Similarly, FS-NH₂ decreased the SP and CGRP mRNA levels in DRG neurons. FS: FSLLRY-NH₂, PAR-2 antagonist peptide; LR: LRGILS-NH_2, control peptide. *P<0.05 compared with control peptide group; n=6 in each group.

Figure 7. Pancreatic cancer model build by human pancreatic cancer cell line SW1990 in male BALB/c nude mice

(A) SW1990 cells (5×10⁶) in a volume of 20 μL were injected into the body of the pancreas, after 3 weeks, tumors were apparent on the pancreas (T). (B) Immunohistochemistry examination confirmed the success planting of SW1990 cells on the pancreas of naïve mice. (C) Hematoxylin and eosin stained section of pancreatic tissues from control mice. Scale bar = 50μm.

Figure 8. Immunofluorescence assay for PAR-2 and PGP 9.5 in pancreatic tissue specimens from naïve mice

Pancreatic cancer tissues of naïve mice were immunostained with antibody to PGP-9.5 and PAR-2. PAR-2 is expressed in the implanted SW1999 orthotropic pancreatic tumor, while PGP 9.5 immuno-active signals scattered cycling the tumor area in pancreatic cancer model. White arrow indicated the PAR-2 and PGP 9.5 co-expressing cells. Scale bar = 50μm.

Figure 9. PAR-2 antagonist alleviated pancreatic cancer pain in nude mice

(A) Body weights of mice that received SW1990 cell or sham injection. From 2 weeks to 4 weeks, tumor-bearing mice weighed significantly less than sham mice. (B) Hunching behavior of mices with injection of SW1990 cell and PAR-2 antagonist peptide. A significant increase in hunching behavior score in tumor-bearing mice group was observed from day 14 after surgery. Intraperitoneal injection of PAR-2 antagonist peptide FS-NH₂ (500μl, 10mM) every day from 14 to 21 days decreased the hunching behavior score in tumor-bearing mice. (C) Mice withdrawal response to mechanical stimulation with electric von Frey hairs in the abdomen. PAR-2 antagonist peptide FS-NH₂ (500μl, 10mM), but not control peptide LS-NH₂ (500μl, 10mM), significantly inhibited the mechanical hyperalgesia in abdomen. (D) Representative EMG graphs of acromiotrapezius muscle of tumor-bearing mice (lower) and sham mice (upper). (E) PAR-2 antagonist peptide FS-NH₂ (500μl, 10mM), but not control peptide LS-NH₂ (500μl, 10mM), significantly inhibited the EMG activity of acromiotrapezius muscle in tumor-bearing mice. T: tumor-bearing mice; FS: FSLLRY-NH₂, PAR-2 antagonist peptide; LR: LRGILS-NH_2, control peptide. # P<0.05 compared with sham group; ## P<0.01 compared with sham group; * P<0.05 compared with control peptide group; **P<0.01 compared with control peptide group. n=6 in each group.