Pancreatic stellate cells contribute pancreatic cancer pain via activation of sHH signaling pathway

Abstract

Abdominal pain is a critical clinical symptom in pancreatic cancer (PC) that affects the quality of life for PC patients. However, the pathogenesis of PC pain is largely unknown. In this study, we show that PC pain is initiated by the sonic hedgehog (sHH) signaling pathway in pancreatic stellate cells (PSCs), which is activated by sHH secreted from PC cells, and then, neurotrophic factors derived from PSCs mediate the pain. The different culture systems were established in vitro, and the expression of sHH pathway molecules, neurotrophic factors, TRPV1, and pain factors were examined. Capsaicin-evoked TRPV1 currents in dorsal root ganglion (DRG) neurons were examined by the patch-clamp technique. Pain-related behavior was observed in an orthotopic tumor model. sHH and PSCs increased the expression and secretion of TRPV1, SP, and CGRP by inducing NGF and BDNF in a co-culture system, also increasing TRPV1 current. But, suppressing sHH pathway or NGF reduced the expression of TRPV1, SP, and CGRP. In vivo, PSCs and PC cells that expressed high levels of sHH could enhance pain behavior. Furthermore, the blockade of NGF or TRPV1 significantly attenuated the pain response to mechanical stimulation compared with the control. Our results demonstrate that sHH signaling pathway is involved in PC pain, and PSCs play an essential role in the process greatly by inducing NGF.

Keywords: pain; pancreatic cancer; pancreatic stellate cells; sHH.

Figure 1. Qualification of DRG and staining of TRPV1, SP, and CGRP in DRG neurons

(A) Identification of DRG extracted from newborn rat by S100 staining. (B–D) Staining of TRPV1, SP, and CGRP in DRG neurons. (a, d, g, j) S100, TRPV1, SP, and CGRP-positive neurons (green) in DRG, respectively. (b, e, h, k) Nuclear staining with DAPI. (c, f, i, l) Merge of staining is shown.

Figure 2. Expression and secretion of TRPV1, SP, and CGRP in DRG

(A) The graph shows the mRNA expression of TRPV1, SP, and CGRP in DRG in different culture systems. The expression levels in co-culture of AsPc-1 cells with PSCs were higher than in other groups (p < 0.05) or in DRG cultured alone as a control. (B) The protein expression of TRPV1, SP, and CGRP in DRG in different culture systems determined by Western blotting. The protein results were similar to mRNA; DRG were cultured alone as a control. (C) Co-culture of AsPc-1 cells and PSCs increased the secretion of pain factors, SP, and CGRP by ELISA, compared with other groups (p < 0.05).

Figure 3. Effect of sHH and cyclopamine on expression and secretion of TRPV1, SP and CGRP in DRG in co-culture system

(A) The cyclopamine-pre group (cyclopamine was used to treat the co-culture of PC cells and PSCs before removing the medium (1 ml) and adding it to DRG) suppressed the mRNA expression of TRPV1, SP and CGRP compared with control and other groups (p < 0.05). However, the sHH-pre group (sHH was used to treat the co-culture of PC cells and PSCs before removing the medium (1 ml) and adding it to DRG) increased the mRNA expression compared to control or the cyclopamine-pre group (p < 0.05). In contrast, the cyclopamine group (cyclopamine was used to treat DRG directly) was not different compared to control. (B) The Western blotting results were similar to mRNA results. (C) Cyclopamine-pre decreased the secretion of SP and CGRP compared with control and sHH-pre; in contrast, sHH-pre enhanced the secretion compared to other groups (p < 0.05). No effect of cyclopamine on the secretion of SP and CGRP was observed (p > 0.05).

Figure 4. PC cells activate the sHH signaling pathway and increase expression of NGF and BDNF in PSCs

(A) Panc-1-Sh-sHH cells and recombinant sHH up-regulated sHH signaling molecules (Gli1, Gli2 and SMO) and increased the expression of NGF and BDNF in PSCs compared with the Panc-1 cells group; furthermore, AsPc-1-Si-sHH and cyclopamine treatment reduced the expression of signal molecules (Gli1, Gli2 and SMO) and NGF and BDNF compared to the AsPc-1 cells group (p < 0.05). However, there was no difference in GDNF among the groups. (B) Panc-1-Sh-sHH cells and recombinant sHH increased the mRNA expression of neurotrophic factors; in contrast, AsPc-1-Si-sHH and cyclopamine reduced their expression (p < 0.05). However, the stimulation had no effect on GDNF for the two cell types. Panc-1-Sh-sHH: Panc-1 cells (low sHH expression) were stably transfected with sHH plasmids; AsPC-1-Si-sHH: AsPc-1 cells (high sHH expression) were transiently transfected with sHH siRNA.

Figure 5. Effect of anti-NGF and anti-BDNF on expression of TRPV1, SP, and CGRP in DRG in co-culture system

(A, B) Recombinant NGF or BDNF increased the mRNA and protein expression of TRPV1, SP and CGRP compared with control, and anti-NGF neutralizing antibody suppressed TRPV1, SP and CGRP expression compared to control. However, anti-GDNF had no effect on the expression of TRPV1, SP, and CGRP. (C) Recombinant NGF enhanced the secretion of SP and CGRP, and anti-NGF reduced the secretion (p < 0.05).

Figure 6. The sHH signaling pathway and NGF potentiated TRPV1 currents in DRG in co-culture system

(A) Representative TRPV1 current evoked by capsaicin (1 μM) application to DRG neurons as a control. (B) TRPV1 current was potentiated by sHH-pre (used to treat the co-culture of PC cells and PSCs) in DRG neurons. DRG neurons were treated with the co-culture medium (1 ml) of PC cells and PSCs that was pretreated with sHH for 12 h before a 2 min application of capsaicin (1 μM) (n = 12 cells, p < 0.05). (C) Cyclopamine-pre treatment attenuated TRPV1 currents in DRG neurons treated with co-culture medium before the application of capsaicin (1 μM) (n = 17 cells, p < 0.05). (D) NGF enhanced TRPV1 currents in DRG neurons treated with NGF before a 2 min application of capsaicin (1 μM) (n = 15 cells, p < 0.05). (E) Anti-NGF treatment attenuated TRPV1 currents (n = 20 cells, p < 0.05). (F) Treatment with combined sHH-pre and NGF obviously increased TRPV1 currents (n = 11 cells, p < 0.05). (G) Treatments of cyclopamine-pre and anti-NGF significantly inhibited the currents (n = 22 cells, p < 0.05). (H) The bar graph shows the mean peak current above shown in DRG neurons compared with the control group.

Figure 7. PSCs aggravated pain behaviors and induced secretion of SP and CGRP in mice

(A) Panc-1 cells or mixed Panc-1 cells and PSCs were co-injected into the pancreases of nude mice (a). After 4 weeks, tumors were apparent (b). (B) The graphs show the average number of responses to mechanical stimulation 4 weeks after inoculation. The response rate was greater in the co-injection of Panc-1 cells and PSCs compared with control (p < 0.05). In contrast, the co-injection of Panc-1-Sh-sHH cells and PSCs had the strongest effect on pain behaviors. (C, D) The secretion of SP and CGRP into serum gradually increased in the following order: PC cells group < mixed PC cells and PSCs group < mixed Panc-1-Sh-sHH and PSCs group.