High‑glucose microenvironment promotes perineural invasion of pancreatic cancer via activation of hypoxia inducible factor 1α

Abstract

Pancreatic cancer (PC) is one of the most lethal diseases, with a 5‑year survival rate of <9%. Perineural invasion (PNI) is a common pathological hallmark of PC and is correlated with a poor prognosis in this disease. Hyperglycemia has been shown to promote the invasion and migration of PC cells; however, the effect of hyperglycemia on the PNI of PC and its underlying mechanism remains unclear. In the present study, Western blotting was utilized to detect the expression of hypoxia inducible factor 1α (HIF1α) and nerve growth factor (NGF). Transwell and wound‑healing assays were performed to detect the influence of hyperglycemia on the invasion and migration ability of PC cells. An in vitro PC‑dorsal root ganglion (DRG) co‑culture system and an in vivo PNI sciatic nerve‑infiltrating tumor model were used to evaluate the severity of PNI in hyperglycemic conditions. In the results, hyperglycemia promoted the invasion/migration ability and elevated the expression of NGF in PC by upregulating HIF1α. Moreover, in vitro short‑term hyperglycemia caused little damage on the DRG axons and accelerated both the PNI of the PC and the outgrowth of the DRGs by increasing the expression of NGF via activation of HIF1α. Indeed, in vivo long‑term hyperglycemia promoted the infiltration and growth of PC, and then disrupted the function of the sciatic nerve in a HIF1α‑dependent manner. In conclusion, a high‑glucose microenvironment promotes PNI of PC via activation of HIF1α.

Keywords: HIF1α; PC; PNI; hyperglycemia.

Figure 1.

Hyperglycemia promotes the invasion and migration of pancreatic cancer cells and affects the outgrowth of DRGs. (A and B) Cell invasive ability of the negative control (0 mM glucose), euglycemia (5.5 mM glucose) and hyperglycemia (25 mM glucose) groups in BxPC-3 and Panc-1 cells assessed using (A) a Transwell assay (crystal violet staining; ×100 magnification), with (B) the associated quantification. (C and D) Cell migration ability of the negative control (0 mM glucose), euglycemia (5.5 mM glucose) and hyperglycemia (25 mM glucose) groups in BxPC-3 and Panc-1 cells assessed using (C) a wound-healing assay (×50 magnification), with (D) the associated quantification. (E) Dorsal root ganglion axon outgrowth in the conditioned media containing 0, 5.5 and 25 mM glucose following intervention for 6, 24 and 48 h. *P<0.05 vs. negative control. Scale bar, 500 µm. ns, not significant.

Figure 2.

Hyperglycemia accelerates the NGF expression and PNI of PC. (A) Pattern diagrams of the PC cell-DRG co-culture model. (B) NGF expression in Panc-1 and BxPc-3 after 0, 5.5 and 25 mM glucose intervention. (C) The quantification of the PC cell invasion index in the euglycemia (5.5 mM glucose) and hyperglycemia (25 mM glucose) groups. (D) The quantification of the DRG outgrowth index in the euglycemia (5.5 mM glucose) and hyperglycemia (25 mM glucose) groups. (E) Micrograph of the PNI in the euglycemia (5.5 mM glucose) and hyperglycemia (25 mM glucose) groups when using the PC cell-DRG co-culture model. *P<0.05 vs. 5.5 mM glucose group. Scale bar, 200 µm. DRG, dorsal root ganglion; NGF, nerve growth factor; PC, pancreatic cancer; PNI, perineural invasion.

Figure 3.

Hyperglycemia upregulates HIF1α, which promotes the invasion and migration of PC cells. (A) HIF1α expression in Panc-1 and BxPc-3 after 0, 5.5 and 25 mM glucose intervention. (B) Disease-free survival and (C) overall survival of patients with PC according to HIF1α expression. (D) The relative expression of HIF1α in normal and PC tissues. (E) The relative expression between HIF1α and NGF. (F and G) Expression of HIF1α and NGF in high-glucose and hypoxic conditions in (F) BxPc-3 and (G) Panc-1. (H and I) Cell invasion and migration ability of the control and 2-ME groups in BxPC-3 and Panc-1 cells assessed by (H) Transwell assay (crystal violet staining; ×100 magnification), and (I) wound-healing assay (×50 magnification), respectively. (J and K) Quantification of (H) and (I), respectively. *P<0.05 vs. control. HIF1α, hypoxia inducible factor 1α; HR, hazard ratio; PAAD, pancreatic adenocarcinoma; TPM, transcripts per million; NGF, nerve growth factor; nor, normoxia (O₂ concentration, 20%); hypo, hypoxia (O₂, concentration 1%); 2-ME, 2-mercaptoethanol; PC, pancreatic cancer.

Figure 4.

Hyperglycemia facilitates the invasion and migration of PC cells via HIF1α. (A) HIF1α and NGF expression in control, si-NC and si-HIF1α groups under normoxic and hypoxic conditions. (B) The quantification of the PC cell invasion index in the control and si-HIF1α groups. (C) The quantification of the DRG outgrowth index in the control and si-HIF1α groups. (D) The micrograph of perineural invasion in the control and si-HIF1α groups using the PC cell-DRG co-culture model. (E) Cell invasive/migration ability of control, high-glucose (25 mM glucose) and high-glucose plus si-HIF1α groups in BxPC-3 and Panc-1 cells assessed using a Transwell assay (crystal violet staining; ×100 magnification) or wound-healing assay (×50 magnification). (F and G) The quantification of (F) PC cell invasion and (G) migration ability in the control, high-glucose (25 mM glucose) and high-glucose plus si-HIF1α groups. *P<0.05 vs. control group or si-NC group, as indicated. Scale bar, 200 µm. HIF1α, hypoxia inducible factor 1α; NGF, nerve growth factor; si, small interfering; NC, negative control; DRG, dorsal root ganglion; ns, not significant; PC, pancreatic cancer; HG, high glucose.

Figure 5.

Hyperglycemia facilitates the PNI of PC via activation of HIF1α in vitro. (A) The micrograph of PNI in the control, hyperglycemia (25 mM glucose) and hyperglycemia plus si-HIF1α groups after intervention for 6, 24 and 48 h. (B) The quantification of the PC cell invasion index in the control, high-glucose (25 mM glucose) and high-glucose plus si-HIF1α groups. (C) The quantification of the DRG outgrowth index in the control, hyperglycemia (25 mM glucose) and hyperglycemia plus si-HIF1α groups. (D) HIF1α and NGF expression in the control, high-glucose (25 mM glucose) and high-glucose plus si-HIF1α groups. (E) Pattern diagrams of high hyperglycemia promoting PNI of PC by activating HIF1α. *P<0.05. Scale bar, 200 µm. ns, not significant; PNI, perineural invasion; HIF1α, hypoxia inducible factor 1α; NGF, nerve growth factor; si, small interfering; NC, negative control; PC, pancreatic cancer; DRG, dorsal root ganglion; HG, high glucose.

Figure 6.

Hyperglycemia promotes the PNI of pancreatic cancer cells in a HIF1α-dependent manner in vivo. (A) The pattern diagram of the establishment of the in vivo PNI sciatic nerve infiltrating tumor model with a background of hyperglycemia. (B) Blood glucose levels of mice in the euglycemia and hyperglycemia groups. (C and D) The quantification of (C) the sciatic nerve score and (D) the distance of paw span in the euglycemia, hyperglycemia and hyperglycemia plus 2-ME groups. (E and F) The macrophotography of (E) tumor size and (F) the quantification of tumor volume in the euglycemia, hyperglycemia and hyperglycemia plus 2-ME groups. The hyperglycemia group was used to compare with the other two groups. *P<0.05 vs. hyperglycemia or STZ group. Scale bar, 1 cm. ns, not significant; STZ, streptozotocin; 2-ME, 2-mercaptoethanol; PNI, perineural invasion.