Lysyl Oxidase Promotes the Formation of Vasculogenic Mimicry in Gastric Cancer through PDGF-PDGFR Pathway

Abstract

Objective: Vasculogenic mimicry (VM) generates an important supplementary form of blood supply in cancer, which many factors regulate. However, the effect of lysyl oxidase (LOX) on VM formation is unclear. In this study, gastric cancer tissues and cells were used to investigate the role of LOX in the formation of VM. Materials and Methods: The samples were collected from 49 patients with a final diagnosis of gastric cancer. According to metastasis (including lymph node metastases and distant metastases), gastric cancer samples were divided into metastasis and non-metastasis groups. Based on the degree of invasion, gastric cancer specimens were divided into T1 + T2 and T3 + T4 groups. The relative expression of LOX was detected using Western blot. The formation of VM was measured by double staining with CD34 and Periodic acid-Schiff (PAS) in gastric cancer tissue slices, and the correlation between LOX and VM was analyzed with Pearson's correlation analysis. Gastric cancer cell line BGC-803 was treated with LOX, β-aminopropionitrile (BAPN, an inhibitor of LOX), and AG1295 or AG1296 (inhibitors of the platelet-derived growth factor receptor). The formation of VM was then measured using PAS staining. The expression of platelet-derived growth factor receptor (PDGFR)α and PDGFRβ in gastric cancer cells was detected by Western blot. Results: In gastric cancer samples, the level of LOX was higher in the metastasis group than in the non-metastasis group (P < 0.05) and in the T3 + T4 group than in the T1 + T2 group (P < 0.05). VM formation was greater in the T3+T4 group than in the T1+T2 group (P < 0.05) and in the metastasis group than in the non-metastasis group (P < 0.05). The expression level of LOX was positively correlated with VM formation (P < 0.01). In gastric cancer cells, LOX concentration was positively correlated with the degree of VM, and BAPN concentration was negatively correlated with the degree of VM (P <0.05). PDGFR levels in the T3+T4 and metastasis groups were relatively higher (P <0.01) and positively correlated with LOX levels in gastric cancer specimens (P < 0.01). The relative expression of PDGFRα and PDGFRβ in gastric cancer cells was up-regulated with increasing LOX and downregulated with increasing BAPN (P < 0.05). With inhibition of PDGFRα and PDGFRβ using AG1295 or AG1296, VM formation in gastric cancer cells decreased (P <0.05), but the number of VM structures increased while LOX was added (P < 0.05). Conclusion: LOX partially promotes the formation of VM in gastric cancer through the PDGF-PDGFR signaling pathway.

Keywords: Gastric cancer; Lysyl oxidase; Platelet-derived growth factor receptor; Vasculogenic mimicry.

Figure 1

Comparison of the level of LOX and the degree of VM in gastric cancer tissues. (A) Images of LOX protein expression in gastric cancer tissues detected by immunohistochemistry(bars: 100 μm, brown indicates LOX positive); (B) Images showing anti-CD34 immunohistochemistry co-stained with a periodic acid-Schiff reagent to detect VM formation (bars: 100 μm); (C) Bar charts show that LOX MOD in metastatic gastric cancer tissues was significantly higher than in nonmetastatic gastric cancer tissues, P < 0.05; (D) Bar charts show that LOX MOD in gastric cancer tissues of the T3 + T4 group was significantly higher than of the T1 + T2, P < 0.05; (E) LOX relative expression was positively correlated with VM in gastric cancer tissues (r = 0.574, P <0.01). (F) Bar graphs show that the number of VM structures in metastatic gastric cancer was higher than in nonmetastatic gastric cancer, P < 0.05; (G) Bar graphs show that the number of VM structures in gastric cancer tissues in the T3+T4 group was higher than in the T1 + T2 group, P < 0.05. The error bars represent the standard deviation (SD).

Figure 2

Comparison of the relative expression of LOX and PDGFR in gastric cancer tissues through WB. (A) Relative expression of LOX: metastatic gastric cancer tissues (Lanes 1-3), nonmetastatic gastric cancer tissues (Lanes 4-5). (B) Relative expression of LOX: T3-T4 group (Lanes 1-2), T1-T2 group (Lanes 3-4). (C) Relative expression of PDGFR: metastatic gastric cancer tissues (Lanes 1-3), nonmetastatic gastric cancer tissues (Lanes 4-5). (D) Relative expression of PDGFR: T3-T4 group (Lanes 1-2), T1-T2 group (Lanes 3-4). (E) Relative expression of LOX in metastatic gastric cancer tissues was higher than in nonmetastatic gastric cancer tissues; the relative expression of LOX in the T3-T4 group was higher than in the T1-T2 group, P < 0.05. (F) Scatter plot showing that the relative expression of LOX was positively correlated with PDGFR in gastric cancer tissue (r_LOX-PGGFR = 0.634, P < 0.0001). (G) Bar charts show that the relative expression of PDGFR in metastatic gastric cancer tissues was higher than in nonmetastatic gastric cancer tissues; the relative expression of PDGFR in the T3-T4 group was higher than in the T1-T2 group, P < 0.05.

Figure 3

LOX significantly improved VM formation in gastric cancer cells. The VM were analyzed by three-dimensional culture of gastric cancer cells. (A) VM formation in gastric cancer cells treated with LOX. The LOX concentration was positively correlated with the number of VM structures. (B) VM formation in gastric cancer cells treated with BAPN. BAPN concentration was negatively correlated with the number of VM structures. The black arrow indicates VM formation (×40).

Figure 4

LOX upregulated PDGFR expression in gastric cancer cells. (A) Results of Western blot show the relative expression of PDGFRα and PDGFRβ in gastric cancer cells treated with exogenous LOX. The LOX concentration was positively correlated with the relative expression of PDGFR. (B) Western blot shows the relative expression of PDGFRα and PDGFRβ in gastric cancer cells treated with BAPN. The concentration of BAPN was negatively correlated with the relative expression of PDGFR. Error bars represent SD, n = 5.

Figure 5

PDGFR inhibitors can suppress VM formation in gastric cancer cells. The VM were analyzed by two-dimensional culture of gastric cancer cells. (A) Different concentrations of PDGFR inhibitors significantly inhibited VM formation in gastric cancer cells (black arrow indicates VM formation; ×40); (B) Concentrations of PDGFR inhibitors were negatively correlated with the number of VM structures.

Figure 6

Blocking PDGFR may partially inhibit the promotion of LOX on VM formation. The VM were analyzed by two-dimensional culture of gastric cancer cells. (A) VM formation of gastric cancer cells treated with LOX and 10 μM AG1295 or 5 μM AG1296 (black arrow indicates VM formation; ×40). The LOX concentration was positively correlated with the number of VM structures. (B) VM formation of gastric cancer cells treated with BAPN and 5 μM AG1296. (C) Comparison of VM formation with or without AG1295 and AG1296 at the same concentration of LOX.