OxLDL promotes lymphangiogenesis and lymphatic metastasis in gastric cancer by upregulating VEGF‑C expression and secretion

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  Figure 1. Plasma oxLDL in patients with gastric cancer is positively correlated with lymph node metastasis. (A) The association between plasma oxLDL and lymph node metastasis in gastric cancer (case numbers: N0=8, N1=4, N2=4, N3a=5 and N3b=7; *P<0.05, compared with N0). (B) The correlation between the plasma oxLDL and the LVD was determined by Pearson’s correlation analysis (n=17; r=0.79 and P<0.05). Plasma oxLDL in patients with gastric cancer was detected via ELISAs. Lymphatic vessels in gastric cancer tissues were stained with D2-40. LVD was calculated using the number of vessels per field. (C) The correlation between the plasma VEGF-C and the LVD. Plasma VEGF-C in patients with gastric cancer was detected by ELISAs (n=17, r=0.87 and P<0.05). (D) Lymphatic vessel in adjacent tissues and gastric cancer tissues were stained with D2-40 by immunohistochemistry. Histograms represent the number of lymphatic tubes per field (for adjacent and gastric cancer tissues n=17). (E) Tube formation of LEC incubated with the condition medium from the culture medium of HGC-27 treated by nLDL, oxLDL or siLOX-1+oxLDL for 24 h (*P<0.05 vs, Ctrl). A total of 2×104 human lymphatic endothelial cells were…

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  Figure 2. OxLDL promotes the lymph node metastasis of gastric cancer cells in vivo. (A) The general morphology of gastric cancer xenografts from nude mice was depicted following treatment with PBS, nLDL or oxLDL for 28 days. (B) The line chart represents the volume of xenografts (the black line represents the control group, the blue line represents the nLDL group and the red line represents the oxLDL group). (C) The histogram represents the weight of the xenografts. (D) The general morphology of popliteal lymph nodes from animal models following treatment with PBS, nLDL or oxLDL for 28 days. (E) The histogram represents the volume of each group of lymph nodes. (F) The histogram represents the weight of each group of lymph nodes. (G) The gastric cancer cells that had metastasized to the homolateral popliteal lymph nodes were imaged by live IVIS following a luciferin injection. Red arrows represent the metastasized gastric cancer cells that were labeled with luciferase beforehand. Prior to the popliteal lymph nodes being imaged, the footpads of mice were obstructed due to strong signal intensity from the footpad, which was the injection site. The nLDL group, oxLDL group and PBS group…

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  Figure 3. OxLDL promotes the expression and secretion of VEGF-C. (A) Plasma oxLDL was correlated with plasma VEGF-C in patients with gastric cancer. Plasma oxLDL and VEGF-C levels in patients with gastric cancer were detected using ELISA kits. The number of patients was 23 (r=0.64 and P<0.05). (B) VEGF-C mRNA levels in HGC-27 cells treated with 50 µg/ml nLDL or oxLDL for 24 h, as measured by reverse transcription-quantitative polymerase chain reaction. A total of three independent experiments were performed. (C) VEGF-C expression levels were upregulated by nLDL and oxLDL in AGS, HGC-27, MGC-803 and SGC-7901 gastric cancer cell lines. (D) OxLDL dose-dependently downregulated VEGF-C expression in gastric cancer cells. (E) OxLDL time-dependently downregulated VEGF-C expression in gastric cancer cells. VEGF-C protein levels in cell lysates were measured by western blotting, with β-actin as the control. (F) To measure the effect of oxLDL on the secretion of VEGF-C by HGC-27 and SGC-7901 gastric cancer cells, VEGF-C was detected in the supernatants using an ELISA kit. *P<0.05, results are presented as the means ± standard deviations. OxLDL, oxidized low-density lipoprotein; Ctrl, control; nLDL,…

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  Figure 4. NF-κB signaling is involved in the regulation of VEGF-C expression by oxLDL. (A) Activation of the NF-κB signaling pathway in HGC-27 cells following oxLDL treatment. IκB/p-IκB, P65/p-P65 and β-actin in gastric cancer cell lysates were measured by western blot analysis. (B) Translocation of NF-κB and P65 following oxLDL treatment; HGC-27 cells were incubated with 50 µg/ml oxLDL for 6 h. (C) The amount of P65 that was translocated into the nucleus following oxLDL treatment was measured by western blotting. HGC-27 cells were incubated with 50 µg/ml nLDL and oxLDL for 12 h. (D) The upregulated VEGF-C level was rescued by the NF-κB inhibitor PDTC. The protein levels were measured via western blotting. (E) VEGF-C levels in the supernatants were detected using an ELISA kit. (F) The knockdown effect of P65 siRNA was verified by western blotting. (G) The upregulated VEGF-C level was rescued by P65 siRNA. P65/p-P65 and VEGF-C protein levels in gastric cancer cell lysates were measured via western blotting. *P<0.05, results are presented as the means ± standard deviations. OxLDL, oxidized low-density lipoprotein; Ctrl, control; nLDL, native LDL; VEGF-C, vascular endothelia growth factor-C;…

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  Figure 5. OxLDL promotes the activation of the NF-κB signaling pathway, as well as the expression and secretion of VEGF-C, through LOX-1. (A) Validation of siRNA efficacy in HGC-27 cells. (B) The effects of oxLDL on NF-κB activity and VEGF-C expression were blocked following the knockdown of LOX-1. VEGF-C protein levels in the cell lysates were measured via western blotting, (C) supernatant VEGF-C levels were detected using an ELISA kit. (D) The regulatory effects of oxLDL on NF-κB activity and VEGF-C expression were blocked following LOX-1 being inhibited by 250 µg/ml Poly I. VEGF-C protein levels in the cell lysates were measured via western blotting, (E) supernatant VEGF-C levels were detected using an ELISA kit. (F) Schematic depicting the mechanism by which oxLDL promotes lymph node metastasis in gastric cancer. Results are presented as the means ± standard deviations. NS, non-significant; IKK, IBκ kinase; OxLDL, oxidized low-density lipoprotein; Ctrl, control; nLDL, native LDL; VEGF-C, vascular endothelia growth factor-C; NF-κB; nuclear factor-κB; p-, phospho-; si, small interfering; LOX, lectin-like oxLDL; IκB, inhibitor of κB α; NCsiRNA, non-specific control siRNA; poly I,…