High-mobility group box-1 contributes tumor angiogenesis under interleukin-8 mediation during gastric cancer progression

Abstract

Many soluble factors are involved in tumor angiogenesis. Thus, it is valuable to identify novel soluble factors for effective control of tumor angiogenesis in gastric cancer (GC). We investigated the role of extracellular high-mobility group box-1 (HMGB1) and its associated soluble factors in the tumor angiogenesis of GC. Clinically, we measured serum levels of HMGB1 and GC-associated cytokines/chemokines using GC serum samples (n = 120), and calculated microvessel density (MVD) by CD34 immunostaining using human GC tissues (n = 27). Then we analyzed the correlation of serum HMGB1 levels with MVD or that with cytokine/chemokine levels by linear regression. As in vitro angiogenesis assay for HMGB1, HUVEC migration and capillary tube formation assay were carried out using different histological types of human GC cells (N87 and KATOIII). CD34-positive microvessels were detected from early GC, but MVD increased according to GC stages, and were closely correlated with serum HMGB1 levels (R = 0.608, P = 0.01). The HUVECs cultured in conditioned media derived from rhHMGB1-treated or HMGB1-TF GC cells showed remarkably enhanced migration and tube formation activities. These effects were abrogated by anti-HMGB1 antibody or HMGB1 siRNA in both N87 and KATOIII cells (all P < 0.05). Among tested cytokines/chemokines, interleukin-8 (IL-8) was the most remarkable cytokine correlated with serum HMGB1 (P < 0.001), and enhanced HUVEC migration and tube formation activities by rhHMGB1 or HMGB1-TF were significantly reversed by IL-8 inhibition. These results indicate overexpressed HMGB1 contributes to tumor angiogenesis through IL-8 mediation, and combined targeting of HMGB1 and IL-8 can control tumor angiogenesis in GC.

Keywords: Angiogenesis; cytokine; gastric cancer; high-mobility group box-1; interleukin-8.

Figure 1

CD34 immunohistochemical staining for microvessels in gastric cancer (GC) tumor sections (tumor angiogenesis) according to GC stage. (a) CD34‐positive microvessels in early GC (EGC); serum high‐mobility group box‐1 (sHMGB1) level, 7.7 ng/mL. (b) CD34‐positive microvessels in locally advanced GC (AGC‐M0); sHMGB1 level, 34.1 ng/mL. (c) CD34‐positive microvessels in highly advanced GC with metastasis (AGC‐M1); sHMGB1 level, 30.7 ng/mL. Microvessel density (MVD) was determined as the average count of the five fields. Scale bar = 100 μm. Magnification, ×200 by light microscopy.

Figure 2

Correlation of serum high‐mobility group box‐1 (sHMGB1) levels with microvessel density (MVD), as determined by quantifying CD34‐positive microvessels in gastric cancer tumor sections. (a) Results of immunohistochemical staining for determining MVD according to sHMGB1 levels. Scale bar = 100 μm. (b) Linear regression analysis to determine the correlation of sHMGB1 levels with MVD. Magnification, ×200 by light microscopy.

Figure 3

In vitro angiogenesis assay in gastric cancer cell lines: HUVEC migration assay. (a) Migratory activity of HUVECs cultured in conditioned media (CM) derived from N87 cells without treatment, or treated with 10 μg/mL recombinant human high‐mobility group box‐1 (rhHMGB1), or 10 μg/mL rhHMGB1 plus 60 μg/mL anti‐HMGB1 antibody for 24 h. (b) Migratory activity of HUVECs cultured in CM derived from untreated, rhHMGB1‐treated, and rhHMGB1 plus anti‐HMGB1 Ab‐treated KATOIII cells for 24 h. (c) Migratory activity of HUVECs cultured in CM derived from mock‐transfected (TF), HMGB1‐TF, and HMGB1‐TF plus HMGB1 siRNA‐treated N87 cells for 24 h. (d) Migratory activity of HUVECs cultured in CM derived from mock‐TF, HMGB1‐TF, and HMGB1‐TF plus HMGB1 siRNA‐treated KATOIII cells for 24 h. Columns, mean number of HUVECs migrating through the inserts in three independent experiments, as observed using DP controller software with an IX71 microscope (magnification, ×100; Olympus Corporation). Staining, 2 μM calcein AM for 30 min. Bars, SE. *P < 0.05; **P < 0.01, Student's t‐test.

Figure 4

In vitro angiogenesis assay in gastric cancer cell lines: HUVEC capillary tube formation assay. (a) Capillary tube formation activity of HUVECs cultured in conditioned medium (CM) derived from untreated, 10 μg/mL recombinant human high‐mobility group box‐1 (rhHMGB1)‐treated, or 10 μg/mL rhHMGB1 plus 60 μg/mL anti‐HMGB1 antibody (Ab)‐treated N87 cells for 24 h. (b) Tube formation activity of HUVECs cultured in CM derived from mock‐transfected (TF), HMGB1‐TF, or HMGB1‐TF plus HMGB1 siRNA‐treated N87 cells for 24 h. (c) Tube formation activity of HUVECs cultured in CM derived from rhHMGB1‐treated KATOIII cells. (d) Tube formation activity of HUVECs cultured in CM derived from HMGB1‐TF KATOIII cells. Columns, mean counts of tube formation in three independent experiments (left). Bars, SE. *P < 0.05; **P < 0.01, Student's t‐test. Images are obtained using DP controller software with an IX71 microscope (Olympus Corporation) (magnification, ×40; right). Staining, 2 μM calcein AM for 30 min.

Figure 5

Linear regression graphs presenting the correlation between serum high‐mobility group box‐1 (HMGB1) levels and serum cyokines/chemokines levels in gastric cancer. The graphs show the positive correlations of serum HMGB1 levels with serum interleukin‐8 (IL‐8), vascular endothelial growth factor (VEGF), and tumor necrosis factor‐α (TNF‐α) levels. Among them, serum IL‐8 levels show the strongest correlation with serum HMGB1 levels.

Figure 6

In vitro angiogenesis assays showing that interleukin‐8 (IL‐8) mediates high‐mobility group box‐1 (HMGB1)‐induced tumor angiogenesis in gastric cancer cells. (a, b) HUVEC migration assay. (a) Significant decline in the migratory activity of HUVECs cultured in CM derived from recombinant human HMGB1 (rhHMGB1; 10 μg/mL) plus IL‐8 siRNA‐treated N87 cells compared with that of HUVECs cultured in CM derived from cells treated with rhHMGB1 alone. (b) Significant decline in the migratory activity of HUVECs cultured in CM derived from HMGB1‐transfected (HMGB1‐TF) plus IL‐8 siRNA‐treated N87 cells compared with that of HUVECs cultured in CM derived from cells treated with HMGB1‐TF alone. (c, d) HUVEC capillary tube formation assay. (c) Significant decline in tube formation activity of HUVECs cultured in CM derived from rhHMGB1 plus IL‐8 siRNA‐treated or HMGB1‐TF plus IL‐8 siRNA‐treated N87 cells compared with that of HUVECs cultured in CM derived from rhHMGB1‐treated or HMGB1‐TF N87 cells (upper panel). Decline in tube formation activity of HUVECs cultured in CM derived from KATOIII cells in which IL‐8 was inhibited by IL‐8 siRNA (lower panel). (d) Images of capillary tube formation of HUVECs obtained using DP controller software with an IX71 microscope (magnification, ×40; Olympus Corporation). Columns, mean of three experiments. Bars, SE. *P < 0.05; **P < 0.01, Student's t‐test.

Figure 7

Schematic figure of a hypothetic model of interaction between extracellular high‐mobility group box‐1 (HMGB1) and interleukin‐8 (IL‐8) during gastric cancer (GC) progression. This interaction may contribute to GC progression at a relatively early stage of GC metastasis, providing a mechanistic link between tumor angiogenesis and epithelial–mesenchymal transition (EMT) through HMGB1 and IL‐8 interaction. AGC‐M0, locally advanced GC without metastasis. AGC‐M1, highly advanced GC with metastasis. EGC, early GC; TNF‐α, tumor necrosis factor‐α.