Figure 1.
Involvement of VEGF-C promoted by Periostin overexpression on the tube formation of TR-LE cells.
(A) Schema shows the strategy to identify Periostin and VEGF-C by comparing the gene expression profile between the parent (MSCC-1 cells) and a highly invasive clone (MSCC-Inv1 cells). (B) Higher expression of VEGF-C mRNA in cells of the highly invasive clone MSCC-Inv1 and VEGF-C expression in periostin-overexpressing cells. HSC4 cells without periostin expression were transduced using a retroviral plasmid encoding hexa-histidine-tagged periostin. Periostin and VEGF-C mRNA expression levels in MSCC-1 cells, MSCC-Inv1 cells, empty vector-transfected HSC4 cells (empty), and periostin-overexpressing HSC4 cells (His-periostin) were examined by RT-PCR. GAPDH expression was used as a loading control. Conditioned media were collected from empty vector-transfected HSC4 cells (empty) and periostin-overexpressing HSC4 cells (His-periostin) after incubation for 4 days. Conditioned media were concentrated and analysed by western blotting for expression of His-periostin and VEGF-C in conditioned media. (C) Conditioned media from periostin-overexpressing cells promotes tube formation of lymphatic endothelial cells. Tube formation of TR-LE cells by adding conditioned media from periostin-overexpressing cells. TR-LE cells were seeded onto matrigel-coated wells in the presence of conditioned media from empty vector-transfected (control CM) or periostin-overexpressing (periostin CM) HSC4 cells. After incubation for 0–9 h, the lengths of the tube-like structures formed were evaluated. The figure shows the cells after incubation for 9 h. (D) The graph shows the tube scores after 0–9 h incubation of control CM or periostin CM. The bars show the average values and SDs from 3 independent experiments.
Figure 2.
Correlation between periostin and VEGF-C in HNSCC.
(A) Immunohistochemical staining for periostin and VEGF-C in HNSCC cases. Representative HNSCC cases (high and low magnification) with periostin and VEGF-C expression are shown. Scale bar is shown in each picture. (B) Graph shows the VEGF-C expression status in HNSCC cases with high or low expression of periostin. (C) Serum level of periostin and VEGF-C in 81 HNSCC patients was examined by ELISA. Serum level of periostin and VEGF-C was compared with tumor stage. Graph shows percentage of periostin or VEGF-C positive cases in different tumor stage (from stage 1 to 4). (D) Serum level of periostin and VEGF-C was compared with lymph node metastasis. Graph shows percentage of periostin or VEGF-C positive cases in cases with or without lymph node metastasis (E) Serum level of periostin was compared with serum level of VEGF-C. Graph shows percentage of VEGF-C positive cases in cases with periostin positive or negative.
Figure 3.
Direct involvement of periostin in tube formation of lymphatic endothelial cells.
(A) TR-LE cells were seeded onto matrigel-coated wells in the presence of periostin (100 or 200 ng/mL) or VEGF-C (100 ng/mL). After incubation for 1–9 h, the lengths of the tube-like structures formed were evaluated. The figure shows cells after incubation for 9 h. (B) The graph shows the tube scores after incubation for 1–9 h. The bars show the average values and SDs from 3 independent experiments. (C) The graph shows the tube score ratios after treatment with VEGF-C or periostin for 9 h. The tube score of control was defined as 1.0. (D) TR-LE cells were seeded onto matrigel-coated wells in the presence of periostin (100 ng/mL) and VEGF-C (100 ng/mL). After incubation for 1–9 h, the lengths of the tube-like structures formed were evaluated. The graph shows the tube score after incubation for 1–9 h. The bars show the average values and SDs from 3 independent experiments. (E) The graph shows the tube score ratios after treatment with VEGF-C and periostin for 9 h. The tube score of control was defined as 1.0. (F) Periostin promotes Src and Akt phosphorylation. Levels of total and phosphorylated forms of Src, Akt, ERK, and FAK after treatment of TR-LE cells with periostin (100 ng/mL) shown by western blotting. β-actin expression was used as a loading control. (G) The lengths of the tube-like structures formed by TR-LE cells incubated for 1–9 h with recombinant periostin (100 ng/mL) with or without SU6656 (400 nM) were evaluated. The upper graph shows the tube scores after incubation for 1–9 h. The bars show the average values and SDs from 3 independent experiments. The lower graph shows the tube score ratio. The tube score of control was defined as 1.0. (H) The lengths of the tube-like structures formed by TR-LE cells incubated for 1–9 h with recombinant periostin (100 ng/mL) with or without LY294002 (10 µM) were evaluated. The upper graph shows the tube scores after incubation for 1–9 h. The bars show the average values and SDs from 3 independent experiments. The lower graph shows the tube score ratio. The tube score of control was defined as 1.0.
Figure 4.
Periostin promotes proliferation and migration of lymphatic endothelial cells.
(A) Proliferation of TR-LE cells after recombinant periostin treatment. Cells were seeded onto fibronectin-coated 24-well plates at 1.5×104/well. After pre-incubation at 33°C for 24 h, the temperature was changed and recombinant periostin (100 ng/mL) was added to the medium. The cells were trypsinized and counted 0, 2, 4, or 6 days after the addition of recombinant periostin. The bars show the average values and SDs from 3 independent experiments. (B) The effect of periostin on cell migration of TR-LE cells. Cells were seeded onto filters pre-coated with 10 µg of fibronectin. The lower compartment contained 0.5 mL of serum-free medium with or without 100 ng/mL recombinant periostin. After incubation for 4 h, cells that had migrated to the lower surfaces of the filters were visualized by hematoxylin staining and counted. The assay was repeated 3 times. The figure shows cells that had migrated to the lower surface of the filter (upper panel). The graph shows the number of cells on the lower surfaces of the filters with or without periostin (100 ng/mL) (lower panel). (C) TR-LE cells were seeded on cover slips coated with PBS or recombinant periostin (200 ng/mL) and allowed to attach for 60, 120, 180, or 240 min. The cells were stained with Alexa Fluor 488-phalloidin antibody and anti-vinculin-FITC antibody. DNA was visualized by 4′,6-diamidino-2-phenylindole (DAPI) staining.
Figure 5.
Periostin promotes lymphangiogenesis in vivo.
(A) Periostin-overexpressing (periostin) and empty vector-transfected (control) HSC2 cells (1×107 cells) were individually injected subcutaneously at 2 sites in each of 5 nude mice. After 1 month, the tumors were resected and stained with an anti-mouse LYVE-1 antibody recognising lymphatic vessels. Representative cases of immunohistochemical staining for LYVE-1 in control and periostin-overexpressing tumors are shown. Arrow shows LYVE-1 positive lymphatic vessels. (B) The LYVE-1 positive lymphatic vessels in control and periostin-overexpressing tumors were counted. The graph shows the average numbers of lymphatic vessels in control and periostin-overexpressing tumors. The bars show the average values and SDs.
Figure 6.
Correlation between periostin expression and lymphatic status in clinical HNSCC cases.
(A) Periostin and D2-40 expression levels were examined by immunohistochemical staining of HNSCC case specimens. A representative case of periostin and D2-40 expression in HNSCC is shown. Arrow shows D2-40 positive lymphatic vessels. (B) The graph shows the comparison between the average numbers of lymph vessels in intratumoral, peritumoral, and total areas in HNSCC cases with high or low expression of periostin. (C) The graph shows the status of lymphatic invasion in HNSCC cases with high or low expression of periostin.
Figure 7.
A model of periostin-promoted lymphangiogenesis.
Periostin expression is upregulated in cancer cells. Periostin in turn upregulates VEGF-C expression in cancer cells. Periostin secreted from the cancer cells promotes migration and tube formation of lymphatic endothelial cells through the activation of Src and Akt. Src may activate VEGFR-2 and VEGFR-3 [36]–[38]. Moreover, Secreted VEGF-C from cancer cells also promotes migration and tube formation of lymphatic endothelial cells. Thus, in addition to activation of VEGF-C signaling pathway, periostin-integrin interaction may trigger the intracellular signaling and activation of certain genes that are involved in tube formation and migration of lymphatic endothelial cells through Src and Akt activation.