Caveolin-1 Knockdown Decreases SMMC7721 Human Hepatocellular Carcinoma Cell Invasiveness by Inhibiting Vascular Endothelial Growth Factor-Induced Angiogenesis

Figure 6

Supernatants from caveolin-1 siRNA-expressing SMMC7721 cells significantly inhibited the formation of endothelial tubular structures. (a) Bright-field views were taken using an Olympus BX40 fluorescent microscope equipped with a photometric CCD camera. Note that compared to cells treated with supernatants from control (CON) and negative control (NC) SMMC7721 cells at 6 h, 12 h, and 24 h fewer, capillary-like tubules developed in cells treated with supernatants from cells with knocked down caveolin-1 (KD). Capillary-like tubule formation was recovered following the addition of recombinant human VEGF (KD + VEGF); capillary-like tubule formation was also rescued if cells were treated with supernatants from KD cells subsequently infected with pGC-FU-caveolin-1 DNA plasmid LV (KD + CAV1). Immunocytochemical labeling of CD34 was performed at 24 h to further examine capillary-like tubule formation. (b) The difference in capillary-like tubule formation between cells treated with KD-derived supernatants and cells treated with supernatants from CON, NC, KD + VEGF, or KD + CAV1 SMMC7721 cells was quantitated. Capillary-like tubular structures were scored by counting the number of tubules in five microscopic fields at a magnification of 200x; values from each well were then averaged. Data represent the mean ± SD from three independent experiments; all samples were run in duplicate. At 6 h, 12 h, and 24 h, the results were as follows: KD versus CON, ; KD versus NC, ; KD + VEGF versus KD, ; KD + CAV1 versus KD, ; KD + VEGF versus KD + CAV1, ).

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