Figure 3.

Multiple mechanisms decreasing HIFα in sulfoquinovosyl-acylpropanediol (SQAP). (a) The presence of the proteasome inhibitor MG-132 decreased the effect of SQAP on HIFα protein levels. HAK1-B cells incubated under either normoxia or 3% O₂ hypoxia were treated with 10 μmo/l SQAP for 24 hours and further incubated for 4 hours in the presence of 10 μmo/l MG-132. (b) HIF1α synthesis at the transcriptional level decreased upon SQAP addition in a dose-dependent manner. HAK-1B cells were incubated with medium containing different dose of SQAP (1 and 10 μmo/l). Cellular RNA was then extracted and analyzed for HIF1α mRNA expression by quantitative real-time polymerase chain reaction, with GAPDH as a control. (c) SQAP decreases NFκB expression in HAK1-B and Huh-7. Western blots for both cell lines exposed to SQAP (1 and 10 μmo/l) under hypoxic conditions are shown. Cells were incubated with SQAP-containing medium for 24 hours, after which the cells were moved to 3% O₂ hypoxic conditions for 24 hours and then lysed with radioimmunoprecipitation buffer. (d) Representative immunohistochemistry images for pimonidazole in HAK1-B tumor treated with SQAP. Scale bar = 200 μm. (e) Western blot analysis of pimonidazole adducts in HAK1-B tumors treated with SQAP. Whole tumors were collected from tumor bearing mice (n = 4 per group). Negative control: HAK1-B cells cultured under normoxia in vitro. Positive control: HAK1-B cell cultured under 3% O₂ hypoxia in vitro. Pimonidazole adducts are shown in the indicated multiple bands. The band densities for each protein were measured and calibrated by β-actin. Average bands densities are represented as mean ± standard deviation (SD). (f) Representative double immunofluorescence images using both CD31 (green) and α-SMA (red) in HAK1-B tumors treated with SQAP. Nuclei were stained with DAPI. Scale bar = 50 μm. Relative number of tumor vessels, α-SMA-positive cells, and tumor vessels covered with α-SMA positive cells in HAK1-B tumors treated with SQAP are shown. The number of tumor vessels and the ratio of pericyte covered vessels are represented as mean ± SD. DAPI, 4’,6-diamidino-2-phenylindole, dihydrochloride.