Fig. 1Effect of hypoxia and Genipin on intracellular level of HIF-1α in HeLa cells. (A) Cells were treated with various concentrations (10–500 µM) of Genipin for 24 h. The cytotoxic effect of Genipin on HeLa cells was determined using the trypan blue dye exclusion assay as described in Materials and Methods section. Error bars represent standard error of the mean (S.E.M.) from three separate experiments. (B) Kinetics of HIF-1α accumulation under hypoxic conditions. (C) Kinetics of HIF-1α accumulation during treatment with Genipin for 8 h under hypoxic conditions. (D) Cells were exposed to hypoxia, cobalt chloride, and DFX along with Genipin for 8 h, and then, harvested. Cells lysates containing equal amounts of protein (20 µg) were separated by SDS-PAGE and immunoblotted with anti-HIF-1α antibody. Actin was used as a loading control. Symbols denote a response that was significantly different from the control (*P < 0.05, **P < 0.01).
Fig. 2Effect of Genipin on HIF-1α accumulation during hypoxia in various cancer cell lines. HepG2, HCT116, LNCaP, and MDA231 cells were exposed to 1% O2alone or were treated with 100 µM Genipin in combination with 1% O2 for 8 h. Cell lysates containing equal amounts of protein (20 µg) were separated by SDS-PAGE and immunoblotted with anti-HIF-1α antibody. Actin was used as a loading control.
Fig. 3Role of ERK and Akt in the accumulation of HIF-1α in HeLa cells. Cells were treated with 100 µM Genipin in combination with 1% O2 in the presence or absence of 10 µM LY294002 for 8 h. Cells were pretreated with PD98059 (10 µM) and LY294002 (10 µM) for 30 min followed by treatment with Genipin and/or hypoxia for 8 h in the presence of PD98059 and LY294002. Cell lysates containing equal amounts of protein (20 µg) were separated by SDS-PAGE and immunoblotted using anti-HIF-1α, anti-phospho-Akt (S473), or anti-Akt antibody. Actin was used as a loading control.
Fig. 4Genipin inhibits production of VEGF and invasion during hypoxia. (A) HeLa cells were exposed to 1% O2 for various time periods (2–24 hours). (B) HeLa cells were treated with various concentrations of Genipin (50, 100, and 250 µM) for 16 h during hypoxia. The concentration of VEGF protein in the culture media was determined by ELISA. The assays were performed in triplicates. The results represent the mean values of VEGF concentrations and error bars represent standard error of the mean from triplicate samples. (C) Invasion indicators are expressed as the mean ± S. D. of three independent experiments. The data was evaluated for statistical significance by Student's t-test. The means noted with an asterisk were statistically different from the matched normoxic control. (D) Western blot analysis shows that Genipin inhibits invasion related proteins in HeLa cells during hypoxic condition. Symbols denote a response that was significantly different from the control (*P < 0.05, **P < 0.01).