Figure 4.

Overexpression of hCAR inhibits colony formation of hepatoma cells in vitro and the growth of hepatoma xenograft in vivo. Normal and hCAR-inducible HepG2 and Hep3B cells were treated with Dox (1 μg/ml) or vehicle control for 72 h followed by a colony formation in soft-agar assay as described in the “Experimental procedures” section. Representative colony images and quantification colony formation are shown in A and B. The histograms represent mean ± SD of the cloning efficiency (%). Data presented are representative images from at least three independent experiments in A and B. For the xenograft experiment, 3 to 5 × 10⁶ HepG2-hCAR or Hep3B-hCAR cells were inoculated into the nude mice for xenograft formation as detailed under the “Experimental procedures” section. The tumor growth curves of HepG2-hCAR (C) and Hep3B-hCAR xenografts (F) were measured in the control-diet and Dox-diet feeding groups during the experimental periods. RT–PCR and Western blotting were used to measure the mRNA and protein expressions of hCAR in the xenograft tumors (D and G). Relative blot densitometry was quantified using ImageJ from three separately prepared cell experiments and normalized to the density of the loading control. Expression of Ki67 was examined by immunohistochemistry (E and H); the scale bar represents 100 μm. Ki67 intensity was quantified using ImageJ and was expressed as mean ± SD (n = 9). ∗p < 0.05 and ∗∗p < 0.01. Dox, doxycycline; hCAR, human constitutive androstane receptor.