Article Figures & Data
Figures
- Fig. 1.
Expression of HIF-1α and β-catenin in hypoxic HCC cell lines and HAL-treated xenografts. Immunoblotting showed that hypoxia increased β-catenin protein levels in PLC/PRF/5 and HepG2 cells (A), and induced its intracellular translocation in Hep3B and MHCC97 cells (B). The induction of HIF-1α expression by hypoxia was detected in all cells with respect to their normoxic controls. Blots were normalized to β-actin protein in each lane. The ratios of each protein relative to matched normoxic cells are listed below each blot. C, immunoblotting of tumor tissues showed that HAL markedly increased HIF-1α expression in MHCC97-R and Hep3B-R xenografts compared with those in sham-operated groups but slightly upregulated β-catenin protein level. Ratios relative to corresponding sham-operated control were normalized to β-actin. D, immunostaining revealed that most β-catenin–positive cells in above tumor tissues showed a cytoplasmic/nuclear accumulation.
- Fig. 2.
Effect of hypoxia on the transcription and stabilization of β-catenin in HCC cells. A, qRT-PCR showed that hypoxia did not significantly increase β-catenin mRNA levels in HCC cells, except in HepG2 cells. B, immunoblotting revealed that the effects of hypoxia on elevated levels of β-catenin in PLC/PRF/5 and HepG2 cells, and on nuclear accumulation of β-catenin in Hep3B and MHCC97 cells, were similar to the effects of the proteasome inhibitor MG132 on β-catenin in these cells. The ratios of each protein relative to nontreated cells were normalized to β-actin. C, alterations of β-catenin in hypoxic HCC cells coincided with p-Akt expression, assayed by immunoblotting, which both were inversely related to changes in GSK-3β levels. The ratios of each protein relative to normoxic cells were normalized to β-actin. D, immunoblotting showed that LY294002 remarkably repressed hypoxia-induced GSK-3β degradation and augmentation of p-Akt. The ratios of each protein relative to normoxic cells were normalized to β-actin.
- Fig. 3.
Effect of silencing β-catenin on the invasion and metastasis of MHCC97 and Hep3B cells under normoxic and hypoxic conditions. A, in vitro Transwell analysis revealed that depletion of β-catenin completely inhibited hypoxia-enhanced invasiveness of MHCC97 and Hep3B cells, and partially impaired their original invasiveness in normoxia. B, quantification of bioluminescence showed that HAL accelerated pulmonary metastasis in MHCC97-R xenografts and increased peritoneal seeding in Hep3B-R xenografts, compared with matched controls. However, the enhanced metastatic potential was abrogated significantly by silencing β-catenin. C, representative bioluminescence images visualized tumor metastatic loci in lung and peritoneum.
- Fig. 4.
Effect of β-catenin knockdown on hypoxia-induced molecular changes consistent with EMT in HCC cells. A, qRT-PCR revealed an augmentation of transcription factors Snail, Slug, and Twist expression in hypoxic MHCC97 and Hep3B cells with respect to normoxic controls. When β-catenin in these cells was knocked down by shRNA, the upregulation of Slug or Twist due to hypoxia were inhibited, whereas the level of Snail mRNA was higher than that in parental cells. B, morphologic changes consistent with EMT (spindle-shaped cells with loss of polarity and increased intercellular separation) were observed in MHCC97 and Hep3B cells in response to hypoxia. C, immunoblotting showed that hypoxia-elicited EMT in MHCC97-Mock and Hep3B-Mock cells, characterized by the loss of E-cadherin and plakoglobin as well as the upregulation of vimentin and N-cadherin, was repressed by silencing β-catenin. The ratios of each protein relative to corresponding vector-transfected cells in normoxia were normalized to β-actin. D, gelatin zymography showed that hypoxia increased the expression of MMP-2 in HCC cells, which was reversed by silencing β-catenin. The activity of MMP-9 was not affected. Ratios relative to vector-transfected cells in normoxia were normalized to β-actin.
- Fig. 5.
Expression patterns of HIF-1α and β-catenin immunostaining in HCC TMAs. Representative examples of H&E staining (A), negative control staining (B), weak cytoplasmic staining (C), moderate cytoplasmic staining (D), and strong nuclear staining (E) for HIF-1α; constitutive expression of β-catenin in normal bile ducts as positive control (F); weak membranous staining (G), strong membranous staining (H), cytoplasmic staining (I), and nuclear staining (J) for β-catenin in cancer tissues.
- Fig. 6.
Prognostic significance assessed by Kaplan-Meier survival estimates and log-rank tests. Comparisons of OS and TTR by HIF-1α (A and B), β-catenin (C and D), and HIF-1α/β-catenin (E and F).
Additional Files
Supplementary Data
Files in this Data Supplement:
Volume 16, Issue 10
