Epithelial mesenchymal transition and hedgehog signaling activation are associated with chemoresistance and invasion of hepatoma subpopulations - PubMed (original) (raw)
Epithelial mesenchymal transition and hedgehog signaling activation are associated with chemoresistance and invasion of hepatoma subpopulations
Xiaoli Chen et al. J Hepatol. 2011 Oct.
Erratum in
- J Hepatol. 2013 Feb;58(2):405
Abstract
Background & aims: Our previous studies showed that CD133, EpCAM, and aldehyde dehydrogenase (ALDH) are useful markers to identify cancer stem cells (CSCs) in hepatocellular carcinoma (HCC) tissues. The present study aims to evaluate chemosensitivity and invasion capability of HCC based on CSC marker profiles, and to explore the underlying molecular mechanisms.
Methods: Hepatoma cell lines were separated into subpopulations according to CD133, EpCAM, and ALDH expression profiles. Epithelial mesenchymal transition (EMT) and hedgehog (Hh) signaling were examined to identify their links with chemoresistance and aggressive invasion.
Results: Well-differentiated cell lines were positive for CD133(+)/ALDH(high) and CD133(+)/EpCAM(+) at 1.5-15% and 2.3-8.3%; whereas, poorly-differentiated cells were almost all negative for these markers. FACS-enriched CD133(+)/ALDH(high) and CD133(+)/EpCAM(+) Hep3B and Huh-7 cells formed more spheroids in vitro. CD133(-)/ALDH(low) HLE cells were more resistant to cisplatin, doxorubicin or sorafenib than their positive counterparts. CD133(-)/EpCAM(-) Huh-7 cells or CD133(-)/ALDH(-) HLE cells exhibited a higher invasion rate than their positive counterparts. HLE and HLF cells acquired EMT in double negative subpopulations. Hh activity in Huh-7 CD133(-)/EpCAM(-) cells was higher than in their positive counterparts, and the inhibition of Hh activity by cyclopamine resulted in reduced cell proliferation.
Conclusions: Well-differentiated CD133(+)/ALDH(high) or CD133(+)/EpCAM(+) cells appear to be a CSC/initiating subpopulation; whereas, in poorly-differentiated hepatoma cells, EMT and enhanced hedgehog signaling activity may be responsible for their chemoresistance and invasion. These findings underscore the significance of EMT and enhanced Hh signaling in liver cancer stem or initiating cells.
Copyright © 2011 European Association for the Study of the Liver. All rights reserved.
Figures
Figure 1. Flow cytometric analysis of hepatoma cells
A. CD133+/ALDHhigh cells in Hepatoma cell lines. B. CD133+/EpCAM+ cells in hepatoma cell lines. CD133+/ALDHhigh and CD133−/ALDHlow or CD133+/EpCAM+ and CD133−/EpCAM− cells were separated by FACS after staining with APC-conjugated anti-CD133 antibody and an Adelfluor kit (A) or with APC-conjugated anti-CD133 and FITC-conjugated anti-EpCAM antibodies (B).
Figure 2. Spheroid formation of hepatoma subpopulations after FACS enrichment
A, B and C: Spheroid cores were formed after seeding on low-attachable plates for 3 weeks, and co-stained with antibodies against AFP and CD133 or PCNA. D. Summary of spheroid formation in Hep3B and Huh-7 subpopulations 3 weeks after FACS enrichment.
Figure 3. Chemosensitivity of FACS-enriched HLE subpopulations
Chemosensitivity of HLE subpopulations to cisplatin (A), doxorubicin (B) and sorafenib (C) was assayed with WST-1 reagent after treatment for 24 hours and expressed as optical density value per 100 μg protein content. *, **, *** p<0.05, 0.01 and 0.005 compared to HLE CD133+/ALDHhigh subpopulation.
Figure 4. Invasion capability and gene expression of TGF-β1 and procollagen type I in hepatoma subpopulations
A. Wound healing after scraping in Huh-7 and HLE subpopulations. B. Matrigel invasion assay of Huh-7 subpopulations. C. Gene expression of TGF-β1 and procollagen type I by quantitative RT-PCR.
Figure 5. Acquisition of EMT in CD133−/ALDHlowHep3B, HLE and HLF cells
FACS-enriched CD133+/ALDHhigh or CD133−/ALDHlow subpopulations were stained with either Cy5-conjugated monoclonal antibodies against E-cadherin or vimentin in red. Cy5-stained images were overlaid with DAPI nuclear counter-staining in blue. Hep3B and HLF (20×), HLE (10×).
Figure 6. Western blot analysis of EMT markers and Hh signaling activity in Huh-7 and HLE subpopulations
A. Western blot image of E-cadherin and snail in Huh-7 and HLE subpopulations. β-actin was used as a loading control. B. Zeb1 levels in various hepatoma subpopulations. TATA box binding protein was used as a nuclear protein loading control. C. PTCH1 levels in the membrane fraction of Huh-7 subpopulations were determined by Western blot analysis with integrin β1 as a loading control. D. GLI2 levels in nuclear fractions of Huh-7 and HLE subpopulations. E. Hh signaling activity after transfection with GLI-lux plasmid and mutated plasmid (GLI-Lux-M) in the presence or absence of cyclopamine (5 μM CPM). Luciferase activity was determined one day after transfection of the plasmids. ** p <0.01 compared to Huh-7 CD133+/EpCAM+ subpopulation. Δ, ΔΔ p <0.05 and 0.01 compared to GLI2-Lux+DMSO. DMSO was used to dissolve CPM, and included in transfection controls. F. Inhibition of cell proliferation by cyclopamine in Huh-7 CD133+/EpCAM+ subpopulation. Cell proliferation was determined after transfection with the GLI-Lux plasmid using the WST-1 reagent and expressed by optical density per 100 μg protein. Fugene-6 was included as a transfection reagent control.
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