Cancer Res Treat.  2008 Jun;40(2):53-61.

Cytoplasmic Trapping of CXCR4 in Hepatocellular Carcinoma Cell Lines

• Kim SW
• Kim HY
• Song IC
• Jin SA
• Lee HJ
• Yun HJ
• Kim S
• Jo DY

Abstract

PURPOSE: The chemokine receptor CXCR4 plays a role in the metastasis and progression of a broad range of malignant tumors; however, its influence on hepatocellular carcinoma (HCC) is not well defined. Thus, we analyzed the expression of CXCR4 and its functions in HCC cell lines in vitro.
MATERIALS AND METHODS
Five HCC cell lines (HepG2, Hep3B, SK-HEP-1, NCI-H630 and PLC/PRF5) were investigated. The CXCR4 expression was analyzed by RT-PCR, Western blotting, flow cytometry and immunofluorescence staining. In addition, the effects of stromal cell-derived factor-1 (SDF-1) on the migration, proliferation and survival of the cells were investigated, as well as the SDF-1-induced phosphorylation of signaling molecules.
RESULTS
All five cell lines had abundant CXCR4 in their cytoplasm, whereas a cell surface CXCR4 expression was only detected in a very small population of PLC/ PRF5 cells. In contrast, SDF-1 bound to all the cells. SDF-1 induced the phosphorylation of AKT and ERK1/2 in the PLC/PRF5 cells and the phosphorylation of Stat3, AKT and ERK1/2 in the Hep3B cells. Nonetheless, SDF-1 did not induce migration or proliferation in any of the cells, nor did it rescue the cells from serum deprivation-induced apoptosis. Recruitment of CXCR4 from the cytoplasm to the cell surface was not elicited by dexamethasone, proinflammatory cytokines or VEGF. Hypoxia increased both the cytoplasmic and cell surface expressions of CXCR4 in only the PLC/PRF5 cells.
CONCLUSIONS
CXCR4 is trapped in the cytoplasm and it is not recruited to the cell surface by standard extrinsic stimuli in the majority of HCC cell lines, and the result of this is a negligible response to SDF-1.

Keyword

Hepatocellular carcinoma; Stromal cell-derived factor-1; CXCR4

MeSH Terms

Anoxia
Apoptosis
Blotting, Western
Carcinoma, Hepatocellular
Cell Line
Cytokines
Cytoplasm
Dexamethasone
Flow Cytometry
Fluorescent Antibody Technique
Neoplasm Metastasis
Phosphorylation
Vascular Endothelial Growth Factor A
Cytokines
Dexamethasone
Vascular Endothelial Growth Factor A

Figure

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  Fig. 1 The CXCR4 expression in the liver cancer cell lines. (A) RT-PCR for CXCR4 and SDF-1 in the liver cancer cells. Bone marrow stromal cells (BMSC) and cells from the myeloma cell line RPMI8226 served as positive controls for SDF-1 and CXCR4, respectively. (B) Western blot analysis of the CXCR4 expression in the liver cancer cells.

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  Fig. 2 Flow cytometric analysis of the CXCR4 expression. (A) The CXCR4 expression was analyzed using antibodies against CXCR4 (12G5). The cell surface expression of CXCR4 was absent or minimal in all of the cells; however, abundant CXCR4 was detected in the cytoplasm of the cells after permeabilization. (B) The cell surface CXCR4 expression in the cells was analyzed using three additional antibodies against CXCR4 (clones 44708, 44716 and 44717). The results were the same as those produced with using 12G5.

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  Fig. 3 Immunofluorescence staining for CXCR4 in the liver cancer cell lines. The cells were incubated with 12G5, reacted with a secondary antibody labeled with FITC and then they were subjected to confocal microscopic examination. Note the abundant cytoplasmic expression of CXCR4 in all of the cells.

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  Fig. 4 Binding of SDF-1 to the liver cancer cell lines. The cells were incubated with biotinylated SDF-1 with (b) or without (a) pretreatment with SDF-1, and then they were reacted with avidin labeled with FITC and at last they were subjected to flow cytometric analysis.

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  Fig. 5 SDF-1-induced phosphorylation of signaling proteins in the liver cancer cell lines. The PLC/PRF5 cells (A) and Hep3B cells (B) were incubated in the presence of 100 ng/ml SDF-1. After the indicated time periods, the cells were subjected to Western blot analysis to evaluate the phosphorylation status of Stat3, AKT and ERK1/2. (C) AMD3100 partially inhibited the SDF-1-induced phosphorylation of AKT in the PLC/PRF5 cells. The cells were incubated with 100 ng/ml SDF-1 and AMD3100 for 10 min.

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  Fig. 6 The cell surface expression of CXCR7 in the liver cancer cells. The cells were incubated with allophycocyanin (APC)-conjugated monoclonal antibody to CXCR7/RDC-1 (clone 11G8) at 4℃ for 30 min and then the cells were analyzed by performing flow cytometry. In parallel, MCF-7 breast cancer cells were analyzed as positive controls.

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  Fig. 7 In vitro effects of SDF-1 on the liver cancer cells. (A) SDF-1 does not induce cellular transmigration. The cells were loaded into the upper chamber of a Transwell and they were allowed to migrate to the lower chamber, which contained 100 ng/ml SDF-1, over a 4-h period. The data is given as the mean±S.D. of the migration index from three independent experiments. (B) SDF-1 does not stimulate cellular proliferation. A modified MTT assay, known as a CCK-8 assay, was used. The data is given as the mean±S.D. of the proliferation index from three independent experiments. (C) SDF-1 does not rescue liver cancer cells from serum deprivation-induced apoptosis. The cells were incubated in serum-free RPMI medium in the presence or absence of 100 ng/ml SDF-1 for 24 h and then they were subjected to flow cytometric analysis.

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  Fig. 8 Dexamethasone and the proinflammatory cytokines (A) and VEGF (B) do not modulate the cell surface expression of CXCR4 in the liver cancer cells. The cells were incubated with 10-5 M dexamethasone, 10 ng/ml IFN-γ, 10 ng/ml TNF-α, 10 ng/ml TGF-1 or 50 ng/ml VEGF in serum-free X-VIVO medium for 24 h and then they were subjected to flow cytometric analysis for CXCR4.

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  Fig. 9 Hypoxia up-regulates the CXCR4 expression in the PLC/PRF5 cells. To induce hypoxia, the cells were incubated in serum-free X-VIVO medium at 1% O2 for 16 h. (A) The cell surface expression of CXCR4 in the five liver cancer cell lines that were subjected to normoxia and hypoxia. (B) The cell surface expression of CXCR4 was enhanced in the PLC/PRF5 cells by hypoxic incubation. (a) Isotype control. (b) Normoxia. (c) Hypoxia. (C) The Western blotting showed the induction of the HIF-1α expression by hypoxia in the PLC/PRF5 cells. (D) Flow cytometric analysis after permeabilization revealed that hypoxia enhanced the cytoplasmic expression of CXCR4. (E) Hypoxia induced the transmigration of PLC/PRF5 cells in response to SDF-1. The data is given as the mean±S.D. of the migration index from three independent experiments. *p<0.05.

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