Figures & data
Figure 1 Cell proliferation, colony formation and xenograft tumor growth in CCT-NPC or parental cells.
Notes: (A) MTT assays for acute exposure to Cd (1 nM, 1 µM and 1 mM). (B) MTT assays following 1 µM Cd treatment for 10 weeks. (C) Effects of chronic Cd exposure on the colonogenic ability in CNE-1/CNE-2 and CCT-CNE1/CCT-CNE2 cells (N=3). (D) Gross appearance of xenograft tumors at 28 days after CNE-1 or CCT-CNE1 cells injection. (E). Tumor growth curves and weight data in transplanted nude mice with CNE-1 and CCT-CNE1 through four weeks; data are mean (± SD) tumor volume (N=5). Each assay was performed in triplicate. *P<0.05; **P<0.01; ***P<0.001, compared with the parental cells.
Abbreviation: CCT-NPC, chronic cadmium-treated nasopharyngeal carcinoma.
Figure 2 CCT-NPC cells acquired metastasis-associated phenotype.
Notes: (A) The gross view of cell invasion assay stained with Giemza and corresponding quantitative analyses of the results for CCT-NPC cells and the control cells. Magnification 200×. (B) The gross view of cell migration assay stained with hematoxylin-eosin and corresponding quantitative analyses of the results for CCT-NPC cells and the control cells. Magnification 200×. (C) Immuno-fluorescence staining for actin filament in CCT-NPC cells and the parental cells. Actin filament cytoskeleton was stained with rhodamine conjugated phalloidin (red) and nuclei with DAPI (blue). Magnification 400×. (D) Expression of the EMT markers E-cadherin, vimentin and N-cadherin in CCT-NPC and NPC cell lines. *P<0.05; **P<0.01; ***P<0.001, compared with the parental cells. *P<0.05; **P<0.01; ***P<0.001, compared with the parental cells.
Abbreviations: CCT-NPC, chronic cadmium-treated nasopharyngeal carcinoma; EMT, epithelial–mesenchymal transition.
Figure 3 Cd treatment activates Wnt/β-catenin signaling and aberrant methylation of the CK1α promoter in NPC cell lines.
Notes: (A) Western blot analysis of total β-catenin and CK1α in cCd-treated NPC cells and controls; β-actin was used as a loading control. (B) Immunofluorescence staining patterns of β-catenin and CK1α in NPC and CCT-NPC cells. Nuclei were stained with DAPI (magnification 400×). (C) Luciferase reporter assays using TOPflash/FOPflash reporter plasmids to assess the activity of Wnt/β-catenin. (D) RT-PCR analysis of relative transcript levels of the β-catenin target genes cyclin E, cyclin D1, c-Myc and c-Jun. *P<0.05; **P<0.01; ***P<0.001.
Abbreviations: CCT-NPC, chronic cadmium-treated nasopharyngeal carcinoma; CK1α, casein kinase 1α; RT-PCR, reverse transcription-PCR.
Figure 4 Hypermethylation of CK1α induces a switch in Wnt/β-catenin signaling and malignant progression in CCT-NPC cells.
Notes: (A) Methylation status of the CK1α promoter analyzed by MS-PCR. (B) Western blot analyses of CK1α and β-catenin in CCT-CNE1 cells following treatment with 50 µM 5-aza-CdR for 48 hours. (C) RT-PCR analysis of relative transcript levels of β-catenin target genes following treatment of cells with 5-aza-CdR (50 µM). (D) Western blot and RT-PCR analyses of the effect of CK1α depletion and (or) 5-aza-CdR treatment on β-catenin expression and downstream gene transcription in CCT-CNE1 cells. (E) MTT assays for cell viability of CCT-CNE1 and CCT-CNE2 cells with increasing concentrations of 5-aza-CdR treatment. (F) Invasion and migration ability of 5-aza-CdR-treated CCT-CNE1 cells. *P<0.05; **P<0.01; ***P<0.001, compared with parental cells. #P>0.05, compared with CK1α treated cells.
Abbreviations: CCT-NPC, chronic cadmium-treated nasopharyngeal carcinoma; CK1α, casein kinase 1α; MS-PCR, methylation specific-PCR; RT-PCR, reverse transcription-polymerase chain reaction.
Figure 5 Hypothesized model for the induction of Cd on Wnt/β-catenin signaling and malignant progression in NPC cells.
Notes: Chronic low-dose Cd treatment of NPC cells induces CK1α promoter hypermethylation that downregulates CK1α expression, leading to accumulation and nuclear translocation of β-catenin thereby activating Wnt/β-catenin signaling to promote malignancy.
Abbreviations: APC, adenomatous polyposis coli; CK1α, casein kinase α; EMT, epithelial–mesenchymal transition; GSK-3β, glycogen synthase kinase 3β; NPC, nasopharyngeal carcinoma; TCF/LEF, T-cell factor/lymphoid enhancer factor; β-Trcp, β-transducin repeats-containing proteins.
