Cancer Res Treat.  2009 Sep;41(3):164-170.

TNF-alpha Downregulates E-cadherin and Sensitizes Response to gamma-irradiation in Caco-2 Cells

• Yi JY
• Jung YJ
• Choi SS
• Chung E

Abstract

PURPOSE
The purpose of the present study was to assess the biological effects of TNF-alpha in Caco-2 well-differentiated colon adenocarcinoma cells and to determine radiation sensitivity in order to develop TNF-alpha into a cancer therapeutic agent. MATERIALS AND METHODS: A cell viability test was conducted via a colorimetric and colony forming assay after 1 day and 3 days of incubation with TNF-alpha. Western blotting analysis and immunofluorescence staining were conducted to explore TNF-alpha-induced morphological and molecular changes in the adhesion molecules, E-cadherin and claudin-4. The effects of gamma-irradiation at a dose of 2 Gy on cell survival were evaluated by a clonogenic assay. The molecular changes in apoptosis-regulatory proteins were assessed by Western blotting. RESULTS: Caco-2 cells were highly resistant to TNF alpha-induced cell death and 2 Gy of gamma-irradiation. However, we observed the downregulation of the adherens junctional protein, E-cadherin and translocation of tight junctional protein, claudin-4 from the membrane to the cytosol induced by TNF-alpha treatment which would indicate cell-cell junction disruptions. These alterations of junctional proteins influenced the regulation of cell death in response to 2 Gy of gamma-irradiation. The combined treatment of TNF-alpha with 2 Gy of gamma-irradiation reduced the survival of Caco-2 cells by down-regulating bcl-xl and activating JNK pathways. CONCLUSION: These results suggest that TNF-alpha might be potentially applied as a therapeutic agent in order to enhance sensitivity to 2 Gy of gamma-irradiation administered in radiotherapy for the treatment of human colon cancer.

Keyword

TNF-alpha; E-cadherin; Claudin-4; Radio-sensitivity; Bcl-xl; Caco-2 cells

MeSH Terms

Adenocarcinoma
Blotting, Western
Caco-2 Cells
Cadherins
Cell Death
Cell Survival
Claudin-4
Colon
Colonic Neoplasms
Cytosol
Down-Regulation
Fluorescent Antibody Technique
Humans
MAP Kinase Signaling System
Membranes
Proteins
Radiation Tolerance
Tumor Necrosis Factor-alpha
Cadherins
Claudin-4
Proteins
Tumor Necrosis Factor-alpha

Figure

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  Fig. 1 Caco-2 cells are resistant to cell death induced by TNF-alpha treatment. (A) Cell viability was not altered by TNF-alpha treatment. Cytotoxicity assays were conducted on TNF-alpha-treated Caco-2 cells at 10 and 100 ng/mL concentrations. Caco-2 cells were seeded at 5×103 cells in 96-well plates and permitted to attach to plates for 2 days, after which TNF-alpha was added. After 3 days of incubation, 10 µL of color reagent was added to each well, and the cells were incubated for 1 h. The mean absorbance at 450 nm in each set of samples was measured. Shown are the mean percentages of cell survival ± S.E. of three independent experiments. (B) Downregulation of E-cadherin was noted in the TNF-alpha (10 ng/mL)-treated Caco-2 cells, but not claudin-4. E-cadherin, and claudin-4 expression levels were assessed via Western blot. Total cell lysates were prepared. (C) Downregulation of E-cadherin and altered claudin-4 translocation were noted in the TNF-alpha (10 ng/mL)-treated Caco-2 cells. Immunofluorescence staining for E-cadherin and claudin-4 was conducted on Caco-2 cells cultured on cover slips. The cells were fixed in 4% paraformaldehyde, permeabilized for 5 min with 0.1% Triton X-100, and antibodies were added at a dilution recommended by the manufacturer. Laser scanning confocal microscopy was conducted with a MRC-1024/ES confocal microscope.

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  Fig. 2 Reduced cell survival of Caco-2 via combined treatment with TNF-alpha and 2 Gy of γ-irradiation. (A) Clonogenic assays were conducted to determine cell survival under the following experimental conditions: cells were treated separately with TNF-alpha (10 ng/mL) and γ-irradiation (2 Gy) or cells were pre-treated with TNF-alpha for 16 hr and then exposed to 2 Gy of γ-irradiation (combined treatment). After 10 days of incubation, the colonies were stained with crystal violet and the number of colonies was counted. The percent of cell survival was obtained by counting the surviving colonies. Shown are the mean percentages of cell survival±S.E. of three independent experiments. (B) The surviving fractions of TNF-alpha treatment for control were obtained to represent relative changes of surviving fractions under various doses of γ-irradiation conditions, 2 Gy, 4 Gy, and 8 Gy following TNF-alpha treatment. Only 2 Gy of γ-irradiation exposure to pre-treated TNF-alpha (10 ng/mL) cells exhibited reduction of cell survival. Shown are the surviving fraction of TNF-alpha treatment divided by the control surviving fraction, indicated below as a table, the mean percentages of cell survival±S.E. of three independent experiments.

• 

  Fig. 3 TNF-alpha activates pro-apoptotic signals and JNKs. (A) The synergistic alterations of apoptotic signaling molecules were noted in the combined treatment of TNF-alpha with 2 Gy of γ-irradiation. Increased cleavage of PARP and downregulation of bcl-xl were assessed in cells treated with TNF-alpha in combination with 2 Gy of γ-irradiation. Total cell lysates were prepared and α-tubulin was utilized as a loading control. (B) TNF-alpha induces JNK activation in response to 2 Gy of γ-irradiation. The phosphorylation of JNK-1, 2, and 3 was detected after the combined treatment of TNF-alpha and 2 Gy of γ-irradiation in Caco-2 cells. The cells were pre-treated with TNF-alpha (10 ng/mL) for 16 hr prior to γ-irradiation and harvested after 6 hrs of exposure. JNK-1, 2, and 3 phosphorylation were only observed in the combined treatment of TNF-alpha and 2 Gy of γ-irradiation. Total cell lysates were prepared and α-tubulin was utilized as the loading control.

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