Identification of CEA-interacting proteins in colon cancer cells and their changes in expression after irradiation

Abstract

Purpose: The serum carcinoembryonic antigen (CEA) level has been recognized as a prognostic factor in colorectal cancer, and associated with response of rectal cancer to radiotherapy. This study aimed to identify CEA-interacting proteins in colon cancer cells and observe post-irradiation changes in their expression.

Materials and methods: CEA expression in colon cancer cells was examined by Western blot analysis. Using an anti-CEA antibody or IgG as a negative control, immunoprecipitation was performed in colon cancer cell lysates. CEA and IgG immunoprecipitates were used for liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis. Proteins identified in the CEA immunoprecipitates but not in the IgG immunoprecipitates were selected as CEA-interacting proteins. After radiation treatment, changes in expression of CEA-interacting proteins were monitored by Western blot analysis.

Results: CEA expression was higher in SNU-81 cells compared with LoVo cells. The membrane localization of CEA limited the immunoprecipitation results and thus the number of CEA-interacting proteins identified. Only the Ras-related protein Rab-6B and lysozyme C were identified as CEA-interacting proteins in LoVo and SNU-81 cells, respectively. Lysozyme C was detected only in SNU-81, and CEA expression was differently regulated in two cell lines; it was down-regulated in LoVo but up-regulated in SNU-81 in radiation dosage-dependent manner.

Conclusion: CEA-mediated radiation response appears to vary, depending on the characteristics of individual cancer cells. The lysozyme C and Rab subfamily proteins may play a role in the link between CEA and tumor response to radiation, although further studies are needed to clarify functional roles of the identified proteins.

Keywords: Carcinoembryonic antigen; Colorectal neoplasms; Lysozyme C; Rab-6B; Radiation.

Fig. 1.

Carcinoembryonic antigen (CEA) expression in human colon cancer cell lines (LoVo and SNU-81). Western blot analysis of CEA was performed in LoVo and SNU-81 cells. CEA expression normalized with β-actin (CEA/β-actin) was higher in SNU-81.

Fig. 2.

SDS-PAGE images of IgG and anti-CEA antibody immunoprecipitates. Lane 1, size marker; lane 2, LoVo whole cell lysates; lane 3, IgG and LoVo whole cell lysate immunoprecipitates; lane 4, anti-CEA antibody and LoVo whole cell lysate immunoprecipitates; lane 5, SNU-81 whole cell lysates; lane 6, IgG and SNU-81 whole cell lysate immunoprecipitates; lane 7, antiCEA antibody and SNU-81 whole cell lysate immunoprecipitates. The stained gel was sliced and numbered as shown in this figure for liquid chromatography-tandem mass spectrometry (LC-MS/ MS) analysis. SDS-PAGE, sodium dodecyl sulfate–polyacrylamide gel electrophoresis; CEA, carcinoembryonic antigen.

Fig. 3.

Expression of CEA and CEA-interacting proteins, lysozyme C and Rab-6B, in LoVo and SNU-81 cells after irradiation. Western blot analysis of CEA, lysozyme C and Rab-6B was performed after 2, 4 and 6 Gy irradiation. The expression of CEA and lysozyme C was much higher in SNU-81 cells. CEA expression was down-regulated in LoVo but it was increased in SNU-81 in radiation dosage-dependent manner. Commercially available antibody failed to detect Rab-6B in Western blot analysis, but presence of Rab-6B and interaction between CEA and Rab-6B was confirmed in CEA IP of LoVo whole cell lysate (Supplementary Fig. S2). CEA, carcinoembryonic antigen.

Fig. 4.

Differential radiation responses between SNU-81 and LoVo cells. The proliferation rate and radiation response of the two cell lines differed. The proliferation rate of LoVo cells was higher than that of SNU-81 cells but was more suppressed after irradiation.