Abstract
Cholangiocarcinoma (CCA), or bile duct cancer, is incurable with a high mortality rate due to a lack of effective early diagnosis and treatment. Identifying cytoplasmic membrane proteins of invasive CCA that facilitate cancer progression would contribute toward the development of novel tumor markers and effective chemotherapy. An invasive CCA cell line (KKU-100) was stimulated using TNF-α and then biotinylated and purified for mass spectrometry analysis. Novel proteins expressed were selected and their mRNAs expression levels were determined by real-time RT-PCR. In addition, the expression of ALCAM was selected for further observation by Western blot analysis, immunofluorescent imaging, and antibody neutralization assay. After comparing the proteomics profile of TNF-α induced invasive with non-treated control cells, over-expression of seven novel proteins was observed in the cytoplasmic membrane of TNF-α stimulated CCA cells. Among these, ALCAM is a novel candidate which showed significant higher mRNA- and protein levels. Immunofluorescent assay also supported that ALCAM was expressed on the cell membrane of the cancer, with increasing intensity associated with TNF-α. This study indicated that ALCAM may be a novel protein candidate expressed on cytoplasmic membranes of invasive CCA cells that could be used as a biomarker for development of diagnosis, prognosis, and drug or antibody-based targeted therapies in the future.
Highlights
Cholangiocarcinoma (CCA) is a malignant tumor of the biliary tract that has a high incidence in Southeast Asia, including Thailand (Srivatanakul et al, 2004; Sripa and Pairojkul, 2008)
Only 7 novel proteins over-expression were observed in the TNF-α treated CCA cell line (ALCAM, decay-accelerating factor (DAF), transmembrane protein 33 (TMM33), progesterone receptor component-2 (PGRC2), PLAK, transmembrane emp24 domain-containing protein 9 (TMED9) and protein disulfide isomerase family A member 3 (PDIA3))
Jiao et al, suggested that up-regulation of activated leukocyte cell adhesion molecule (ALCAM) was associated with severe clinical manifestations including metastasis and castration resistant prostate cancers (CRPC) (Jiao et al, 2012)
Summary
Cholangiocarcinoma (CCA) is a malignant tumor of the biliary tract that has a high incidence in Southeast Asia, including Thailand (Srivatanakul et al, 2004; Sripa and Pairojkul, 2008). Identifying cytoplasmic membrane proteins of invasive CCA that facilitate cancer progression would contribute toward the development of novel tumor markers and effective chemotherapy. The expression of ALCAM was selected for further observation by Western blot analysis, immunofluorescent imaging, and antibody neutralization assay. Results: After comparing the proteomics profile of TNF-α induced invasive with non-treated control cells, over-expression of seven novel proteins was observed in the cytoplasmic membrane of TNF-α stimulated CCA cells. Immunofluorescent assay supported that ALCAM was expressed on the cell membrane of the cancer, with increasing intensity associated with TNF-α. Conclusions: This study indicated that ALCAM may be a novel protein candidate expressed on cytoplasmic membranes of invasive CCA cells that could be used as a biomarker for development of diagnosis, prognosis, and drug or antibody-based targeted therapies in the future
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