Bcl‐2 is overexpressed and alters the threshold for apoptosis in a cholangiocarcinoma cell line

Cholangiocarcinoma is a malignant neoplasm originating from cholangiocytes. The mechanisms responsible for oncogenesis of cholangiocytes are unknown. Resistance to apoptosis, especially by altered expression of B‐cell lymphoma/leukemia 2 (Bcl‐2) family members, has been implicated as a mechanism contributing to malignant transformation. Thus, our aim was to test the hypothesis that altered expression of Bcl‐2 family members by cholangiocarcinoma cells renders them resistant to apoptosis. We compared the apoptotic threshold and expression of the Bcl‐2 protein family members, Bcl‐2, Bcl‐XL, and Bax, in two human cell lines: 1) nonmalignant human cholangiocytes immortalized by transfection with the simian virus 40 (SV 40) large T antigen; and 2) a malignant human cholangiocarcinoma cell line. Apoptosis was induced pharmacologically using beauvericin. Bcl‐2, Bcl‐x long, and Bax protein expression were evaluated by immunoblot analysis, and Bcl‐2 expression was modulated using antisense technology. The cholangiocyte and malignant/nonmaligant phenotype of both cell lines was verified using both in vitro and in vivo approaches. Beauvericin induced apoptosis of nonmalignant cholangiocytes in a concentration‐ (0 to 25 µmol/L) and time‐ (0 to 6 hours) dependent manner. In contrast, malignant cholangiocytes were resistant to apoptosis. Although expression of Bcl‐x long and Bax protein were similiar in the two cell lines, Bcl‐2 protein expression was 15‐fold greater in malignant than in nonmalignant cholangiocytes. An 18 mer bcl‐2 antisense oligonucleotide reduced expression of Bcl‐2 protein by 50% and increased the rate of beauvericin‐induced apoptosis more than threefold in the malignant cells. Our results support the hypothesis that resistance to apoptosis by overexpression of Bcl‐2 may be a feature of cholangiocarcinoma.