Applied Biochemistry and Biotechnology

, Volume 166, Issue 4, pp 942–951 | Cite as

Apoptosis Mechanism of Human Cholangiocarcinoma Cells Induced by Bile Extract from Crocodile

  • Jin-He Kang
  • Wen-Qing Zhang
  • Wei Song
  • Dong-Yan Shen
  • Shan-Shan Li
  • Ling Tian
  • Yan Shi
  • Ge Liang
  • You-Xiong Xiong
  • Qing-Xi ChenEmail author


Animal bile is popularly used as a traditional medicine in China, and bile acids are their major bioactive constituents. In the present study, effects of bile extract from crocodile gallbladder on QBC939 cell growth, cell cycle, and apoptosis were investigated by MTT assay, inverted microscopy, fluorescence microscopy, transmission electron microscopy, scanning electron microscopy, PI single- and FITC/PI double-staining flow cytometry, and western blotting. Our data have revealed that bile extract inhibited cells growth significantly, and the cell cycle was arrested in G1 phase. Bile extract induced QBC939 cell apoptosis, which was associated with collapse of the mitochondrial membrane potential and increase of ROS. In bile extract-treated cells, it was observed that the expression of bcl-2 decreased and cytochrome c released to cytosol, but the expression of bax remained unchanged. The data indicated that mitochondrial pathway might play an important role in bile extract-induced apoptosis in QBC939 cells. These results provide significant insight into the anticarcinogenic action of bile extract on cholangiocarcinoma cells.


QBC939 cells Apoptosis Bcl-2 Cytochrome c Crocodile Bile 





Reactive oxygen species


Mitochondrial transmembrane potential


Propidium iodide


Fetal bovine serum


3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide


Horseradish peroxidase


Enhanced chemiluminescence


Transmission electron microscopy


Scanning electron microscope


Rhodamine 123


2,7-Dichlorofluorescein diacetate





The present investigation was supported by Grant 81072014 of the Natural Science Foundation of China, National Foundation for fostering talents of basic science (J1030626) and supported by Sriracha Tiger Zoo Co., Ltd. Sriracha Thailand.


  1. 1.
    Mahmoud, N. N., Dannenberg, A. J., Bilinski, R. T., Mestre, J. R., Chadburn, A., Churchill, M., et al. (1999). Administration of an unconjugated bile acid increases duodenal tumors in a murine model of familial adenomatous polyposis. Carcinogenesis, 20, 299–303. doi: 10.1093/carcin/20.2.299.CrossRefGoogle Scholar
  2. 2.
    Martinez, J. D., Stratagoules, E. D., LaRue, J. M., Powell, A. A., Gause, P. R., Craven, M. T., et al. (1998). Different bile acids exhibit distinct biological effects: The tumor promoter deoxycholic acid induces apoptosis and the chemopreventive agent ursodeoxycholic acid inhibits cell proliferation. Nutrition and Cancer, 31, 111–118. doi: 10.1080/01635589809514689.CrossRefGoogle Scholar
  3. 3.
    Tatsumura, T., Sato, H., Yamamoto, K., & Ueyama, T. (1981). Ursodeoxycholic acid prevents gastrointestinal disorders caused by anticancer drugs. The Japanese Journal of Surgery, 11, 84–89. doi: 10.1007/BF02468874.CrossRefGoogle Scholar
  4. 4.
    Alberts, D. S., Martinez, M. E., Hess, L. M., Einspahr, J. G., Green, S. B., Bhattacharyya, A. K., et al. (2005). Phase III trial of ursodeoxycholic acid to prevent colorectal adenoma recurrence. Journal of the National Cancer Institute, 97, 846–853. doi: 10.1093/jnci/dji144.CrossRefGoogle Scholar
  5. 5.
    Tint, G. S., Dayal, B., Batta, A. K., Shefer, S., Joanen, T., Larry, M., et al. (1980). Biliary bile acids, bile alcohols, and sterols of Alligator mississippiensis. Journal of Lipid Research, 21, 110–117.Google Scholar
  6. 6.
    Yeh, Y. H., Wang, D. Y., Liau, M. Y., Wu, M. L., Deng, J. F., Noguchia, T., et al. (2003). Bile acid composition in snake bile juice and toxicity of snake bile acids to rats. Comparative Biochemistry and Physiology, 136, 277–284. doi: 10.1016/S1532-0458(03)00230-8.Google Scholar
  7. 7.
    Malhi, H., & Gores, G. J. (2006). Cholangiocarcinoma: Modern advances in understanding a deadly old disease. Journal of Hepatology, 45, 856–867. doi: 10.1016/j.jhep.2006.09.001.CrossRefGoogle Scholar
  8. 8.
    Patel, T. (2002). Worldwide trends in mortality from biliary tract malignancies. BMC Cancer, 2, 10. doi: 10.1186/1471-2407-2-10.CrossRefGoogle Scholar
  9. 9.
    Lazaridis, K. N., & Gores, G. J. (2005). Cholangiocarcinoma. Gastroenterology, 128, 1655–1667. doi: 10.1053/j.gastro.2005.03.040.CrossRefGoogle Scholar
  10. 10.
    Gatto, M., Bragazzi, M. C., Semeraro, R., Napoli, C., Gentile, R., Torrice, A., et al. (2010). Cholangiocarcinoma: Update and future perspectives. Digestive and Liver Disease, 42, 253–260. doi: 10.1016/j.dld.2009.12.008.CrossRefGoogle Scholar
  11. 11.
    Hu, Y., Yang, Y., You, Q. D., Liu, W., Gu, H. Y., Zhao, L., et al. (2006). Oroxylin A induced apoptosis of human hepatocellular carcinoma cell line HepG2 was involved in its antitumor activity. Biochemical and Biophysical Research Communications, 351, 521–527. doi: 10.1016/j.bbrc.2006.10.064.CrossRefGoogle Scholar
  12. 12.
    Han, P., Kang, J. H., Li, H. L., Hu, S. X., Lian, H. H., Qiu, P. P., et al. (2009). Antiproliferation and apoptosis induced by tamoxifen in human bile duct carcinoma QBC939 cells via upregulated p53 expression. Biochemical and Biophysical Research Communications, 385, 251–256. doi: 10.1016/j.bbrc.2009.05.059.CrossRefGoogle Scholar
  13. 13.
    Farnebo, M., Bykov, V. J., & Wiman, K. G. (2010). The p53 tumor suppressor: A master regulator of diverse cellular processes and therapeutic target in cancer. Biochemical and Biophysical Research Communications, 396, 85–89. doi: 10.1016/j.bbrc.2010.02.152.CrossRefGoogle Scholar
  14. 14.
    Mork, C. N., Faller, D. V., & Spanjaard, R. A. (2005). A mechanistic approach to anticancer therapy: Targeting the cell cycle with histone deacetylase inhibitors. Current Pharmaceutical Design, 11, 1091–1104. doi: 10.2174/1381612053507567.CrossRefGoogle Scholar
  15. 15.
    Tompson, C. B. (1995). Apoptosis in the pathogenesis and treatment of disease. Science, 267, 1456–1462. doi: 10.1126/science.7878464.CrossRefGoogle Scholar
  16. 16.
    Green, D. R. (1998). Apoptotic pathways: The roads to run. Cell, 94, 695–698.CrossRefGoogle Scholar
  17. 17.
    Green, D. R., & Reed, J. C. (1998). Mitochondria and apoptosis. Science, 281, 1308–1312. doi: 10.1126/science.281.5381.1309.Google Scholar
  18. 18.
    Zong, W. X., Li, C., Hatzivassiliou, G., Lindsten, T., Yu, Q. C., Yuan, J., et al. (2003). Bax and Bak can localize to the endoplasmic reticulum to initiate apoptosis. The Journal of Cell Biology, 162, 59–69. doi: 10.1083/jcb.200302084.CrossRefGoogle Scholar
  19. 19.
    Circu, M. L., & Aw, T. Y. (2010). Reactive oxygen species, cellular redox systems, and apoptosis. Free Radical Biology & Medicine, 48, 749–762. doi: 10.1016/j.freeradbiomed.2009.12.022.CrossRefGoogle Scholar
  20. 20.
    Sakon, S., Xue, X., Takekawa, M., Sasazuki, T., Okazaki, T., Kojima, Y., et al. (2003). NF-kappaB inhibits TNF-induced accumulation of ROS that mediate prolonged MAPK activation and necrotic cell death. The EMBO Journal, 22, 3898–3909. doi: 10.1093/emboj/cdg379.CrossRefGoogle Scholar
  21. 21.
    Chen, Y., & Gibson, S. B. (2008). Is mitochondrial generation of reactive oxygen species a trigger for autophagy? Autophagy, 16, 246–248.Google Scholar

Copyright information

© Springer Science+Business Media, LLC 2011

Authors and Affiliations

  • Jin-He Kang
    • 1
  • Wen-Qing Zhang
    • 1
  • Wei Song
    • 1
  • Dong-Yan Shen
    • 1
  • Shan-Shan Li
    • 1
  • Ling Tian
    • 1
  • Yan Shi
    • 1
  • Ge Liang
    • 1
  • You-Xiong Xiong
    • 2
  • Qing-Xi Chen
    • 1
    Email author
  1. 1.Key Laboratory of the Ministry of Education for Coastal and Wetland Ecosystems, School of Life Sciences, College of Environment and EcologyXiamen UniversityXiamenChina
  2. 2.Sriracha Tiger Zoo Co., Ltd.SrirachaThailand

Personalised recommendations