Cytotoxicity, Toxicity, and Anticancer Activity of Zingiber Officinale Roscoe Against Cholangiocarcinoma

  • Published : 2012.09.30


Cholangiocarcinoma (CCA) is an uncommon adenocarcinoma which arises from the epithelial cells of the bile ducts. The aim of the study was to investigate the cytotoxicity, toxicity, and anticancer activity of a crude ethanolic extract of ginger (Zingiber officinale Roscoe) against CCA. Cytotoxic activity against a CCA cell line (CL-6) was assessed by calcein-AM and Hoechst 33342 assays and anti-oxidant activity was evaluated using the DPPH assay. Investigation of apoptotic activity was performed by DNA fragmentation assay and induction of genes that may be involved in the resistance of CCA to anticancer drugs (MDR1, MRP1, MRP2, and MRP3) was examined by real-time PCR. To investigate anti-CCA activity in vivo, a total of 80 OV and nitrosamine (OV/DMN)-induced CCA hamsters were fed with the ginger extract at doses of 1000, 3000, and 5000 mg/kg body weight daily or every alternate day for 30 days. Control groups consisting of 10 hamsters for each group were fed with 5-fluorouracil (positive control) or distilled water (untreated control). Median $IC_{50}$ (concentration that inhibits cell growth by 50%) values for cytotoxicity and anti-oxidant activities of the crude ethanolic extract of ginger were 10.95, 53.15, and $27.86{\mu}g/ml$, respectively. More than ten DNA fragments were visualized and up to 7-9 fold up-regulation of MDR1 and MRP3 genes was observed following exposure to the ethanolic extract of ginger. Acute and subacute toxicity tests indicated absence of any significant toxicity at the maximum dose of 5,000 mg/kg body weight given by intragastric gavage. The survival time and survival rate of the CCA-bearing hamsters were significantly prolonged compared to the control group (median of 54 vs 17 weeks). Results from these in vitro and in vivo studies thus indicate promising anticancer activity of the crude ethanolic extract of ginger against CCA with the absence of any significant toxicity. Moreover, MDR1 and MRP3 may be involved in conferring resistance of CCA to the ginger extract.


  1. Woods D, Attwell A, Ross K, Theron G (2012). Text messages as a learning tool for midwives. South African Medical J, 102, 100-1.
  2. Ames BN, Gold LS, Willett WC (1995). The causes and prevention of cancer. Proc Nat Acad Sci, 92, 5258-65.
  3. Bibby MC (2004). Orthotopic models of cancer for preclinical drug evaluation: advantages and disadvantages. Eur J Cancer, 40, 852-7.
  4. Bode AM, Ma WY, Surh YJ, Dong Z (2001). Inhibition of epidermal growth factor-induced cell transformation and activator protein 1 activation by [6]-gingerol. Cancer Res, 61, 850-3.
  5. Boonmars T, Wu Z, Boonjaruspinyo S, et al (2009). Alterations of gene expression of RB pathway in Opisthorchis viverrini infection-induced cholangiocarcinoma. Parasitol Res, 105, 1273-81.
  6. Cao L, Duchrow M, Windhovel U, et al (1998). Expression of MDR1 mRNA and encoding P-glycoprotein in archival formalin-fixed paraffin-embedded gall bladder cancer tissues. Eur J Cancer, 34, 1612-7.
  7. Chaimuangraj S, Thamavit W, Tsuda H, Moore MA (2003). Experimental investigation of Opisthorchiasis-associated cholangiocarcinoma induction in the Syrian hamster - pointers for control of the human disease. Asian Pac J Cancer Prev, 4, 87-93.
  8. Chauhan DP (2002). Chemotherapeutic potential of curcumin for colorectal cancer. Curr Pharm Res, 8, 1695-706.
  9. Chen CY, Liu TZ, Liu YW, et al (2007). 6-shogaol (alkanone from ginger) induces apoptotic cell death of human hepatoma p53 mutant Mahlavu subline via an oxidative stress-mediated caspase-dependent mechanism. J Agri Food Chem, 55, 948-54.
  10. Choudhury D, Das A, Bhattacharya A, Chakrabarti G (2010). Aqueous extract of ginger shows antiproliferative activity through disruption of microtubule network of cancer cells. Food Chem Toxicol, 48, 2872-80.
  11. Chrubasik S, Pittler MH, Roufogalis BD (2005). Zingiberis rhizoma: a comprehensive review on the ginger effect and efficacy profiles. Phytomed, 12, 684-701.
  12. Courtois A, Payen L, Lagadic D, Guillouzo A, Fardel O (1999). Evidence for a multidrug resistance-associated protein 1 (MRP1)-related transport system in cultured rat liver biliary epithelial cells. Life Sci, 64, 763-74.
  13. Fava G, Marucci L, Glaser S, et al (2005). Gamma-Aminobutyric acid inhibits cholangiocarcinoma growth by cyclic AMPdependent regulation of the protein kinase A/extracellular signal-regulated kinase 1/2 pathway. Cancer Res, 65, 11437-46.
  14. Gigliozzi A, Fraioli F, Sundaram P, et al (2000). Molecular identification and functional characterization of Mdr1a in rat cholangiocytes. Gastroenterol, 119, 1113-22.
  15. Gottesman MM, Fojo T, Bates SE (2002). Multidrug resistance in cancer: role of ATP-dependent transporters. Nat Rev Cancer, 2, 48-58.
  16. Haswell-Elkins MR, Mairiang E, Mairiang P, et al (1994). Cross sectional study of Opisthorchis viverrini infection and cholangiocarcinoma in communities within a high-risk area in northeast Thailand. Int J Cancer, 59, 505-9.
  17. Hejna M, Pruckmayer M, Radrer M (1998). The role of chemotherapy and radiation in the management of biliary cancer: a review of the literature. Eur J Cancer, 34, 977-86.
  18. Hoblinger A, Grunhage F, Sauerbruch T, Lammert F (2009). Association of the c.3972C>T variant of the multidrug resistance-associated protein 2 gene (MRP2/ABCC2) with susceptibility to bile duct cancer. Digest, 80, 36-9.
  19. Hsu YL, Kuo PL, Tzeng TF, et al (2008). Huang-lian-jie-dutang, a traditional Chinese medicine prescription, induces cell-cycle arrest and apoptosis in human liver cancer cells in vitro and in vivo. J Gastroenterol Hepatol, 23, 290-9.
  20. Huang MT, Lysz T, Ferraro T, et al (1991). Inhibitory effects of curcumin on in vitro lipooxygenase and cyclooxygenase activities in mouse epidermis. Cancer Res, 51, 813-9.
  21. Iki K, Tsujiuchi T, Majima T, et al (1998). Increased telomerase activity in intrahepatic cholangiocellular carcinomas induced by N-nitrosobis(2-oxopropyl)amine in hamsters. Cancer Lett, 131, 185-90.
  22. Imray CH, Newbold KM, Davis A, Lavelle-Jones M, Neoptolemos JP (1992). Induction of cholangiocarcinoma in the Golden Syrian hamster using methylazoxymethyl acetate. Eur J Sur Oncol, 18, 373-8.
  23. Jan YY, Yeh TS, Yeh JN, Yang HR, Chen MF (2007). Expression of epidermal growth factor receptor, apomucins, matrix metalloproteinases, and p53 in rat and human cholangiocarcinoma: appraisal of an animal model of cholangiocarcinoma. Ann Sur, 240, 89-94.
  24. Jimeno A, Viqueira BR, ONE AUTHOR, et al (2005). Epidermal growth factor receptor dynamics influences response to epidermal growth factor receptor targeted agents. Cancer Res, 65, 3003-10.
  25. Johnson JI, Decker S, Zaharevitz D, et al (2001). Relationships between drug activity in NCI preclinical in vitro and in vivo models and early clinical trials. Br J Cancer, 84, 1424-31.
  26. Kim EC, Min JK, Kim TY, et al (2005a). [6]-Gingerol, a pungent ingredient of ginger, inhibits angiogenesis in vitro and in vivo. Biochem Biophys Res Com, 335, 300-8.
  27. Kim SO, Kundu JK, Shin YK, et al (2005b). [6]-Gingerol inhibits COX-2 expression by blocking the activation of p38 MAP kinase and NF-kappaB in phorbol ester-stimulated mouse skin. Oncogene, 224, 2558-67.
  28. Kiuchi F, Shibuya M, Sankawa U (1982) Inhibitors of prostaglandin biosynthesis from ginger. Chem Pharm Bull (Tokyo), 30, 754-7.
  29. Kiuchi F, Iwakami S, Shibuya M, Hanaoka F, Sankawa U (1992). Inhibition of prostaglandin and leukotriene biosynthesis by gingerols and diarylheptanoids. Chem Pharm Bull, 40, 387-91.
  30. Lai GH, Zhang Z, Shen XN, et al (2005). ErbB-2/neu transformed rat cholangiocytes recapitulate key cellular and molecular features of human bile duct cancer. Gastroenterol, 129, 2047-57.
  31. Larkin A, O'Driscoll L, Kennedy S, et al (2004). Investigation of MRP-1 protein and MDR-1 P-glycoprotein expression in invasive breast cancer: a prognostic study. Int J Cancer, 112, 286-94.
  32. Lee CH (2004). Reversing agents for ATP-binding cassette (ABC) transporters: application in modulating multidrug resistance (MDR). Curr Med Chem Anticancer Agents, 4, 43-52.
  33. Lee E, Park KK, Lee JM, et al (1998). Suppression of mouse skin tumor promotion and induction of apoptosis in HL-60 cells by Alpinia oxyphylla Miquel (Zingiberaceae). Carcinogen, 19, 1377-81.
  34. Lee HS, Seo EY, Kang NE, Kim WK (2008). [6]-Gingerol inhibits metastasis of MDA-MB-231 human breast cancer cells. J Nutr Biochem, 19, 313-9.
  35. Ling H, Yang H, Tan SH, Chui WK, Chew EH (2010). 6-Shogaol, an active constituent of ginger, inhibits breast cancer cell invasion by reducing matrix metalloproteinase-9 expression via blockade of nuclear factor-$\kappa B$ activation. Br J Pharm, 161, 1763-77.
  36. Liu ZH, He YP, Zhou Y, Zhang P, Qin H (2010). Establishment and identification of the human multi-drugresistant cholangiocarcinoma cell line QBC939/ADM. Mol Biol Rep, 5, 121-30.
  37. Lonning PE (2003). Study of suboptimum treatment response: lessons from breast cancer. Lancet Oncol, 4, 177-85.
  38. Mahavorasirikul W, Viyanant V, Chaijaroenkul W, Itharat A, Na- Bangchang K (2010). Cytotoxic activity of Thai medicinal plants against human cholangiocarcinoma, laryngeal and hepatocarcinoma cells in vitro. BMC Com Altern Med, 10, 55-62.
  39. Maronpot RR, Giles HD, Dykes DJ, Irwin RD (1991). Furaninduced hepatic cholangiocarcinomas in Fischer 344 rats. Toxicol Pathol, 19, 561-70.
  40. Mosconia S, Berettab GD, Labiancaa R, et al (2009). Cholangiocarcinoma. Crit Rev Oncol Hematol, 69, 259-70.
  41. Moungjaroen J, Nimmannit U, Callery PS, et al (2006). Reactive oxygen species mediate caspase activation and apoptosis induced by lipoic acid in human lung epithelial cancer cells through Bcl-2 down-regulation. J Pharmacol Exp Ther, 319, 1062-9.
  42. Naus PJ, Henson R, Bleeker G, et al (2007). Tannic acid synergizes the cytotoxicity of chemotherapeutic drugs in human cholangiocarcinoma by modulating drug efflux pathways. J Hepatol, 46, 222-9.
  43. Nita ME, Tominaga O, Nagawa H, Tsuruo T, Muto T (1998). Dihydropyrimidine dehydrogenase but not thymidylate synthase expression is associated with resistance to 5- fluorouracil in colorectal cancer. Hepatogastroentero1, 45, 2117-22.
  44. OECD (2004.) Acute oral toxicity, guideline 420, The OECD Guideline for Testing of Chemical, 1-14.
  45. Park KK, Chun KS, Lee JM, Lee SS, Surh YJ (1998.) Inhibitory effects of [6]-gingerol, a major pungent principle of ginger, on phorbol ester-induced inflammation, epider.mal ornithine decarboxylase activity and skin tumor promotion in ICR mice. Cancer Lett, 129, 139-44.
  46. Pereira MM, Haniadka R, Chacko PP, Palatty PL, Baliga MS (2011). Zingiber officinale Roscoe (ginger) as an adjuvant in cancer treatment: a review. J BUON, 16(3), 414-424.
  47. Phuphathanaphong, L (1979). Thai medicinal plants. 2nd ed., Department of Forestry, Bangkok.
  48. Rost D, Konig J, Weiss G, et al (2001). Expression and localization of the multidrug resistance proteins MRP2 and MRP3 in human gallbladder epithelia. Gastroenterol, 121, 1203-8.
  49. Sausville EA, Burger AM (2006). Contributions of human tumor xenografts to anticancer drug development. Caner Res, 66, 3351-4.
  50. Schoonen WG, de Roos JA, Westerink WM, Débiton E (2005). Cytotoxic effects of 110 reference compounds on HepG2 cells and for 60 compounds on HeLa, ECC-1 and CHO cells. II Mechanistic assays on NAD(P)H, ATP and DNA contents. Toxicol in Vitro, 19, 491-503.
  51. Shukla Y, Singh M (2007). Cancer preventive properties of ginger: a brief review. Food Chem Toxicol, 45, 683-90.
  52. Sirica AE, Zhang Z, Lai GH, et al (2008). A novel "patient-like" model of cholangiocarcinoma progression based on bile duct inoculation of tumorigenic rat cholangiocyte cell lines. Hepatol, 47, 1178-90.
  53. Somchai P, Yusuke H, Ning M, Puangrat Y, Reiji S (2004). Mechanism of NO-mediated oxidative and nitrative DNA damage in hamsters infected with Opisthorchis viverrini: a model of inflammation-mediated carcinogenesis. Nitric Oxide, 11, 175-83.
  54. Sriamporn S, Pisani P, Pipitgool V, Suwanrungruang K (2004). Prevalence of Opisthorchis viverrini infection and incidence of cholangiocarcinoma in Khon Kaen, Northeast Thailand. Trop Med Int Hlth, 9, 588-94.
  55. Stavrovskaya AA (2000). Cellular mechanisms of multidrug resistance of tumor cells. Biochem (Mosc), 65, 95-106.
  56. Surh TJ (2003). Cancer chemoprevention with dietary phytochemicals. Nat Rev Cancer, 3, 768-80.
  57. Surh YJ, Park KK, Chun KS, et al (1999). Anti-tumor-promoting activities of selected pungent phenolic substances present in ginger. J Environ Pathol Toxicol Oncol, 18, 131-9.
  58. Surh YJ, Lee JY, Choi KJ, Ko SR (2002). Effects of selected ginsenosides on phorbol ester-induced expression of cyclooxygenase-2 and activation of NF-kappaB and ERK1/2 in mouse skin. Ann New York Acad Sci, 973, 396-401.
  59. Suzuki F, Kobayashi M, Komatsu Y, Kato A, Pollard RB (1997). Keishi-ka-kei-to, a traditional Chinese herbal medicine, inhibits pulmonary metastasis of B16 melanoma. Anticancer Res, 17, 873-8.
  60. Szabo MR, Iditoiu C, Chambre D, Lupea AX (2007). Improved DPPH determination for antioxidant activity spectrophotometric assay. Chem, 61, 214-6.
  61. Tengchaisri T, Chawengkirttikul R, Rachaphaew N, et al (1998). Antitumor activity of TPL against cholangiocarcinoma growth in vitro and in hamsters. Cancer Lett, 133, 169-75.
  62. Tepsiri N, Chaturat L, Sripa B, et al (2005). Drug sensitivity and drug resistance profiles of human intrahepatic cholangiocarcinoma cell lines. World J Gastroenterol, 11, 2748-53.
  63. Tesana S, Takahashi Y, Sithithaworn P, et al (2007). Ultrastructural and immunohistochemical analysis of cholangiocarcinoma in immunized Syrian golden hamsters infected with Opisthorchis viverrini and administered with dimethylnitrosamine. Parasitol Int, 49, 239-51.
  64. Tew KD (1994). Glutathione-associated enzymes in anticancer drug resistance. Cancer Res, 54, 4313-20.
  65. Thamavit N, Bhamarapravati S, Sahaphong S, Vajrasthira T, Angsubhakorn S (1978). Effects of dimethylnitrosamineon induction of cholangiocarcinoma in Opisthorchis viverrini infected Syrian golden hamsters. Cancer Res, 38, 4634-9.
  66. Thamavit W, Pairojkul C, Tiwawech D, et al (1993). Promotion of cholangiocarcinogenesis in the hamster liver by bile duct ligation after dimethylnitrosamine initiation. Carcinogen, 14, 2415-7.
  67. Vapiwala N, Mick R, Hampshire MK, Metz JM, DeNittis AS (2006). Patient initiation of complementary and alternative medical therapies (CAM) following cancer diagnosis. Cancer J, 12, 467-74.
  68. Voskoglou-Nomikos T, Pater JL, Seymour L (2003). Clinical predictive value of the in vitro cell line, human xenograft, and mouse allograft preclinical cancer models. Clinical Cancer Res, 9, 4227-39.
  69. Wang H, Nair MG, Strasburg GM, et al (1999). Antioxidant and antiinflammatory activities of anthocyanins and their aglycon, cyanidin, from tart cherries. J Nat Prod, 62, 294-6.
  70. Weng CH, Wu CF, Huang HW, Ho CT (2010). Anti-invasion effects of 6-shogaol and 6 gingerol, two active components in ginger, on human hepatocarcinoma cells. Molecular Nutr Food Res, 54, 1618-27.
  71. Xu X, Kobayashi S, Qiao W, et al (2006). Induction of intrahepatic cholangiocellular carcinoma by liver-specific disruption of Smad4 and Pten in mice. J Clin Invest, 116, 1843-52.
  72. Young LC, Campling BG, Voskoglou-Nomikos T, et al (1999). Expression of multidrug resistance protein-related genes in lung cancer: correlation with drug response. Clin Cancer Res, 5, 673-80.

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