Elsevier

Food and Chemical Toxicology

Volume 48, Issues 8–9, August–September 2010, Pages 2265-2272
Food and Chemical Toxicology

Redox modulation and human bile duct cancer inhibition by curcumin

https://doi.org/10.1016/j.fct.2010.05.059Get rights and content

Abstract

Curcumin, a major component from tumeric and well-known dietary spice, possesses various pharmacological effects. The cancer chemoprevention effect is suggested to act through its pro-oxidant property. The study was to clarify effects of curcumin on cholangiocarcinoma cells, a cancer of the bile duct that refractory to chemotherapeutic drugs. We examined time-course of oxidant formation in relation to antitumor and the adaptive antioxidant response of the cells. Curcumin induced antiproliferation and apoptosis in KKU-M214 CCA cells with concentration- and time- dependent manners. The antiproliferative effect of curcumin was observed at concentrations as low as 3 μM and was not necessarily associated with oxidative stress, while induction of apoptosis required significant production of superoxide anion, suppression of cellular redox and collapse of mitochondrial transmembrane potential. Western blot analysis showed a temporal relationship between the suppression of nuclear NF-κB with Bcl-XL protein levels. Up-regulation of p53 and Bax was associated with marked oxidative stress and apoptosis. Curcumin also induced Nrf2 protein expression with up-regulation of γ-glutamylcysteine ligase mRNA and increased cellular antioxidant, glutathione. The study suggests that curcumin could be developed into an effective chemoprevention against CCA.

Introduction

Epidemiological and animal studies have shown that phytochemicals present in several diet may be effective for the prevention and treatment of malignancy. Cancer chemoprevention and antitumor activity of various phytochemicals, including curcumin, tea catechin or isothiocyanates may be associated with anti-inflammatory and cytoprotective effects in normal cells, and induction of cell cycle arrest and apoptotic cell death in transformed cells (Nair et al., 2007, Surh, 2003). In particular, curcumin, a product of spice turmeric, Curcuma longa Linn., has significant antioxidant, anti-inflammatory, cytoprotective, and antitumor activities in vitro and in some preclinical animal models (Barve et al., 2008, Sandur et al., 2007, Sharma et al., 2005, Sompamit et al., 2009). The safety of curcumin is well recognized by the fact that several populations in the world consume curcumin as a dietary spice for centuries (Aggarwal and Sung, 2009, Sharma et al., 2005). Curcumin has been shown to inhibit the proliferation of a wide variety of tumor cells, including non-small cell lung carcinoma cells (Shishodia et al., 2003), mammary epithelial carcinoma cells (Choudhuri et al., 2005), colon adenocarcinoma cells (Rashmi et al., 2005), and pancreatic carcinoma cells (Lee et al., 2005). The antitumor effects have been suggested to depend partly on the suppression of the nuclear factor-kappa B (NF-κB) signaling pathway (Aggarwal and Sung, 2009, Surh, 2003), whereas its downstream genes are ultimately involved with several critical processes leading to carcinogenesis, including proinflammation, proliferation, angiogenesis, antiapoptosis and drug resistance (Aggarwal and Sung, 2009). The target genes down-regulated by curcumin include cyclooxygenase-2, cyclin-D1, c-myc, IAP, Bcl-2, Bcl-XL, VEGF, MMP-9, and ICAM-1. These down-regulated NF-κB gene products of curcumin and other plant polyphenols may lead to the chemosensitizing and radiosensitizing effects (Garg et al., 2005). Curcumin can induce apoptosis through modulation of the mitochondrial permeability transition causing the release of AIF, cytochrome C and finally caspase 3 activation (Thayyullathil et al., 2008). The tumor suppressor p53 protein plays an important role in the induction of cell cycle arrest and apoptosis by transactivation-dependent and -independent effects (Vaseva and Moll, 2009). On the other hand, oxidative stress also activates the nuclear factor-E2-related factor 2 (Nrf2), then binds with the cis-acting antioxidant response element (ARE) and modulates downstream antioxidant genes, such as γ-glutamylcysteine ligase (GCL), glutathione transferases, and heme oxygenase-1 (Kensler et al., 2007, Lu, 2009). This adaptive antioxidant response may be implicated in cancer resistance (Chen et al., 2008, Wang et al., 2008). Previous study showed that curcumin induced increased cellular GSH levels (Sandur et al., 2007) and this may be related to antioxidative stress response.

How curcumin mediated anti-inflammatory and proapoptotic effects is the subject of intense research. Curcumin has been suggested to act through its pro-oxidant/ antioxidant effects because formation of reactive oxygen species by curcumin and curcumin derivatives correlates with their apoptotic activity on tumor cells (Mishra et al., 2005, Syng-Ai et al., 2004). The induction of reactive oxygen species leads to change in cellular GSH, the main redox buffer, which may, in turn, modulates several redox-sensitive enzymes and transcription factors leading to growth suppression, apoptotic cell death (Mishra et al., 2005, Syng-Ai et al., 2004). The antiproliferation and induction of apoptosis are usually supposed to be resulted from a similar insult, i.e. oxidative stress. This may not be always accurate. The roles of ROS and redox status in cancer inhibition are needed clarification. Moreover, several of curcumin’s effects may depend on cell type; for instance, curcumin has high efficacy in cells with high constitutive NF-κB activity and low expression of Bcl-XL (Aggarwal and Sung, 2009, Jang and Surh, 2004).

Cholangiocarcinoma (CCA), a malignant tumor of the biliary tract, presents a formidable difficulty in diagnosis and treatment (Blechacz and Gores, 2008, Khan et al., 2002). The worldwide incidence and mortality rates of intrahepatic CCA are increasing (Khan et al., 2002). Current adjuvant or palliative chemotherapy has not been shown to substantially improve survival in patients (Blechacz and Gores, 2008, Khan et al., 2005, Lazaridis and Gores, 2005). There is a need to improve chemotherapy to circumvent drug resistance in CCA because it is evidently involved in the expression of multi-drug resistance genes as well as the up-regulation of antiapoptotic genes and the down-regulation of proapoptotic genes (Blechacz and Gores, 2008).

In this study, the effects of curcumin on the induction of antiproliferation, apoptosis and the adaptive antioxidant response in CCA cells were analyzed on time-course association with pro-oxidant activity and modulation of redox-sensitive transcription factors and enzymes to establish characteristics of curcumin induced tumor inhibition.

Section snippets

Cell culture and cytotoxicity assay

Cultured cell line, KKU-M214, established in our institute by Dr. B. Sripa of Department of Pathology, Faculty of Medicine, was derived from human intrahepatic cholangiocarcinoma tissues. The CCA cells were routinely cultured in Ham’s F12 media supplemented with 4 mM L-glutamine, 1 mM sodium pyruvate, 12.5 mM N-2-hydroxyethylpiperazine-N′-2-ethanesulfonic acid (HEPES; pH 7.3), 100 U/mL penicillin, 100 μg/mL streptomycin sulfate, and 10% fetal calf serum as previously described (Buranrat et al., 2007

Cytotoxicity of curcumin in CCA cells

KKU-M214 cells were tested against curcumin at varied concentrations. The IC50 values for cytotoxicity of curcumin was 5.9 ± 2.6 μM. The antiproliferative effect of curcumin is shown in Fig. 1A. At concentrations as low as 3 μM curcumin, the inhibition of cell proliferation was apparent at 9 h; at higher concentrations, marked growth suppression was observed as early as 3 h (Fig. 1A). An induction of apoptotic cell death was demonstrated at concentrations ⩾10 μM (Fig. 1B), whereas curcumin at the

Discussion

The chemoprevention of cancer may involve the inhibition of proliferation and the induction of apoptosis in malignant or premalignant cells. Although the aim of chemopreventive agents is to target cancer cells, some of the agents’ effects may induce cellular defense mechanisms; for instance, they may induce antioxidant and xenobiotic detoxifying enzymes or inhibit xenobiotic activating enzymes in normal cells (Nair et al., 2007, Surh, 2003). Our study showed that the induction of cell death by

Conflict of Interest

The authors declare that there are no conflicts of interest.

Acknowledgements

This work was supported by the Thailand Research Fund (TRF), National Science and Technology Development Agency (NSTDA), research funding from Khon Kaen University. Sarinya Kongpetch was supported by the Royal Golden Jubilee Ph.D. program. Benjaporn Buranrat was supported by the Office of the Commission on Higher Education.

References (35)

  • K. Sompamit et al.

    Curcumin improves vascular function and alleviates oxidative stress in non-lethal lipopolysaccharide-induced endotoxaemia in mice

    Eur. J. Pharmacol.

    (2009)
  • F. Thayyullathil et al.

    Rapid reactive oxygen species (ROS) generation induced by curcumin leads to caspase-dependent and -independent apoptosis in L929 cells

    Free Radic. Biol. Med.

    (2008)
  • F. Tietze

    Enzymic method for quantitative determination of nanogram amounts of total and oxidized glutathione: applications to mammalian blood and other tissues

    Anal. Biochem.

    (1969)
  • Y. Tsujimoto et al.

    Mitochondrial membrane permeability transition and cell death

    Biochim. Biophys. Acta

    (2006)
  • A.V. Vaseva et al.

    The mitochondrial p53 pathway

    Biochim. Biophys. Acta

    (2009)
  • A. Barve et al.

    Murine prostate cancer inhibition by dietary phytochemicals–curcumin and phenyethylisothiocyanate

    Pharm. Res.

    (2008)
  • B. Blechacz et al.

    Cholangiocarcinoma: advances in pathogenesis, diagnosis, and treatment

    Hepatology

    (2008)
  • Cited by (0)

    1

    Present address: Faculty of Science and Technology, Loei Rajabhat University, Loei, Thailand.

    2

    Present address: School of Pharmacy, Naresuan University Phayao, Phayao, Thailand.

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