Elsevier

Free Radical Biology and Medicine

Volume 45, Issue 10, 15 November 2008, Pages 1403-1412
Free Radical Biology and Medicine

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

https://doi.org/10.1016/j.freeradbiomed.2008.08.014Get rights and content

Abstract

Evidence that curcumin may have anticancer activities has renewed interest in its potential to prevent and treat disease. In this study, we show that curcumin-mediated rapid generation of reactive oxygen species (ROS) leads to apoptosis by modulating different apoptotic pathways in mouse fibroblast L929 cells. We show for the first time that curcumin-induced rapid ROS generation causes the release of apoptosis inducing factor (AIF) from the mitochondria to the cytosol and nucleus, hence, leading to caspase 3-independent apoptosis. However, our studies also show that curcumin induces the release of cytochrome c from mitochondria, causing activation of caspase 3, and concomitant PARP cleavage, which is the hallmark of caspase-dependent apoptosis. Furthermore, curcumin-induced ROS generation leads to the induction of the proapoptotic protein p53 and its effector protein p21 and down-regulation of cell cycle regulatory proteins such as Rb and cyclin D1 and D3. Both glutathione (GSH) and N-acetylcysteine (NAC) pretreatment resulted in the complete inhibition of curcumin-induced ROS generation, AIF release from mitochondria, and caspase activation. Additionally, pretreatment of L929 cells with these antioxidants completely blocked the induction of p53-dependent p21 accumulation. In conclusion, our data show that in addition to caspase 3 activation, curcumin-induced rapid ROS generation leads to AIF release, and the activation of the caspase-independent apoptotic pathway.

Introduction

Curcumin, also known as diferuloymethane, is the major yellow pigment extracted from turmeric (Curcuma longa) and it is commonly used as a flavoring agent in food. Curcumin has been reported to possess anti-inflammatory and antioxidant activities [1], and it has been reported as a potent inhibitor of mutagenesis and carcinogenesis [2], [3]. Several biological effects of curcumin have been reported. These include cell shrinkage, chromatin condensation, and oxidative DNA damage [4]. Other reported signaling effects of curcumin include: inhibition of nitric oxide synthase, receptor tyrosine kinase, protein kinase C, Akt, NFκB, and cyclin D1 and activation of p53 [5], [6], [7], [8], [9]. Importantly, curcumin has been shown to induce apoptosis in different malignant cancer cell lines [10].

It is now well established that apoptosis or programmed cell death is characterized by cell shrinkage, cytoplasmic, nuclear, and chromatin condensation, membrane blebbing, protein fragmentation and DNA degradation, and finally breakdown of the cell into smaller units or apoptotic bodies [11]. Many proteins are involved in this complex process. Caspases, a family of cysteine-dependent aspartate-directed proteases, play a critical role in the initiation and execution of apoptosis [12]. Among this family of caspases, caspase 3, in particular, is believed to be one of the most commonly involved caspases in the execution of apoptosis in various cell types [13]. More recently, caspase-independent apoptogenic proteins, such as apoptosis inducing factor (AIF), endonuclease G (EndoG), and second mitochondria-derived activator of caspase (smac) have been identified and characterized [14], [15], [16].

Apoptosis inducing factor is a mitochondrial protein whose translocation in the cytosol, as well as in the nucleus, mediates caspase-independent apoptosis in a number of model systems [17]. The human full-length AIF (613 amino acids) has no effect on apoptosis. The amino terminal 102 amino acids of AIF, namely the mitochondrial localization sequence, once removed, yield a mature AIF molecule. On the initiation of apoptosis, the mature AIF releases into the cytosol and the nucleus, inducing chromatin condensation and large DNA fragmentation (50 kbp). During apoptosis, EndoG, like AIF, translocates to the nucleus where it causes oligonucleosomal DNA fragmentation [16]. Subsequent studies have demonstrated that EndoG catalyzes both high molecular DNA cleavage and oligonucleosomal DNA breakdown. Moreover, EndoG cooperates with exonuclease and Dnase1 to facilitate DNA processing [18].

Recently, it has been reported that curcumin-induced apoptosis is due to the production of reactive oxygen species (ROS) [19]. These free radicals are the by-products of normal cellular oxidative processes, and they are generated in the mitochondria and from other sources. They inflict serious damage to lipids, proteins, and DNA [20], and they have been suggested as regulating the process involved in the initiation of apoptotic signaling [21]. Indeed, ROS have been demonstrated to perform certain functions in the early stages of apoptosis, and to induce the depolarization of the mitochondrial membrane, which eventually results in an increase in the level of other proapoptotic molecules in the cytosol [22]. Although, ROS may constitute a direct cause of mitochondrial dysfunction, it remains controversial as to what is its role during apoptosis. Additionally, the role of ROS in Fas-mediated apoptosis has been demonstrated in a variety of cell types [23]. Taken together, the molecular events associated with the role of ROS in the cytotoxic mechanisms have yet to be clarified. The tumor suppressor p53 is known to be an important regulator of apoptotic cell death. It has been shown in several cell lines that the growth arrest mediated by p53 is dependent on the transcriptional activation of p21 [24], mitochondrial cytochrome c release, and caspase activation [25].

To date, the mechanism of ROS-triggered cell death induced by curcumin is poorly understood. In order to shed more light on the mechanism of ROS-mediated apoptosis induced by curcumin, we investigated the effects of this compound on mouse fibroblast L929 cells. Our study shows that in L929 cells curcumin induces a very rapid and significant ROS generation, which in turn leads to multiple apoptotic signal including both caspase-dependent and -independent pathways. Additionally, we have found that curcumin activates p53-dependent p21 accumulation and down-regulation of cell cycle regulatory proteins. Moreover, our study shows that antioxidants such as GSH and NAC inhibit curcumin-induced cell death, by inhibiting ROS generation.

Section snippets

Reagents

Curcumin, glutathione, N-acetylcysteine, 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide (MTT), proteinase K, RNase, glutathione assay chemicals, bovine liver catalase, super oxide dismutase, anti-rabbit IgG, and anti-mouse IgG were purchased from Sigma Chemical Co. (St. Louis, MO). Oxidation-sensitive DCFH-DA (D-399) and -insensitive 5-(and -6)-carboxy-2′,7′-dichlorofluorescein diacetate (C-369) were purchased from Molecular Probes (Eugene, OR). RPMI 1640 GlutaMAX medium, fetal

Effect of curcumin on L929 cell viability

The effect of different doses (0–100 μm) of curcumin on cell morphology was examined. During a 24-h period, curcumin treatment caused L929 cell shrinkage, rounding and partial detachment, thus demonstrating the cytotoxic effects of curcumin on L929 cells as shown in Fig. 1A. On assaying the effect of curcumin on cell viability by MTT assay, we observed a decrease in cell viability (Fig. 1B). At 80 μM curcumin concentration, significant loss of viability can be detected during the 0- to 24-h

Discussion

In order to better understand the mechanism of ROS-mediated apoptosis induced by curcumin, we investigated the effects of this compound on mouse fibroblast L929 cells. This study shows that curcumin induces a very rapid and significant ROS generation in L929 cells, leading to multiple apoptotic signals, including caspase-dependent and -independent pathways. Noteably, curcumin activates p53-dependent p21 accumulation, Rb dephosphorylation, and down-regulation of cell cycle regulatory proteins

Concluding remarks

In conclusion, we have demonstrated that curcumin-induced apoptosis in L929 cells involves at least three different signaling pathways, both caspase-dependent and caspase-independent, with ROS being a prime initiating signaling candidate. A molecular understanding of the mechanism of action of curcumin is a prerequisite in establishing its therapeutic relevance. We feel that our study has contributed to this understanding. However, the significance of activation of multiapoptotic signaling by

Acknowledgments

This work was financially supported by grants from The Terry Fox Foundation for Cancer Research (UAE-05-98), The Sheikh Hamdan Award for Medical Sciences, and grants from the Faculty of Medicine and Health Sciences, United Arab Emirates University. We thank Prof. Yusuf A. Hannun (Department of Biochemistry and Molecular Biology, MUSC, USA) for reviewing this manuscript.

References (43)

  • D.W. Voehringer

    BCl-2 and glutathione: alterations in cellular redox state that regulate apoptosis sensitivity

    Free Radic. Biol. Med.

    (1999)
  • T. Choudhuri et al.

    Curcumin selectively induces apoptosis in deregulated cyclin D1-expressed cells at G2 phase of cell cycle in a p53-dependent manner

    J. Biol. Chem.

    (2005)
  • R.J. Anto et al.

    L-929 Cells harboring ectopically expressed RelA resist curcumin-induced apoptosis

    J. Biol. Chem.

    (2000)
  • R.J. Anto et al.

    Anti-inflammatory activity of natural and synthetic curcuminoids

    Pharm. Pharmacol. Commun.

    (1998)
  • M.A. Azuine et al.

    , Protective role of aqueous turmeric extract against mutagenicity of direct-acting carcinogens as well as benzo[a]pyrene-induced genotoxicity and carcinogenicity

    J. Cancer Res. Clin. Oncol.

    (1992)
  • C.V. Rao et al.

    Chemoprevention of colon carcinogenesis by dietary curcumin, a naturally occurring plant phenolic compound

    Cancer Res.

    (1995)
  • N. Sreejayan et al.

    Curcuminoids as potent inhibitors of lipid peroxidation

    J. Pharm. Pharmacol.

    (1994)
  • S. Aggarwal et al.

    Curcumin (diferuloylmethane) down-regulates expression of cell proliferation and antiapoptotic and metastatic gene products through suppression of IκBα kinase and Akt activation

    Mol. Pharmacol.

    (2006)
  • A. Mukhopadhyay et al.

    Curcumin-induced suppression of cell proliferation correlates with down-regulation of cyclin D1 expression and CDK4-mediated retinoblastoma protein phosphorylation

    Oncogene

    (2002)
  • M.C. Jiang et al.

    Differential regulation of p53, c-myc, bcl-2 and bax protein expression during apoptosis induced by widely divergent stimuli in human hepatoblastoma cells

    Oncogene

    (1996)
  • R.J. Anto et al.

    Curcumin (diferuloylmethane) induces apoptosis through activation of caspase-8, BID cleavage and cytochrome c release: its suppression by ectopic expression of Bcl-2 and Bcl-xl

    Carcinogenesis

    (2002)
  • Cited by (218)

    • Curcumin encapsulation in functional PLGA nanoparticles: A promising strategy for cancer therapies

      2022, Advances in Colloid and Interface Science
      Citation Excerpt :

      P53 protein, for example, is inactivated in more than 50% of human cancers, compromising an important effector of apoptosis cascade induction [116]. Curcumin exerts important effects on this hallmark, either by inducing the release of proapoptotic factors (e.g., Bax and cytochrome c), through mitochondrial activation, or by contrarily inhibiting the expression of antiapoptotic proteins (e.g., Bcl-2, Bcl-xL) and caspase inhibitors [117–126]. In order to evolve from the initial neoplastic state and expand to macroscopic size tumors, cancer cells rely on the fourth hallmark capability: enabling replicative immortality.

    View all citing articles on Scopus
    View full text