Cancer Letters

Cancer Letters

Volume 272, Issue 1, 8 December 2008, Pages 77-90
Cancer Letters

DNA damage and endoplasmic reticulum stress mediated curcumin-induced cell cycle arrest and apoptosis in human lung carcinoma A-549 cells through the activation caspases cascade- and mitochondrial-dependent pathway

https://doi.org/10.1016/j.canlet.2008.06.031Get rights and content

Abstract

Curcumin, a major component of the Curcuma species, is known to have antioxidant, anti-inflammatory properties and induce apoptosis of cancer cells, however, the precise molecular mechanisms of apoptosis in vitro are unclear. In this study, we showed that curcumin, a plant product containing the phenolic phytochemical, caused DNA damage and endoplasmic reticulum (ER) stress and mitochondrial-dependent-induced apoptosis through the activation of caspase-3 at a treatment concentration of 30 μM in human lung cancer A-549 cells. In contrast, treatment with 5–10 μM of curcumin did not induce significant apoptosis, but rather induced G2/M-phase arrest in A-549 cells. Flow cytometric analysis indicated that curcumin directly increased intracellular oxidative stress based on the cell permeable dye, 2′,7′-dichlorodihydrofluorescein diacetate (DCFH-DA) acting as an indicator of reactive oxygen species (ROS) generation. GADD153 and GRP78 were increased by curcumin which was indicative of ER stress. Curcumin increased Ca2+ levels and the mitochondrial membrane potentialΨm), was decreased in A-549 cells. Overall, our results demonstrated that curcumin treatment causes cell death by activating pathways inducing G2/M-phase arrest and apoptosis.

Introduction

Cell cycle is a series of tightly integrated events which allows the cell to grow, proliferate and die, and it serves to protect the cell from genotoxic stress. Cell cycle can be divided into G0/G1-, S- and G2/M-phase based on DNA content. The cyclins, cyclin-dependent kinases (CDKs) and even some of the inhibitors of cyclins and CDKs are involved in cell cycle. CDKs promote the cell to move from one phase to the next phase (G0/G1 to S, S to G2/M or G2 to M) of the cell cycle. If agents affect the cyclin and/or CDKs, it may lead to cell cycle arrest. If a cell continues to cycle with its damaged DNA intact, the apoptotic machinery is triggered and the cell will undergo apoptosis.

Numerous naturally occurring substances are recognized as antioxidants, cancer preventive agents, or even as a cancer therapy drug such as Taxol [1]. Those bioactive substances exert their anticancer activity through blocking cell cycle progression and triggering tumor cell apoptosis. Therefore, an effective strategy for anticancer activity of certain agents is to cause cell cycle arrest and the induction of apoptosis in tumor cells [2], [3]. Curcumin (diferuloylmethane), a phenolic compound from the plant Curcuma longa (Linn), is a widely used flavoring agent in food [4]. Curcumin inhibits cell proliferation and induces apoptosis in human leukemia, prostate cancer, and non-small cell lung cancer cell lines [5], [6], [7]. Curcumin has been shown to have anti-tumor activity in the colon, skin, stomach, duodenum, soft palate, and breasts of rodents [8], [9], [10]. In a recent report on a phase I clinical trial of oral curcumin in colon cancer patients, high tolerability of the drug was observed and it was suggested developing curcumin as an oral cancer preventive or therapeutic agent [11].

The anti-tumor activity of curcumin is attributed to its ability to induce apoptosis via caspase-3 activation [12]. Although there have been extensive investigations on potential mechanisms of curcumin as an anti-tumor agent, the exact mechanism associated with DNA damage and ER stress associated with GADD153 and GRP78 are yet to be established. Therefore, in the present study, we focused on the effect of curcumin on lung cancer cells and also investigated the role of GADD153 and GRP78 in the induction of apoptosis in human lung cancer A-549 cells.

Section snippets

Chemicals and reagents

Curcumin, propidium iodide (PI), Triton X-100, Tris–HCl, Trypan blue, ribonuclease-A were obtained from Sigma Chemical Co. (St. Louis, MO, USA). Potassium phosphates, dimethyl sulfoxide (DMSO), and TE buffer were purchased from Merck Co. (Darmstadt, Germany). 90% Ham’s F12K medium with 2 mM l-glutamine adjusted to contain 1.5 g/l sodium bicarbonate + 10% fetal bovine serum, penicillin–streptomycin, and trypsin–EDTA were obtained from Gibco BRL (Grand Island, NY, USA).

Cell culture

Human lung carcinoma A-549 cell

Curcumin-induced morphological changes decreased the percentage of viable A-549 cells

A-549 cells were treated with or without different concentrations of curcumin for different time periods and morphological changes were examined using phase-contrast microscopy. Cell viability was determined by flow cytometry. Curcumin-induced morphological changes (Fig. 1A), apoptotic bodies were observed and some substances in the cytosol were released to the media and effects were time-dependent effect (Fig. 1A). Curcumin ranging from a concentration of 10–40 μM decreased the percentage of

Discussion

Several studies have demonstrated curcumin-induced cell cycle arrest and apoptosis in human cancer cell lines but the exact role of DNA damage and ER stress associated with induction of apoptosis by curcumin is still unclear. Therefore, in the present study we focused on the role of DNA damage and ER stress (hallmarks of ER stress are GADD153 and GRP78) associated apoptosis in human lung cancer A-549 cells exposed to curcumin. Overall our results showed that curcumin-induced G2/M arrest and

Acknowledgements

This work was supported by Grants NSC-94-2745-B-039-002-URD and NSC 95-2745-B-039-002-URD from the National Science Council of Taiwan.

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