Elsevier

Archives of Oral Biology

Volume 51, Issue 10, October 2006, Pages 913-921
Archives of Oral Biology

Induction of early apoptosis and ROS-generation activity in human gingival fibroblasts (HGF) and human submandibular gland carcinoma (HSG) cells treated with curcumin

https://doi.org/10.1016/j.archoralbio.2006.03.016Get rights and content

Summary

Objective

Curcumin [1] is well known to possess apoptosis-inducing activity in some cancer cells, but little is known about its activity in normal cells of oral origin, such as HGF. The aim of the present study was to clarify the relationship between early apoptosis in HGF and the induction of reactive oxygen species (ROS) generation by curcumin.

Design

We treated HGF and HSG cells with curcumin [1] and the related compounds biseugenol [2], eugenol [3], α-diisoeugenol [4], and isoeugenol [5] and measured cell survival (MTT method), ROS generation (DCFH-DA staining), and induction of early apoptosis. Early apoptosis was detected by monitoring loss of mitochondrial membrane potentialΨm) by JC-1 staining and externalization of phosphatidylserine (PS) on the cell surface by annexin V-FITC/PI staining combined with flow cytometry.

Results

The cytotoxic activities of curcumin [1] and [4] were similar and were nearly 10- to 100-fold stronger than those of the other compounds. Only curcumin was able to induce ROS generation and early apoptosis. Loss of ΔΨm, PS externalization and ROS generation were significantly more pronounced in HGF cells than in HSG cells at curcumin concentrations lower than about 15 μM, and were inhibited by the addition of the antioxidants N-acetyl-l-cysteine and glutathione.

Conclusion

The potent PS externalization and loss of ΔΨm in curcumin-treated HGF cells appears to be mediated by ROS generation.

Introduction

Curcumin [1], 1,7-bis(4-hydroxy-3-methoxyphenol)-1,6-heptadiene-3,5-dione, obtained from the rhizome of the plant Curcuma longa, is a naturally occurring pigment and a component of the spice called turmeric. Curcumin has attracted considerable interest in the last few years because it possesses anti-inflammatory,1 anticarcinogenic,2, 3 and antioxidant4, 5 properties. Although numerous studies have shown that curcumin induces apoptosis in cancer cells,6, 7 the precise mechanism of this activity has not been elucidated. On the other hand, although curcumin in foods may directly affect the oral mucosa and gingiva as well as the gastrointestinal tract, the cytotoxicity of curcumin toward cells derived from oral tissues and the induction of apoptosis in such cells have been little studied.

Previous studies on the biological activity of curcumin and related compounds have demonstrated that antioxidant/prooxidant properties are enhanced by the presence of a methoxy group in the ring.8, 9, 10, 11 Bhaumic et al.12 and Morin et al.13 previously reported that curcumin treatment caused enhanced generation of superoxide, loss of mitochondrial membrane potential (ΔΨm), and cytochrome c release to the cytosol, with the concomitant externalization of phosphatidylserine (PS) residues on the cell surface. However, the causal link between the induction of cytotoxicity/apoptosis by curcumin and its ROS-generation activity has not been sufficiently investigated. In addition, the difference between normal cells and cancer cells in terms of induction of apoptosis and ROS generation remains unclear.

Thus, to clarify the role of ROS generation in the induction of early apoptosis, we examined the cytotoxicity, the ability to induce early apoptosis (loss of ΔΨm and PS externalization), and the ROS-generation activity of compounds [1], [2], [3], [4] and [5] in cells of oral origin. These dose- and time-response studies used both cancer cells (human submandibular carcinoma cell line; HSG) and normal cells (primary culture of human gingival fibroblasts; HGF).

Section snippets

Reagents

Curcumin (1,7-bis-(4-hydroxy-3-methoxyphenol)-1,6-heptadiene-3,5-dione), eugenol (4-allyl-2-methoxyphenol), and isoeugenol (4-(1-propenyl)-2-meth oxyphenol) were obtained from Tokyo Kasei Chem. Co., Tokyo, Japan and used without further purification. Biseugenol (3,3-dimethoxy-5,5′-di-2-propenyl-1,1′-diol) and α-diisoeugenol (r-1-ethyl-5-hydroxy-t-3-(4-hydroxy-3-methoxyphenyl)-6-methoxy-c-methylindan) were synthesized as previously described.14 The chemical structures of the test compounds used

Cytotoxicity

We examined the survival of HSG cells (A) and HGF cells (B) after treatment with curcumin [1], biseugenol [2], eugenol [3], α-diisoeugenol [4] and isoeugenol [5] at concentrations of 0.1 μM–1 mM (Fig. 2). The 50% cytotoxic concentration (CC50, mM) against HSG cells declined in the order [3] (0.287) > [2] (0.182) > [5] (0.059) > [1] (0.004)  [4] (0.003). In contrast, the CC50 against HGF cells declined in the order [2] (0.330) > [3] (0.215) > [5] (0.052) > [1] (0.003)  [4] (0.002). The cytotoxicity of curcumin

Discussion

In the present study, early apoptotic events characterized by loss of ΔΨm (Fig. 4) and PS externalization (Fig. 6) in curcumin-treated HSG and HGF cells paralleled ROS generation (Fig. 3) in terms of dose-response and time-response. Also, these early apoptotic events were inhibited by the antioxidants NAC and GSH (Fig. 7B and C), strongly suggesting that apoptotic events induced by curcumin are mediated by ROS generation. It has previously been reported that low levels of ROS are able to

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