Direct inhibitory effect of curcumin on Src and focal adhesion kinase activity
Introduction
Curcumin (diferuloylmethane), a popular dietary spice in the East, is a well-known agent with anti-inflammatory, antioxidant, and anticarcinogenic properties. The major constituent of tumeric powder extracted from the rhizomes of Curcuma longa Lin. gives the unique flavor and yellow color to curry [1]. As a β-diketone moiety containing polyphenolic compound, curcumin possesses two ferulic acid molecules linked via a methylene bridge at the C atoms of the carboxyl groups (Fig. 1A) [2]. Animal experiments have shown that in addition to its anti-inflammatory and antioxidant properties, curcumin also displays chemopreventive activity in various tissues [2]. Although it is still unclear which part of the agent is crucial for biological activity, the speculated candidates include the hydroxyl groups of the benzene rings, the double bonds in the alkene part of the molecule, and/or the central β-diketone moiety. To date, the inhibitory effects of curcumin on various signaling proteins including cyclooxygenase, ornithine decarboxylase, nitric oxide synthase, transcription factors, matrix metalloproteinases, and protein kinases have been reported [2]. Conceivably, through inhibition of these molecules, curcumin can effectively suppress or revert tumor formation and retard metastasis.
The protein TK is a large and diverse multigene family that historically defines the prototypical class of oncogenes involved in most animal malignancies [3]. Phosphorylation mediated by TK is an important post-translational modification that conveys signals concerning a variety of physiologic activities such as proliferation, differentiation, adhesion, transformation, and mobility. C-Src, encoded by the cellular homologue of v-src, is a ubiquitously expressed cytoplasmic TK whose overexpression and enhancement of enzymatic activity have been strongly implicated in human tumors [4]. Tyr-416 and -527 are the two well-documented Src phosphorylation sites that are located within the kinase domain and the C-terminal regulatory region, respectively. While self-mediated phosphorylation of the former enhances the catalytic activity of Src, phosphorylation of the latter that mediated by CSK (a C-terminal Src kinase) downregulates Src activity. The lack of Tyr-527 in v-Src renders v-Src to be constitutively active [5]. A number of signaling proteins such as FAK [6], cortactin [7], [8], and Shc [9] have been identified as the substrates for Src family kinases. Previous studies have demonstrated that Shc Tyr-317 is an Src-mediated residue whose phosphorylation, which is associated with the SH2 domain of Grb2, propagates signaling to the proline-rich target (i.e. SOS) and leads to the activation of Ras and triggers the ERK kinase cascade [10], [11].
As a substrate for Src and a unique cytoplasmic TK localized at focal contacts, FAK is an important player in integrin signal transduction [12]. Upon integrin engagement, FAK becomes activated and autophosphorylated at Tyr-397, which confers the binding site for Src family kinases [13], [14]. Through the formation of a complex with FAK, Src mediates the phosphorylation of FAK at Tyr-407, -576/577, -863 and -925, and either enhances the enzymatic activity of FAK [15], [16], [17] or provides the binding site for the Grb2 SH2 domain and triggers Ras signaling [18]. Recently, accumulated evidence has indicated that the formation and activation of the Src–FAK bipartite kinase complex is important in cell spreading, migration, and survival [12].
The inhibitory effect of curcumin on EGFR and p185neu has been well studied [19], [20]. However, unlike these two receptor TKs, the influence of curcumin on the activity of Src was poorly documented. In an attempt to address this question, we investigated the biological responses to curcumin and the molecular mechanisms involved in these responses in cells transformed with v-Src. Interestingly, following curcumin treatment, the tyrosyl phosphorylation of Shc, cortactin, and FAK progressively disappeared with a concomitant decrease of Src kinase activity. Notably, we observed that curcumin could directly inhibit the activity of both Src and FAK, which has not been revealed before. And through downregulation of the activity of these two kinases, curcumin effectively inhibited the proliferation and migration of v-Src transformed cells.
Section snippets
Cell lines and drug treatment
The clonal C3H10T1/2 murine fibroblast cell line expressing v-Src (IV5) was a generous gift provided by Dr. Sarah J. Parsons and its derivation and maintenance were previously described [21]. For drug treatment, cells were incubated with or without curcumin. Their lysates were then harvested and analyzed.
Antibodies
Src-specific mouse mAb GD11 and anti-cortactin mAb (4F11) were provided by Dr. Sarah J. Parsons in University of Virginia. Polyclonal FAK antibody is directed against the C-terminal region of
Curcumin inhibits the proliferation and v-Src kinase activity in IV5 cells
Since curcumin (Fig. 1A) is a well-established food constituent with chemopreventive properties and its effect on Src is poorly described, thus, we utilized cells transformed with v-Src (IV5) to evaluate the possibility that curcumin might inhibit the proliferation of IV5 cells. As shown in Fig. 1B, compared to control cells, curcumin significantly inhibited cellular growth of IV5 cells in a dose-dependent manner (the ic50 for 24 hr growth inhibition is approximately 15 μM). Because tyrosyl
Discussion
By suppressing, retarding, or reversing the process of carcinogenesis in cell culture and animal models, curcumin has been well established as a dietary constituent with chemopreventive properties. Although a variety of target proteins have been reported, we are the first to report on the inhibitory effect of curcumin on both Src and FAK that results in reduced cellular growth and movement in v-Src-transformed cells.
Despite that phosphorylation mediated by TK is a crucial post-translational
Acknowledgements
We thank Dr. Sarah J. Parsons for providing the monoclonal Src antibody (GD11) and Suparat Charoenfuprasert for her assistance in preparation of this manuscript. These studies were supported by National Science Council grants to M.-C.M. (NSC 91-2311-B-040-002) and grants of NHRI (NHRI-EX-91-8932SL) and MOE Program for Promoting Academic Excellence of Universities (91-B-FA09-1-4) to T.-H.L.
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