Phosphotyrosine profiling of curcumin-induced signaling

Curcumin, a bioactive compound derived from the rhizome Curcuma longa has been known for its chemopreventive and chemotherapeutic potential [1, 2]. It is a polyphenol compound with an aromatic ring structure connected by two ?, ?-unsaturated carbonyl groups and has been extensively studied for its diverse range of biological activities, including anti-inflammatory, anti-oxidant, analgesic and antiseptic properties [3–6]. It has attracted widespread attention as a potential therapeutic agent because of its pharmacological effects. The anti-tumor activity of curcumin is thought to be mediated through multiple mechanisms. At the molecular level, curcumin is known to induce apoptosis in a wide array of cancer cells including human colon, stomach, liver, breast, and prostate cancers [7–11]. It is known to mediate its effects by inhibition of anti-apoptotic markers such as Bcl-2, Bcl-xL, Survivin, and increased expression of pro-apoptotic factors such as Bax, Bad, Bak, PUMA, Bim, Noxa and TRAIL-R1 [12–14]. Curcumin has also been shown to inhibit cellular proliferation by downregulating several oncogenes such as EGFR, HER-2, PI3K/AKT, MAPK and upregulating the expression of various tumor suppressor genes such as p21WAF1/CIP1, p27KIP1 and p53 [15–19]. Furthermore, in vivo studies using animal models of skin and oral cancer have shown that curcumin inhibits tumor initiation and progression [20, 21].

Curcumin mediates it effect by targeting multiple cell growth signaling pathways, including PI3K-AKT, mTOR, EGFR and TGF-? signaling, amongst others [22–25]. It has been reported to cause a dose and time-dependent decrease in the phosphorylation of AKT and mTOR leading to decreased cellular proliferation and survival [26]. Curcumin has also been reported to induce the suppression of NF-?? and I?? activation in melanoma cells and inhibit JNK signaling and STAT3 activation which in turn decreases the expression of pro-survival proteins [27–29]. Currently, information pertaining to curcumin-mediated tyrosine phosphoproteome signaling is minimal and the detailed signaling mechanism responsible for various biological effects of curcumin remains elusive. Understanding the signaling pathways responsible for its anti-neoplastic activity will provide avenues to identify novel therapeutic targets for cancers.

Aberrant activation of signaling pathways mediated by kinases is a common phenomenon in multiple malignancies. Tyrosine kinases regulate various cellular processes such as cell proliferation, differentiation, motility, cell cycle homeostasis, transcriptional regulation, and metabolism through reversible phosphorylation [30]. Although several studies have been carried out to characterize curcumin-induced alterations in cellular proteome of neuroblastoma [31], breast [32], gastric [11] and cervical cancers [33]; no effort have been made to study the changes in tyrosine signaling mediated by curcumin using quantitative phosphoproteomics approach.

In this study, we carried out SILAC-based quantitative proteomic analysis of CAL 27 cells (a HNSCC cell line) to investigate the tyrosine signaling in response to curcumin. Previous studies have reported curcumin-induced apoptosis and decreased cell proliferation in CAL 27 [34, 35]. Combining SILAC with anti-phosphotyrosine antibody-based enrichment and high resolution mass spectrometry analysis enabled identification of 627 unique phosphorylation sites mapping to 359 proteins including several novel curcumin-regulated phosphorylation events. Further, bioinformatics analysis identified perturbations in pathways regulating focal adhesions and actin cytoskeleton in curcumin-treated cells suggesting that curcumin may mediate its anti-proliferative effects through these pathways.