Quercetin inhibits HGF/c-Met signaling and HGF-stimulated melanoma cell migration and invasion

Cao et al. Molecular Cancer Quercetin inhibits HGF/c-Met signaling and HGF- stimulated melanoma cell migration and invasion Hui-Hui Cao 0 Chi-Yan Cheng 0 Tao Su 0 Xiu-Qiong Fu 0 Hui Guo 0 Ting Li 0 Anfernee Kai-Wing Tse 0 Hiu-Yee Kwan 0 Hua Yu 0 Zhi-Ling Yu 0 0 Jockey Club School of Chinese Medicine Building, 7 Baptist University Road, Kowloon Tong , Kowloon , China Background: Melanoma is notorious for its propensity to metastasize, which makes treatment extremely difficult. Receptor tyrosine kinase c-Met is activated in human melanoma and is involved in melanoma progression and metastasis. Hepatocyte growth factor (HGF)-mediated activation of c-Met signaling has been suggested as a therapeutic target for melanoma metastasis. Quercetin is a dietary flavonoid that exerts anti-metastatic effect in various types of cancer including melanoma. In a previous report, we demonstrated that quercetin inhibited melanoma cell migration and invasion in vitro, and prevented melanoma cell lung metastasis in vivo. In this study, we sought to determine the involvement of HGF/c-Met signaling in the anti-metastatic action of quercetin in melanoma. Methods: Transwell chamber assay was conducted to determine the cell migratory and invasive abilities. Western blotting was performed to determine the expression levels and activities of c-Met and its downstream molecules. And immunoblotting was performed in BS3 cross-linked cells to examine the homo-dimerization of c-Met. Quantitative real-time PCR analysis was carried out to evaluate the mRNA expression level of HGF. Transient transfection was used to overexpress PAK or FAK in cell models. Student's t-test was used in analyzing differences between two groups. Results: Quercetin dose-dependently suppressed HGF-stimulated melanoma cell migration and invasion. Further study indicated that quercetin inhibited c-Met phosphorylation, reduced c-Met homo-dimerization and decreased c-Met protein expression. The effect of quercetin on c-Met expression was associated with a reduced expression of fatty acid synthase. In addition, quercetin suppressed the phosphorylation of c-Met downstream molecules including Gab1 (GRB2-associated-binding protein 1), FAK (Focal Adhesion Kinase) and PAK (p21-activated kinases). More importantly, overexpression of FAK or PAK significantly reduced the inhibitory effect of quercetin on the migration of the melanoma cells. Conclusions: Our findings suggest that suppression of the HGF/c-Met signaling pathway contributes to the anti-metastatic action of quercetin in melanoma. Quercetin; Melanoma; Migration; Invasion; Metastasis; c-Met - Background The incidence and mortality rates of melanoma have increased world-wide in the last 30 years [1]. Melanoma is notorious for its propensity to metastasize. Early stage melanoma is readily treatable, but advanced metastatic melanoma becomes resistant to treatment. It is reported that the long-term survival rate for patients with metastatic melanoma is only 5% [2]. Currently available chemotherapeutic approaches for melanoma often carry tolerance, low response rate [3] and high toxicity [4,5]. New targeted therapies with high response rate and low toxicity are urgently needed for managing malignant melanoma. Recently, the role of receptor tyrosine kinase c-Met in melanoma pathogenesis has been gaining interest. c-Met is a cell surface receptor consists of a 50-kDa extracellular chain and a 140-kDa membrane-spanning chain, and is synthesized from a single-chain 170-kDa precursor [6]. Binding of HGF (hepatocyte growth factor), the only known endogenous ligand of c-Met [7], to c-Met leads to c-Met homo-dimerization and auto-phosphorylation. The phosphorylated regions of c-Met then act as the multifunction docking site for adaptor molecules which propagate a signaling cascade through a number of effector proteins [8]. Dysregulation of c-Met has been found in many types of cancer, which usually correlated with a poor prognosis [9]. Interestingly, abnormal activation of c-Met signaling is implicated in the acquisition of tumorigenic and metastatic phenotypes in tumors [10,11]. Examinations indicated that c-Met was expressed and activated in melanoma tissues and cell lines [12]. Studies showed that overexpression of c-Met was associated with melanoma growth and metastasis [13,14]. Constitutive activation of c-Met signaling has been reported to promote melanoma metastasis in mice [15,16], while inhibition of c-Met signaling with a specific small molecule tyrosine kinase inhibitor reduced growth and metastasis of experimental human melanoma [17,18]. Blockade of c-Met signaling with the specific small interfering (si) RNA also induced melanoma cell differentiation and prevented melanoma metastasis in a mouse model [17,18]. These studies suggest that c-Met is a therapeutic target for melanoma metastasis. The dietary flavonoid quercetin (3,3,4,5,7-pentahydroxyflavone) is a bioactive compound that wildly distributed in the plant kingdom. It possesses low intrinsic toxicity and does not have carcinogenic activity in vivo [19]. Besides, it has a relatively high oral bioavailability [20]. Quercetin has many biological functions including anti-melanoma activity [21]. Several studies showed that quercetin inhibited melanoma growth [22-24] and metastasis [25,26]. Moreover, quercetin also inhibited HGF-induced c-Met phosphorylation in human medulloblastoma cell line DAOY [27], and suppressed HGFstimulated migration and invasion in DAOY cells [27] and human hepatoma HepG2 cells [28]. Our published data [29] demonstrated that quercetin inhibited melanoma cell migration and invasion in vitro and prevented melanoma lung metastasis in vivo. Here, we show that quercetin inhibits HGF/c-Met signaling manifested by suppressing c-Met phosphorylation, interfering c-Met dimerization, reducing c-Met protein expression and attenuating the activities of downstream molecules including Gab1, FAK and PAK, which contributes to the anti-metastatic action of quercetin in melanoma. Results Quercetin suppressed HGF-stimulated melanoma cell migration and invasion The effects of quercetin on HGF-stimulated melanoma cell migration and invasion were determined by the Transwell chamber assays. As shown in Figure 1A, HGF significantly enhanced the migratory abilities in melanoma A2058 and A375 cells. After a 24-h stimulation with HGF, the numbers of A2058 and A375 cells that migrated through the membranes were 4.3-fold and 1.8-fold more than that under unstimulated condition, respectively. A 48-h stimulation with HGF also caused a significant increase in cell migration, whereas treatment with quercetin reduced cell migratory abilities in a dose-dependent manner. In parallel, a Matrigel invasion assay showed that stimulation with HGF significantly increased the invasiveness of melanoma cells at both 24 h- and 48 h-incubation periods, and this effect was dose-dependently reverted by quercetin treatments (Figure 1B). Under a (...truncated)