Background
Radix Glycyrrhiza (Licorice) has been used to treat cancer in China for thousands of years. Licorice contains various types of flavonoids and triterpenes, and many of them have been found to possess anti-tumorigenic activities. Isoliquiritigenin (ISL), one of the flavonoids in licorice, can inhibit growth of diverse cancer types including breast, lung, cervical, ovary, prostate and colon cancer by inducing apoptosis, autophagy and causing DNA damage [
1‐
6]. ISL has also been shown to exhibit inhibitory effect on migration of breast and prostate cancer cells [
7‐
9]. Recently, ISL has been shown to potently suppress imflammatory responses by blocking Toll-like receptor 4 signaling and inflammasome activation [
10,
11]. As tumor-promoting inflammation is recognized as a hallmark of cancer [
12], ISL may also elicit its tumor-suppressive effect through immune regulation. In xenograft models, ISL was reported to effectively suppress breast and lung tumorigenesis [
13,
14]. Although anti-tumorigenic effect of ISL has been associated with the interference of various signaling pathways including JNK/AP1, PI3K/Akt and VEGF/VEGF2 [
8,
14,
15], direct target of ISL remains to be characterized.
Src is member of Src family kinase (SFK) and can be activated through the engagement of diverse cellular receptor types such as integrins, growth factor receptors and G protein-coupled receptors [
16]. It is critically involved in a wide spectrum cellular events including cell migration and cell growth. For example, Src phosphorylates cortactin and focal adhesion kinase (FAK) to facilitate cytoskeletal reorganization and focal adhesion turnover, two key steps in cell migration [
17‐
19]. Another example is that Src phosphorylates Yes-associated protein 1, leading to the activation of Hippo signaling pathway and subsequent hyperproliferation in skin squamous carcinoma cells [
20]. Numerous studies have convincingly demonstrated the importance of Src in tumor progression and metastasis [
21]. For instance, Src often interacts with EGFR and mediate EGFR-associated events [
22]. Since oncogenic mutations and aberrant activation of EGFR occurs frequently in various tumor types including non-small cell lung carcinomas (NSCLC) [
23,
24], SFK inhibitors are currently used to treat various malignancies [
25,
26].
With the aid of 3 well-migratory lung cancer cell lines, we compared various components in licorice for their effect on cell migration. Among all tested components, we showed that ISL was the only compound effectively inhibiting cell migration and its motility-inhibitory effect was through its ability to impair cytoskeleton reorganization and focal adhesion assembly. To elucidate mechanism underlying ISL action, we showed that ISL reduced cortactin’s Tyr421-phosphorylation, FAK’s Tyr861- and Tyr925-phosphorylation, indicating that ISL interfered with Src function because they are known to be Src phosphorylation sites [
17,
19,
27,
28]. Intriguingly, ISL was unable to inhibit Src activity in cell-free kinase assay but it decreased the amount of Tyr416-phosphorylated Src in lung cancer cells at a concentration as low as 3 μM. This observation raised the possibility that ISL inhibits Src through its cellular metabolites. In fact, 2, 4, 2′, 4′-tetrahydroxychalcone (THC), one of two main metabolites from ISL (naringenin chalcone was the other) not only decreased Tyr416 phosphorylation of Src in lung cancer cells but also inhibited Src activity in cell-free kinase assay. Moreover, THC suppressed cell migration as well as cytoskeleton reorganization/focal adhesion assembly in similar extent of ISL and specific Src inhibitor PP1. Importantly, both ISL and THC subdued tumor outgrowth and metastasis of H1299 lung cancer cells. In conclusion, this study uncovers that ISL exerts its anti-tumorigenic effect by targeting Src signaling pathway through its metabolite THC.
Discussion
Licorice has long been used as a medical herb in China while is widely used for culinary purposes in Western countries. Various components isolated from licorice have been shown to exhibit anti-tumorigenic effect. For example, glycyrhetinic acid inhibits growth of melanoma [
37], androgen dependent prostate cancer [
38] and breast cancer cells [
39], whereas glabridin induces apoptosis in human hepatoma cells [
40]. With the aid of H1299 lung cancer cell lines, we found that several compounds from licorice (18α-glycyrhetinic acid, liquiritin, licochalcon A and ISL) were able inhibit cell growth at the concentration of 30 μM or higher (Additional file
1: Figure S2). However, ISL was able to deter cell migration at concentration < 10 μM and actually was the only compound capable of effectively doing this (Additional file
1: Figure S3, 1A and B). The observation that ISL more potently inhibited cell migration than cell growth is also in stark contrast from other components of licorice in which they were only effective in cell growth suppression. The capability of ISL to negatively regulate both cell migration and growth clearly supports the notion that ISL is the main biologically active components for licorice’s anti-tumorigenic effects [
41,
42].
Coordinated cytoskeleton reorganization and focal adhesion assembly are essential for cell migration [
43]. While serum elicited actin reorganization and assembly of paxillin in the focal adhesion plaque (Fig.
2a and
b and Additional file
1: Figure S5), treatment of ISL abolished these two serum-induced events in lung cancer cells (Fig.
2a and
b and Additional file
1: Figure S5), suggesting that ISL deters cell migration by impairing cytoskeleton reorganization. To define the underlying mechanism associated with ISL action, we analyzed the phosphorylation status of proteins associated with cytoskeleton and identified that Tyr421 phosphorylation of cortactin, Tyr861 and Tyr925 phosphorylation of FAK were most affected by ISL (Additional file
1: Table S1 and Fig.
3a). Since these sites are known to be phosphorylated by Src [
17,
19,
28], we speculated that ISL interferes with Src function, leading to the deterrence of cell migration. This notion is supported by our observations that 1) Similar to ISL, Src specific inhibitor PP1 also blocked serum-induced cytoskeleton reorganization and focal adhesion assembly (Fig.
3b and Additional file
1: Figure S5); and 2) ISL diminished Tyr416-phosphorylation of Src in lung cancer cells (Fig.
3c). As Src signaling pathway is well documented for its role in cell migration [
25], our study thus links inhibitory role of ISL in cell migration to its capability to impair Src signaling pathway.
Butein, a chalcone structurally similar to ISL (Additional file
1: Figure S11), has been previously shown to inhibit Src activity [
44]. Intriguingly, cell-free Src kinase assay showed that ISL was unable to inhibit Src activity (Additional file
1: Figure S6). A previous study identified butein as a metabolite of ISL in liver microsome [
45]. This raised the possibility that ISL might block Src activity through its metabolite in lung cancer cells. Although butein was not detected, we identified THC and naringenin chlcone as main ISL metabolites in ISL-treated H1299 cells (Fig.
4a). Importantly, THC blocked Src activity both in cell-free kinase assay and in various cell lines (Fig.
4b and
d). Comparison of structures between butein and ISL reveals an additional hydroxyl group on ring II (Additional file
1: Figure S13). Since THC also contains an additional hydroxyl group on ring II while inactive naringenin chalcone does not (Additional file
1: Figure S11), we reason that additional hydroxyl group on ring II may be critical for the ability of THC to act as a Src inhibitor. One observation that has to be noted is that the inhibitory effect of THC for Src phosphorylation in lung cancer cells was in the same degree as that of ISL despite that it is an ISL metabolite (Figs.
3c and
4b). Although we do not truly understand why THC was not more effective than ISL for reducing Src phosphorylation, we speculate that stability of THC in culture medium may be less than ISL, thus affecting its potency. In addition, we cannot rule out the possibility that THC might not get into cells as efficiently as ISL.
ISL has been shown to inhibit prostate cancer cell migration in a mechanism possibly involving in decreased JNK/AP-1 signaling [
8]. ISL has also been reported to inhibit breast cancer cell migration through downregulating cyclooxygenase-2 (COX-2) signaling [
7]. However, these studies did not identify the direct target(s) of ISL. Here, we demonstrated that ISL deterred cell migration by inhibiting Src activity through its metabolite THC (Fig.
5). Earlier studies revealed that Src mediates RalA- and advanced glycation end product-induced AP1 activation [
46,
47]. COX-2-facilitated cell migration was shown to be associated with Src signaling [
48]. Taken together, it is most likely that ISL inhibits Src activity, leading to the impairment of AP-1 and COX-2 signaling.
A series of investigations has presented evidences that the increase of Src activity is accompanied with the progression of many cancers [
49,
50] and the activation status of Src can be used as an indicator for tumor progression [
51]. Because of the importance of Src in tumor progression and metastasis [
52], Src inhibitors have been developed and several of them (AZD0530, dasatinib, ponatinib, etc) have even been approved to treat malignancies [
53]. Blocking Src activity is apparently effective in suppressing lung tumorigenesis as we observed that AZD0530 effectively deterred both lung tumor outgrowth and metastasis (Additional file
1: Figure S12). Given the capability of ISL and THC to interfere with Src signaling, they are expected to possess anti-tumorigenic effects. In fact, both ISL and THC suppressed tumor outgrowth and lung metastasis of lung cancer cells in athymic nude mice (Figs.
6c–
e,
7b and
c). As remarkable reduction in the intensities of phosphorylated cortactin and FAK staining was detected in excised tumor specimens from these mice (Fig.
7d), these results clearly support the notion that tumor-suppressive effect of ISL and THC is associated with their ability to inhibit Src activity.