The characteristics that define successful cancers include not only the capacity for sustained, self-sufficient proliferation, but also increased resistance to apoptosis, metabolic reprogramming, acquisition of migration and invasion capabilities, and pro-angiogenic potential [1
]. In this context, human triple-negative breast cancers (TNBCs) represent a major challenge in treatment owing to their inherent resistance to chemotherapy and high capacity for metastatic spread [2
]. Similarly, non-small cell lung cancer (NSCLC), which accounts for the great majority (~85%) of all lung cancers, is relatively resistant to chemotherapy and is associated with poor prognosis, with an expected survival of fewer than two years in patients with advanced disease [4
Interleukin-6 (IL-6) is a proinflammatory cytokine with pleiotropic functions in regulating the growth and differentiation of different types of cancer cells, including breast, colon, and lung cancers [5
]. The binding of IL-6 to its receptor, IL-6Rα, triggers a heterodimeric association with the signal-transducing receptor gp130 to form a signaling complex that initiates the phosphorylation and activation of Janus kinases JAK1 and JAK2 [8
]. This catalyzes the tyrosine phosphorylation of the transcription factor STAT3 (signal transducer and activator of transcription 3), which dimerizes and translocates to the nucleus, thereby initiating a complex transcriptional set that promotes cell growth and inhibits apoptosis [9
High levels of IL-6 are expressed in malignant breast cancers where, together with breast stromal fibroblasts, drive both autocrine and paracrine growth through the IL-6/IL-6R/STAT3 positive feedback loop [10
]. IL-6 has also been implicated in the malignant transformation of breast cancer stem cells and in the enhancement of cancer cell metastatic potential and epithelial to mesenchymal transition [13
]. Similarly, patients with lung adenocarcinoma usually have elevated levels of IL-6, which are associated with poor prognosis [16
]. Constitutively, activated tyrosine (Y705)-phosphorylated STAT3 (p-STAT3) has been demonstrated in many human breast [12
] and lung [7
] cancer cell lines, in 40–50% of primary human breast cancers [12
], and 22–65% of non-small cell lung cancers [20
], making it an attractive target for the development of anti-cancer therapies. The induction of STAT3 transcriptional activity increases the expression of many genes involved in cancer cell proliferation, survival, migration, invasion, angiogenesis, and metastasis [21
]. Given the central role of the IL-6-STAT3 pathway in the regulation of breast and lung cancer progression and metastasis, blockade of its various components may potentially lead to new therapeutic modalities.
Previous work from our group demonstrated the potential use of manuka honey (MH) as a modulatory anti-cancer agent [22
]. The treatment of human breast cancer cells with MH led to a dose and time-dependent inhibition of the transcriptional activity of STAT3 [23
]. The potency of MH in inhibiting this critical signaling pathway in cancer cells was demonstrated by the fact that as low as 0.03% solution (w/v) of MH (equivalent to a concentration of 0.3mg/mL) was sufficient to cause a significant reduction in p-STAT3 levels [23
]. Importantly, inhibition of p-STAT3 was accompanied by a reduction in IL-6 secretion, hence depriving breast cancer cells of this critical growth-promoting factor. In this context, MH was able to inhibit the migration, invasion and angiogenic potential of breast cancer cells. These findings identify multiple functional pathways affected by MH in human breast cancer and highlight the IL-6/STAT3 signaling pathway as a potentially critical target in this process [23
]. However, the precise mechanism by which MH inhibits p-STAT3 activation remains to be elucidated.
In addition to the IL-6/STAT3 pathway, the diverse properties of cancer cells are regulated by signaling through multiple receptors, including platelet-derived growth factor receptor (PDGF-R), epidermal growth factor receptor (EGF-R), fibroblast growth factor receptor (FGF-R), vascular endothelial growth factor receptor (VEGF-R) and insulin-like growth factor-1 receptor (IGF-1R). Src family kinases are critical mediators in all of these receptor-signaling pathways and play an important role in tumor resistance [24
]. Moreover, Src kinases are intricately involved in integrin signaling, thereby regulating tumor metastasis [26
]. STAT activation is also known to be induced by growth factor receptors, such as EGF-R, and the Src family of kinases, particularly c-Src [28
]. Given the central role played by c-Src in mediating signaling from a multitude of other receptors on cancer cells, the potential effect of MH on Src activity remains unknown.
In the present study, we demonstrate that MH-induced inhibition of oncogenic p-STAT3 in human TNBC cell line MDA-MB-231 and NSCLC cell line A549 is associated with decreased levels of gp130 and p-JAK2 proteins, two critical upstream components of the IL-6R signaling pathway. Importantly, MH had no effect on p-Src levels in both cancer cell lines. Furthermore, using recombinant proteins, we demonstrate that MH binds directly and specifically to IL-6Rα, interfering with the binding of IL-6 ligand. Thus, we identify the IL-6Rα chain as a direct target of MH. Finally, molecular docking studies identified potential binding sites of MH flavonoids on IL-6Rα. Our findings represent the first demonstration of the ability of MH to act as an antagonist of a key pro-oncogenic pathway through binding to the IL-6Rα protein expressed by human breast and lung cancer cells.
We previously identified p-STAT3 as an early molecular target of MH in cancer. MH causes a rapid loss of p-STAT3 in human breast cancer cells, reducing their proliferation, migration, and invasiveness [23
]. In the present study, we demonstrate that exposure to MH leads to a decline in p-STAT3 levels in human A549 lung cancer cells with very similar kinetics to that observed in MDA-MB-231 cells. Moreover, we show that the loss of p-STAT3 is accompanied by a decrease in the levels of gp130 and p-JAK2, two upstream regulators of p-STAT3 activity in breast (MDA-MB-231) and lung (A549) cancer cells. Importantly, our current findings demonstrate, and for the first time, that MH binds directly and competitively to IL-6Rα protein, competing out the binding of IL-6 ligand. To the extent of the limited receptor types tested herein, the binding of MH to IL-6Rα appears to be specific, as no binding to the closely related IL-11Rα or to IL-8R was evident. Moreover, exposure to MH had no effect on the levels of a constitutively active p-Src kinase in MDA-MB-231 and A549 cancer cells. Given that p-Src is induced by different types of growth factors, hormone, and integrin receptors in cancer cells, the lack of its inhibition by MH suggests that the latter does not interfere with ligand binding to multiple receptor types, including PDGF-R, EGF-R, FGF-R, VEGF-R, and IGF-1R [34
]. We conclude that the capacity of MH to inhibit p-STAT3 is most likely a consequence of its ability to bind directly to IL-6Rα protein and interfere with the JAK-STAT3 signaling pathway. However, the possibility that MH may also interfere with other pro-tumorigenic receptors in cancer cells cannot be completely excluded.
The IL-6 signaling pathway plays an important role in linking chronic inflammation to tumorigenesis, being directly involved in both cancer initiation and progression [39
]. The functional activity of IL-6 is dysregulated in a variety of malignancies, including breast, lung, pancreatic, colorectal, gastric, blood, and skin cancers, and high serum IL-6 levels are associated with bad prognosis in cancer patients [40
]. There are two well-characterized ways in which IL-6R signaling takes place. Classical signaling is initiated by the binding of IL-6 to the membrane-bound IL-6Rα chain, which recruits gp130 to form a hexameric IL-6/IL-6R/gp130 complex, ultimately leading to the phosphorylation and activation of STAT3 [43
]. The membrane form of IL-6Rα is expressed only on hepatocytes and some immune cells, such as neutrophils and monocytes/macrophages, and has been suggested to be primarily important for the anti-inflammatory and regenerative activities of IL-6 [44
]. The second type of signaling, termed trans-signaling, is dependent on the release of a soluble form of the IL-6Rα (sIL-6Rα) by either alternative splicing of the IL-6Rα transcript or by proteolytic cleavage of the membrane-bound form. Trans-signaling is initiated when a complex is formed between IL-6 and sIL-6Rα, which subsequently binds to the membrane-expressed gp130 unit and triggers downstream events in the pathway [44
]. Given that gp130 is ubiquitously expressed in many cell types, including cancer cells, trans-signaling is the predominant form of IL-6 signaling in inflammation and cancer [45
]. Since MDA-MB-231 and A549 cancer cells lack surface expression of IL-6Rα (unpublished data), it is likely that MH exerts its anti-tumor effect via binding to sIL-6Rα, inhibiting its association with the IL-6 cytokine and ultimately preventing the triggering of the IL-6 trans-signaling pathway.
There is mounting evidence showing that blocking of IL-6 trans-signaling not only inhibits tumor initiation in animal models but can also interfere with the growth of established tumors [45
]. An inhibitory mAb, Tocilizumab that binds human IL-6R and blocks its binding to IL-6 has been developed and is currently approved for the treatment of autoimmune conditions [50
]. Moreover, several studies have highlighted the potential of using inhibitors of IL-6R in preclinical cancer models [51
]. The current study is the first to demonstrate the ability of natural compounds, such as MH, to bind sIL-6R and interfere with IL-6 trans-signaling in cancer cells.
The potential contribution of flavonoid compounds to the binding of the IL-6Rα protein was also investigated. The data revealed that with the exception of pinocembrin, each of the other major flavonoids in MH (luteolin, quercetin, chrysin, and galangin) is able to bind IL-6Rα and, in the 5–50 μM concentration range, resulting in a moderate, albeit statistically significant, inhibition (30–35%) in the binding of the cognate IL-6 ligand. Molecular docking studies confirmed that, due to the similarity in their structure, each flavonoid compound could bind IL-6Rα at a site that would be predicted to affect ligand binding. Functional studies demonstrated that flavonoid compounds also have a differential capacity to inhibit p-STAT3. In terms of relative IC50, luteolin, galangin and chrysin were most effective in inhibiting p-STAT3 (IC50 of 3.5 μM, 4.4 μM, and 7.7 μM, respectively), while quercetin and pinocembrin were the least effective (IC50 of 51 μM and 70.2 μM, respectively). The fact that pinocembrin exhibited the lowest efficacy in p-STAT3 inhibition correlates well with our inability to detect any binding between this flavonoid and IL-6Rα at the maximum dose used (50 μM). It is important to state that the IC50 estimates based on densitometry quantification of p-STAT3 levels by Western blots are only semi-quantitative. As such, our data serve to solely illustrate the relative potency of the different flavonoids in p-STAT3 inhibition. Taken together, these findings suggest flavonoid compounds may well be responsible for MH-mediated inhibition of the IL-6/STAT3 signaling pathway in human breast and lung cancer cells.
We undertook a comparative analysis of the IC50
for p-STAT3 inhibition of each flavonoid compound when used alone or as part of the MH mixture. This analysis revealed that much higher concentrations of each major flavonoid are needed to affect p-STAT3 levels in breast cancer cells when used in pure form in comparison with the whole MH solution. In fact, based on the known concentration of each flavonoid compound in MH, we estimate that MH as a mixture is 2000 to 160,000-fold more efficacious in blocking STAT3 activity than any flavonoid used individually. These findings are in line with previously published data by other investigators showing that a combination of polyphenols or mixtures of polyphenols with vitamins, amino acids, and other micronutrients exhibited superior anti-cancer activity than individual compounds [55
The capacity of some flavonoids to inhibit p-STAT3 activity has been described. A high dose of quercetin (66 μM) was found to reduce p-STAT3 levels in lung cancer cells after a long incubation period (12–24 h) by an indirect effect on NF-κB activation and IL-6 production [56
]. Similarly, high concentrations (40–80 μM) of chrysin were recently shown to inhibit p-STAT3 via the production of ROS in human bladder cancer cells [57
]. This was observed mostly after 24 h of incubation with chrysin, suggesting an indirect effect. Of note, no evidence for ROS generation was observed in MH-treated MDA-MB-231 human breast cancer cells [23
], indicating that inhibition of p-STAT3 in these cells is largely ROS-independent. Furthermore, several studies reported the capacity of luteolin to inhibit STAT3 in different types of cancer cells, including breast, stomach, lung, liver, pancreas, cervix, and bile duct cancers [58
]. Mechanistically, luteolin appears to directly bind to Hsp90, a molecular chaperone that stabilizes p-STAT3 [59
]. This, in turn, inhibits Hsp90 and promotes the degradation of p-STAT3 [58
]. Whether a similar mechanism could underlie the inhibition observed in MH-treated breast cancer cells is unknown. It is interesting to note; however, that luteolin-mediated inhibitory effects on different cancer cell types were observed at 20–50 μM concentration range (~6–14 μg/mL), which are at least 400–1000-fold higher than the concentration of luteolin in 1% (w/v) MH solution used in the present study [23
]. Given the above findings with the different flavonoids, it is not unreasonable to propose that flavonoids could exert their effects through interfering with IL-6R signaling.
In addition to IL-6, breast and lung cancer cells are known to rely on autocrine signaling by IL-11 for their tumor-associated functions, including survival, invasion, and metastasis [31
]. IL-6 and IL-11 cytokines are closely related and signal exclusively through the signal-transducing receptor gp130 to activate the JAK-STAT3 pathway [65
]. Moreover, available evidence indicates that both IL-6 and IL-11 cytokines are produced constitutively at equivalent levels by breast cancer cells [66
]. Our findings suggest that MH interacts selectively with IL-6Rα and interferes with its ligand binding. Nevertheless, in our model system, exposure of breast cancer cells to MH leads to a rapid loss of >80% of activated p-STAT3 protein [23
], which is also associated with decreased gp130 and p-JAK2 levels. The fact that the substantial loss of p-STAT3 is seen under conditions affecting only IL-6/IL-6R signaling suggests that the contribution of IL-11/IL-11R to maintaining constitutive p-STAT3 levels in these cancer cells is relatively small. Alternatively, these findings may suggest that MH components could act at additional levels downstream of the IL-6R signaling pathway. A case in point is the previous demonstration that luteolin could also interact with Hsp90 and promote the degradation of p-STAT3 [59
]. Therefore, studies in which the effect of MH or its flavonoid components is tested on cancer cells in the presence of exogenous IL-6 or IL-11 could further our understanding of the molecular targets within breast and lung cancer cells.
In conclusion, the present findings identify IL-6Rα as a selective target for MH and its flavonoid constituents. This is based on three lines of evidence. First, MH binds to and interferes with the ligand binding to IL-6Rα, but not the closely related IL-11Rα or IL-8R proteins. Second, in silico docking studies reveal favorable binding to IL-6Rα at sites predicted to affect ligand binding. Third, MH does not inhibit tyrosine phosphorylation of c-Src kinase, another major proto-oncogene in a variety of human cancers. Since p-Src is induced by a large group of growth factor, integrin and hormonal receptors [34
], the absence of any inhibition by MH suggests a lack of association with these other receptors/signaling pathways. Identification of the molecular targets of MH and characterization of the mechanisms underlying its inhibitory effects on cancer cells will lead to a better understanding of the applicability of, and the most appropriate approach for the use of, MH and/or its bioactive constituents as therapeutic agents in cancer treatment [68