A Unique Anti-Cancer 3-Styrylchromone Suppresses Inflammatory Response via HMGB1-RAGE Signaling

Abstract

Background: High mobility group box 1 (HMGB1)-receptor for advanced glycation endo-products (RAGE) axis serves as a key player in linking inflammation and carcinogenesis. Recently, papaverine was revealed to suppress the HMGB1-RAGE inflammatory signaling pathway and cancer cell proliferation. Therefore, a dual suppressor targeting this axis is expected to become a new type of therapeutic agent to treat cancer. Methods: Papaverine 3D pharmacophore mimetic compounds were selected by the LigandScout software from our in-house, anti-cancer chemical library and assessed for their anti-inflammatory activities by a HMGB1-RAGE-mediated interleukin-6 production assay using macrophage-like RAW264.7 cells. Molecular-biological analyses, such as Western blotting, were performed to clarify the mechanism of action. Results: A unique 6-methoxy-3-hydroxy-styrylchromone was found to possess potent anti-inflammatory and anti-cancer activities via the suppression of the HMGB1-RAGE-extracellular signal-regulated kinase 1/2 signaling pathway. Furthermore, the 3D pharmacophore-activity relationship analyses revealed that the hydroxyl group at the C4' position of the benzene ring in a 3-styryl moiety was significant in its dual suppressive effects. Conclusions: These findings indicated that this compound may provide a valuable scaffold for the development of a new type of anti-cancer drug possessing anti-inflammatory activity and as a tool for understanding the link between inflammation and carcinogenesis.

Keywords: 3-styrylchromone; ERK 1/2; HMGB1; RAGE; carcinogenesis; inflammation; tumor microenvironment.

Figure 1

Schematic illustrating the functional role of receptor for the advanced glycation endo-products (RAGE)-RAGE ligand signaling in linking inflammation to carcinogenesis. In a microenvironment of tumor, where cancer cells up-regulate NF-κB expression by hypoxia-inducible factor-1α (HIF-1α), an inflammatory tumor microenvironment (TME) is established by the secretion of pro-inflammatory cytokines, such as IL-6 and TNF-α, and thereby the release of RAGE ligands, such as HMGB1 and S100s, from recruited macrophages and endothelial cells. The RAGE-RAGE ligand signaling activates inflammation in feedforward loops, resulting in the recruitment of NK and T cells, and further myeloid-derived suppressor cells (MDSCs). MDSCs suppress NK and T cells, leading to T cell tolerance and impaired anti-cancer immunity.

Figure 2

Structures of papaverine and five 6M3SC derivatives.

Figure 3

Anti-inflammatory activities of 6M3SC derivatives in HMGB1-stimulated RAW264.7 cells. (a) The cells were treated with indicated concentrations of each compound: 0 (open circle), 1 (open triangle), 2 (closed square), 3 (closed circle), and 4 (closed triangle). The IL-6 production (% of control) was measured using the IL-6 ELISA system, as described in the Materials and Methods section. Values are mean ± standard error (SE) for four independent experiments, and the bars indicate the SE values. (b) Cell viability was determined by the WST-8 assay, as described in the Material and Methods section. The data are presented as mean ± SE of four independent experiments and * p < 0.05 was accepted as a significant difference compared with untreated samples for compound 3.

Figure 4

Suppressive effect of compound 3 on the HMGB1-induced activation of ERK 1/2. RAW264.7 cells were pretreated with compound 3 at the indicated concentrations for 2 h and then stimulated with HMGB1 (5 μg/mL) for 18 h. Cell lysates were subjected to Western blotting using phospho-ERK 1/2 (P-ERK 1/2) and total ERK 1/2 antibodies as described in the Materials and Methods section. GAPDH served as a loading control. The panels depicted a typical immunoblot (a) and values presented as mean ratio of three independent experiments of each P-ERK 1/2, ERK 1/2, and GAPDH band intensities were calculated by densitometric analysis (b). * p < 0.05 was accepted as a significant difference.

Figure 5

Structural characteristics of four 6M3SC derivatives analyzed by the LigandScout software. The 3D pharmacophore models and 2D characteristics of four 6M3SC compounds were analyzed by the LigandScout software. AR, Aromatic; H, hydrophobic; HBA, hydrogen-bond acceptor; HBD, hydrogen-bond donor. Oxygen and nitrogen atoms are illustrated with red and blue sticks, respectively, and the carbon atoms of these compounds are depicted by gray sticks. Aromatic rings, double bonds, and methoxy groups are illustrated with blue circles, yellow, and green, respectively.

Figure 6

Designing a new lead scaffold for the development of novel dual pharmaceuticals possessing anti-cancer and anti-inflammatory activities. The two sections of the 3-styrylchromone molecule, the heterocyclic chromone, and 3-styryl benzene ring moieties are subjected to structure optimization studies for functional groups of R1 and R2, respectively.

Figure 7

Schematic illustrating the suppressive effect of 6M3HSC (compound 3) against the RAGE-RAGE ligand signaling pathway. A potential mechanism of the suppressive effect of this compound 3 on HMGB1-RAGE-ERK 1/2 signaling pathway is shown. The interactions of multi-ligands with RAGE up-regulate the RAGE downstream signaling pathway. Consequently, RAGE-regulated genetic programs of pro-inflammation and carcinogenesis (TEM and oncogenesis) or cell repair and tissue regeneration (homeostasis and ontogenesis) start driving by the action of transcription factors, such as NF-κB and AP-1. RAGE structure includes the variable (V), constant (C₁ and C₂) domains, the transmembrane region (TM), and the cytoplasmic tail (CT).