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Tumor-associated macrophages (TAM) of M2 phenotype promote tumor proliferation and are associated with a poor prognosis in patients with glioblastoma. We screened the natural compounds possessing an inhibitory effect on M2 polarization in human monocyte-derived macrophages. Among 130 purified natural compounds examined, corosolic acid significantly inhibited the expression of CD163, one of the phenotype markers of M2 macrophages, and also suppressed the secretion of IL-10, one of the anti-inflammatory cytokines preferentially produced by M2 macrophages, thus suggesting that corosolic acid suppresses M2 polarization of macrophages. Furthermore, corosolic acid inhibited the proliferation of glioblastoma cells, U373 and T98G, and the activation of signal transducer and activator of transcription-3 (STAT3) and nuclear factor-kappa B (NF-κB) in both human macrophages and glioblastoma cells. These results indicate that corosolic acid suppresses the M2 polarization of macrophages and tumor cell proliferation by inhibiting both STAT3 and NF-κB activation. Therefore, corosolic acid might be a potential new tool for tumor prevention and therapy. (Cancer Sci 2011; 102: 206–211)
Macrophages infiltrating in cancer tissues are referred to as tumor-associated macrophages (TAM) and they are closely involved in the development of the tumor microenvironment.(1–3) Tumor-associated macrophages are considered to belong to alternatively activated macrophages (M2) because of their anti-inflammatory functions.(4,5) In many kinds of tumors, the presence of TAM is associated with a poor prognosis for patients.(3,6,7)
Macrophage subpopulations have a different type of receptor expression and cytokine production.(5,8–10) Classically activated macrophages (M1 macrophages) have the IL-12high, IL-23high and IL-10low phenotypes and produce nitrogen intermediates and inflammatory cytokines, such as IL-1β, tumor necrosis factor-α (TNF-α) and IL-6.(5,8–11) In contrast, alternatively activated macrophages (M2 macrophages) have the IL-12low, IL-23low and IL-10high phenotypes and also show high expression of several receptors such as the class A scavenger receptor (SR-A, CD204), the mannose receptor, CD163, dectin-1 and DC-SIGN.(5,8–11) Furthermore, it is well known that M1 macrophages are potent effecter cells integrated in Th1 responses, which kill microorganisms and tumor cells and produce copious amounts of proinflammatory cytokines. In contrast, M2 macrophages regulate inflammatory responses and adaptive type I immunity, scavenge debris and promote angiogenesis, tumor progression, tissue remodeling and repair.
We previously demonstrated CD163 to be a useful marker for detecting M2 cells on paraffin-embedded surgical specimens.(12) In human glioblastoma, the proportion of CD163-positive M2 TAM are closely involved in tumor cell proliferation and are also associated with a poor prognosis, whereas the total number of macrophages is not.(13) These observations therefore indicate the significance of macrophage differentiation in tumor development.
Signal transducer and activator of transcription 3 (STAT3) is involved in the tumor microenvironment and tumor development due to its association with immunosuppression, angiogenesis and cancer cell proliferation.(14) Therefore, STAT3 is considered to be an important target molecule for anti-cancer therapy, and many researchers have so far reported the importance of various STAT3 inhibitors in anti-cancer therapy.(15) STAT3 signaling in macrophages is well known to be involved in the regulation of immune responses in the murine model,(16,17) and STAT3 activation is essential for macrophage differentiation toward the M2 phenotype.(18) Furthermore, NF-κB activation plays an important role in macrophage differentiation toward the M2 phenotype(19) and cancer cell proliferation.(20,21)
In this study, we prepared 130 purified compounds from natural products and measured their inhibitory effect on M2 polarization in human monocyte-derived macrophages (HMDM) in order to identify potentially useful candidate agents for cancer immunotherapy.
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It is well known that TAM play an important role in cancer growth. The TAM release many proangiogenic cytokines and growth factors, such as vascular endothelial growth factor (VEGF), epidermal growth factor (EGF), colony stimulation factor-1 (CSF-1), platelet-derived growth factor (PDGF) and basic fibroblast growth factor to promote tumor progression. They also produce arginase-1, IL-10 and transforming growth factor-β (TGF-β), which inhibit the antitumor function of T cells and natural killer cells.(1–3) In our recent study, we revealed that macrophages account for a major share of the cells that infiltrate glioblastomas and their polarization toward an M2 phenotype is significantly associated with a poor prognosis in those patients.(13) We also found that tumor-derived factors induce STAT3 activation in TAM, thereby leading them to differentiate themselves into M2 macrophages (unpublished data). Therefore, it is speculated that the inhibition of macrophage polarization toward the M2 phenotype could thus possibly be a new strategy for anticancer therapy.
In the present study, we used CD163 as a marker of the M2 phenotype of macrophages. CD163, a member of the scavenger receptor cysteine-rich protein superfamily, is a receptor for the hemoglobin–haptoglobin (Hb-Hp) complex, erythroblasts, TNF-like weak inducer of apoptosis (TWEAK), and porcine reproductive and respiratory syndrome virus.(31–34) CD163 also binds bacteria and induces the production of proinflammatory cytokines.(35) It is reported that binding of the Hb-Hp complex to CD163-bearing cells elicited potent interleukin-10 secretion and this was inhibited by the anti-CD163 antibody.(36) It is also reported that CD163 upregulates HO-1 expression.(37) These data indicate that CD163 is actively involved in the anti-inflammatory function of M2 macrophages, although the precise ligand–receptor–effector pathway is not clear yet.
In this study, we prepared 130 purified compounds from natural products and screened their inhibitory effect on the M2 polarization of human monocyte-derived macrophages. In this screening, we identified several natural compounds, such as corosolic acid, tigogenin and aucubin, which all have an inhibitory effect on CD163 expression, which is a maker of the M2 phenotype (Fig. 1). Among these compounds, corosolic acid significantly inhibited CD163 expression (Fig. 1). Therefore, we chose corosolic acid as a candidate agent for anticancer therapy.
Corosolic acid (Fig. 2A), a triterpenoid compound, is contained in several plants such as banaba leaves, Eriobotrya japonica leaves and apples. The compound possesses various biological properties, including anti-diabetic, anti-obesity and anti-oxidative activities.(38–40)
The current study demonstrated that corosolic acid inhibited IL-10- and TCS-induced CD163 expression and TCS-induced IL-10 secretion and enhanced IL-6 and IL-12 secretion reduced by TCS treatment in human macrophages (Fig. 2), whereas corosolic acid did not affect cell survival in HMDM (Fig. 3E), thus suggesting that corosolic acid changes M2 polarization to M1 polarization in human macrophages.
In a previous study, STAT3 and NF-κB activation were revealed to contribute to the M2 polarization of macrophages.(18,41) Corosolic acid significantly suppressed JAK-STAT3 and NF-κB activation in human macrophages (Fig. 4A–C), thus indicating that corosolic acid inhibits M2 polarization by suppressing the JAK-STAT and NF-κB signaling pathway. Recently, it is known that STAT3 and NF-κB activation also correlate with tumor cell proliferation, and various STAT3 inhibitors have been reported by many researchers with the aim of identifying a new anticancer therapy.(15) Corosolic acid inhibited cell proliferation in U373 glioblastoma cells by suppressing both STAT3 and NF-κB activation (Fig. 4D). Furthermore, corosolic acid also suppresses STAT3 activity on osteosarcoma cells and ovarian carcinoma cells (data not shown, manuscript in preparation), and enhanced the anticancer effect of CDDP (Fig. S1), suggesting that the combination of corosolic acid and anticancer agents might be useful for anticancer therapy.
Recently, corosolic acid has been reported to induce apoptosis through the mitochondrial pathway and caspase activation in human cervical carcinoma HeLa cells.(26) In the present study, corosolic acid also induced caspase activation in human glioblastoma cells (Fig. 3F). Therefore, corosolic acid might induce tumor apoptosis by both induction of caspase activation and inhibition of STAT3 and NF-κB activation. Furthermore, corosolic acid also inhibits macrophage polarization of the M2 phenotype associated with tumor proliferation. These data suggest that corosolic acid might be a potentially useful new compound for anticancer therapy.