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Inhibition of tumor growth by a newly-identified activator for epidermal fatty acid binding protein.

Rao E, Singh P, Zhai X, Li Y, Zhu G, Zhang Y, Hao J, Chi YI, Brown RE, Cleary MP, Li B - Oncotarget (2015)

Bottom Line: Although EI-05 is unable to bind E-FABP directly, it significantly increases E-FABP expression in macrophages during inflammation.Stimulation of macrophages with EI-05 remarkably enhances lipid droplet formation and IFNβ production, which further promotes the anti-tumor activity of macrophages.Altogether, these results suggest that EI-05 may represent a promising drug candidate for anti-tumor treatment through enhancing E-FABP activity and IFNβ responses in macrophages.

View Article: PubMed Central - PubMed

Affiliation: The Hormel Institute, University of Minnesota, Austin, MN, USA.

ABSTRACT
Our previous studies have demonstrated that expression of epidermal fatty acid binding protein (E-FABP) in tumor associated macrophages (TAMs) promotes macrophage anti-tumor activity by enhancing IFNβ responses in tumor models. Thus, E-FABP represents a new protective factor in enhancing tumor immune surveillance against tumor development. Herein, we report the compound 5-(benzylamino)-2-(3-methylphenyl)-1,3-oxazole-4-carbonitrile (designated EI-05) as a novel E-FABP activator for inhibition of mammary tumor growth. EI-05 was selected from the ZINC compound library using molecular docking analysis based on the crystal structure of E-FABP. Although EI-05 is unable to bind E-FABP directly, it significantly increases E-FABP expression in macrophages during inflammation. Stimulation of macrophages with EI-05 remarkably enhances lipid droplet formation and IFNβ production, which further promotes the anti-tumor activity of macrophages. Importantly, administering EI-05 in vivo significantly inhibits mammary tumor growth in a syngeneic mouse model. Altogether, these results suggest that EI-05 may represent a promising drug candidate for anti-tumor treatment through enhancing E-FABP activity and IFNβ responses in macrophages.

No MeSH data available.


Related in: MedlinePlus

EI-05 treatment promotes IFNβ production in macrophagesGM-BMMs were activated by LPS (10 ng/ml) in the presence or absence of indicated concentrations of EI-05 for 24 h. IFNβ expression in macrophages was quantified by qPCR (A). IFNβ protein levels in cultural supernatants were measured by ELISA (B) (***, p < 0.001 as compared to the control group). (C) Flow cytometric analysis of 7-AAD and annexin V expression on GM-BMMs treated with indicated concentrations of EI-05 for 24 h. (D-E) E-FABP WT and KO macrophage cell lines were treated activated by LPS (10ng/ml) in the presence of EI-05 or DMSO control for 3h. Expression of E-FABP (D) and IFNβ (E) was analyzed by realtime PCR (**, p < 0.01 as compared to WT macrophages). (F) Confocal microscopy analysis of lipid droplet formation (BODIPY) in BM-GMMs with designated treatment with LPS (10 ng/ml), EI-05 (20 μM) or Triacsin C (5 μM). (G) Measurement of IFNβ levels in cultural supernatants of GM-BMMs with indicated treatment.
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Figure 3: EI-05 treatment promotes IFNβ production in macrophagesGM-BMMs were activated by LPS (10 ng/ml) in the presence or absence of indicated concentrations of EI-05 for 24 h. IFNβ expression in macrophages was quantified by qPCR (A). IFNβ protein levels in cultural supernatants were measured by ELISA (B) (***, p < 0.001 as compared to the control group). (C) Flow cytometric analysis of 7-AAD and annexin V expression on GM-BMMs treated with indicated concentrations of EI-05 for 24 h. (D-E) E-FABP WT and KO macrophage cell lines were treated activated by LPS (10ng/ml) in the presence of EI-05 or DMSO control for 3h. Expression of E-FABP (D) and IFNβ (E) was analyzed by realtime PCR (**, p < 0.01 as compared to WT macrophages). (F) Confocal microscopy analysis of lipid droplet formation (BODIPY) in BM-GMMs with designated treatment with LPS (10 ng/ml), EI-05 (20 μM) or Triacsin C (5 μM). (G) Measurement of IFNβ levels in cultural supernatants of GM-BMMs with indicated treatment.

Mentions: As E-FABP expression in TAMs can promote IFNβ responses [8], we next analyzed whether EI-05 treatment impacts IFNβ production in macrophages. Indeed, addition of EI-05 greatly enhanced IFNβ mRNA levels in LPS-activated GM-BMMs (Figure 3A) in a dose-dependent manner. Similarly, IFNβ protein levels in the culture supernatants were also positively elevated in response to increasing concentrations of EI-05 (Figure 3B). As leaking DNA from cellular damage can induce IFNβ production [17], we analyzed the cytotoxicity of EI-05 on macrophages, and demonstrated a minimal impact of EI-05 on macrophage death (Figure 3C), suggesting that a specific effect of IFNβ production was induced by EI-05. When we measured IFNβ production using E-FABP WT and KO macrophages, we found that EI-05 treatment promoted E-FABP and IFNβ production in the WT cells, but not in the E-FABP KO cells (Figure 3D, 3E), indicating an E-FABP-dependent effect for EI-05-induced IFNβ production in macrophages. In our previous studies, we have shown that E-FABP-promoted lipid droplet (LD) formation was positively associated with IFNβ production [8]. It is likely that EI-05 treatment may promote IFNβ production through E-FABP-promoted LD formation. To this end, we measured the impact of EI-05 on LD formation in macrophages. Confocal microscope analysis showed that EI-05 greatly upregulated LD formation in macrophages (Figure 3F). In agreement with our previous results, EI-05-enhanced LD formation and IFNβ production were dramatically inhibited by Tracsin C, a specific LD inhibitor (Figure 3G), further indicating the importance of LDs in mediating the production of IFNβ in macrophages. Of note, EI-05 treatment did not affect the expression of other FABP members, such as L-FABP and A-FABP, and the production of other tumor-related cytokines, such as TNF-α, IL-6, IL-10, IL-12, iNOS, etc (Figure 4A–4H). When we further analyzed the production of IFNβ and other cytokines by peritoneal macrophages (PEMs), we confirmed that that EI-05 also enhanced IFNβ production in these physiologic populations (Figure 4I, 4J). These results indicate that EI-05 treatment greatly promotes E-FABP-mediated IFNβ production in macrophages.


Inhibition of tumor growth by a newly-identified activator for epidermal fatty acid binding protein.

Rao E, Singh P, Zhai X, Li Y, Zhu G, Zhang Y, Hao J, Chi YI, Brown RE, Cleary MP, Li B - Oncotarget (2015)

EI-05 treatment promotes IFNβ production in macrophagesGM-BMMs were activated by LPS (10 ng/ml) in the presence or absence of indicated concentrations of EI-05 for 24 h. IFNβ expression in macrophages was quantified by qPCR (A). IFNβ protein levels in cultural supernatants were measured by ELISA (B) (***, p < 0.001 as compared to the control group). (C) Flow cytometric analysis of 7-AAD and annexin V expression on GM-BMMs treated with indicated concentrations of EI-05 for 24 h. (D-E) E-FABP WT and KO macrophage cell lines were treated activated by LPS (10ng/ml) in the presence of EI-05 or DMSO control for 3h. Expression of E-FABP (D) and IFNβ (E) was analyzed by realtime PCR (**, p < 0.01 as compared to WT macrophages). (F) Confocal microscopy analysis of lipid droplet formation (BODIPY) in BM-GMMs with designated treatment with LPS (10 ng/ml), EI-05 (20 μM) or Triacsin C (5 μM). (G) Measurement of IFNβ levels in cultural supernatants of GM-BMMs with indicated treatment.
© Copyright Policy - open-access
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4480718&req=5

Figure 3: EI-05 treatment promotes IFNβ production in macrophagesGM-BMMs were activated by LPS (10 ng/ml) in the presence or absence of indicated concentrations of EI-05 for 24 h. IFNβ expression in macrophages was quantified by qPCR (A). IFNβ protein levels in cultural supernatants were measured by ELISA (B) (***, p < 0.001 as compared to the control group). (C) Flow cytometric analysis of 7-AAD and annexin V expression on GM-BMMs treated with indicated concentrations of EI-05 for 24 h. (D-E) E-FABP WT and KO macrophage cell lines were treated activated by LPS (10ng/ml) in the presence of EI-05 or DMSO control for 3h. Expression of E-FABP (D) and IFNβ (E) was analyzed by realtime PCR (**, p < 0.01 as compared to WT macrophages). (F) Confocal microscopy analysis of lipid droplet formation (BODIPY) in BM-GMMs with designated treatment with LPS (10 ng/ml), EI-05 (20 μM) or Triacsin C (5 μM). (G) Measurement of IFNβ levels in cultural supernatants of GM-BMMs with indicated treatment.
Mentions: As E-FABP expression in TAMs can promote IFNβ responses [8], we next analyzed whether EI-05 treatment impacts IFNβ production in macrophages. Indeed, addition of EI-05 greatly enhanced IFNβ mRNA levels in LPS-activated GM-BMMs (Figure 3A) in a dose-dependent manner. Similarly, IFNβ protein levels in the culture supernatants were also positively elevated in response to increasing concentrations of EI-05 (Figure 3B). As leaking DNA from cellular damage can induce IFNβ production [17], we analyzed the cytotoxicity of EI-05 on macrophages, and demonstrated a minimal impact of EI-05 on macrophage death (Figure 3C), suggesting that a specific effect of IFNβ production was induced by EI-05. When we measured IFNβ production using E-FABP WT and KO macrophages, we found that EI-05 treatment promoted E-FABP and IFNβ production in the WT cells, but not in the E-FABP KO cells (Figure 3D, 3E), indicating an E-FABP-dependent effect for EI-05-induced IFNβ production in macrophages. In our previous studies, we have shown that E-FABP-promoted lipid droplet (LD) formation was positively associated with IFNβ production [8]. It is likely that EI-05 treatment may promote IFNβ production through E-FABP-promoted LD formation. To this end, we measured the impact of EI-05 on LD formation in macrophages. Confocal microscope analysis showed that EI-05 greatly upregulated LD formation in macrophages (Figure 3F). In agreement with our previous results, EI-05-enhanced LD formation and IFNβ production were dramatically inhibited by Tracsin C, a specific LD inhibitor (Figure 3G), further indicating the importance of LDs in mediating the production of IFNβ in macrophages. Of note, EI-05 treatment did not affect the expression of other FABP members, such as L-FABP and A-FABP, and the production of other tumor-related cytokines, such as TNF-α, IL-6, IL-10, IL-12, iNOS, etc (Figure 4A–4H). When we further analyzed the production of IFNβ and other cytokines by peritoneal macrophages (PEMs), we confirmed that that EI-05 also enhanced IFNβ production in these physiologic populations (Figure 4I, 4J). These results indicate that EI-05 treatment greatly promotes E-FABP-mediated IFNβ production in macrophages.

Bottom Line: Although EI-05 is unable to bind E-FABP directly, it significantly increases E-FABP expression in macrophages during inflammation.Stimulation of macrophages with EI-05 remarkably enhances lipid droplet formation and IFNβ production, which further promotes the anti-tumor activity of macrophages.Altogether, these results suggest that EI-05 may represent a promising drug candidate for anti-tumor treatment through enhancing E-FABP activity and IFNβ responses in macrophages.

View Article: PubMed Central - PubMed

Affiliation: The Hormel Institute, University of Minnesota, Austin, MN, USA.

ABSTRACT
Our previous studies have demonstrated that expression of epidermal fatty acid binding protein (E-FABP) in tumor associated macrophages (TAMs) promotes macrophage anti-tumor activity by enhancing IFNβ responses in tumor models. Thus, E-FABP represents a new protective factor in enhancing tumor immune surveillance against tumor development. Herein, we report the compound 5-(benzylamino)-2-(3-methylphenyl)-1,3-oxazole-4-carbonitrile (designated EI-05) as a novel E-FABP activator for inhibition of mammary tumor growth. EI-05 was selected from the ZINC compound library using molecular docking analysis based on the crystal structure of E-FABP. Although EI-05 is unable to bind E-FABP directly, it significantly increases E-FABP expression in macrophages during inflammation. Stimulation of macrophages with EI-05 remarkably enhances lipid droplet formation and IFNβ production, which further promotes the anti-tumor activity of macrophages. Importantly, administering EI-05 in vivo significantly inhibits mammary tumor growth in a syngeneic mouse model. Altogether, these results suggest that EI-05 may represent a promising drug candidate for anti-tumor treatment through enhancing E-FABP activity and IFNβ responses in macrophages.

No MeSH data available.


Related in: MedlinePlus