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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 has no major impact on other tumor-related molecules in macrophages(A-H) GM-BMMs were cultured with indicated treatment (EI-05, 20 μM; LPS 10 ng/ml) for 24 h. The expression of L-FABP, A-FABP and other tumor-related cytokines were quantified by qPCR. PEMs were cultured with indicated treatment (EI-05, 20 μM; LPS 10 ng/ml) for 24 h. Cells were lyzed for mRNA extraction and PCR analysis of relative RNA levels IFNβ (I). The protein levels of IFNβ in the cultural supernatants were measured by ELISA (J) (***, p < 0.001 as compared to the LPS+DMSO group).
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Figure 4: EI-05 treatment has no major impact on other tumor-related molecules in macrophages(A-H) GM-BMMs were cultured with indicated treatment (EI-05, 20 μM; LPS 10 ng/ml) for 24 h. The expression of L-FABP, A-FABP and other tumor-related cytokines were quantified by qPCR. PEMs were cultured with indicated treatment (EI-05, 20 μM; LPS 10 ng/ml) for 24 h. Cells were lyzed for mRNA extraction and PCR analysis of relative RNA levels IFNβ (I). The protein levels of IFNβ in the cultural supernatants were measured by ELISA (J) (***, p < 0.001 as compared to the LPS+DMSO group).

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 has no major impact on other tumor-related molecules in macrophages(A-H) GM-BMMs were cultured with indicated treatment (EI-05, 20 μM; LPS 10 ng/ml) for 24 h. The expression of L-FABP, A-FABP and other tumor-related cytokines were quantified by qPCR. PEMs were cultured with indicated treatment (EI-05, 20 μM; LPS 10 ng/ml) for 24 h. Cells were lyzed for mRNA extraction and PCR analysis of relative RNA levels IFNβ (I). The protein levels of IFNβ in the cultural supernatants were measured by ELISA (J) (***, p < 0.001 as compared to the LPS+DMSO group).
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Figure 4: EI-05 treatment has no major impact on other tumor-related molecules in macrophages(A-H) GM-BMMs were cultured with indicated treatment (EI-05, 20 μM; LPS 10 ng/ml) for 24 h. The expression of L-FABP, A-FABP and other tumor-related cytokines were quantified by qPCR. PEMs were cultured with indicated treatment (EI-05, 20 μM; LPS 10 ng/ml) for 24 h. Cells were lyzed for mRNA extraction and PCR analysis of relative RNA levels IFNβ (I). The protein levels of IFNβ in the cultural supernatants were measured by ELISA (J) (***, p < 0.001 as compared to the LPS+DMSO group).
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