CPX-351 activates the protective AhR pathway. C57BL/6 mice were treated intravenously with the “7 + 3” combination or CPX-351 every 3 days for 3 times and assessed for (A) AhR-related genes expression in the ileum, (B) AhR immunofluorescence, and (C) IL-22 production in colon. Yellow arrows indicate AhR+ cells. AhR–/– mice were treated as above and assessed for (D) weight loss (daily measured), (E) intestinal histopathology (PAS staining) with the relative histology score, (F) bacterial translocation to mesenteric lymph nodes, and (G) Il10 gene expression in the colon. (H) Viability of CaCo-2 cells exposed to cytarabine, daunorubicin, alone, or in combination, or CPX-351 by MTT assay. (I) ZO-1 expression in differentiated CaCo-2 cells exposed as above for 24 hours. (J) Transepithelial electrical resistance (TEER) and (K) apparent permeability coefficient (Papp) of CaCo-2 cells exposed to 10 μM cytarabine, 2 μM dauno, 10 μM CPX-351, and 10 μM empty liposomes in the presence of 25 ng/mL lipopolysaccharide (LPS). The dotted line indicates untreated cells. (L) AhR-related gene expression by reverse transcription polymerase chain reaction and (M) AhR nuclear translocation by immunofluorescence in CaCo-2 cells treated as above for 4 hours (FICZ was used as control). (N) AhR activity by the luciferase assay on the reporter cell line H1L6.1c3. (O) C57BL/6 mice were concurrently treated with empty liposomes or indole-3-aldehyde (3-IAld) and the “7 + 3” combination and evaluated for (P) weight loss, (Q) bacterial translocation to mesenteric lymph nodes, and (R) Il22 and Il10 gene expression in the colon. Photographs were taken with a high-resolution microscope (Olympus BX51); original magnification ×40 (B,E; scale bars, 100 μm) and ×100 (I,M; scale bars, 20 μm). For immunofluorescence, nuclei were counterstained with Hoechst. Numbers refer to colocalization coefficients to quantify the overlap degree of AhR and Hoechst. Data are representative of 2 or 3 independent experiments. Each in vivo experiment includes 6 mice per group. Data are represented as mean ± SEM. ∗P < .05; ∗∗P < .01; ∗∗∗P < .001; ∗∗∗∗P < .0001; CPX-351- vs “7 + 3”–treated mice or cells; “7 + 3”- or liposome or 3-IAld vs none (untreated) cells; “7 + 3”- vs FICZ-treated cells; CPX-351 vs liposome-treated cells; liposome + 3-IAld vs 3-IAld- or liposome-treated cells; “7 + 3” + liposome or “7 + 3” + 3-IAld- vs “7+3”–treated mice. One-way or 2-way ANOVA, Bonferroni multiple comparison test.

CPX-351 activates the protective AhR pathway. C57BL/6 mice were treated intravenously with the “7 + 3” combination or CPX-351 every 3 days for 3 times and assessed for (A) AhR-related genes expression in the ileum, (B) AhR immunofluorescence, and (C) IL-22 production in colon. Yellow arrows indicate AhR+ cells. AhR–/– mice were treated as above and assessed for (D) weight loss (daily measured), (E) intestinal histopathology (PAS staining) with the relative histology score, (F) bacterial translocation to mesenteric lymph nodes, and (G) Il10 gene expression in the colon. (H) Viability of CaCo-2 cells exposed to cytarabine, daunorubicin, alone, or in combination, or CPX-351 by MTT assay. (I) ZO-1 expression in differentiated CaCo-2 cells exposed as above for 24 hours. (J) Transepithelial electrical resistance (TEER) and (K) apparent permeability coefficient (Papp) of CaCo-2 cells exposed to 10 μM cytarabine, 2 μM dauno, 10 μM CPX-351, and 10 μM empty liposomes in the presence of 25 ng/mL lipopolysaccharide (LPS). The dotted line indicates untreated cells. (L) AhR-related gene expression by reverse transcription polymerase chain reaction and (M) AhR nuclear translocation by immunofluorescence in CaCo-2 cells treated as above for 4 hours (FICZ was used as control). (N) AhR activity by the luciferase assay on the reporter cell line H1L6.1c3. (O) C57BL/6 mice were concurrently treated with empty liposomes or indole-3-aldehyde (3-IAld) and the “7 + 3” combination and evaluated for (P) weight loss, (Q) bacterial translocation to mesenteric lymph nodes, and (R) Il22 and Il10 gene expression in the colon. Photographs were taken with a high-resolution microscope (Olympus BX51); original magnification ×40 (B,E; scale bars, 100 μm) and ×100 (I,M; scale bars, 20 μm). For immunofluorescence, nuclei were counterstained with Hoechst. Numbers refer to colocalization coefficients to quantify the overlap degree of AhR and Hoechst. Data are representative of 2 or 3 independent experiments. Each in vivo experiment includes 6 mice per group. Data are represented as mean ± SEM. ∗P < .05; ∗∗P < .01; ∗∗∗P < .001; ∗∗∗∗P < .0001; CPX-351- vs “7 + 3”–treated mice or cells; “7 + 3”- or liposome or 3-IAld vs none (untreated) cells; “7 + 3”- vs FICZ-treated cells; CPX-351 vs liposome-treated cells; liposome + 3-IAld vs 3-IAld- or liposome-treated cells; “7 + 3” + liposome or “7 + 3” + 3-IAld- vs “7+3”–treated mice. One-way or 2-way ANOVA, Bonferroni multiple comparison test.

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