Discovery of a Novel Microtubule Targeting Agent as an Adjuvant for Cancer Immunotherapy

Figure 2

Lead compound #1, a fused naphthalene, enhances LPS induced cytokine production and sustains NF-B signaling. (a) EC₅₀ for IL-12 and IL-6 production. BMDCs were treated for 18 h with graded concentrations of #1 (from 10 μM) in the presence of 0.5 ng/mL LPS. 0.5 % DMSO was used as a negative control. IL-12 and IL-6 secretion levels were measured by ELISA. EC₅₀ for IL-12 and IL-6 were 0.23 μM and 0.15 μM, respectively. (b) Immunoblot analysis for NF-B signaling pathway in THP-1 cells. THP-1 cells were treated with compound #1 (5 μM) in the presence or absence of 4 ng/mL LPS at indicated time periods; p-NF-B and t-NF-B indicate phospho-NF-B and total-NF-B, respectively. (c) Relative intensity of bands was calculated using ImageJ software. Band intensities were normalized to vehicle 4 h. (d) Nuclear translocation of NF-B by #1. After 2 h and 8 h treatment with 5 μM #1, THP-1 cells were fixed and stained for NF-B (green) and nuclear DNA (blue). The overlap in the merged images appears pale green. Arrowheads indicate nuclear translocation. Bars indicate 10 μm. (e) NF-B targeted gene expression analysis. THP-1 cells were treated with 5 μM #1 plus 4 ng/mL LPS for 4 h and 16 h. Expressions of IL8, IL1B, CCL2, and IL23A were examined by QuantiGene plex assay and the expression was normalized to HPRT mean fluorescence intensity (MFI) (=100). P<0.05, P<0.01, and P<0.0001 by one-way ANOVA with Tukey’s post hoc test. Data are presented as mean ± SD of triplicate and are representative of two independent experiments showing similar results.

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