Fig. 3.

SPA70 reduces the metabolism and enhances the efficacy of chemotherapeutic agents. a, b SPA70 reduces the metabolism of paclitaxel (PTX) in primary human hepatocytes (PHHs). In a, PHHs were pretreated for 72 h with 0.1% DMSO, 1.6 µM RIF or 0.5 µM or 5 µM PTX in the presence or absence of 10 µM SPA70 before receiving 3.3 µM midazolam (MDZ) and being incubated for a further 4 h. The concentrations of 1′-OH MDZ (an MDZ metabolite) in the medium for each treatment were determined using LC/MS/MS. In b, PHHs were treated with 1 µM PTX in the presence or absence of 10 µM SPA70. The concentrations of PTX metabolites, 3-p-OH PTX (generated by CYP3A4) and 6α-OH PTX (generated by CYP2C8), were determined using LC/MS/MS. Each data point represents the compound concentration measured after 24 or 48 h of treatment. Data are expressed as the mean and 95% confidence interval (CI). c, d hPXR levels inversely correlate with the sensitivity of cancer cells to PTX. In c, LS180 cells with (hPXR overexpression) or without (parental cells) stable exogenous expression of hPXR were treated with PTX in the presence or absence of 10 µM SPA70 for 96 h before a CellTiter-Glo assay was performed. The vehicle was DMSO. In d, SNU-C4 cells treated with pooled siRNAs (siPXR_pool) or individual siRNAs (siPXR_1, 2, 3 and 4) targeting hPXR, or with nontargeting controls (siNT) with or without SPA70, were treated with PTX for 96 h before a CellTiter-Glo assay was performed. The ‘% normalized growth’ was determined by normalizing the luminescent signal from compound-treated cells to that from DMSO-treated cells. The drug concentration is expressed using a log scale. Final DMSO concentration was 0.2% in a–d. The half-maximal inhibitory concentration (IC₅₀) is indicated. Data are presented as the means ± S.E.M. (n = 4 in c and n = 3 in d). *P < 0.05, **P < 0.01 or ****P < 0.0001 for each group relative to the parental cells/vehicle in c or siNT-treated cells in d. Statistical significance was calculated by one-way analysis of variance with Tukey’s multiple-comparison test