Figure 2 from ZSTK3744, a Novel Aryl Hydrocarbon Receptor Agonist, Exhibits Efficacy against Chemotherapy-Resistant Triple-Negative Breast Cancer

Screening of antitumor agents for chemotherapy-resistant TNBC cells. A, The chemical structures of ZSTK3744 and ZSTK3341, which were screened from our compound library for their cell growth–inhibitory activity against chemotherapy-resistant cells. B, Parental MM468, MM468/AR, and MM468/PR cells were treated with ZSTK3744, ZSTK3341, adriamycin, paclitaxel, or eribulin at the indicated concentrations for 72 hours. Cell viability was assessed using the Cell Counting Kit-8 assay (mean ± SD of quadruplicate experiments). The mRNA levels of CYP1A1 (C) and TIPARP (D) were measured by qRT-PCR using cDNA synthesized from total RNA extracted from MM468 cells treated with DMSO or ZSTK3744. Data are normalized to control genes 18S rRNA and presented as mean ± SD from quadruplicate experiments. Statistical analysis was performed using the Mann–Whitney U test (*, P < 0.05 vs. DMSO for all time points). E, MM468 cells were treated with ZSTK3744 or in combination with the AhR antagonist CH223191 (1 and 10 μmol/L) for 72 hours, and cell viability was assessed using a Cell Counting Kit-8 assay (mean ± SD of quadruplicate experiments). Statistical analysis was performed using one-way ANOVA, followed by a Dunnett test (*, P < 0.05 vs. DMSO for all concentrations). F, AhR and GAPDH protein expression levels were analyzed using Western blotting in parental T47D (wild type) and AhR-KO T47D cells. GAPDH was used as a loading control. G, CYP1A1 and CYP1B1 activity induced by ZSTK3341 at the indicated dose in CYP1A1-KO T47D and parental cells. Enzyme activity was assessed using the P450-Glo CYP1A1 Assay System. CYP1A1/CYP1B1 activity was measured based on chemiluminescence intensity, which was determined using SpectraMax iD3. The results are presented as mean ± SD of quadruplicate experiments. Statistical analysis was performed using the Mann–Whitney U test (*, P < 0.05 vs. parental T47D cells). H and I, T47D wild type (blue), CYP1A1-KO T47D (green), and AhR-KO T47D (orange) cells were treated with ZSTK3341 (H) and ZSTK3744 (I) at the indicated concentrations for 72 hours, and cell viability was assessed using a Cell Counting Kit-8 assay (mean ± SD of quadruplicate experiments). Statistical analysis was performed using the Mann–Whitney U test (*, P < 0.05; **, P < 0.01 vs. respective CYP1A1-KO T47D cells at each concentration; #P < 0.05, ##P < 0.01 vs. respective AhR-KO T47D cells at each concentration). J, AhR peptide was captured on the sensor chip, followed by ZSTK3744 injection at the indicated concentration. wt, wild type.