Table 1.
Differential antiproliferative activity of AFP464 in human breast cancer cell lines.
Table 2.
Sensitivity of MCF-7 and MDA-MB-231 (with stable transfection of vector, wild type ERα, or mutant ERα) cell lines to AFP464 in the absence or presence of 17β-estradiol (E2) or endoxifena.
Figure 1.
Cytoplasmic localization of the AhR was associated with the sensitivity of breast cancer cell lines to AFP464.
(A) Western blot shows constitutive expression of ERα and AhR in AFP464-sensitive (MCF-7, SUM44, MDA-MB-468, and BT20) and -resistant (MDA-MB-231, and Hs578T) human breast cancer cell lines (Lanes 1–6, respectively). (B) Immunofluorescence staining of the cellular localization of ERα and AhR in AFP464-sensitive and -resistant human breast cancer cell lines. The blue DAPI, red TRITC, and green FITC staining indicates positive staining for the nucleus, AhR, and ERα, respectively.
Figure 2.
Immunofluorescence staining of AhR and γ-H2AX (a biomarker for DNA double-strand breaks) showed that stable transfection of ERα to MDA-MB-231 cells resulted in translocalization of the AhR from the nucleus to the cytoplasm and rendered cells sensitive to AFP464.
The AF-sensitive, ERα-positive MCF-7 cell line was used as a positive control. The AhR was localized predominantly in the cytoplasm in MCF-7 and MDA-MB-231/wtERα cells, but was localized in the nucleus in the parental or empty vector-transfected MDA-MB-231 cells. Nuclear γ-H2AX foci formed in MDA-MB-231/wtERα cells but not in the parental or empty vector-transfected cells when the cell lines were treated with AFP464 at their respective IC50 values for 24 h. The blue DAPI, red TRITC, and green FITC staining indicates positive staining for the nucleus, AhR, and γ-H2AX foci, respectively.
Figure 3.
Vorinostat sensitized mesenchymal-like TNBC MDA-MB-231 and Hs578T cells to AFP464 by, at least in part, reactivating ERα expression and restoring AhR responsiveness to AF, as indicated by transcriptional induction of CYP1A1.
(A) Fraction-affected (Fa) versus combination index (CI) plots of sequential or simultaneous treatment with vorinostat (V) and AFP464 (AFP) in MDA-MB-231 and Hs578T cells. AFP464 and vorinostat were combined at a fixed concentration ratio of 5∶1 for both cell lines. The open triangles represent experimental CI values, and the solid lines represent simulated CI values, both of which were calculated by Calcusyn software based on the mean cell proliferation data from two independent experiments. CI >1.0, CI = 1.0, and CI <1.0 indicates antagonistic, additive, and synergistic effects, respectively. (B) Western blot of ERα and AhR in control and vorinostat-treated MDA-MB-231 and Hs578T cells. Cells were grown in phenol red-free medium supplemented with charcoal-stripped FBS for 1 week prior to the treatment. MDA-MB-231 and Hs578T cells were treated with vorinostat at their respective IC50 values (2.5 and 8 µM) for 6, 12, or 24 h (Lanes 3, 4, and 5, respectively) followed by incubation in fresh medium containing E2 (100 nM) for an additional 24 h. (C) Immunofluorescence staining of ERα and AhR in control and vorinostat-treated MDA-MB-231 and Hs578T cells. The cells were treated in the same way as for western blot analysis. The blue DAPI, red TRITC, and green FITC staining indicates positive staining for the nucleus, AhR, and ERα, respectively. (D) Real-time RT-PCR determination of relative mRNA levels of ERα, CYP1A1, and SULT1A1 in MDA-MB-231 and Hs578T cells treated with vorinostat (V) and AFP464 (AFP) at their respective IC50 values, each alone or in sequential combination. (E) Real-time RT-PCR demonstrated that transient knockdown of ERα in the vorinostat-pretreated MDA-MB-231 and Hs578T cells diminished AhR-dependent transcriptional induction of CYP1A1 and SULT1A1 after AFP464 treatment. MDA-MB-231 and Hs578T cells were pretreated with vorinostat at their respective IC50 values (2.5 or 8 µM) for 24 and 12 h, respectively, and then the cells were incubated with the ERα siRNA:transfection reagent (1∶8) mixture complexes in fresh drug-free medium for 4 h. After that, the cells were re-covered in fresh complete medium for 2 h and then treated with AFP464 at their respective IC50 values (25 or 20 µM) for 24 h.
Figure 4.
Antitumor activity of AFP464 and vorinostat, alone or in combination, in xenograft models using basal A subtype MDA-MB-468 and basal B subtype (or mesenchymal-like TNBC) MDA-MB-231 cells.
(A) Tumor growth in the control and drug treatment groups. Data are expressed as the median tumor volume of 10–14 tumors. *Kruskal-Wallis test, the median tumor volume in the combined treatment group was significantly different from those in the control and AFP464-only treatment groups in the MDA-MB-231 xenograft model, P<0.05. **Kruskal-Wallis test, the median tumor volume for each AFP464 treatment group was significantly different from that of the control group in the MDA-MB-468 xenograft model, P<0.01. (B) Western blot of ERα protein expression in the tumor tissues that were collected from the control and vorinostat/AFP464-treated mice. MDA-MB-231 and MDA-MB-231/wtERα cell lines were used as the ERα-negative and -positive controls, respectively. Lanes 1 and 2 were whole cell lysates from MDA-MB-231 and MDA-MB-231/wtERα cell lines, respectively; lanes 3–5 are MDA-MB-231 xenograft tumor tissue lysates obtained from mice treated with the vehicle solution (control), vorinostat 50 mg/kg, or a combination of vorinostat (50 mg/kg) and AFP464 (35 mg/kg), respectively. (C) Immunohistochemical staining for AhR and ERα in the tumor tissues that were collected from the same mice as those used for the western blot analysis.