Fig. 6

Genetically and pharmacologically targeting Mcl-1 induces cell death also in trastuzumab-resistant Her2-positive BC cells. a Both genetic depletion of Mcl-1 (siMCL1) and EU-5346 induces apoptosis in trastuzumab-resistant HCC-1954 BC cells. BC cells were transfected with siMCL1 (left) for 2 days or treated with EU-5346 (right) for 3 days and then exposed to hypoxia for 6 hours. b EU-5346 inhibits proliferation in Her2 inhibitor-sensitive and inhibitor-resistant BC cells in a dose-dependent manner. c EU-5346 inhibits spheroid formation in both trastuzumab-sensitive (BT-474) and trastuzumab-resistant (HCC-1954) BC cells. d Combining trastuzumab with siMCL1 in Her2 inhibitor-resistant Her2-positive BC cells does not induce synergistic effects. HCC-1954 cells (5.5 × 10³ cells) were seeded per well in agar-coated 96-well plates prior to siMCL1 transfection. c, d Spheroid formation was assessed in EU-5346-treated or siMCL1-treated and/or trastuzumab-treated and control cells using an inverted fluorescence light microscope. Photographs of spheroid formation (10× magnification) are representative of each group and three independent experiments (left). Spheroid volumes were calculated as described in Materials and methods. Data represent mean ± SD (right). *p <0.05 and **p < 0.001 by Student’s t test. e, f siMCL1 e and EU-5346 f but not siHER2 e induces apoptosis in multidrug/Her2 inhibitor-resistant patient BC cells. BC cells were transfected with siMCL1 or siHER2 e for 2 days or treated with EU-5346 f for 3 days and then exposed to hypoxia for 6 hours. a, e, f Whole-cell extracts were analyzed by immunoblotting with indicated antibodies. Immunoblotting for Erk2 confirmed equal protein loading. g EU-5346 inhibits proliferation in multidrug/Her2 inhibitor-resistant patient BC cells in a dose-dependent manner. b, g BC cells were treated with EU-5346 for 3 days under hypoxic conditions. [³H]-thymidine was added during the last 8 hours. Data represent mean ± SD for triplicate samples. Results shown are representative of three independent experiments. Erk2 extracellular signal-regulated kinase 2, Her2 human epidermal growth factor receptor 2, Hif hypoxia-inducible factor, kD kilodalton, Mcl-1 myeloid cell leukemia-1, PARP Poly (ADP-ribose) polymerase