Figure 5
From: FTY720 in resistant human epidermal growth factor receptor 2-positive breast cancer
FTY720 overcomes resistance to trastuzumab by influencing the regulation of apoptosis and autophagy. (a) HCC1954 cells were collected and prepared 24 h after FTY720 treatment. Cell morphology was assessed using an electron microscope. (b) Autophagy-related proteins, p62 and LC3-II, were analyzed after HCC1954 cells were treated with FTY720, rapamycin, and bafilomycin A1. (c) The protein stability test was performed using cycloheximide chase assays after treatment with the indicated drugs in HCC1954 cells. Lysates were collected every hour up to 5 h after cycloheximide treatment. (d) mRNA levels of p62 in HCC1954 cells were evaluated using reverse transcription quantitative real-time PCR 0 and 2 h after FTY720 incubation. (e) Cells were treated with 3-methyladenine or bafilomycin A1 with or without FTY720. (f) Cells were treated with the pan caspase inhibitor Z-VAD-FMK, FTY720, or a combination of both drugs. In the combination group, cells were pretreated with Z-VAD-FMK for 1 h followed by FTY720 treatment. (g) Lysates were collected and analyzed 24 h after FTY720 treatment. In the combination group, cells were treated with Z-VAD-FMK 1 h before FTY720. Numbers under cleaved caspase-3 section indicate the intensity of the blot pointed by the arrow. All experiments were repeated at least three times except for images captured using the electron microscope. During Western blot analysis, equal loading of proteins was verified using beta-actin, and numbers under each Western blot indicate the intensity of the protein relative to that at 0 h or of the control. During evaluation of antiproliferative effects, the percentage of WST-1 absorbance in cells was measured 72 h after drug incubation. Each plot indicates the mean value, whereas error bars indicate the standard error of the mean. The concentrations of FTY720, rapamycin, bafilomycin A1, 3-methyladenine, and Z-VAD-FMK were 10 μM, 100 nM, 1 nM, 20 μM, and 20 μM, respectively. P value: **P < 0.01 and ***P < 0.001. CHX: cycloheximide, F plus VAD: FTY720 plus Z-VAD-FMK, n.s.: not significant.
