Figures & data
Figure 1. Combination of cladribine and entinostat significantly induces growth inhibition of MM cells, and is synergistic over a wide range of concentrations. Human MM cells were plated onto 96-well plates with fresh RPMI1640 medium (2.5% FBS) or same medium containing indicated concentrations of cladribine (Clad) or entinostat (Ent) or their combinations (Combo) with a fixed ratio for 48 hrs. The percentages of surviving cells as compared to controls, defined as 100% survival, were determined by reduction of MTS. Data shows the representative of three independent experiments. Bars, SD. P values vs single agent treatments. The combination index (CI) curves were calculated using Calcusyn software according to the Chou–Talalay equation. (a) RPMI8226, (b) U266, (c) MM1.R.
Figure 2. Combination of cladribine and entinostat causes a profound mitotic catastrophe in MM cells. (a) MM cells were cultured with RPMI1640 (2.5% FBS) in the absence or presence of entinostat (Ent), cladribine (Clad) alone or their combinations for 48 hrs. Cells were collected and subjected to cytospin onto cell slides followed by Giemsa staining and examination under a light microscope (×200 magnification). Arrows indicate the cells with mitotic catastrophe. (b) Cells that showed atypical mitotic figures, multi-nucleation, atypical chromosome clusters, and/or apoptosis were counted against normal cells, and reported in percentage. Data shows the representative of three independent experiments. Bars, SD.
Figure 3. Entinostat and cladribine block cell cycle progression. U226 cells and MM1.R were cultured with RPMI1640 (2.5% FBS) in the absence or presence of entinostat (Ent), cladribine (Clad) alone or the combinations of entinostat and cladribine for 48 hrs. (a) Cells were harvested and subjected to flow cytometric analysis of cell cycle distribution. The percentages of RPMI8226 cells in each cell cycle phase were presented in the table underneath. (b) The bar graph reflects the percentage of cells in G1, S, or G2/M phase of the cell cycle for all three MM cell lines (RPMI8226, U266, and MM1.R). Data shows the representative of three independent experiments.
Figure 4. The combinations of cladribine and entinostat markedly alter expression of several key molecular markers critical for G1-S transition. MM cells were cultured with RPMI1640 (2.5% FBS) in the absence or presence of entinostat (Ent), cladribine (Clad) alone or their combinations for 48 hrs. Cells were collected and subjected to western blot analyses with specific antibody directed against Cyclin D1, E2F-1, p21waf−1, p27kip−1, or β-actin. The densitometry analyses of p21waf-1 and Cyclin D1 signals are shown underneath, and the arbitrary numbers indicate the intensities of each cell line relative to controls, defined as 1.0. (a) RPMI8226; (b) U226; (c) MM1.R.
Figure 5. Combination of cladribine and entinostat significantly promotes MM cells undergoing apoptosis. MM cells were cultured with RPMI1640 (2.5% FBS) in the absence or presence of either entinostat (Ent) or cladribine (Clad) alone, or their combinations for 48 hrs. Cells were collected and subjected to apoptotic ELISA or western blot analyses with specific antibody directed against PARP (F-PARP, full length PARP; C-PARP, cleaved PARP), caspase-3 (F-Casp-3, full length caspase-3; C-Casp-3, cleaved caspase-3), caspase-8 (F-Casp-8, full length caspase-8; C-Casp-8, cleaved caspase-8), caspase-9 (F-Casp-9, full length caspase-9; C-Casp-9, cleaved caspase-9), or β-actin. Bars, SD. (a) RPMI8226; (b) U226; (c) MM1.R.
Figure 6. The combinations of cladribine and entinostat potently influence expression of the molecular markers involving in DNA damage response. MM cells were cultured with RPMI1640 (2.5% FBS) in the absence or presence of entinostat (Ent), cladribine (Clad) alone or their combinations for 48 hrs. Cells were collected and subjected to western blot analyses with specific antibody directed against P-H2A.X, P-Chk1, Chk1, P-Chk2, Chk2, or β-actin. (a) RPMI8226; (b) U226; (c) MM1.R.
