Down-regulation of FLT3 expression and its downstream molecules in EGC-treated AML cells.

<p>MOLM-13, MOLM-14, MV4-11 and KOCL-48 cells at a density of 1×10⁵ cells/ml were treated with indicated concentration of EGC or DMSO alone as control for 8 hours. Total cell lysates were subjected to western blot analysis with indicated antibodies.</p

Isobolograms of simultaneous exposure to EGCG and PKC412 in MOLM-13, MOLM-14, MV4-11 and KOCL-48 cell lines.

<p>The isobolograms shown are representative of at least three independent experiments. Each point represents the mean value of at least three independent experiments. The combination of EGCG with PKC412 showed additive effect (MOLM-13 and MOLM-14) and antagonism effect (MV4-11 and KOCL-48).</p

EGCG, EGC and ECG suppressed FLT3 expression through Hsp90.

<p>MOLM-13 cells at a density of 1×10⁵ cells/ml were treated with 60 µM EGCG (A, lane 4), 100 µM EGC, 200 µM ECG (B, lane 2, 4) or DMSO alone as control for 8 hours. Total cell lysates were immunoprecipitated with anti-Hsp90. Precipitated protein were subjected to western blot analysis with anti-FLT3 and anti Hsp90. MOLM-13 cells treated with 2 µM 17-AAG was used as control (A, lane 2).</p

Down-regulation of FLT3 expression and its downstream molecules in ECG-treated AML cells.

<p>MOLM-13, MOLM-14, MV4-11 and KOCL-48 cells at a density of 1×10⁵ cells/ml were treated with indicated concentration of ECG or DMSO alone as control for 8 hours. Total cell lysates were subjected to western blot analysis with indicated antibodies.</p

Down-regulation of FLT3 expression and its downstream molecules in EGCG-treated AML cells.

<p>MOLM-13, MOLM-14, MV4-11, KOCL-48 and THP-1 cells at a density of 1×10⁵ cells/ml were treated with indicated concentration of EGCG or DMSO alone as control for 8 hours. Total cell lysates were subjected to western blot analysis with indicated antibodies.</p

Mean values of observed data and predicted minimum and maximum values of the combination of EGCG and PKC412.

<p>Mean values of observed data and predicted minimum and maximum values of the combination of EGCG and PKC412.</p

EGCG, EGC and ECG induced apoptosis in MOLM-14 cells.

<p>MOLM-14 cells at a density of 1×10⁵ cells/ml were treated with 60 µM EGCG, 100 µM EGC, 200 µM ECG or DMSO alone as control for 16 hours. Total cell lysates were subjected to western blot analysis with indicated antibodies (A) or staining with PE-Annexin V and analyzed by FACS Calibur. Collected data were analyzed by FlowJo software (B).</p

Purine Analog-Like Properties of Bendamustine Underlie Rapid Activation of DNA Damage Response and Synergistic Effects with Pyrimidine Analogues in Lymphoid Malignancies

<div><p>Bendamustine has shown considerable clinical activity against indolent lymphoid malignancies as a single agent or in combination with rituximab, but combination with additional anti-cancer drugs may be required for refractory and/or relapsed cases as well as other intractable tumors. In this study, we attempted to determine suitable anti-cancer drugs to be combined with bendamustine for the treatment of mantle cell lymphoma, diffuse large B-cell lymphoma, aggressive lymphomas and multiple myeloma, all of which are relatively resistant to this drug, and investigated the mechanisms underlying synergism. Isobologram analysis revealed that bendamustine had synergistic effects with alkylating agents (4-hydroperoxy-cyclophosphamide, chlorambucil and melphalan) and pyrimidine analogues (cytosine arabinoside, gemcitabine and decitabine) in HBL-2, B104, Namalwa and U266 cell lines, which represent the above entities respectively. In cell cycle analysis, bendamustine induced late S-phase arrest, which was enhanced by 4-hydroperoxy-cyclophosphamide, and potentiated early S-phase arrest by cytosine arabinoside (Ara-C), followed by a robust increase in the size of sub-G1 fractions. Bendamustine was able to elicit DNA damage response and subsequent apoptosis faster and with shorter exposure than other alkylating agents due to rapid intracellular incorporation via equilibrative nucleoside transporters (ENTs). Furthermore, bendamustine increased the expression of ENT1 at both mRNA and protein levels and enhanced the uptake of Ara-C and subsequent increase in Ara-C triphosphate (Ara-CTP) in HBL-2 cells to an extent comparable with the purine analog fludarabine. These purine analog-like properties of bendamustine may underlie favorable combinations with other alkylators and pyrimidine analogues. Our findings may provide a theoretical basis for the development of more effective bendamustine-based combination therapies.</p></div

Bendamustine induces apoptosis faster than other alkylating agents but does not exert sufficient cytotoxicity against all tumors.

<p>A) We cultured the indicated cell lines with various concentrations of bendamustine and measured cell proliferation with the MTT reduction assay after 72 hours. IC50 and IC80 values are defined as the concentrations of drugs that produce 50 and 80% inhibition of cell growth, respectively. The means ± S.D. (bars) of three independent experiments are shown. B) HBL-2 cells were cultured in the absence (−) or presence (+) of the IC50 value of bendamustine (BDM), harvested at the indicated time points, and stained with propidium iodide in preparation for cell cycle analysis. C) HBL-2 cells were cultured in the absence (None) or presence of IC50 values of 4-OHCY or chlorambucil (CB), harvested at the indicated time points, and stained with propidium iodide in preparation for cell cycle analysis. Columns indicate the quantification of cells in each phase of the cell cycle obtained with the ModFitLT 2.0 program. The means ± S.D. (bars) of three independent experiments are shown. <i>P</i>-values were calculated by one-way ANOVA with the Student-Newman-Keuls multiple comparisons test. Asterisks denote <i>p</i><0.05 against the untreated control.</p

Cell cycle effects of the combination of bendamustine with 4-OHCY or cytosine arabinoside.

<p>(A) HBL-2 cells were cultured with bendamustine alone, cytosine arabinoside alone or their combination for 48 hours. (B) HBL-2 cells were cultured with bendamustine alone, 4-OHCY alone or their combination for 48 hours. Cell cycle profiles were obtained by flow cytometry as described in Materials and Methods. The size of the sub-G1 fraction was calculated by analyzing DNA histograms with the ModFitLT 2.0 program. The data shown are representative of multiple independent experiments with various concentrations of the drugs.</p