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

Quantitative analysis of the combination of bendamustine and other drugs in lymphoid malignancies.

<p>*Mean values of observed data (S.D. not shown).</p><p>**Mean values of the predicted minimum values for an additive effect (S.D. not shown).</p><p>***Mean values of the predicted maximum values for an additive effect (S.D. not shown).</p>#<p>Overall effect of drug combination (see Materials and Methods for the method of evaluation).</p

The selection of suitable drugs to be combined with bendamustine using isobologram.

<p>Cells were cultured with various concentrations of bendamustine in combination with (A) 4-hydroperoxy-cyclophosphamide (4-HC), (B) cytosine arabinoside (araC), (C) doxorubicin (DOX) and (D) methotrexate (MTX) for 4 days (Namalwa and HBL-2) or 7 days (U266). Isobolograms were generated from dose-response curves of each combination as described previously <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0090675#pone.0090675-Furukawa1" target="_blank">[31]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0090675#pone.0090675-Koyama1" target="_blank">[32]</a>. The results of data quantification and statistical analysis are shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0090675#pone-0090675-t001" target="_blank">Table 1</a>.</p

Purine analog-like properties of bendamustine.

<p>(A) Effects of dilazep (left panel) and NBTI (right panel) on cytotoxicity of the indicated drugs at IC50 against HBL-2 (upper panel) and Namalwa (lower panel) cells. (B) <i>ENT1</i> mRNA expression in HBL-2 and Namalwa cells treated with the indicated drugs. The y-axes indicate relative gene expression against the expression levels of the untreated control being set at 1.0. 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. (C) HBL-2 and Namalwa cells were cultured in the absence (Control) or presence of IC50 values of the indicated drugs. Whole cell lysates were isolated after 48 hours and subjected to immunoblot analysis for the expression of ENT1, ENT2 and GAPDH (internal control). The data shown are representative of multiple independent experiments.</p

Expression level of <i>REG3A</i> in B-LCLs of each <i>REG3A</i> rs7588571 genotype.

<p><b>A. Semi-quantitative reverse transcription PCR for <i>REG3A</i></b>. Total RNA was extracted from three B-LCLs for each genotype and reverse transcribed. The cDNA equivalent to the same amount of total RNA was subjected to reverse trancription PCR. Panc-1, which is a pancreatic cancer cell line expressing <i>REG3A</i>, was used as a positive control. <i>GAPDH</i> was used as an internal control. <b>B. Comparison of <i>REG3A</i> expression level among rs7588571 genotypes</b>. The intensity of each PCR band was quantified, and expression levels of <i>REG3A</i> were normalized to <i>GAPDH</i> expression levels. The normalized expression level of <i>REG3A</i> in the GG genotype was used as a reference. The relative expression levels of <i>REG3A</i> were compared among the three rs7588571 genotypes using one-way ANOVA (left) and between the GG and non-GG genotypes using a t-test (right). Data of two independent experiments (mean ± S.E.M.) each performed in triplicate are shown. <b>C. Western blot for REG3A protein</b>. Lysate was extracted from three B-LCLs for each genotype that were used in Fig 1A. The same amount of lysate was applied in SDS-PAGE. Panc-1 was used as a positive control. The β-actin was used as a loading control. <b>D. Comparison of expression level of REG3A protein among rs7588571 genotypes</b>. The expression level of REG3A protein was quantified. The expression level of REG3A protein in the GG genotype was used as a reference. The relative expression levels of REG3A protein were compared among the three rs7588571 genotypes using one-way ANOVA (left) and between the GG and non-GG genotypes using a t-test (right). Data of two independent experiments (mean ± S.E.M.) are shown.</p

Patient characteristics categorized by rs7588571 genotype.

<p>Patient characteristics categorized by rs7588571 genotype.</p

Univariate and multivariate analyses of risk factors for extensive chronic GVHD.

<p>Univariate and multivariate analyses of risk factors for extensive chronic GVHD.</p