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

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

Inhibition of 20 S proteasome activity by homopiperazine derivatives.

<p>A. Purified erythrocyte-derived 20 S proteasome was incubated in the absence (control) or presence of the indicated HPDS at 5 µM. Chymotrypsin-like, caspase-like and trypsin-like activities were determined by measuring fluorescence generated from the cleavage of specific substrates. Results are represented as relative fluorescence units (RFU) with control set at 100%. 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 indicate p<0.05 against corresponding controls. B. RPMI8226 cells were treated with or without 5 µM HPDs, and analyzed for proteolytic activities as described above. C. RPMI8226 cells were cultured in the absence (control) or presence of 10 µM HPDs for 24 hours, and subjected to immunoblotting for ubiquitinated proteins and GAPDH (internal control).</p

Statistics of crystallographic analysis.

a<p>Completeness and <i>R-</i>merge, are given for overall data and for the highest resolution shell.</p><p>The highest resolution shells for the dataset were 2.54–2.50 Å.</p>b<p><i>R</i>merge = ∑ | <i>I_i</i> – <<i>I</i>> |/∑|<i>I_i</i>|; where <i>I_i</i> is intensity of an observation and <<i>I</i>> is the mean value of that reflection and the summations are over all equivalents.</p>c<p><i>R-work</i> = ∑<i>_h</i> || <i>Fo(h)</i> | – | <i>Fc(h)</i> ||/∑<i>_hFo(h)</i>; where <i>Fo</i> and <i>Fc</i> are the observed and calculated structure factor amplitudes, respectively. The <i>R-free</i> was calculated with 5% of the data excluded from the refinement.</p

Cytotoxic effects of K-7174 on bortezomib-resistant MM cells.

<p>We established wild-type (WT) and mutant (mutant) sublines from RPMI8226 by transducing with wild-type and mutated <i>PSMB5</i> cDNA, respectively, and analyzed the expression of VENUS by flow cytometry (A) and proteasome ß5 subunit by immunoblotting (B). The signal intensities of ß5 subunit (PSMB5) were quantified, normalized to those of the corresponding GAPDH, and shown as relative values in the panel B. C. Cell proliferation was measured by MTT assays after culturing each subline with either K-7174 or bortezomib at the indicated doses for 72 hours. Results are represented as relative absorbance with untreated control set at 100%. 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 indicate p<0.01 against the WT subline. D. Each subline was cultured with either K-7174 or bortezomib (Bort) at the indicated doses for 48 hours. Whole cell lysates were subjected to immunoblotting for cellular protein ubiquitination, proteasome ß5 subunit (PSMB5) and GAPDH (internal control).</p

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

Comparison of K-7174 and bortezomib in cytotoxic activity and proteasome binding.

<p>A. Cell proliferation was measured by MTT assays after culturing with serially diluted K-7174, K-10487 and bortezomib for 72 hours. Absorbance at 450 nm was analyzed with a microplate reader, and expressed as a percentage of the value of corresponding untreated cells. The IC₅₀ value was defined as the concentration of each drug that produces 50% inhibition of cell growth. The means ± S.D. (bars) of three independent experiments are shown. Asterisks indicate “not determined”. B. Overall crystallographic structures showing the folds of ß1 to ß7 subunits in the proteasome bound with K-7174 (left panel) and bortezomib (right panel). Mutation sites observed in bortezomib-resistant cells are circled. C. Structure of the proteasome in complex with bortezomib (PDB cord 2F16) overlapped that with K-7174 described here. Only the protein atoms of the bortezomib-bound form are shown. Bortezomib-resistant mutant residues (Ala49, Thr21, Cys52 and Met45) are colored red and shown as a space-filling diagram.</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