Patulin Acts Synergistically with Doxorubicin and Bortezomib in Cytotoxicity against Hematological Malignancies

Abstract

Patulin (4-Hydroxy-4H-furo 3,2-C-pyran-2(6H)-one) is a first-in-class mycotoxin under development as a novel chemotherapeutic agent. The mechanism of action of Patulin has been reported to include activation of mitogen activated protein kinases (MAPKs) and generation of reactive oxygen species (ROS). We have previously shown Patulin to have activity against myeloma, leukemia, and lymphoma cell lines, as well as primary tumor cells in clinical samples from patients with these diagnoses (Wang BLOOD 2007). Moreover, we have shown that Patulin specifically and potently targets tumor cells over normal cells and effectively killed primary tumor cells of patients with refractory illness. The aim of this study was to determine whether Patulin acts synergistically with the topoisomerase inhibitor doxorubicin or the proteasome inhibitor bortezomib. We hypothesize that drugs with non-cross-reactive modes of action could be complementary. Human tumor cell lines from B-cell lymphoma (DAUDI), T-Cell leukemia and lymphoma (Jurkat and H9, respectively), and myeloid leukemia (HL60) malignancies were tested for their sensitivity to single agents Patulin, bortezomib, and doxorubicin as well as Patulin in combination with the latter two agents (Patulin and bortezomib, P + B; Patulin and doxorubicin, P + D). Cells were treated with a range of concentrations of each single agent and the drugs in combination over 24 hours. Following treatment, cell metabolic activity was assessed using a microculture tetrazolium (MTT) assay and cell viability was assessed by flow cytometry using Annexin V and propidium iodide (PI) staining. Dose-effect curves, median effect plots, and combination index (CI) values were generated in the Compusyn software program for each target cell population. Median-effect doses (IC50s) of individual drugs were interpolated using the y-intercept of median-effect plots. Three dose-effect data points were used to create a range of CI values at different fractions of affect (fa). The lower and upper values of the CI range were used to characterize drug combinations as synergistic, antagonistic, or additive based on Chou's Symbols for Synergism and Antagonism using CI analysis (Table 1). The role for ROS in the mechanism of action of Patulin was confirmed by flow cytometry showing increased levels of ROS in cell lines following Patulin exposure. Preincubation of cell lines with N-acetyl cysteine (NAC) or concurrent exposure to Patulin and NAC abrogated the cytotoxic activity of the mycotoxin. H9 cells were most sensitive to the effects of Patulin, with an IC50 of 1.2 μM. Combinations of P + B acted synergistically against Jurkat, H9, DAUDI, and HL60 tumor cells; however, P + B also demonstrated moderate antagonism against the Jurkat and H9 cell lines (Table 2). Likewise, combinations of P + D interacted synergistically against Jurkat, H9, DAUDI, and HL60 tumor cell lines while simultaneously demonstrating strong antagonism against the H9 cell line. Patulin kills leukemia and lymphoma cells via generation of intracellular ROS. Synergy of Patulin with either bortezomib or doxorubicin in leukemia and lymphoma cell lines indicates a distinct mechanism of action for the mycotoxin and compared to other chemotherapeutics and supports the rationale for continued development of Patulin as a novel chemotherapeutic mycotoxin.TABLE 1. Chou's Symbols for Synergism and Antagonism using CI AnalysisCIDescription< 0.1Very Strong Synergism0.1–0.3Strong Synergism0.3–0.7Synergism0.7–0.85Moderate Synergism0.85–0.90Slight Synergism0.90–1.10Nearly Additive1.10–1.20Slight Antagonism1.20–1.45Moderate Antagonism1.45–3.3Antagonism3.3–10Strong Antagonism> 10Very Strong AntagonismTABLE 2. IC50 of single agents patulin, bortezomib, and doxorubicin and CI in hematological cancer cell linesTumor cell lineIC50 of patulin (μM)IC50 of bortezomib (nM)IC50 of doxorubicin (μM)CI: P + BCI: P + DT-CellJurkat1.1656005.20.6 – 1.20.1 – 1.2H91.2140.330.4 – > 10.3 – > 1B-CellDAUDI0.981.10.19< 0.1 – 0.1< 0.1 – 0.3MyeloidHL601.70.420.07< 0.1 – 0.1< 0.1