Phloridzin docosahexaenoate, a novel flavonoid derivative, suppresses growth and induces apoptosis in T-cell acute lymphoblastic leukemia cells

Abstract

The overall clinical outcome in T-cell acute lymphoblastic leukemia (T-ALL) can be improved by minimizing risk for treatment failure using effective pharmacological adjuvants. Phloridzin (PZ), a flavonoid precursor found in apple peels, was acylated with docosahexaenoic acid (DHA) yielding a novel ester known as phloridzin docosahexaenoate (PZ-DHA). Here, we have studied the cytotoxic effects of PZ-DHA on human leukemia cells using in vitro and in vivo models. The inhibitory effects of PZ-DHA were tested on human Jurkat T-ALL cells in comparison to K562 chronic myeloid leukemia (CML) cells and non-malignant murine T-cells. PZ-DHA, not PZ or DHA alone, reduced cell viability and ATP levels, increased intracellular LDH release, and caused extensive morphological alterations in both Jurkat and K562 cells. PZ-DHA also inhibited cell proliferation, and selectively induced apoptosis in Jurkat and K562 cells while sparing normal murine T-cells. The cytotoxic effects of PZ-DHA on Jurkat cells were associated with caspase activation, DNA fragmentation, and selective down-regulation of STAT3 phosphorylation. PZ-DHA significantly inhibited Jurkat cell proliferation in zebrafish larvae; however, the proliferation of K562 cells was not affected in vivo. We propose that PZ-DHA-induced cytotoxic response is selective towards T-ALL in the presence of a tumor-stromal microenvironment. Prospective studies evaluating the combinatorial effects of PZ-DHA with conventional chemotherapy for T-ALL are underway.

Keywords: Phloridzin; STAT3; apoptosis; cytotoxicity; docosahexaenoic acid; flavonoid derivative; leukemia; proliferation; zebrafish.

Figure 1

PZ-DHA is more potent than the parent compounds, PZ and DHA. A. Cell viability was measured using MTS assay after treating Jurkat and K562 cells with 10, 25, 50, 75, and 100 µM PZ, DHA, or PZ-DHA for 12, 24, and 48 h. Doxorubicin and imatinib were used as positive controls for Jurkat and K562 cell lines, respectively. B. Cellular ATP levels were measured using CellTiter-Glo luminescent assay following treatment with test compounds (100 µM) for 24 h. C. The release of LDH into culture media was measured following treatment with test compounds (100 µM) for 24 h. Data represent mean ± SEM (n=3). *P < 0.05 and **P < 0.001, compared among means (ANOVA, Tukey’s test).

Figure 2

PZ-DHA suppresses leukemia cell proliferation. A. Oregon Green 488 fluorescent dye-stained Jurkat and K562 cells were treated with vehicle or test compounds (30 µM) for 72 h. Cells were then harvested and fluorescence intensity was quantified by flow cytometry relative to the non-proliferative control (baseline). Histograms from a representative experiment are shown. B. Bar graphs (derived from A show mean number of cell division in each treatment normalized to the non-proliferative control. Data represent mean ± SD (n=3). *P < 0.05, compared to vehicle control (ANOVA, Dunnett’s test).

Figure 3

PZ-DHA induces apoptosis in leukemia cells while sparing normal murine T-cells. A. Jurkat and K562 cells were treated with vehicle or test compounds (50 µM) for 24 h and photographed using phase contrast microscopy at 400 × magnification. Photos are representative of three independent experiments. B. Flow cytometry analysis with Annexin V/PI dual staining was used to evaluate apoptosis induction in Jurkat and K562 cell lines treated with vehicle or test compounds (50 µM) for 24 h. Cytograms are representative of one of three independent experiments. C. Bar graphs (derived from A show % of apoptotic/necrotic (Annexin V, PI and Annexin V/PI positive) cells. D. Bar graphs showing % apoptotic/necrotic normal murine T-cells, Jurkat, and K562 cells treated with vehicle or test compounds at 25, 50, and 75 µM treatment doses. Data represent mean ± SD (n=3). *P < 0.05 and **P < 0.001, compared among means (ANOVA, Tukey’s test).

Figure 4

PZ-DHA-induced apoptosis is associated with caspase activation and DNA fragmentation in Jurkat cells. A. Jurkat and K562 cells were treated with vehicle, test compounds (50 µM), or 2.5 µM staurosporine, STS (positive control) for 18 h and caspase activity was detected using Caspase-Glo 3/7 assay. Luminescence units were recorded. B. Jurkat and K562 cells were treated with vehicle or test compounds (50 µM) for 24 h and DNA fragmentation was quantified by ELISA assay. Absorbance values were measured at 370 nm. Data represent mean ± SD (n=3). *P < 0.05 and **P < 0.001, compared to vehicle control (ANOVA, Dunnett’s test).

Figure 5

PZ-DHA selectively inhibits inducible phosphorylation of STAT3 protein in Jurkat cells. Jurkat cells were treated with vehicle (V) or indicated concentrations of PZ-DHA for 24 h before 1 h IFN-α incubation. Relative expression levels of (A) phospho-STAT1 (p-STAT1) and STAT1, (B) phospho-STAT3 (p-STAT3) and STAT3, and (C) phospho-STAT5 (p-STAT5) and STAT5 were measured by Western blot analysis. Actin was used as loading control. Relative graphical representations of p-STAT expression (normalized to STAT) determined using densitometry are shown. Data represent mean ± SD (n=3). **P < 0.001, compared to vehicle control (ANOVA, Dunnett’s test).

Figure 6

PZ-DHA suppresses in vivo proliferation of Jurkat cells in xenotransplanted zebrafish larvae. A. Representative brightfield and fluorescent images (5 × objective) of zebrafish larvae injected with fluorescently labeled Jurkat cells and monitored in the presence of vehicle or 75 µM PZ-DHA for 72 h. B. The effect of PZ-DHA on Jurkat cell proliferation was quantified by dissociating untreated and treated groups of larvae at baseline (0-hour post-treatment, hpt) and 72 hpt. Bar graph depicts fold change in cell number at 72 hpt relative to baseline. Data represent mean ± SD (n=3). *P < 0.05, compared to vehicle control (ANOVA, Dunnett’s test).