Ethyl Pyruvate Combats Human Leukemia Cells but Spares Normal Blood Cells

Abstract

Ethyl pyruvate, a known ROS scavenger and anti-inflammatory drug was found to combat leukemia cells. Tumor cell killing was achieved by concerted action of necrosis/apoptosis induction, ATP depletion, and inhibition of glycolytic and para-glycolytic enzymes. Ethyl lactate was less harmful to leukemia cells but was found to arrest cell cycle in the G0/G1 phase. Both, ethyl pyruvate and ethyl lactate were identified as new inhibitors of GSK-3β. Despite the strong effect of ethyl pyruvate on leukemia cells, human cognate blood cells were only marginally affected. The data were compiled by immune blotting, flow cytometry, enzyme activity assay and gene array analysis. Our results inform new mechanisms of ethyl pyruvate-induced cell death, offering thereby a new treatment regime with a high therapeutic window for leukemic tumors.

Fig 1. Growth inhibition of leukemia cells by EP and EL.

(A, B) THP-1 cells (5000 cells/well) were seeded (start) and cultured at 37°C / 5% CO₂ in the absence or presence of increasing concentrations of EP ort EL for 24 h. After that, cell proliferation was evaluated using the WST-1 assay. Data represent the mean ± SD of three independent experiments. (C, D) Reactivation studies show treatment of THP-1 cells by EP and EL for defined time intervals followed by medium replenishment. THP-1 cells (10⁴ cells/mL) were cultured in 75 ml-flasks in the absence and presence of graded concentrations of EP or EL for 24 h. After certain time points aliquots of cell suspension were removed for vitality testing by the trypan blue exclusion test. Reactivation of growth inhibition was analyzed by suspending cells at day 2 in fresh medium without inhibitors. The ordinate shows the number vital cells only. Data represent the mean ± SD (n = 3). (E-H) Colony formation assay: K562 and THP-1 cells (2.5 x 10⁵ each) were contacted with increasing concentrations of EP for 24 h in RPMI-FCS before seeded into a 0.3% agar layer of Petri-dishes in the absence of EP. Colony formation was check 14 days after incubation. (E) K562 cells; (F) THP-1 cells. (G, H) Statistic analysis of counted cell colonies (n = 3).

Fig 2. Effect of EP and EL at cellular ATP content of THP-1 and K562 cells.

(A), THP-1 cells were seeded (5000 cells/well) and cultured in the presence of increasing concentrations of EP (0–20 M) and (B) EL (0–20 M) for 6h and 24 h, respectively. Cellular ATP content was determined using the CellTiter-Glo® Luminescent Cell Viability Assay. Data represent the mean ± SD of three independent experiments (n = 12). (C) Effect of EP on ATP content in K562 cells. Microscopic inspection of THP-1 cells grown in medium for 24 h in absence (D) or presence (E) of 10 mM EP (magnification: x20). (F) Enlargement of an aspect of E.

Fig 3. Analysis of glycolytic/paraglycolytic enzymes in THP-1 cells and PBMCs.

(A-D) THP-1 cells and PBMCs were cultured RPMI 1640 medium containing 10% FCS in the presence of increasing concentrations of EP and EL for 24 h. Cell extracts were prepared from washed cells and analyzed for protein content and GLO1 activity. (E), Western blot analysis for GLO1 in THP1 cells treated with increasing concentrations of EP (0–20 mM). GLO1 in PBMC extract is shown for comparison. GAPDH was used as internal loading control. (F) Expression of GLO1-–mRNA in THP-1 cells treated with EP. (G) THP-1 cells were treated without and with 15 mM EP for 6 h followed by measurement of enzyme activity of GLO1, LDH and PK in the cytosolic extract. (H) V/S plot of activity of purified LDH-1 (SigmaAldrich, Germany) in dependence on increasing concentrations of EP (Michaelis-Menten-Plot); Insert depicts the Lineweaver-Burg-Plot. (I) LDH isoenzymes distribution in diverse tumor cell extracts as analyzed by agar gel electrophoresis.

Fig 4. EP/EL induces necrosis/apoptosis of THP-1 cells but do not impair innate immune functions of normal white blood cells.

(A) Flow cytometric analysis of THP-1 cells and blood monocytes upon treatment with EP and EL. THP-1 cells and blood monocytes were treated with EP and EL at increasing concentrations (1 to 20 mM) for 12 h at 37°C, then stained with annexin-V-FITC/PI, and apoptosis/necrosis was determined by flow cytometry. Results of four independent experiments are shown. The columns depict the mean percentage of vital cells, apoptotic cells, and necrotic/late apoptotic cells. Apoptotic cells are annexin-V-positive only, whereas necrotic cells are PI-positive and annexin-V-positive. (B) Time-dependent effects of EP on death induction of THP-1 cells and blood monocytes. THP-1 cells (upper row) and PBMCs (lower row) were treated with 20 mM EP for 3, 12 and 24 h and apoptosis/necrosis is measured. Controls represent untreated cells (n = 3). (C) Effect of EP at phagocytic and burst activity of blood cells. Heparinized human whole blood was incubated with increasing concentrations of EP at 37°C and 6 h, and a sample without stimulus served as negative background control. Blood was further incubated with fluorescein-labeled opsonized E. coli bacteria for 10 min at 4°C or 37°C, respectively, to measure phagocytic activity. Burst activity was analyzed by treatment of blood with 2 x 10⁸ opsonized, non-labelled E. coli for 10 min at 4°C and 37°C in the presence of dihydrorhodamine 123. The mean fluorescence intensity (MFI) of monocytes and granulocytes were identified by flow cytometry (n = 3).

Fig 5. Impact of EL on cell cycle.

(A) THP-1 cells were treated with EL for 24-h, stained by propidium iodine and subjected to cell cycle analysis by flow cytometry. All data represent mean ±SD. Statistical analyses were conducted among control (0 mM EL) and treated groups in G0/G1, S and G2/M phases separately, and different notations in the bar charts indicate statistical significance (n = 3). (B) THP-1 cells were exposed to 20mM EL for 24 h and mRNA expression was analyzed using a 96-well PCR-array of WNT-related transcripts. Data of 3 independent experiments demonstrated as the arithmetic mean of relative gene regulation factor. The asterisks represent significant differences relative to untreated controls (P was calculated in the Pair-Wise Fixed Reallocation Randomisation Test using REST 2009).*, P<0.05; **, P<0.01.

Fig 6. EP and EL inactivate GSK-3β by induced phosphorylation.

Used Cell lines THP-1 (A-F), K562 (F),1321N1 (A), PC-3 (A) and LNCaP (F) were cultured at 37°C / 5% CO₂ in the absence or presence of increasing concentrations of EP, EL or lithium for different times (10 min to 120 min (C) and 24 h (B, D-G), respectively. After that cells were harvested, lysed and subjected to immunoblots using specific antibodies against non-phosphorylated and phosphorylated proteins. Beta-actin and GPDH were used as loading control. (G) Cell proliferation of THP-1 cells in the presence of increasing concentration of lithium. Band intensities of selected blots were analysed by E.A.S.Y. Win 32 software and displayed in S Fig 1.

Fig 7. Dual regulation of cell death and survival by EP and EL.

Induction of cell death by necrosis/apoptosis in tumor cells exhibiting a high glycolytic throughput is primary mediated by depletion of ATP due to inhibition of PK and LDH by EP while inhibition of GLO1/GLO2 yields to accumulation of the cell toxic MGO. EL is not inhibitory to these enzymes and therefore not toxic to cells. EL predominantly stimulates pivotal cell pathways primarily through induction of phosphorylation of GSK-3ß. Which mechanism may dominate depends on the degree of glycolysis, expression of monocarboxylate transporters (MCT) for EP/EL and the type of LDH isoenzyme in the cells.