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. 2015 Sep 11;10(9):e0136878.
doi: 10.1371/journal.pone.0136878. eCollection 2015.

G2/M Cell Cycle Arrest and Tumor Selective Apoptosis of Acute Leukemia Cells by a Promising Benzophenone Thiosemicarbazone Compound

Maia Cabrera  1 , Natalia Gomez  2 , Federico Remes Lenicov  3 , Emiliana Echeverría  1 , Carina Shayo  4 , Albertina Moglioni  5 , Natalia Fernández  2 , Carlos Davio  2
Affiliations

G2/M Cell Cycle Arrest and Tumor Selective Apoptosis of Acute Leukemia Cells by a Promising Benzophenone Thiosemicarbazone Compound

Maia Cabrera et al. PLoS One. .

Abstract

Anti-mitotic therapies have been considered a hallmark in strategies against abnormally proliferating cells. Focusing on the extensively studied family of thiosemicarbazone (TSC) compounds, we have previously identified 4,4'-dimethoxybenzophenone thiosemicarbazone (T44Bf) as a promising pharmacological compound in a panel of human leukemia cell lines (HL60, U937, KG1a and Jurkat). Present findings indicate that T44Bf-mediated antiproliferative effects are associated with a reversible chronic mitotic arrest caused by defects in chromosome alignment, followed by induced programmed cell death. Furthermore, T44Bf selectively induces apoptosis in leukemia cell lines when compared to normal peripheral blood mononuclear cells. The underlying mechanism of action involves the activation of the mitochondria signaling pathway, with loss of mitochondrial membrane potential and sustained phosphorylation of anti-apoptotic protein Bcl-xL as well as increased Bcl-2 (enhanced phosphorylated fraction) and pro-apoptotic protein Bad levels. In addition, ERK signaling pathway activation was found to be a requisite for T44Bf apoptotic activity. Our findings further describe a novel activity for a benzophenone thiosemicarbazone and propose T44Bf as a promising anti-mitotic prototype to develop chemotherapeutic agents to treat acute leukemia malignancies.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Fig 1
Fig 1. Chemical structure of 4,4’-dimethoxybenzophenone thiosemicarbazone (T44Bf).
Fig 2
Fig 2. Cell cycle distribution after T44Bf treatment in human acute leukemia cell lines.
Synchronized G0/G1 cells were exposed to T44Bf at the indicated concentrations or to 0.05% (v/v) DMSO, vehicle control group for 15h. Cell cycle distribution was calculated as described in Material and Methods. Data represent the mean ± SD (n > 3). *P < 0.05
Fig 3
Fig 3. Pro-apoptotic activity of T44Bf in human acute leukemia cell lines.
Cells in exponential growth were exposed to T44Bf at the indicated concentrations or to 0.05% (v/v) DMSO, vehicle control group. (A) After 24h of treatment cells were analyzed to detect exposed phosphatidylserine by annexin V binding assay. The graphic shows the different cell subpopulations according to the annexin V/PI staining pattern: cells labeled with only annexin V (early apoptosis), cells labeled with annexin V and PI (late apoptosis), and cells labeled only with PI (necrotic cells). (B) Determination of cleaved caspase 3 and PARP by Western blot. Equal amounts of protein were subjected to SDS–PAGE with anti-caspase 3 and PARP antibodies. Data are representative of at least three independent experiments. (C) Caspase 3 enzimatic activity induced by T44Bf in human acute leukemia cell lines. Cells were treated with T44Bf in the indicated concentrations and time, and caspase 3 protease activity measured as described in Materials and Methods and expressed as OD405nm values. Data are presented as mean ± SD from four independent experiments. *P<0.05.
Fig 4
Fig 4. Compound selectivity assessed in normal peripheral blood mononuclear cells (PBMC).
Phosphatidylserine exposure was measured by annexin V binding assay in monocytes, unstimulated lymphocytes or Phytohemagglutinin A activated (i.e. proliferating) lymphocytes after 24h treatment with T44Bf at the indicated concentrations or to 0.05% (v/v) DMSO vehicle control group. Data represent the mean ± SD (n > 3). *P<0.05.
Fig 5
Fig 5. T44Bf effects on mitochondrial membrane potential and Bcl-2 family protein levels.
(A) Cells treated for 3, 5, 6 and 7h with T44Bf or 0.05% (v/v) DMSO vehicle control group, were evaluated for changes in fluorescence intensity of DiOC6 probe by flow cytometry. The bar graph shows differences among treatment times. (B) The representative graphic shows Bcl-2, Bad, Bax and Bcl-xL protein assessment by Western blot. Equal amounts of protein were subjected to SDS–PAGE with anti-Bcl-2 family protein antibodies. Blots were subjected to densitometry analysis using ImageJ software. Data are presented as mean ± SD respect to control of at least four independent experiments. *P < 0.05
Fig 6
Fig 6. ERK time-course phosphorylation after T44Bf treatment.
(A) Representative blot of ERK 1/2 phosphorylation following exposure of HL60 cells to 10 μM T44Bf at different times. Equal amounts of protein were subjected to SDS–PAGE with anti-pERK 1/2 antibody. Blots were stripped and incubated with ERK total antibody and α-tubulin as loading control. Bar plot showing arbitrary units obtained from densitometry measurement. Data are expressed as mean ± SD of three independent experiments. *P < 0.05. (B) Effect of MEK inhibitor U0126 on T44Bf-induced apoptosis after 12h in the HL60 cell line. Blot of cleaved caspase 3 and PARP after T44Bf treatment in the presence of 10 μM U0126. Graphic is representative of three independent experiments.
Fig 7
Fig 7. Indirect immunofluorescence microscopy in HL60 and U937 cells treated with T44Bf.
Synchronized cells were treated with T44Bf (10 μM and 20 μM) or Vincristine (200 nM) for 15h. Cells were fixed and stained to detect α-tubulin (red) and counterstained by Hoechst for DNA (blue). Mounted slides were visualized under 1000X magnification on a Nikon Eclipse E200 fluorescence microscope. Yellow arrows indicate extra spindle poles and green arrows indicate unaligned chromosomes. Images are representative of three independent experiments.
Fig 8
Fig 8. Cell cycle progression after T44Bf removal.
Cells were exposed to 10 (HL60) and 20 μM (U937, KG1a and Jurkat) of T44Bf for 15h. Then, the drug-containing medium was removed and replaced by fresh culture medium for 2h. Cells were stained with PI and analyzed by flow cytometry. Data are expressed as mean ± SD of three independent experiments. *P<0.05.
Fig 9
Fig 9. Cyclin A, Cyclin B1and Cdc2 evaluation by western blot.
(A)Total lysates of cells exposed to 10 (HL60) and 20 μM (U937, KG1a and Jurkat) T44Bf for 15 and 17h were analyzed for Cyclin A by Western blot. Densitometry for Cyclin A blot was performed using Image J software. Arbitrary units represent protein level analysis respect to load control β-actin. (B) Cells were exposed to 10 (HL60) and 20 μM (U937, KG1a and Jurkat) T44Bf for 15h. Then, the drug-containing medium was replaced by fresh culture medium for 2 and 5h. Total cell lysates were analyzed by Western blot for Cyclin B1 and Cdc2. Densitometry for Cyclin B1 and Cdc2 blots were performed using Image J software. Arbitrary units represent protein level analysis respect to load control α-tubulin. *P < 0.05.
Fig 10
Fig 10. Schematic diagram of T44Bf mechanism of action in human acute leukemia cells.
T44Bf induces chronic prometaphase arrest, associated to SAC permanent activation through a currently unknown mechanism. This is evidenced by Cyclin A downregulation, increased Cyclin B1 levels and condensed chromosomes visualization. This chronic arrest leads to an apoptotic response involving mitochondrial signaling pathway activation and up-regulation of ERK pathway, which is a requisite for T44Bf-induced cell death.
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References

    1. McCulloch EA. Stem cells in normal and leukemic hemopoiesis (Henry Stratton Lecture, 1982). Blood. 1983;62(1):1–13. - PubMed
    1. Bonnet D, Dick J. Human acute myeloid leukemia is organized as a hierarchy that originates from a primitive hematopoietic cell. Nature medicine. 1997;3(7):730–7. - PubMed
    1. Schimmer A, Hedley D, Penn L, Minden M. Receptor- and mitochondrial-mediated apoptosis in acute leukemia: a translational view. Blood. 2001;98(13):3541–53. - PubMed
    1. Döhner H, Estey E, Amadori S, Appelbaum F, Büchner T, Burnett A, et al. Diagnosis and management of acute myeloid leukemia in adults: recommendations from an international expert panel, on behalf of the European LeukemiaNet. Blood. 2010;115(3):453–74. 10.1182/blood-2009-07-235358 - DOI - PubMed
    1. Tallman M, Gilliland D, Rowe J. Drug therapy for acute myeloid leukemia. Blood. 2005;106(4):1154–63. - PubMed

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This work was supported by grants from the Universidad de Buenos Aires UBACyT 20020100100601 CD; Agencia Nacional de Promoción Científica y Tecnológica: Prestamo BID-PICT-2013-2050 CD. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.