Synthetic phosphoethanolamine has in vitro and in vivo anti-leukemia effects

Abstract

Background: We recently showed that synthetic phosphoethanolamine reduces tumour growth and inhibits lung metastasis in vivo. Here, we investigated its anti-leukaemia effects using acute promyelocytic leukaemia (APL) as a model.

Methods: Cytotoxic effects of Pho-s on leukaemia cells were evaluated by MTT assay. Leukaemic cells obtained from hCG-PML-RARa transgenic mice were transplanted to NOD/SCID mice. After the animals were diagnosed as leukaemic, treatment started with Pho-s using all-trans retinoid acid or daunorubicin as positive control or and saline control. Cell morphology and immunophenotyping were used to detect the undifferentiated blast cells in the spleen, liver and bone marrow. The induction of apoptosis in vitro and in malignant leukaemic clones was evaluated.

Results: Synthetic phosphoethanolamine is cytotoxic and induces apoptosis through the mitochondrial pathway in vitro to leukaemia cell lines. In vivo Pho-s exhibits anti-proliferative effects in APL model reducing the number of CD117(+) and Gr-1(+) immature myeloid cells in the BM, spleen and liver. Synthetic phosphoethanolamine impairs the expansion of malignant clones CD34(+)/CD117(+), CD34(+) and Gr-1(+) in the BM. In addition, Pho-s induces apoptosis of immature cells in the spleen and liver, a notable effect.

Conclusion: Synthetic phosphoethanolamine has anti-leukaemic effects in an APL model by inhibiting malignant clone expansion, suggesting that it is an interesting compound for leukaemia treatment.

Figure 1

Effects of Pho-s on the cell viability of KG-1, K562 and Jukart cells. Cells were seeded into 96-well plates at 10⁴ cells per well and treated with Pho-s for 24 h. Cell viability was assessed by MTT assay and is expressed as the percentage of cells comparing the optical density (540 nm) of the treated cells with the optical density of the untreated cells. The data are representative of three independent experiments performed in triplicate.

Figure 2

Pho-s induces mitochondrial membrane depolarisation and caspase-3 activity in leukemic cell lines. (A) Representative histograms of cells stained with Mitotracker red are shown. There is a substantial reduction in fluorescence emission, indicating a decrease of ΔmΨ in the leukaemic cells treated with Pho-s (black line) as compared with untreated cells (red line). (B) A substantial increase in apoptotic cells was observed after 8 h of treatment with Pho-s. (C) The increase in caspase-3 activity correlates with the increase in the frequency of apoptotic cells evaluated after the treatment. The data are representative of three independent experiments performed in triplicate. Significant differences are indicated as: *P<0.05 statistically different between Pho-s versus untreated.

Figure 3

Haematological and cytological analysis of leukaemic cells. To confirm the success of the transplant, the mice were bled from the tail 15 days after transplantation. (A) The increases in the total number of leukocytes, as well as thrombocytopenia and anaemia, are evidences of the progression of the disease. (B) An increase in the number of undifferentiated cells, characteristic of blast cells, infiltrated in the bone marrow, spleen and liver was observed.

Figure 4

Immunophenotyping by flow cytometry of CD117⁺ and Gr-1⁺ cells infiltrated in the spleen (A) and liver (B) of NOD/SCID leukaemic mice. The bar diagram shows the percentage of CD117⁺ and Gr-1⁺cells in the parenchyma from untreated mice and from mice treated with 40 mg kg⁻¹ Pho-s, 80 mg kg⁻¹ Pho-s, 1 mg kg⁻¹ ATRA or 10 mg kg⁻¹ DA (the last two, positive controls) and WT mice. The data are expressed as mean ±s.d. from five mice. *P<0.05; **P<0.01 and ***P<0.001 versus untreated mice, One-way analysis of variance (ANOVA) with Tukey–Kramer post-hoc test.

Figure 5

Immunophenotyping by flow cytometry of CD45⁺, CD117⁺ and CD34⁺ cells infiltrated in the BM of NOD/SCID leukaemic mice. The bar diagram shows CD45 and CD117 (A) and CD34 (B) expression in the BM from untreated mice and from mice treated with 40 mg kg⁻¹ Pho-s, 80 mg kg⁻¹ Pho-s, 1 mg kg⁻¹ ATRA or 10 mg kg⁻¹ DA (the last two, positive controls). The data are expressed as mean ±s.d. from five mice. *P<0.05; **P<0.01 and ***P<0.001 versus untreated mice, one-way analysis of variance (ANOVA) with Tukey–Kramer post-hoc test.

Figure 6

Immunophenotyping by flow cytometry of CD117⁺ and CD34⁺ cells infiltrated in BM of NOD/SCID leukaemic mice. The bar diagram shows CD117 and CD34 expression in the BM from untreated mice and from mice treated with 40 mg kg⁻¹ Pho-s, 80 mg kg⁻¹ Pho-s, 1 mg kg⁻¹ ATRA or 10 mg kg⁻¹ DA (the last two, positive controls). The data are expressed as mean ±s.d. from five mice. **P<0.01 and ***P<0.001 versus untreated mice, one-way analysis of variance (ANOVA) with Tukey–Kramer post-hoc test.

Figure 7

Evaluation of cell death by apoptosis using annexin V-FITC/PI staining. (A) Dot plot profile of cells from untreated mice and from mice treated with 40 mg kg⁻¹ Pho-s, 80 mg kg⁻¹ Pho-s, 1 mg kg⁻¹ ATRA or 10 mg kg⁻¹ DA (the last two, positive controls). The apoptotic cells selected at the gate to CD117⁺ infiltrates in the spleen were stained with V-FITC/PI and analysed by flow cytometry. (B) Left panel: The percentage of apoptotic CD117⁺ cells is shown in the bar diagram as mean ±s.d. from five mice. Right panel: The percentage of apoptotic cells is shown in relation to the total number of CD117⁺ infiltrates in the spleen. The values were ***P<0.001 versus untreated mice, one-way analysis of variance (ANOVA) with Tukey–Kramer post-hoc test.

Figure 8

Evaluation of cell death by apoptosis using annexin V-FITC/PI staining. (A) Dot plot profile of cells from untreated mice and from mice treated with 40 mg kg⁻¹ Pho-s, 80 mg kg⁻¹ Pho-s, 1 mg kg⁻¹ ATRA or 10 mg kg⁻¹ DA (the last two, positive controls). Apoptotic cells selected at the gate for CD117⁺ infiltrates in the liver were stained with V-FITC/PI and analysed by flow cytometry. (B) Left panel: The percentage of apoptotic CD117⁺ cells is shown in the bar diagram as mean ±s.d. from five mice. Right panel: The percentage of apoptotic cells is shown in relation to the total number of CD117⁺ infiltrates in the liver. The values were **P<0.001 versus untreated mice, one-way analysis of variance (ANOVA) with Tukey–Kramer post-hoc test.

Figure 9

Evaluation of cell death by apoptosis using annexin V-FITC/PI staining. (A) Dot plot profile of cells from untreated mice and from mice treated with 40 mg kg⁻¹ Pho-s, 80 mg kg⁻¹ Pho-s, 1 mg kg⁻¹ ATRA or 10 mg kg⁻¹ DA (the last two, positive controls). Apoptotic cells selected at the gate for Gr-1⁺ infiltrates in the spleen were stained with V-FITC/PI and analysed by flow cytometry. (B) Left panel: The percentage of apoptotic Gr-1⁺ cells is shown in the bar diagram as mean ±s.d. from five mice. Right panel: The percentage of apoptotic cells is shown in relation to the total number of Gr-1⁺ infiltrates in the spleen. The values were ***P<0.001 versus untreated mice, one-way analysis of variance (ANOVA) with Tukey–Kramer post-hoc test.

Figure 10

Evaluation of cell death by apoptosis using annexin V-FITC/PI staining. (A) Dot plot profile of cells from untreated mice and from mice treated with 40 mg kg⁻¹ Pho-s, 80 mg kg⁻¹ Pho-s, 1 mg kg⁻¹ ATRA or 10 mg kg⁻¹ DA (the last two, positive controls). The percentage of apoptotic cells selected into the gate to Gr-1⁺ infiltrates in the liver were stained with V-FITC/PI and analysed by flow cytometry. (B) Left panel: The percentage of apoptotic Gr-1⁺ cells is shown in the bar diagram as mean±s.d. from five mice. Right panel: The percentage of apoptotic cells is shown in relation to the total number of Gr-1⁺ infiltrates in the liver. The values were ***P<0.001 versus untreated mice, one-way analysis of variance (ANOVA) with Tukey–Kramer post-hoc test.