Limits...
Repositioning of bromocriptine for treatment of acute myeloid leukemia

View Article: PubMed Central - PubMed

ABSTRACT

Background: Treatment for acute myeloid leukemia (AML) has not significantly changed in the last decades and new therapeutic approaches are needed to achieve prolonged survival rates. Leukemia stem cells (LSC) are responsible for the initiation and maintenance of AML due to their stem-cell properties. Differentiation therapies aim to abrogate the self-renewal capacity and diminish blast lifespan.

Methods: An in silico screening was designed to search for FDA-approved small molecules that potentially induce differentiation of AML cells. Bromocriptine was identified and validated in an in vitro screening. Bromocriptine is an approved drug originally indicated for Parkinson’s disease, acromegaly, hyperprolactinemia and galactorrhoea, and recently repositioned for diabetes mellitus.

Results: Treatment with bromocriptine reduced cell viability of AML cells by activation of the apoptosis program and induction of myeloid differentiation. Moreover, the LSC-enriched primitive AML cell fraction was more sensitive to the presence of bromocriptine. In fact, bromocriptine decreased the clonogenic capacity of AML cells. Interestingly, a negligible effect is observed in healthy blood cells and hematopoietic stem/progenitor cells.

Conclusions: Our results support the use of bromocriptine as an anti-AML drug in a repositioning setting and the further clinical validation of this preclinical study.

Electronic supplementary material: The online version of this article (doi:10.1186/s12967-016-1007-5) contains supplementary material, which is available to authorized users.

No MeSH data available.


Bromocriptine treatment had an anti-leukemia activity in AML cell lines. a HL-60, KG-1, MonoMac-1 and Kasumi-1 AML cell lines were treated with 0.1, 1 and 10 µM bromocriptine for 48 h. Relative number of live cells refer to vehicle control-treated samples is represented. b HL-60 cells were treated with vehicle or 10 µM bromocriptine for 48 h. Frequency of Annexin-V-positive cells measured by flow cytometry is represented. c HL-60 cells were treated with bromocriptine at the concentrations indicated. Frequency of CD11b-positive cells detected by flow cytometry is represented. d HL-60 cells were treated with bromocriptine at 10 µM for 48 h. A representative picture of May–Grünwald–Giemsa-stained cells is shown. e HL-60 cells were cultured with HS-5 stroma cells at indicated concentration. Cell viability was measured 48 h after treatment by flow cytometry. Bars represent mean values of at least 3 experiments performed in triplicates. Error bars represent SEM. *p < 0.05; **p < 0.01; ***p < 0.005; ****p < 0.001
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

License 1 - License 2
getmorefigures.php?uid=PMC5015257&req=5

Fig1: Bromocriptine treatment had an anti-leukemia activity in AML cell lines. a HL-60, KG-1, MonoMac-1 and Kasumi-1 AML cell lines were treated with 0.1, 1 and 10 µM bromocriptine for 48 h. Relative number of live cells refer to vehicle control-treated samples is represented. b HL-60 cells were treated with vehicle or 10 µM bromocriptine for 48 h. Frequency of Annexin-V-positive cells measured by flow cytometry is represented. c HL-60 cells were treated with bromocriptine at the concentrations indicated. Frequency of CD11b-positive cells detected by flow cytometry is represented. d HL-60 cells were treated with bromocriptine at 10 µM for 48 h. A representative picture of May–Grünwald–Giemsa-stained cells is shown. e HL-60 cells were cultured with HS-5 stroma cells at indicated concentration. Cell viability was measured 48 h after treatment by flow cytometry. Bars represent mean values of at least 3 experiments performed in triplicates. Error bars represent SEM. *p < 0.05; **p < 0.01; ***p < 0.005; ****p < 0.001

Mentions: AML is characterized by the accumulation of transformed immature myeloid blasts which have lost their ability to normally differentiate. As targeted therapies aiming to force AML blast cells to terminally differentiate will ultimately result in cell death, a PMA (phorbol myristate acetate)-induced differentiation-associated gene expression profile was identified and interrogated against the Connectivity Map database (https://www.broadinstitute.org/cmap/) [12]. In order to identify potential drugs that selectively induced differentiation of AML cells, the results obtained were filtered at a P value <0.05 and a connectivity score >0.75 in HL-60 (AML cell line) but <0.5 in PC3 (prostate cancer cell line) and MCF7 (breast cancer cell line), at a concentration <10 µM (Additional file 3: Figure S1). Bromocriptine, a FDA- and EMA-approved drug for treatment of Parkinson’s disease [19], pituitary tumors [20], hyperprolactinemia [21] and type II diabetes [22], was identified as a potential differentiation-inducing drug for AML blasts. Four different AML cell lines (HL-60, AML FAB M2; KG-1, AML FAB M0/1; MonoMac-1, AML FAB M5 MLL-AF9 positive; Kasumi-1, AML FAB M2 t(8;21) positive) were treated at different doses of bromocriptine for 48 h. As shown in Fig. 1a, bromocriptine treatment resulted in at least 50 % cell viability reduction at 10 µM concentration. Interestingly, bromocriptine-treated AML cells were highly positive for the early apoptosis marker Annexin-V (Fig. 1b, Additional file 4: Figure S2A and data not shown), indicating that bromocriptine activated the cell death program in AML. In concordance with the in silico screening performed to identify bromocriptine, treatment with this drug induced the surface expression of the myeloid-associated differentiation marker CD11b (Fig. 1c, Additional file 4: Figure S2B and data not shown) and morphological changes compatible with terminal differentiation (Fig. 1d).Fig. 1


Repositioning of bromocriptine for treatment of acute myeloid leukemia
Bromocriptine treatment had an anti-leukemia activity in AML cell lines. a HL-60, KG-1, MonoMac-1 and Kasumi-1 AML cell lines were treated with 0.1, 1 and 10 µM bromocriptine for 48 h. Relative number of live cells refer to vehicle control-treated samples is represented. b HL-60 cells were treated with vehicle or 10 µM bromocriptine for 48 h. Frequency of Annexin-V-positive cells measured by flow cytometry is represented. c HL-60 cells were treated with bromocriptine at the concentrations indicated. Frequency of CD11b-positive cells detected by flow cytometry is represented. d HL-60 cells were treated with bromocriptine at 10 µM for 48 h. A representative picture of May–Grünwald–Giemsa-stained cells is shown. e HL-60 cells were cultured with HS-5 stroma cells at indicated concentration. Cell viability was measured 48 h after treatment by flow cytometry. Bars represent mean values of at least 3 experiments performed in triplicates. Error bars represent SEM. *p < 0.05; **p < 0.01; ***p < 0.005; ****p < 0.001
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC5015257&req=5

Fig1: Bromocriptine treatment had an anti-leukemia activity in AML cell lines. a HL-60, KG-1, MonoMac-1 and Kasumi-1 AML cell lines were treated with 0.1, 1 and 10 µM bromocriptine for 48 h. Relative number of live cells refer to vehicle control-treated samples is represented. b HL-60 cells were treated with vehicle or 10 µM bromocriptine for 48 h. Frequency of Annexin-V-positive cells measured by flow cytometry is represented. c HL-60 cells were treated with bromocriptine at the concentrations indicated. Frequency of CD11b-positive cells detected by flow cytometry is represented. d HL-60 cells were treated with bromocriptine at 10 µM for 48 h. A representative picture of May–Grünwald–Giemsa-stained cells is shown. e HL-60 cells were cultured with HS-5 stroma cells at indicated concentration. Cell viability was measured 48 h after treatment by flow cytometry. Bars represent mean values of at least 3 experiments performed in triplicates. Error bars represent SEM. *p < 0.05; **p < 0.01; ***p < 0.005; ****p < 0.001
Mentions: AML is characterized by the accumulation of transformed immature myeloid blasts which have lost their ability to normally differentiate. As targeted therapies aiming to force AML blast cells to terminally differentiate will ultimately result in cell death, a PMA (phorbol myristate acetate)-induced differentiation-associated gene expression profile was identified and interrogated against the Connectivity Map database (https://www.broadinstitute.org/cmap/) [12]. In order to identify potential drugs that selectively induced differentiation of AML cells, the results obtained were filtered at a P value <0.05 and a connectivity score >0.75 in HL-60 (AML cell line) but <0.5 in PC3 (prostate cancer cell line) and MCF7 (breast cancer cell line), at a concentration <10 µM (Additional file 3: Figure S1). Bromocriptine, a FDA- and EMA-approved drug for treatment of Parkinson’s disease [19], pituitary tumors [20], hyperprolactinemia [21] and type II diabetes [22], was identified as a potential differentiation-inducing drug for AML blasts. Four different AML cell lines (HL-60, AML FAB M2; KG-1, AML FAB M0/1; MonoMac-1, AML FAB M5 MLL-AF9 positive; Kasumi-1, AML FAB M2 t(8;21) positive) were treated at different doses of bromocriptine for 48 h. As shown in Fig. 1a, bromocriptine treatment resulted in at least 50 % cell viability reduction at 10 µM concentration. Interestingly, bromocriptine-treated AML cells were highly positive for the early apoptosis marker Annexin-V (Fig. 1b, Additional file 4: Figure S2A and data not shown), indicating that bromocriptine activated the cell death program in AML. In concordance with the in silico screening performed to identify bromocriptine, treatment with this drug induced the surface expression of the myeloid-associated differentiation marker CD11b (Fig. 1c, Additional file 4: Figure S2B and data not shown) and morphological changes compatible with terminal differentiation (Fig. 1d).Fig. 1

View Article: PubMed Central - PubMed

ABSTRACT

Background: Treatment for acute myeloid leukemia (AML) has not significantly changed in the last decades and new therapeutic approaches are needed to achieve prolonged survival rates. Leukemia stem cells (LSC) are responsible for the initiation and maintenance of AML due to their stem-cell properties. Differentiation therapies aim to abrogate the self-renewal capacity and diminish blast lifespan.

Methods: An in silico screening was designed to search for FDA-approved small molecules that potentially induce differentiation of AML cells. Bromocriptine was identified and validated in an in vitro screening. Bromocriptine is an approved drug originally indicated for Parkinson&rsquo;s disease, acromegaly, hyperprolactinemia and galactorrhoea, and recently repositioned for diabetes mellitus.

Results: Treatment with bromocriptine reduced cell viability of AML cells by activation of the apoptosis program and induction of myeloid differentiation. Moreover, the LSC-enriched primitive AML cell fraction was more sensitive to the presence of bromocriptine. In fact, bromocriptine decreased the clonogenic capacity of AML cells. Interestingly, a negligible effect is observed in healthy blood cells and hematopoietic stem/progenitor cells.

Conclusions: Our results support the use of bromocriptine as an anti-AML drug in a repositioning setting and the further clinical validation of this preclinical study.

Electronic supplementary material: The online version of this article (doi:10.1186/s12967-016-1007-5) contains supplementary material, which is available to authorized users.

No MeSH data available.