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Synergistic apoptotic effect of Mcl-1 inhibition and doxorubicin on B-cell precursor acute lymphoblastic leukemia cells

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Abstract

Background

Myeloid cell leukemia-1 (MCL-1) is a component of the Bcl-2 anti-apoptotic family that plays a key role in cell proliferation and differentiation. Despite tremendous improvements toward identification of the role of MCL-1 in leukemia progression, the functional significance and molecular mechanism behind the effect of MCL-1 overexpression on the proliferation of B-cell precursor acute lymphoblastic leukemia (BCP-ALL) has not been clarified. In addition, less well appreciated is the effect of MCL-1 inhibition on the potentiation of doxorubicin-induced apoptosis in BCP-ALL cell lines. In the present study, we aimed to shed light on the anti-cancer properties of S63845, a potent Mcl-1 inhibitor, in BCP-ALL cell lines either alone or in combination with a chemotherapeutic drug.

Methods and results

Mononuclear cells from patients with Pre-B ALL and BCP-ALL cell lines were treated with S63845 in presence or absence of doxorubicin, induction of apoptosis was evaluated using Annexin-V/PI staining kit. mRNA and protein expression levels were assessed by qRT-PCR and western blot analysis, respectively. Our results declared that inhibition of Mcl-1 impairs cell growth and induces apoptosis in pre-B ALL cells through activation of caspase-3 and up-regulation of a repertoire of pro-apoptotic Bcl-2 family. Additionally, S63845 acts synergically with doxorubicin to induce apoptosis in BCP-ALL cell lines.

Conclusions

Our data declared that MCL-1 inhibition alone or in combination with a chemotherapeutic agent is considered an appealing strategy for the induction of apoptosis in BCP-ALL cells.

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Abbreviations

S:

S63845 (Mcl-1 inhibitor)

Dox:

Doxorubicin

qRT-PCR:

Quantitative-real time polymerase chain reaction

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Acknowledgements

This study was derived from the thesis of E Ebrahimi and supported by a grant from Iran University of Medical Science.

Funding

This work was supported by the Grant No. 32662 from Iran University of Medical Sciences.

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Authors and Affiliations

  1. Department of Hematology and Blood Banking, Faculty of Allied Medicine, Iran University of Medical Science, Hemmat Highway, 1449614535, Tehran, Iran

    Elham Ebrahimi, Rima Manafi Shabestari & Majid Safa

  2. Department of Hematology and Blood Banking, School of Allied Medical Sciences, Shahid Beheshti University of Medical Sciences, Tehran, Iran

    Davood Bashash

  3. Cellular and Molecular Research Center, Iran University of Medical Sciences, Tehran, Iran

    Majid Safa

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  1. Elham Ebrahimi
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Contributions

MS conceived and designed the research. EEB and RMS conducted the experiment. MS and DB contributed reagent or analytical tools. MS, RMS and DB analyzed data. EEB and RMS wrote the manuscript. MS, DB and RMS revised the manuscript. All authors read and approved the final manuscript.

Corresponding author

Correspondence to Majid Safa.

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Conflict of interest

Elham Ebrahimi, Rima Manafi Shabestari, Davood Bashash, and Majid Safa declares that they have no conflict of interest.

Ethical approval

The procedure performed in present study involving human participants was in accordance with the ethical standards of the Medical Ethic Committee of Iran university of medical science and with the 1964 Helsinki declaration and its later amendments or comparable ethical standards.

Informed consent

Informed consent was obtained from all patients and healthy donors included in the study. All authors involved in this study are agree to publish the manuscript in your worthy journal.

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Supplementary Information

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11033_2021_7021_MOESM1_ESM.tif

Supplementary Fig. 1 (a) NALM-6 and (b) SUP-B15 cells were treated with elevating concentrations of S63845 for 24 and 48 hr and cell viability was evaluated with trypan blue method. Dose-effect curve of diverse doses of S63845 in (c) NALM-6 and (d) SUP-B15 cells was measured using MTT assay and expressed using compusyn software. Supplementary material 1 (TIF 302.5 kb)

11033_2021_7021_MOESM2_ESM.tif

Supplementary Fig. 2 The apoptotic cell graphs for NALM-6 (a) and SUP-B15 cells (b) assessed by flowcytometery. Following the treatment of (c) NALM-6 and (d) SUP-B15 cells with mentioned doses of S63845 for 24 hr, caspase-3 activity assay was performed using a colorimetric caspase-3 kit (n=3, *P<0.05, **P<0.01, compared to control cells). Supplementary material 2 (TIF 395.6 kb)

11033_2021_7021_MOESM3_ESM.tif

Supplementary Fig. 3 (a) NALM-6 and (b) SUP-B15 cells were treated with diverse concentrations of doxorubicin and metabolic activity of cells was measured using MTT assay. The Dose-effect curve of different concentrations of Doxorubicin in was depicted using compusyn software. Supplementary material 3 (TIF 151.8 kb)

11033_2021_7021_MOESM4_ESM.tif

Supplementary Fig. 4 (a) NALM-6 cells were incubated with 500 nM concentrations of S63845 in the presence or absence of 200 nM doxorubicin for 24 hr. (b) SUP-B15 cells were cultured in medium containing 60 nM S63845 either with or without 20 nM doxorubicin for 24 hr. Total RNA was extracted and cDNA was prepared. BAX and BCL-2 mRNA expression levels were measured by quantitative reverse transcription polymerase chain reaction (qRT-PCR) and normalized to the expression of ACTIN (n=3, *P<0.05, **P<0.01 compared to Dox treated cells). Supplementary material 4 (TIF 181.2 kb)

11033_2021_7021_MOESM5_ESM.tif

Supplementary Fig. 5 (a) NALM-6 cells and (b) SUP-B15 cells were treated with indicated doses of MCL-1 S63845 and doxorubicin for 24 hr. Following extraction of total RNA and cDNA preparation, gene expression of C-MYC, hTERT, and MCL-1 was measured by qRT-PCR. All values were normalized to ACTIN. (n=3; *p<0.05, compared to cells treated with doxorubicin alone). Supplementary material 5 (TIF 296.1 kb)

11033_2021_7021_MOESM6_ESM.tif

Supplementary Fig. 6 Mononuclear cells from healthy donors (a) and patients with BCP-ALL (b) were treated with doxorubicin in present or absence of S63845 for 24hr. (*p<0.05, compared to cells treated with doxorubicin alone). Supplementary material 6 (TIF 1394.3 kb)

Supplementary material 7 (DOCX 14.8 kb)

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Ebrahimi, E., Shabestari, R.M., Bashash, D. et al. Synergistic apoptotic effect of Mcl-1 inhibition and doxorubicin on B-cell precursor acute lymphoblastic leukemia cells. Mol Biol Rep 49, 2025–2036 (2022). https://doi.org/10.1007/s11033-021-07021-5

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