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.2019 Oct 1;9(1):14135.
doi: 10.1038/s41598-019-50509-1.

Antileukemic activity of novel adenosine derivatives

Affiliations

Antileukemic activity of novel adenosine derivatives

Anastazja Poczta et al. Sci Rep..

Abstract

The present study investigated the effect of cladribine (CLA) and six of its derivatives containing a formamidine group at position 6 (CLA-FDM, CLA-FPAZ, CLA-FPIR, CLA-FPIP, CLA-FHEX, and CLA-FMOR) on acute promyelocytic, lymphoblastic, and acute monocytic leukemia cells. The role of ATR kinase in deoxycytidine kinase (dCK) activation in response to DNA damage was assessed. The presence of DNA lesions was assessed by measurement phosphorylation of H2AX and by using the alkaline comet assay with proteinase K post-treatment following assessment of the cell cycle. Apoptotic events such as alterations in intracellular calcium concentration, caspase-3/7 activity and increased sub-G1 cell population were measured. CLA derivatives were highly effective against leukemic cells, showing high cytotoxicity, causing DNA fragmentation, and inducing DNA-protein cross-links in leukemic cells. CLA-FMOR showed the highest efficacy. CLA derivatives increased the levels of intracellular calcium ions, caspase-3/7 and the percentage of sub-G1 apoptotic cells and blocked cells in the S phase of the cell cycle to a greater extent than free CLA. The selective ATR inhibitor VE-821 significantly suppressed the increase in dCK activity and decreased basal dCK activity. The present results suggested that ATR kinase controls dCK activity in response to synthetic CLA derivatives.

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Conflict of interest statement

The authors declare no competing interests.

Figures

Figure 1
Figure 1
Concentration dependent cytotoxic effect of cladribine (CLA) and its derivatives (panel left: CLA-FMOR, CLA-FPIP and CLA-FPIR; panel right: CLA-FHEX, CLA-FDMF, CLA-FPAZ) on HL-60, MOLT-4, THP-1 and HMEC-1 cells growth measured by XTT assay. Cells without any treatment were used as controls and taken as 100%. Results are presented as the mean of six independent experiments. Error bars signify standard deviation, * indicate statistically significant changes between samples incubated with the drugs compared with control cells (P < 0.05). Statistical analysis was performed by ANOVA test with the Tukey post hoc test for multiple comparisons.
Figure 2
Figure 2
(a) Comparison of the cytotoxicity (designated as IC50) of cladribine (CLA) and its derivatives in HL-60, MOLT-4, THP-1 and HMEC-1 cell lines. (b) Effect of ATR inhibitor (VE-821) on cytotoxicity of CLA and its derivatives. For comparison the effect of VE-821 cells were preincubated with or without 10 μM VE-821 and cells viability was measured by the XTT assay after 48 h of incubation. Samples without VE-821 are presented as color lines and samples with VE-821 as gray lines. Cells without any treatment were used as controls and taken as 100%. Results are presented as the mean ± SD of six independent experiments. Error bars signify standard deviation, * indicate statistically significant changes between samples incubated with the drugs compared with control cells (P < 0.05). Statistical analysis was performed by ANOVA test with the Tukey post hoc test for multiple comparisons.
Figure 3
Figure 3
(a) The dCK activity in THP-1, MOLT-4, and HL-60 cell lines incubated with cladribine and its derivatives for 24 h in comparison to the activity in control cells. Cells without any treatment were used as controls and taken as 100%. In some experiments (marked on the X axis as: + VE-821), the cells were preincubated with VE-821 for 1 h at 37 °C before addition of the drugs and further incubation for 24 h. (b) Differences in the level of basic dCK activity between the tested cell lines. Error bars signify standard deviation, * indicate statistically significant changes between samples incubated with the drugs compared with control cells (P < 0.05). Symbol + indicate statistically significant changes between samples incubated with the compounds and VE-821 or caspase 3 inhibitor (P < 0.05);# indicate statistically significant changes between samples incubated with cladribine and new derivatives (P < 0.05). Statistical analysis was performed by ANOVA test with the Tukey post hoc test for multiple comparisons.
Figure 4
Figure 4
Effect of cladribine and its derivatives on intracellular Ca2 + levels in three cell lines: THP-1, MOLT-4, and HL-60. Cells were incubated with the different compounds for 2, 24, and 48 h. In some experiments (presented as white bars), the cells were preincubated with ATR inhibitor (VE-821) for 1 h at 37 °C before the addition of drugs and incubation for an additional 2–48 h. Cells, after 24 h incubation with compounds, stained with the Fluo-4-AM probe were visualized under an inverted fluorescence microscope (Olympus IX70, Japan; magnification: 400 × ). Some cells display abnormal morphology: cells with protrusions from the plasma membrane (blebbing), marked by the orange arrows. Error bars signify standard deviation, * indicate statistically significant changes between samples incubated with the drugs compared with control cells (P < 0.05). Symbol + indicate statistically significant changes between samples incubated with the compounds and VE-821 or caspase 3 inhibitor (P < 0.05);# indicate statistically significant changes between samples incubated with cladribine and new derivatives (P < 0.05). Statistical analysis was performed by ANOVA test with the Tukey post hoc test for multiple comparisons.
Figure 5
Figure 5
Caspase-3/7 gene expression as an indicator of apoptosis in three cell lines: THP-1, MOLT-4, and HL-60. Cells without any treatment were used as controls and taken as 100%. The cells were treated (a) 24 h and (b) 48 h with the compounds in the presence or absence of VE-821 and Z-FA-FMK inhibitors. Error bars signify standard deviation, * indicate statistically significant changes between samples incubated with the drugs compared with control cells (P < 0.05). Symbol + indicate statistically significant changes between samples incubated with the compounds and VE-821 or caspase 3 inhibitor (P < 0.05);# indicate statistically significant changes between samples incubated with cladribine and new derivatives (P < 0.05). Statistical analysis was performed by ANOVA test with the Tukey post hoc test for multiple comparisons.
Figure 6
Figure 6
The level of phosphorylation of H2AX (γH2AX) induced by cladribine and its derivatives (CLA-FMOR, CLA-FPIP and CLA-FPIR) in HL-60, MOLT-4 and THP-1 cell lines. Cells were incubated with the compounds for 48 h. In some experiments, the cells were preincubated with ATR inhibitor (VE-821) for 1 h at 37 °C before the addition of CLA or CLA derivatives. Error bars signify standard deviation, * indicate statistically significant changes between samples incubated with the drugs compared with control cells (P < 0.05). Symbol + indicate statistically significant changes between samples incubated with the compounds and VE-821 or caspase 3 inhibitor (P < 0.05);# indicate statistically significant changes between samples incubated with cladribine and new derivatives (P < 0.05). Statistical analysis was performed by ANOVA test with the Tukey post hoc test for multiple comparisons.
Figure 7
Figure 7
Some cells display abnormal morphology: cells with p Cell cycle phase distribution presented as a percentage share of phase subG1, S, G1, G2/M in HL-60, MOLT-4, and THP-1 cells (2–48 h) treated with the compounds in the presence or absence of the ATR inhibitor (VE-821). Individual phases were identified based on cellular DNA content quantified by flow cytometry. Representative histograms of DNA content after treatment with each compound for 48 h. * indicate statistically significant changes between samples incubated with the drugs compared with control cells (P < 0.05). Symbol + indicate statistically significant changes between samples incubated with the compounds and VE-821 or caspase 3 inhibitor (P < 0.05);# indicate statistically significant changes between samples incubated with cladribine and new derivatives (P < 0.05). Statistical analysis was performed by ANOVA test with the Tukey post hoc test for multiple comparisons.
Figure 8
Figure 8
Fragmentation of DNA after treatment the cells with CLA and its derivatives, measured as a percentage of the DNA in the comet tail. Samples were preincubated with ATR inhibitor, VE-821 (a,c,e) or treated with proteinase K (b,d,f) in HL-60, MOLT-4, and THP-1 cells for 2, 24, and 48 h. Representative images of comets. Characteristic comet tails with damaged DNA were visualized under a fluorescence microscope after electrophoresis and gel staining with DAPI. (g) Error bars signify standard deviation, * indicate statistically significant changes between samples incubated with the drugs compared with control cells (P < 0.05). Symbol + indicate statistically significant changes between samples incubated with the compounds and VE-821 or caspase 3 inhibitor (P < 0.05);# indicate statistically significant changes between samples incubated with cladribine and new derivatives (P < 0.05). Statistical analysis was performed by ANOVA test with the Tukey post hoc test for multiple comparisons.
Figure 9
Figure 9
Proposed model of the molecular and cellular responses to the new CLA derivatives. Action of cladribine in leukemia cells: transport of 2-CdA to the cell and the mechanisms underlying the cellular response to genotoxic stress induced by new CLA analogs. DNA strand breaks lead to the activation of the DNA damage response pathway. DNA damage, ssDNA and dsDNA, are recognized by ATR. The consequence of this process is the formation of large clusters of proteins within the DNA damage necessary to activate effector proteins. P53 lead to cell arrest in the cycle and consequently to damage repair or apoptosis.
Figure 10
Figure 10
Structure of the tested compounds.
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