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. 2021 May 21;19(6):285.
doi: 10.3390/md19060285.

Anti-Leukemic Properties of Aplysinopsin Derivative EE-84 Alone and Combined to BH3 Mimetic A-1210477

Affiliations

Anti-Leukemic Properties of Aplysinopsin Derivative EE-84 Alone and Combined to BH3 Mimetic A-1210477

Sungmi Song et al. Mar Drugs. .

Abstract

Aplysinopsins are a class of marine indole alkaloids that exhibit a wide range of biological activities. Although both the indole and N-benzyl moieties of aplysinopsins are known to possess antiproliferative activity against cancer cells, their mechanism of action remains unclear. Through in vitro and in vivo proliferation and viability screening of newly synthesized aplysinopsin analogs on myelogenous leukemia cell lines and zebrafish toxicity tests, as well as analysis of differential toxicity in noncancerous RPMI 1788 cells and PBMCs, we identified EE-84 as a promising novel drug candidate against chronic myeloid leukemia. This indole derivative demonstrated drug-likeness in agreement with Lipinski's rule of five. Furthermore, EE-84 induced a senescent-like phenotype in K562 cells in line with its cytostatic effect. EE-84-treated K562 cells underwent morphological changes in line with mitochondrial dysfunction concomitant with autophagy and ER stress induction. Finally, we demonstrated the synergistic cytotoxic effect of EE-84 with a BH3 mimetic, the Mcl-1 inhibitor A-1210477, against imatinib-sensitive and resistant K562 cells, highlighting the inhibition of antiapoptotic Bcl-2 proteins as a promising novel senolytic approach against chronic myeloid leukemia.

Keywords: BH3 mimetics; aplysinopsin analogs; chronic myeloid leukemia; indole alkaloids; marine source.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Scheme 1
Scheme 1
Synthetic pathway for the preparation of aplysinopsin EE-115 and its analogs EE-31, EE-80, EE-84, and EE-92. Reagents and conditions: (a) benzyl chlorides, NaH, DMF; (b) oxalyl chloride, dry ethyl ether, heating; (c) the reactant amines 2-cyanoacetohydrazide and 1-(2-amino-5-methyl-4,5,6,7-tetrahydrobenzo[b]thiophen-3-yl)ethan-1-one, dry THF, TEA, stirring, 3 h; (d) POCl3 DMF, 0 °C, NaOH; (e) piperidine, reflux, 4 h.
Figure 1
Figure 1
Chemical structures. (A) Aplysinopsin (EE-115) and analogs (EE-31, EE-80, EE-84, EE-92). (B) A-1210477.
Figure 2
Figure 2
Differential anti-leukemic effects observed by CFA after aplysinopsin treatments (AE). Representative pictures from three independent experiments of clonogenic assays after 48 h pretreatment of EE-84 (A) K562, (B) KBM5, (C) MEG01, (D) KBM5IR, (E) K562IR cells are shown on the left. Corresponding quantifications (number of colonies, the total surface area of colonies, and average size of colonies) are indicated on the right. Statistical analysis was performed by one-way ANOVA, followed by Sidak’s multiple comparisons test. Differences were considered significant when * p < 0.05, ** p < 0.01, *** p < 0.001 compared to control. n/s: not significant.
Figure 2
Figure 2
Differential anti-leukemic effects observed by CFA after aplysinopsin treatments (AE). Representative pictures from three independent experiments of clonogenic assays after 48 h pretreatment of EE-84 (A) K562, (B) KBM5, (C) MEG01, (D) KBM5IR, (E) K562IR cells are shown on the left. Corresponding quantifications (number of colonies, the total surface area of colonies, and average size of colonies) are indicated on the right. Statistical analysis was performed by one-way ANOVA, followed by Sidak’s multiple comparisons test. Differences were considered significant when * p < 0.05, ** p < 0.01, *** p < 0.001 compared to control. n/s: not significant.
Figure 3
Figure 3
Percentage of cell death after increasing concentrations of EE-84 treatment in K562 cells. (A) Representative microscopy images of Hoechst 33342/PI double-stained nuclei of K562 cells exposed to different concentrations of aplysinopsin derivatives at indicated times are shown. The percentage of apoptotic cells was calculated from three independent experiments. (B) Results obtained after Annexin V APC/PI staining and quantification by FACS. Ima: imatinib (1 μM). (C) For cell cycle analysis, K562 cells were treated with 10, 30, and 50 µM EE-84 for 48 and 72 h. Cell cycle phase distribution was determined by FACS. All data were expressed as mean ± SD of three independent experiments. Statistical analysis was performed by two-way ANOVA, followed by Dunnett’s multiple comparisons test (Microscopy, Cell cycle); by Sidak’s multiple comparisons test (Annexin V/PI); * p < 0.05, ** p < 0.01, *** p < 0.001 compared to controls.
Figure 4
Figure 4
Effect of aplysinopsin derivatives on healthy cell models. (A) Differential toxicity (RPMI 788/K562) of EE-84 was determined by the trypan blue exclusion test. (B) Toxicity test of EE-84 in nonproliferating and proliferating PBMCs. After 24, 48, and 72 h of EE-84 treatment, viability was determined by the trypan blue exclusion test. Zebrafish toxicity assays were performed by treatment with aplysinopsins at indicated concentrations. (C) Representative pictures of zebrafish are shown. Zebrafish viability (D), relative body length (E), and heartbeats/min (F) were measured. Data represents a total of 15 fish per group. Trypan blue exclusion test data represents the mean ± SD of three independent experiments. Statistical analysis: two-way ANOVA with Dunnett’s multiple comparison test (trypan blue exclusion test); * p < 0.05, ** p < 0.01, *** p < 0.001 compared to controls. Ordinary one-way ANOVA followed by Dunnett’s post hoc test (Zebrafish assay) revealed significant differences indicated by *** p < 0.001 compared to control. n/s: not significant.
Figure 5
Figure 5
Morphological changes of K562 cells treated with EE-84. (A) K562 cells were treated with 50 μM EE-84 for 24, 48, 72, and 96 h. Light microscopy at magnification 200× was used for morphological observation. (B) K562 cells were treated with indicated concentrations of EE-84 for up to 96 h. At the indicated time points, cells were harvested and analyzed by flow cytometry using forward (relative cell sizes; FSC-H) and side scatter (granularity; SSC-H) measurements. For morphological analysis of EE-84, exposed K562 cell debris were excluded. (C) SA-β-gal staining of the K562 cells treated with 50 μM of EE-84 for 72 h. The quantitative analysis of the incidence of the cells with positive β-gal staining. Doxo: doxorubicin (80 nM). (D) Transmission electron microscopy at magnifications of 8000× or 10,000× (whole cells) and 20,000× or 25,000× (cell details). Cells were treated with 30 μM EE-84 and were exposed for up to 72 h. Phagophores, autophagosomes, injured mitochondria, and incidence of mitophagy were highlighted using red, black, blue, and yellow arrows, respectively. DMSO-treated K562 cells at 24 h were used as control. (E) Measurement of mitochondrial function in K562 cells. The experiments were conducted using the Seahorse XFp Cell Mito Stress test, and the OCR measurement is visualized. Results are the mean ± SD of three independent experiments. Statistical analysis was performed by two-way ANOVA, followed by Dunnett’s multiple comparisons test (Cell size by FACS); by Tukey’s multiple comparisons test (Senescence); by Sidak’s multiple comparisons test (Seahorse XFp Cell Mito Stress test); * p < 0.05, ** p < 0.01, *** p < 0.001 compared to controls.
Figure 5
Figure 5
Morphological changes of K562 cells treated with EE-84. (A) K562 cells were treated with 50 μM EE-84 for 24, 48, 72, and 96 h. Light microscopy at magnification 200× was used for morphological observation. (B) K562 cells were treated with indicated concentrations of EE-84 for up to 96 h. At the indicated time points, cells were harvested and analyzed by flow cytometry using forward (relative cell sizes; FSC-H) and side scatter (granularity; SSC-H) measurements. For morphological analysis of EE-84, exposed K562 cell debris were excluded. (C) SA-β-gal staining of the K562 cells treated with 50 μM of EE-84 for 72 h. The quantitative analysis of the incidence of the cells with positive β-gal staining. Doxo: doxorubicin (80 nM). (D) Transmission electron microscopy at magnifications of 8000× or 10,000× (whole cells) and 20,000× or 25,000× (cell details). Cells were treated with 30 μM EE-84 and were exposed for up to 72 h. Phagophores, autophagosomes, injured mitochondria, and incidence of mitophagy were highlighted using red, black, blue, and yellow arrows, respectively. DMSO-treated K562 cells at 24 h were used as control. (E) Measurement of mitochondrial function in K562 cells. The experiments were conducted using the Seahorse XFp Cell Mito Stress test, and the OCR measurement is visualized. Results are the mean ± SD of three independent experiments. Statistical analysis was performed by two-way ANOVA, followed by Dunnett’s multiple comparisons test (Cell size by FACS); by Tukey’s multiple comparisons test (Senescence); by Sidak’s multiple comparisons test (Seahorse XFp Cell Mito Stress test); * p < 0.05, ** p < 0.01, *** p < 0.001 compared to controls.
Figure 6
Figure 6
EE-84 induced autophagy and ER stress. (A) K562 cells were treated with 30 μM EE-84 for 24, 48, and 72 h. Western blot analysis of the LC3B protein. Β-actin was used as a loading control. Quantification of LC3B protein bands through normalization by β-actin protein bands. (B) Diff-Quik staining of K562 cells after 48 h of EE-84 treatment (30 μM) with or without Baf-A1 post-treatment. Baf-A1: bafilomycin A1 (40 nM). (C) ER stress-related proteins were detected by Western blot analysis. Β-actin was used as a loading control. After quantifying the bands, p-eiF2α levels were normalized to total eIF2α, and all other proteins were normalized to β-actin. TSG: thapsigargin (300 nM). (D) Measurement of glycolytic capacity in K562 cells. 2-DG: 2-deoxy-d-glucose. The experiments were conducted using the Seahorse XFp Glycolysis Stress test, and the flow chart and bar graph show the measurement of ECAR. Results are the mean ± SD of three independent experiments. Statistical analysis was performed by two-way ANOVA, followed by Tukey’s multiple comparisons test (autophagy Western blot, Diff-Quik staining); by Dunnett’s multiple comparisons test (ER stress Western blot); by Sidak’s multiple comparisons test (Seahorse XFp Glycolysis Stress test); * p < 0.05, ** p < 0.01, *** p < 0.001 compared to controls.
Figure 6
Figure 6
EE-84 induced autophagy and ER stress. (A) K562 cells were treated with 30 μM EE-84 for 24, 48, and 72 h. Western blot analysis of the LC3B protein. Β-actin was used as a loading control. Quantification of LC3B protein bands through normalization by β-actin protein bands. (B) Diff-Quik staining of K562 cells after 48 h of EE-84 treatment (30 μM) with or without Baf-A1 post-treatment. Baf-A1: bafilomycin A1 (40 nM). (C) ER stress-related proteins were detected by Western blot analysis. Β-actin was used as a loading control. After quantifying the bands, p-eiF2α levels were normalized to total eIF2α, and all other proteins were normalized to β-actin. TSG: thapsigargin (300 nM). (D) Measurement of glycolytic capacity in K562 cells. 2-DG: 2-deoxy-d-glucose. The experiments were conducted using the Seahorse XFp Glycolysis Stress test, and the flow chart and bar graph show the measurement of ECAR. Results are the mean ± SD of three independent experiments. Statistical analysis was performed by two-way ANOVA, followed by Tukey’s multiple comparisons test (autophagy Western blot, Diff-Quik staining); by Dunnett’s multiple comparisons test (ER stress Western blot); by Sidak’s multiple comparisons test (Seahorse XFp Glycolysis Stress test); * p < 0.05, ** p < 0.01, *** p < 0.001 compared to controls.
Figure 7
Figure 7
EE-84 sensitized CML cells to BH3 mimetics. (A) EE-84 increased expression levels of antiapoptotic protein Mcl-1 compared to DMSO-treated control. K562 cells were treated with varying concentrations of EE-84 for 24 h. The effects on Mcl-1 and β-actin were determined by Western blot analysis. Western blot data interpreted in terms of fold changes in protein expression compared to control at the corresponding time point. The values represented the average of three independent experiments. (B) Cotreatment analysis of 20 and 30 μM EE-84 with 10 μM A-1210477 using Hoechst 33258/PI staining showed an increase in apoptotic cell death compared to single treatments of compounds after 24 h in K562 cells. (C) The plot representing the fraction affected-combination index (Fa-CI) of the treatment of K562 cells with EE-84 and A-1210477 after 24 h was obtained with Compusyn software. (D) The optimal compound combination of 30 μM EE-84 and 10 µM A-1210477 was analyzed by Hoechst/PI staining. z-VAD-fmk was used as a pan-caspase inhibitor. (E) The compound combination of 30 μM EE-84 and 10 μM A-1210477 treated K562 cells after 24 h. After 24 h of treatment, the type of cell death triggered by EE-84 with or without A-1210477 was characterized by FACS after Annexin V APC/propidium iodide (PI) staining. Pictures are representative of three independent experiments (left panel), and the corresponding quantification (right panel) is shown. Ima: imatinib (1 μM). (F) K562IR cell viability by trypan blue exclusion assay. (G) Annexin V APC/PI staining of K562IR analyzed by FACS. Results are the mean ± SD of three independent experiments. (H) Mitochondrial membrane potential (MMP) of the combination of 30 μM EE-84 and 10 µM A-1210477 was analyzed by FACS, and the percentage of cells exhibiting MMP loss was calculated (right panel). z-VAD-fmk was used as a pan-caspase inhibitor. Eto: etoposide (100 μM). (I) Differential anti-leukemic effects observed by CFA after EE-84 treatments with A-1210477 on K562. Corresponding quantifications (the number of colonies, the total surface area of colonies, and the average size of colonies) are indicated on the right. Results are the mean ± SD of three independent experiments. Statistical analysis was performed by one-way or two-way ANOVA, followed by Dunnett’s multiple comparisons test (Microscopy), by Tukey’s multiple comparisons test (colony formation assay, MMP, Annexin V/PI); * p < 0.05, ** p < 0.01, *** p < 0.001 compared to controls, & p < 0.05, && p < 0.01, &&& p < 0.001 between indicated conditions.
Figure 7
Figure 7
EE-84 sensitized CML cells to BH3 mimetics. (A) EE-84 increased expression levels of antiapoptotic protein Mcl-1 compared to DMSO-treated control. K562 cells were treated with varying concentrations of EE-84 for 24 h. The effects on Mcl-1 and β-actin were determined by Western blot analysis. Western blot data interpreted in terms of fold changes in protein expression compared to control at the corresponding time point. The values represented the average of three independent experiments. (B) Cotreatment analysis of 20 and 30 μM EE-84 with 10 μM A-1210477 using Hoechst 33258/PI staining showed an increase in apoptotic cell death compared to single treatments of compounds after 24 h in K562 cells. (C) The plot representing the fraction affected-combination index (Fa-CI) of the treatment of K562 cells with EE-84 and A-1210477 after 24 h was obtained with Compusyn software. (D) The optimal compound combination of 30 μM EE-84 and 10 µM A-1210477 was analyzed by Hoechst/PI staining. z-VAD-fmk was used as a pan-caspase inhibitor. (E) The compound combination of 30 μM EE-84 and 10 μM A-1210477 treated K562 cells after 24 h. After 24 h of treatment, the type of cell death triggered by EE-84 with or without A-1210477 was characterized by FACS after Annexin V APC/propidium iodide (PI) staining. Pictures are representative of three independent experiments (left panel), and the corresponding quantification (right panel) is shown. Ima: imatinib (1 μM). (F) K562IR cell viability by trypan blue exclusion assay. (G) Annexin V APC/PI staining of K562IR analyzed by FACS. Results are the mean ± SD of three independent experiments. (H) Mitochondrial membrane potential (MMP) of the combination of 30 μM EE-84 and 10 µM A-1210477 was analyzed by FACS, and the percentage of cells exhibiting MMP loss was calculated (right panel). z-VAD-fmk was used as a pan-caspase inhibitor. Eto: etoposide (100 μM). (I) Differential anti-leukemic effects observed by CFA after EE-84 treatments with A-1210477 on K562. Corresponding quantifications (the number of colonies, the total surface area of colonies, and the average size of colonies) are indicated on the right. Results are the mean ± SD of three independent experiments. Statistical analysis was performed by one-way or two-way ANOVA, followed by Dunnett’s multiple comparisons test (Microscopy), by Tukey’s multiple comparisons test (colony formation assay, MMP, Annexin V/PI); * p < 0.05, ** p < 0.01, *** p < 0.001 compared to controls, & p < 0.05, && p < 0.01, &&& p < 0.001 between indicated conditions.
Figure 7
Figure 7
EE-84 sensitized CML cells to BH3 mimetics. (A) EE-84 increased expression levels of antiapoptotic protein Mcl-1 compared to DMSO-treated control. K562 cells were treated with varying concentrations of EE-84 for 24 h. The effects on Mcl-1 and β-actin were determined by Western blot analysis. Western blot data interpreted in terms of fold changes in protein expression compared to control at the corresponding time point. The values represented the average of three independent experiments. (B) Cotreatment analysis of 20 and 30 μM EE-84 with 10 μM A-1210477 using Hoechst 33258/PI staining showed an increase in apoptotic cell death compared to single treatments of compounds after 24 h in K562 cells. (C) The plot representing the fraction affected-combination index (Fa-CI) of the treatment of K562 cells with EE-84 and A-1210477 after 24 h was obtained with Compusyn software. (D) The optimal compound combination of 30 μM EE-84 and 10 µM A-1210477 was analyzed by Hoechst/PI staining. z-VAD-fmk was used as a pan-caspase inhibitor. (E) The compound combination of 30 μM EE-84 and 10 μM A-1210477 treated K562 cells after 24 h. After 24 h of treatment, the type of cell death triggered by EE-84 with or without A-1210477 was characterized by FACS after Annexin V APC/propidium iodide (PI) staining. Pictures are representative of three independent experiments (left panel), and the corresponding quantification (right panel) is shown. Ima: imatinib (1 μM). (F) K562IR cell viability by trypan blue exclusion assay. (G) Annexin V APC/PI staining of K562IR analyzed by FACS. Results are the mean ± SD of three independent experiments. (H) Mitochondrial membrane potential (MMP) of the combination of 30 μM EE-84 and 10 µM A-1210477 was analyzed by FACS, and the percentage of cells exhibiting MMP loss was calculated (right panel). z-VAD-fmk was used as a pan-caspase inhibitor. Eto: etoposide (100 μM). (I) Differential anti-leukemic effects observed by CFA after EE-84 treatments with A-1210477 on K562. Corresponding quantifications (the number of colonies, the total surface area of colonies, and the average size of colonies) are indicated on the right. Results are the mean ± SD of three independent experiments. Statistical analysis was performed by one-way or two-way ANOVA, followed by Dunnett’s multiple comparisons test (Microscopy), by Tukey’s multiple comparisons test (colony formation assay, MMP, Annexin V/PI); * p < 0.05, ** p < 0.01, *** p < 0.001 compared to controls, & p < 0.05, && p < 0.01, &&& p < 0.001 between indicated conditions.
Figure 8
Figure 8
Caspase-dependent induction of apoptosis in K562 cells by EE-84 in synergism with the Mcl-1 inhibitor A-1210477. (A) K562 cell viability was determined by the measurement of cellular ATP content using the CellTiter-Glo assay. K562 cells were pretreated at the concentration of 50 μM z-VAD-fmk for 1 h before exposure to the compounds. (B) Effects of single or combination treatments of EE-84 and A-1210477 with or without z-VAD-fmk on caspase-3/7 activity in K562 cells. Three separate experiments were performed for both CellTiter-Glo and caspase-3/7 assays, and each condition was measured in triplicates. Values are the mean ± SD of three independent experiments. The asterisk indicates a value significantly different from the control. Statistical analysis was performed by one-way or two-way ANOVA, followed by Dunnett’s multiple comparisons test (ATP assay, caspase-3/7 assay); *** p < 0.001 compared to controls; &&& p < 0.001 between indicated conditions; ### p < 0.001 compared to corresponding treatment without z-VAD-fmk. (CE) Western blots showing expression levels of apoptosis-related proteins after single or combination treatments of EE-84 and A-1210477 in K562 cells. (C) Results of expression levels of initiator caspases, caspase-8 and -9; (D) expression levels of effector caspases, caspase-3 and -7, and PARP-1; (E) expression levels of antiapoptotic proteins Mcl-1 and Bcl-xL are displayed. Protein expression levels of procaspase-8, -9, -3, and -7 are quantified and expressed in fold changes relative to control. Protein expression levels of cleaved PARP-1, Mcl-1, and Bcl-xL are quantified and expressed in fold changes relative to control. The values represent the average of three independent experiments.

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