Anticancer efficacy of 3-(4-isopropyl) benzylidene-8-ethoxy, 6-methyl, chroman-4-one (SBL-060), a novel, dual, estrogen receptor-Akt kinase inhibitor in acute myeloid leukemia cells

Estrogen receptor (ER) α is expressed in a subset of patient-derived acute myeloid leukemia (AML) cells, whereas Akt is predominantly expressed in most types of AML. Targeting AML with dual inhibitors is a novel approach to combat the disease. Herein, we examined a novel small molecule, 3-(4-isopropyl) benzylidene-8-ethoxy,6-methyl, chroman-4-one (SBL-060), capable of targeting AML cells by inhibiting ERα and Akt kinase. The chemical properties of SBL-060 were identified by proton nuclear magnetic resonance (1H-NMR), 13C-NMR, and mass spectroscopy. In silico docking was performed using an automated protocol with AutoDock-VINA. THP-1 and HL-60 cell lines were differentiated using phorbol 12-myristate 13-acetate. ERα inhibition was assessed using ELISA. The MTT assay assessed cell viability. Flow cytometry was performed for cell cycle, apoptosis, and p-Akt analyses. Chemical analysis identified the compound as 3-(4-isopropyl) benzylidene-8-ethoxy,6-methyl, chroman-4-one, which showed high binding efficacy toward ER, with a ΔGbinding score of −7.4 kcal/mol. SBL-060 inhibited ERα, exhibiting IC50 values of 448 and 374.3 nM in THP-1 and HL-60 cells, respectively. Regarding inhibited cell proliferation, GI50 values of SBL-060 were 244.1 and 189.9 nM for THP-1 and HL-60 cells, respectively. In addition, a dose-dependent increase in sub G0/G1 phase cell cycle arrest and total apoptosis was observed after treatment with SBL-060 in both cell types. SBL-060 also dose-dependently increased the p-Akt-positive populations in both THP-1 and HL-60 cells. Our results indicate that SBL-060 has excellent efficacy against differentiated AML cell types by inhibiting ER and Akt kinase, warranting further preclinical evaluations.


Introduction
Acute myeloid leukemia (AML) is an aggressive form of hematologic malignancy induced by myeloid cell (immature) accretion in the blood/bone marrow [1]. AML can be attributed to unusual genetic mutations that accumulate with chromosomal translocations and/or epigenetic modifications [2]. These factors render the disease unique from patient to patient, consequently posing a challenge to treat. Current therapies to treat AML include combined cytarabine and anthracycline, affording an average success rate of 35%-45% in patients aged <60 years and 10%-15% in patients aged >60 years [3]. Therefore, novel agents that are more efficacious against AML are urgently needed. The 3-benzylidene chroman-4-one class of compounds shares a close homology with naturally occurring bioactive compounds such as flavanones, flavones, chromones, and coumarins. Given the common occurrence of basic side chains in therapeutically active compounds, it is deemed worthwhile to incorporate basic groups into chromanones and evaluate their biological activities. Hence, we synthesized such analogs to screen their bioactivities.
AML is often characterized by myeloblast-induced clonal expansion [4]. As this form of the disease has a stem cellderived hematopoietic origin, uncontrolled accumulation of these cells can be fatal [5]. Estrogen receptor (ER) α and Erβ are encoded by ESR1 and ESR2 genes, respectively, and mediate estrogen signaling [6]. In addition, the pattern of distribution of these ERs has recently gained momentum as novel targets in various diseases [1]. In terms of malignancies, it has been reported that Erα expression increases with for potential involvement in disease etiology and progression [7]. Therefore, targeting ERs in AML cells could afford a reasonable target to combat disease progression. Tumor progression and hence is regarded as a valid target to control rapidly proliferating cancer cells [8]. Although AML is not directly linked to sex hormones, the relationship between estrogen, ERs, and AML has been documented [2]. Accumulated evidence suggests that males are twice as likely to be diagnosed with AML than females, suggesting the involvement of estrogen in the disease [3]. The expression of ERs in differentiated AML cells and aberrant hematopoiesis has been assessed. In contrast, inhibitors of Akt kinase can reportedly control the proliferation of AML cells [4]. However, targeting AML cells using a dual ER and Akt inhibitor has not been evaluated. Our previous work shows structure activity relationship of 3-Benzylidene Chroman-4-one analogues to be possess antimicrobial and anticancer efficacies targeting ER and Akt [5]. Accordingly, the present study focused on the efficacy of one such novel small molecule, 3-(4-isopropyl) benzylidene-8-ethoxy,6-methyl, chroman-4-one (SBL-060), against AML cells by utilizing computational and in vitro approaches.

Materials
Chemicals were purchased from Sigma-Aldrich Aldrich USA. THP-1 (acute monocytic leukaemia) and HL-60 (acute promyelocytic leukaemia) cell lines were obtained from the American Type Culture Collection (Rockville, MD, USA). Annexin V and cell cycle assay reagents were purchased from Merck Millipore, CA, USA. Anti-p-Akt-PE antibody was obtained from eBioscience (Thermo Scientific Corp., MA, USA). The ER-alpha ELISA kit was purchased from BioVision Corp., CA, USA.

Structure retrieval and processing
The X-ray crystal structures of ERs (PDBid: 1UOM) and AKT (PDB # 6HHG) were retrieved from the PDB databank (www. rcsb.org). Before docking, all receptor structures were processed by removing external water and adding hydrogens using BIOVIA-Discovery Studio Visualizer. In addition, the SBL-060 structure in SDF format was converted to SYBYL-TRIPOS (mol2) format using BIOVIA-Discovery Studio Visualizer. Likewise, all target unknown ligand structures were prepared using the BIOVIA-Discovery Studio Visualizer prior to docking analysis.

Computational docking analysis
Docking was performed using the SiBDOCk (automated docking submission module) developed by SiBIOLEAD (https://sibiolead.com/). Briefly, a docking box was generated based on the information obtained from respective protein structures, complexed with known inhibitors by selecting two amino acid residues on either side of the active site. The AutoDock-VINA program was used with the standard docking mode. The top ligands were ranked based on the docking scores. Protein-ligand interactions were inferred using the "Receptor-ligand Interactions" module in BIOVIA-Discovery Studio Visualizer.
Cell culture, ER inhibition and cell proliferation assays RPMI-1640 medium was used to culture THP-1 and HL-60 cells. Cell culture was performed using standard protocols. The growth medium contained 10% fetal bovine serum (FBS), 100 U/mL of penicillin, and 100 U/mL streptomycin. Differentiation with phorbol 12-myristate 13-acetate (PMA) can reportedly increase ER expression in AML cells [6]. Prior to the in vitro assays, both THP-1 and HL-60 cells were differentiated by adding 50 ng/mL PMA and further incubated for 48 h [7]. Subsequently, the cells were centrifuged and re-plated to perform the different assays. For ER-ELISA, differentiated THP-1 and HL-60 cells were plated in 24-well plates, treated with different concentrations of SBL-060, and incubated for 24 h. Cellular supernatants were collected and centrifuged for 20 min at 1000 g at 4°C to remove debris, and a clear solution was used as the sample. ELISA was performed according to the manufacturer's instructions. Cell proliferation was assessed using the MTT assay, as described previously [8]. Briefly, THP-1 and HL-60 cells at a concentration of 5 × 10 3 cells/well were grown in 96-well tissue culture plates in regular growth medium. The cells were then treated with different concentrations of the test compound for 24 h. After removing the medium, 100 µL of MTT (1 mg/mL) was added as a medium replacement and incubated for 4 h. Formazan products were dissolved in 200 µL dimethyl sulfoxide (DMSO), and absorbance was measured at 560 nm. Percent inhibition was calculated using GraphPad Prism 6.0 (GraphPad Software, Inc., La Jolla, USA).

Flow cytometry analysis for cell cycle and apoptosis
The cell cycle assay was performed using the cell cycle assay kit according to the manufacturer's instructions. Differentiated THP-1 and HL-60 cells, at a density of 5 × 10 5 cells per well in a 6-well plate, were treated with 100, 200, and 400 nM SBL-060 and further incubated for 72 h. After washing cells with phosphate-buffered buffer (PBS) twice, 50 µL of cell cycle assay reagent was added, followed incubation for 15 min in the dark. After washing with PBS to remove excess staining reagent, the cells were resuspended in Hank's balanced salt solution (HBSS) buffer. Then, 10,000 events were acquired on a Guava easyCyte TM flow cytometer, and data were analyzed with ExpressPro Software from Millipore (Burlington, CA, USA). The percentage of cell populations in the sub G 0 .G 1 phase of the cell cycle was determined. The apoptosis assay was performed using an Annexin V detection kit in accordance with the manufacturer's instructions. After differentiation, both AML cell lines were treated as indicated for the cell cycle assay and incubated for 48 h. Then, 0.25 µg/mL Annexin V reagent was added to cells for 15 min in the dark. After two washes using sterile PBS, cells were resuspended in kit buffer containing 0.5 µg/mL propidium iodide. Next, 10,000 events were acquired on a Guava easyCyte TM flow cytometer. Data analysis was performed using InCyte software to differentiate between healthy and apoptotic cells (early and late apoptosis) and presented using GraphPad Prism version 6.0.
Akt inhibition assay by flow cytometry Differentiated THP-1 and HL-60 cells were plated in 6-well plates with complete media and treated with 100, 200 or 400 nM SBL-060 for 4 h. Then, cells were twice-washed with PBS, followed by the addition of 0.25 μg/mL PEconjugated, anti-p Akt antibody and incubation for 20 min in the dark. After a couple of washes in PBS, the cells were resuspended in HBSS buffer, and 10,000 events were acquired using a Guava easyCyte TM flow cytometer. Data were analyzed using InCyte software from Millipore (Burlington, CA, USA). The percentage of positive p-Akt cells was estimated.

Statistical analysis
Statistical analyses were performed using GraphPad Prism 6.0. Results are expressed as mean ± standard error (SE). Data were analyzed using ANOVA followed by multiple comparisons. Statistical significance was set at p ≤ 0.05.

Inhibition of ER and cell proliferation by SBL-060 in differentiated AML cells
To mimic ERα expression in AML, we used PMA to induce ER and subsequently assessed the activity of SBL-060. We observed that SBL-060 dose-dependently inhibited ERα in differentiated AML cells, with IC 50 (half maximal inhibitory concentration) values of 488.0 and 374.3 nM in THP-1 and HL-60 cells, respectively (Fig. 3a). Additionally, SBL-060 inhibited the proliferation of THP-1 and HL-60 cells, with GI 50 (half maximal growth inhibitory concentration) values of 244.1 and 189.9 nM, respectively (Fig. 3b). Cytarabine was used as a standard and the activity is provided as Suppl. Fig. 1.

SBL-060 increased the sub G 0 /G 1 cell populations in AML cells
To assess the efficacy of SBL-060 in cellular functional assays, we examined the effect of SBL-060 on cell cycle changes and apoptosis induction in PMA-differentiated THP-1 and HL-60 cells. We selected three doses (low and high) based on the proliferation inhibition results of SBL-060 in these cells. Following treatment with SBL-060, we observed a dosedependent increase in the sub G 0 /G 1 phase in both AML cell lines (Fig. 4a). Compared with untreated control cells, THP-1 cells showed an increase in the sub G 0 /G 1 population from 2.35% to 7.91% following treatment with 100 nM SBL-060 (Fig. 4a). Following treatment with 200 and 400 nM SBL-060, the percentage of THP-1 cells in the sub G 0 /G 1 phase increased to 11.56% and 16.09%, respectively (Fig. 4a). Similarly, untreated HL-60 cells exhibited a sub G 0 /G 1 population of 3.66%, and treatment with 100, 200, and 400 nM SBL-160 increased the percentage to 10.58%, 13.41%, and 19.11%, respectively (Fig. 4a).

Induction of total apoptosis in THP-1 and HL-60 cells by SBL-060
On analyzing total apoptosis in AML cells, both early and late phase apoptosis were increased with different SBL-060 doses in both cell lines (Fig. 4b).
SBL-060 bound and inhibited the Akt enzyme Next, we determined whether SBL-060 could inhibit the Akt enzyme, known to be predominantly expressed during AML malignancy. Prior to the in vitro testing, we performed computational in silico docking to assess the binding affinity of SBL-060 toward Akt. Based on our findings, SBL-060 presented an excellent binding affinity to the enzyme, exhibiting a docking score of −11.2 kcal/mol (Fig. 5a). Based on the PLIP analysis, the chief interacting amino acids were ASN54, SER205, LEU210, TRP80, LYS268, THR82, VAL271, TYR326, ILE84, and ARG 273 (Figs. 5b and 5c).

Discussion
ER and Akt are widely activated in breast cancer types and remains as an attracting target to control breast cancer proliferations [9,10]. We have earlier shown novel 3-Benzylidene Chroman-4-one analogues to effectively control breast cancer proliferation targeting ER and Akt enzyme [5]. Accumulated evidence suggests a strong correlation between ERα expression in a subset of patient-derived AML cells [11,12]. Methylation of ERα is frequently observed in normal AML karyotypes, which leads to repressed ERα gene transcription [13]. In addition, hypermethylation of ERα has been shown to improve AML survival rates [14], which indirectly indicates that ERα expression is positively correlated with disease progression. Remarkably, of the 54 genes known to drive AML progression, 35% are reportedly associated and regulated by ERα and E2 [15]. Attempts to regulate AML using ERα antagonists, such as tamoxifen, were found to be successful [16]. The binding position and computational docking score of SBL-060 were satisfactory, necessitating further in vitro evaluations of the compound. Consistent with computational observations, SBL-060 effectively inhibited ERα in a dose-dependent manner. This activity was also supported by GI 50 values of SBL-060 in the nano-molar range, as determined by antiproliferative assays against THP-1 and HL-60 cells.
Cell cycle and apoptosis assays were performed to further evaluate the effects of SBL-060 on the functionality of AML cells. The cell cycle is considered an important checkpoint for rapidly proliferating cells to determine life and death [17]. An increase in the sub G 0 /G 1 phase of AML cells has been associated with successful treatment of AML in vitro [18]. Furthermore, benzylidene chroman-4-ones were shown to induce sub G 0 /G 1 phase accumulation during cancer cell treatments [19]. Therefore, our data regarding the dosedependent increase in the sub G 0 /G 1 phase of AML cells following SBL-060 treatment agreed with the aforementioned reports. Moreover, ERα antagonists have been shown to control AML progression via induction of apoptosis [20]. Consistent with these studies, SBL-060 successfully induced dose-dependent apoptosis in both AML cell types examined.
Protein kinase B (Akt) is a serine-threonine kinase of the PI3K family [21]. Akt is a pivotal regulator of several key cell survival functions, including growth, cell division, angiogenesis, and suppression of apoptosis [22]. Akt dysregulation is frequently observed in numerous human cancer types, and several drug discovery efforts have focused on the development of Akt inhibitors [23]. In most patients with AML, constitutional Akt phosphorylation reportedly occurs via the PI3K-Akt-mTOR pathway, an important signal for AML cell survival [24,25]. Furthermore, studies have revealed a close homology between benzylidene compounds and target Akt for modulating cancer proliferation [26]. Therefore, computational screening of SBL-060 against the Akt enzyme should be further explored. Our docking scores strongly suggest the involvement of Akt inhibition by SBL-060. This computational efficacy was undoubtedly translated in our in vitro findings, exhibiting the dose-dependent inhibition of Akt phosphorylation by SBL-060 in both AML cell types. Previous studies have shown that cell cycle alterations and apoptosis induction can occur by inhibiting Akt kinase in AML cells [27][28][29]. The results of the present study corroborate those of previous reports, suggesting the efficacy of SBL-060 against AML cells by inhibiting Akt, thus hindering the regular cell cycle and inducing cellular apoptosis.

Conclusion
SBL-060 effectively controlled the proliferation of AML cells by inhibiting ERα and Akt kinase to induce apoptosis. The dual inhibitory efficacy of SBL-060 could be further explored to develop an effective chemotherapeutic agent against AML.
Availability of Data and Material: The datasets generated during and/or analyzed during the current study are available from the corresponding author on reasonable request.
Ethics Approval: This study does not use animals or human subjects and therefore does not require ethical approval.
Author Contribution: The authors confirm contribution to the paper as follows: Funding, experimental, data analysis and initial draft: Mesfer Al Shahrani; Experimental, statistical analysis and initial draft, conceptualization, final manuscript: Prasanna Rajagopalan; Analysis and interpretation of results, data curing: Mohammad Abohassan; Data analysis, supervision: Mohammad Alshahrani; Study supervision, data analysis, manuscript draft: Yasser Alraey; Initial draft, data analysis, revision of manuscript: Reem M. Gahtani; Experimental, statistical analysis, manuscript preparation: Suresh Radhakrishnan; Data curing, Manuscript draft, supervision: Khlood Dagreery. All authors reviewed the results and approved the final version of the manuscript.