Department of Clinical Laboratory Sciences, College of Applied Medical Sciences, King Khalid University, Abha, Saudi Arabia.
Department of Periodontics and Community Dental Sciences, College of Dentistry, King Khalid University, Saudi Arabia.
J Food Biochem. 2022 Aug;46(8):e14178. doi: 10.1111/jfbc.14178. Epub 2022 Apr 22.
Punica granatum (Pomegranate fruit) and its constituents are proven effective against various cancer types. However, a kinome-wide screening for the active phytochemicals against kinases is not reported. This study aims in validating pomegranate fruit extract (PFE) against acute myeloid leukemia (AML) cells, and computationally identifying the phytochemicals interacting with active kinases. PFE was made with Soxhlet extractor using absolute ethanol. Gas-chromatography-mass spectroscopy (GC-MS) for phytochemical identification and MTT assay for cytotoxicity in AML (THP-1, TF-1 and HL-60) cells were performed. Apoptosis, CDK5 and CDK8 were assessed with flow cytometry. Kinase profiling was performed using In silico kinome screening. GC-MS analysis revealed 38 bioactive phytochemicals in PFE including pyrazoles, aldehydes, phenols, esters, pyranosides, and octadecadienoic acids. The extract inhibited the AML cell proliferations with GI values of 195.5 μg/ml, 289.1 μg/ml, and 353.5 μg/ml in THP-1, THP-1, and HL-60 cells, respectively. PFE also exhibited a dose-responsive increase in apoptotic cell populations when treated to the AML cells. Computational screening and modeling predicted three critical constituents, viz., Deoxyartemisinin, 3-Methyl-3-phenyl-3H-indazole, and 8-fluoro-5,6-dimethoxy-3,4-dihydro-2H-naphthalen-1-one of pomegranate extract to interact mainly with cyclin-dependent kinases, including CDK5 and CDK8. Proteinand ligand docking predicted binding energies, and binding pose for top candidate lead molecules. In vitro assay exhibited the anticancer properties of PFE in AML cells. Computational kinome screening predicted top three PFE constituents targeting CDKs which may be responsible for the demonstrated anticancer efficacy of the extract against AML. This hypothesis further aligned with observed efficacy of PFE to inhibit CDK5 and CDK8 in all AML cells tested. PRACTICAL APPLICATIONS: Though Punica granatum (Pomegranate fruit) and its constituents are proven effective against various cancer types, a kinome-wide screening for the active phytochemicals against kinases is not reported. In this study, we have conducted GC/MS characterization of the active phytochemicals of PFE and have performed a kinome-wide screening for all the 38 identified compounds toward 310 active kinases commonly expressed in cancers. These observations warrant isolation and further evaluation of these phytochemicals or their analogues as effective CDK inhibitors against AML proliferation. Further, the computational methods used in this study will throw light on literature for new options of kinome panel screening of active phytochemicals or small molecules.
石榴（石榴果实）及其成分已被证明可有效对抗多种癌症类型。然而，针对激酶的活性植物化学物质的全激酶组筛选尚未见报道。本研究旨在验证石榴果提取物（PFE）对急性髓系白血病（AML）细胞的作用，并通过计算鉴定与活性激酶相互作用的植物化学物质。使用 Soxhlet 提取器用无水乙醇制备 PFE。进行气相色谱-质谱联用（GC-MS）分析以鉴定植物化学物质，并用 MTT 测定法测定 AML（THP-1、TF-1 和 HL-60）细胞的细胞毒性。通过流式细胞术评估细胞凋亡、CDK5 和 CDK8。使用计算机激酶组筛选进行激酶谱分析。GC-MS 分析显示 PFE 中含有 38 种生物活性植物化学物质，包括吡唑、醛、酚、酯、吡喃糖苷和十八碳二烯酸。该提取物在 THP-1、THP-1 和 HL-60 细胞中分别以 195.5μg/ml、289.1μg/ml 和 353.5μg/ml 的 GI 值抑制 AML 细胞增殖。当用 PFE 处理 AML 细胞时，还观察到细胞凋亡群体的剂量依赖性增加。计算机筛选和建模预测，石榴提取物的三种关键成分，即去氧青蒿素、3-甲基-3-苯基-3H-吲唑和 8-氟-5,6-二甲氧基-3,4-二氢-2H-萘-1-酮，主要与细胞周期蛋白依赖性激酶相互作用，包括 CDK5 和 CDK8。蛋白配体对接预测了候选先导分子的结合能和结合构象。体外试验显示 PFE 在 AML 细胞中的抗癌特性。计算机激酶组筛选预测了 PFE 的三种主要成分针对 CDK 的靶点，这可能是提取物对 AML 表现出抗癌功效的原因。这一假设与 PFE 抑制所有测试的 AML 细胞中的 CDK5 和 CDK8 的观察效果进一步吻合。实际应用：虽然 Punica granatum（石榴果实）及其成分已被证明可有效对抗多种癌症类型，但针对激酶的活性植物化学物质的全激酶组筛选尚未见报道。在这项研究中，我们对 PFE 的活性植物化学物质进行了 GC/MS 表征，并对 38 种已鉴定化合物进行了全激酶组筛选，以研究它们对 310 种常见于癌症中的活性激酶的作用。这些观察结果证明，需要对这些植物化学物质或其类似物进行分离和进一步评估，以作为有效的 CDK 抑制剂来抑制 AML 增殖。此外，本研究中使用的计算方法将为文献提供新的选择，以进行激酶组筛选，寻找活性植物化学物质或小分子。
