Biomedical and Pharmaceutical Sciences, Chapman University School of Pharmacy, 9401 Jeronimo Road, Irvine, CA, 92618, USA.
Dr. Panjwani Center for Molecular Medicine and Drug Research, International Center for Chemical and Biological Sciences, University of Karachi, Karachi, 75270, Pakistan.
BMC Complement Med Ther. 2020 Jan 13;20(1):5. doi: 10.1186/s12906-019-2804-1.
Nigella sativa (NS), a member of family Ranunculaceae is commonly known as black seed or kalonji. It has been well studied for its therapeutic role in various diseases, particularly cancer. Literature is full of bioactive compounds from NS seed. However, fewer studies have been reported on the pharmacological activity of proteins. The current study was designed to evaluate the anticancer property of NS seed proteins on the MCF-7 cell line.
NS seed extract was prepared in phosphate-buffered saline (PBS), and proteins were precipitated using 80% ammonium sulfate. The crude seed proteins were partially purified using gel filtration chromatography, and peaks were resolved by SDS-PAGE. MTT assay was used to screen the crude proteins and peaks for their cytotoxic effects on MCF-7 cell line. Active Peaks (P1 and P4) were further studied for their role in modulating the expression of genes associated with apoptosis by real-time reverse transcription PCR. For protein identification, proteins were digested, separated, and analyzed with LC-MS/MS. Data analysis was performed using online Mascot, ExPASy ProtParam, and UniProt Knowledgebase (UniProtKB) gene ontology (GO) bioinformatics tools.
Gel filtration chromatography separated seed proteins into seven peaks, and SDS-PAGE profile revealed the presence of multiple protein bands. Among all test samples, P1 and P4 depicted potent dose-dependent inhibitory effect on MCF-7 cells exhibiting IC values of 14.25 ± 0.84 and 8.05 ± 0.22 μg/ml, respectively. Gene expression analysis demonstrated apoptosis as a possible cell killing mechanism. A total of 11 and 24 proteins were identified in P1 and P4, respectively. The majority of the proteins identified are located in the cytosol, associate with biological metabolic processes, and their molecular functions are binding and catalysis. Hydropathicity values were mostly in the hydrophilic range.
Our findings suggest NS seed proteins as a potential therapeutic agent for cancer. To our knowledge, it is the first study to report the anticancer property of NS seed proteins.
黑种草（NS），毛茛科植物，俗称黑种子或黑孜然。它已被广泛研究用于治疗各种疾病，特别是癌症。文献中充满了 NS 种子的生物活性化合物。然而，关于蛋白质的药理活性的研究较少。本研究旨在评估 NS 种子蛋白对 MCF-7 细胞系的抗癌特性。
用磷酸盐缓冲盐水（PBS）制备 NS 种子提取物，并用 80%硫酸铵沉淀蛋白质。使用凝胶过滤色谱法对粗种子蛋白进行部分纯化，并用 SDS-PAGE 分离峰。MTT 法筛选粗蛋白和峰对 MCF-7 细胞系的细胞毒性作用。活性峰（P1 和 P4）进一步研究其对与凋亡相关基因表达的调节作用，实时逆转录 PCR。为了进行蛋白质鉴定，使用 LC-MS/MS 对蛋白质进行消化、分离和分析。数据分析使用在线 Mascot、ExPASy ProtParam 和 UniProt Knowledgebase（UniProtKB）基因本体（GO）生物信息学工具进行。
凝胶过滤色谱将种子蛋白分离成七个峰，SDS-PAGE 图谱显示存在多个蛋白质条带。在所有测试样品中，P1 和 P4 对 MCF-7 细胞显示出强烈的剂量依赖性抑制作用，IC 值分别为 14.25±0.84μg/ml 和 8.05±0.22μg/ml。基因表达分析表明凋亡可能是一种细胞杀伤机制。在 P1 和 P4 中分别鉴定出 11 种和 24 种蛋白质。鉴定出的大多数蛋白质位于细胞质中，与生物代谢过程有关，其分子功能是结合和催化。亲水性值大多在亲水范围内。
我们的研究结果表明 NS 种子蛋白是一种有潜力的癌症治疗剂。据我们所知，这是首次报道 NS 种子蛋白的抗癌特性。
