Biomedical and Pharmaceutical Sciences, Chapman University School of Pharmacy, 9401 Jeronimo Road, Irvine, CA, 92618, USA.
Jamil-ur-Rahman Center for Genome Research, International Center for Chemical and Biological Sciences, University of Karachi, Karachi, 75270, Pakistan.
BMC Complement Med Ther. 2024 May 20;24(1):195. doi: 10.1186/s12906-024-04495-1.
The burden of breast cancer, the second leading cause of death worldwide, is increasing at an alarming rate. Cuscuta, used in traditional medicine for different ailments, including cancer, is known for containing phytochemicals that exhibit anticancer activity; however, the bioactivities of proteins from this plant remain unexplored. This study aimed to screen the cytotoxic potential of proteins from the crude herbal product of Cuscuta epithymum(L.) (CE) harvested from the host plants Alhagi maurorum and Medicago sativa.
The proteins from CE were extracted using a salting-out method, followed by fractionation with a gel filtration chromatography column. Gel-free shotgun proteomics was subsequently performed for protein characterization. The viability assay using MTT was applied to deduce the cytotoxic potential of proteins against MCF-7 breast cancer cells, with further exploration of the effect of treatment on the expression of the apoptotic mediator BCL2-associated X protein (BAX) and B-cell lymphoma protein 2 (BCL-2) proteins, using western blotting to strengthen the findings from the in vitro viability assay.
The crude proteins (CP) of CE were separated into four protein peaks (P1, P2, P3, and P4) by gel filtration chromatography. The evaluation of potency showed a dose-dependent decline in the MCF-7 cell line after CP, P1, P2, and P3 treatment with the respective IC values of 33.8, 43.1, 34.5, and 28.6 µg/ml. The percent viability of the cells decreased significantly upon treatment with 50 µg/ml CP, P1, P2, and P3 (P < 0.001). Western-blot analysis revealed upregulation of proapoptotic protein BAX in the cells treated with CP, P3 (P < 0.01), and P2 (P < 0.05); however, the antiapoptotic protein, BCL-2 was downregulated in the cells treated with CP and P3 (P < 0.01), but no significant change was detected in P2 treated cells. The observed cytotoxic effects of proteins in the CP, P1, P2, and P3 from the in vitro viability assay and western blot depicted the bioactivity potential of CE proteins. The database search revealed the identities of functionally important proteins, including nonspecific lipid transfer protein, superoxide dismutase, carboxypeptidase, RNase H domain containing protein, and polyribonucleotide nucleotidyltransferase, which have been previously reported from other plants to exhibit anticancer activity.
This study indicated the cytotoxic activity of Cuscuta proteins against breast cancer MCF-7 cells and will be utilized for future investigations on the mechanistic effect of active proteins. The survey of CE proteins provided substantial data to encourage further exploration of biological activities exhibited by proteins in Cuscuta.
乳腺癌是全球第二大死亡原因，其负担正在以惊人的速度增加。菟丝子在传统医学中用于治疗各种疾病，包括癌症，其含有的植物化学物质具有抗癌活性；然而，这种植物的蛋白质的生物活性仍未得到探索。本研究旨在筛选从宿主植物骆驼刺和紫花苜蓿中收获的菟丝子粗草药（CE）的蛋白质的细胞毒性潜力。
使用盐析法从 CE 中提取蛋白质，然后用凝胶过滤色谱柱进行分级。随后进行无胶 shotgun 蛋白质组学分析以进行蛋白质特征描述。使用 MTT 活力测定法推断蛋白质对 MCF-7 乳腺癌细胞的细胞毒性潜力，进一步探讨治疗对凋亡介质 BCL2 相关 X 蛋白（BAX）和 B 细胞淋巴瘤蛋白 2（BCL-2）蛋白表达的影响，使用 Western blot 加强体外活力测定结果。
CE 的粗蛋白（CP）通过凝胶过滤色谱分离成四个蛋白峰（P1、P2、P3 和 P4）。效力评估显示，CP、P1、P2 和 P3 处理 MCF-7 细胞系后，IC 值分别为 33.8、43.1、34.5 和 28.6μg/ml，细胞活力呈剂量依赖性下降。当用 50μg/ml CP、P1、P2 和 P3 处理时，细胞的存活率显著下降（P<0.001）。Western blot 分析显示 CP、P3（P<0.01）和 P2（P<0.05）处理的细胞中促凋亡蛋白 BAX 上调；然而，CP 和 P3 处理的细胞中抗凋亡蛋白 BCL-2 下调（P<0.01），但 P2 处理的细胞中未检测到明显变化。从体外活力测定和 Western blot 观察到 CP、P1、P2 和 P3 中的蛋白质的细胞毒性作用表明 CE 蛋白质具有生物活性潜力。数据库搜索显示了功能重要蛋白质的身份，包括非特异性脂质转移蛋白、超氧化物歧化酶、羧肽酶、RNase H 结构域包含蛋白和多核苷酸核苷转移酶，这些蛋白质以前已从其他植物中报道具有抗癌活性。
本研究表明菟丝子蛋白对乳腺癌 MCF-7 细胞具有细胞毒性活性，并将用于进一步研究活性蛋白的作用机制。CE 蛋白质的调查为进一步探索菟丝子中蛋白质表现出的生物活性提供了大量数据。
