Department of Biochemistry, Faculty of Pharmacy, Near East University, Nicosia, Cyprus; Experimental Health Research Center of Health Sciences, Near East University, Nicosia, Cyprus.
Department of Pharmaceutical Botany, Faculty of Pharmacy, Near East University, Nicosia, Cyprus.
Gene. 2019 Feb 20;686:213-219. doi: 10.1016/j.gene.2018.11.047. Epub 2018 Nov 17.
Colchicum pusillum belongs to the family Colchicaceae that particularly rich in tropolonic alkaloids. The aim of this study was to investigate the cytotoxicity and in vitro anticancer activity of Colchicum pusillum ethanolic extract on Colo-320 primer and Colo-741 metastatic colon adenocarcinoma cell lines.
Colchicum pusillum was collected and extracted with ethanol. Different concentrations of Colchicum pusillum extract were incubated for 24 h and 48 h with Colo-320 and Colo-741 cells. Cell growth and cytotoxicity were measured by 3‑(4,5‑dimethylthiazol‑2‑yl)‑2,5‑diphenyltetrazolium bromide (MTT) assays. Anticancer and antiproliferative activities of Colchicum pusillum were investigated by immunocytochemistry using antibodies directed against to β-catenin, Ki-67, LGR-5 Ki-67, DKK1, Frizzled-4, Wnt4, Wnt7a and caspase3 in Colo-741 cells.
All concentrations of Colchicum pusillum extract had toxic effect in Colo-320 cells. Because of this, we used Colchicum pusillum extract at 20 μg/ml for evaluate anticancer activities only in Colo-741 cells. As a result of immunohistochemical staining, β-catenin, LGR-5 and caspase-3 immunoreactivities were significantly increased while Wnt7a immunostaining intensity was decreased in Colo-741 cells. Conclusion We conclude that Colchicum pusillum extract increased β-catenin and LGR-5 via Wnt/β-catenin pathway in colon cancer cells. Interestingly, it decreased other signaling molecule, Wnt7a which is assumed to play protective role during carcinogenesis. Also, it increased significantly caspase-3 immunoreactivity showing that apoptotic pathways were triggered.
秋水仙属于秋水仙科，该科植物富含托品烷生物碱。本研究旨在研究秋水仙乙醇提取物对结肠癌细胞 Colo-320 原代和 Colo-741 转移细胞系的细胞毒性和体外抗癌活性。
采集秋水仙并以乙醇提取。将不同浓度的秋水仙提取物与 Colo-320 和 Colo-741 细胞共孵育 24 和 48 小时。通过 3-(4,5-二甲基噻唑-2-基)-2,5-二苯基四氮唑溴盐 (MTT) 测定法测量细胞生长和细胞毒性。通过免疫细胞化学法用针对β-连环蛋白、Ki-67、LGR-5、Ki-67、DKK1、Frizzled-4、Wnt4、Wnt7a 和 caspase3 的抗体研究秋水仙对 Colo-741 细胞的抗癌和抗增殖活性。
秋水仙提取物的所有浓度在 Colo-320 细胞中均有毒性作用。因此，我们仅在 Colo-741 细胞中使用 20μg/ml 的秋水仙提取物来评估抗癌活性。免疫组织化学染色结果显示，β-连环蛋白、LGR-5 和 caspase-3 的免疫反应性显著增加，而 Wnt7a 的免疫染色强度降低。结论：我们得出的结论是，秋水仙提取物通过 Wnt/β-连环蛋白途径增加结肠癌细胞中的β-连环蛋白和 LGR-5。有趣的是，它降低了另一种信号分子 Wnt7a，该分子在癌变过程中可能发挥保护作用。此外，caspase-3 的免疫反应性显著增加，表明凋亡途径被触发。
