Kabir Syed Rashel, Rahman Md Musfikur, Amin Ruhul, Karim Md Rezaul, Mahmud Zahid Hayat, Hossain M Tofazzal
Department of Biochemistry and Molecular Biology, Faculty of Science, Rajshahi University, Rajshahi, 6205, Bangladesh.
Bangladesh Council of Scientific and Industrial Research Laboratories, Rajshahi, Bangladesh.
Tumour Biol. 2016 Jun;37(6):8437-44. doi: 10.1007/s13277-015-4735-x. Epub 2016 Jan 5.
Recently, a lectin was purified from the potato cultivated in Bangladesh locally known as Sheel. In the present study cytotoxicity of the lectin against Ehrlich ascites carcinoma (EAC) cells was studied by MTT assay in vitro in RPMI-1640 medium and 8.0-36.0 % cell growth inhibition was observed at the range of 2.5-160 μg/ml protein concentration when incubated for 24 h. The lectin-induced apoptosis in EAC cells was confirmed by fluorescence and optical microscope. The apoptotic cell death was also confirmed by using caspase inhibitors. Cells growth inhibition caused by the lectin (36 %) was remarkably decreased to 7.6 and 22.3 % respectively in the presence of caspase-3 and -8 inhibitors. RT-PCR was used to evaluate the expression of apoptosis-related genes Bcl-X, p53, and Bax. An intensive expression of Bcl-X gene was observed in untreated control EAC cells with the disappeared of the gene in Sheel-treated EAC cells. At the same time, Bax gene expression appeared only in Sheel-treated EAC cells and the expression level of the p53 gene was increased remarkable after the treatment of EAC cells with the lectin. The lectin showed strong agglutination activity against EAC cells. Flow cytometry was used to study the cell cycle phases of EAC cells and it was observed that the lectin arrested the G2/M phase. In conclusion, Sheel lectin inhibited EAC cells growth by inducing apoptosis.
最近，从孟加拉国当地种植的名为“Sheel”的土豆中纯化出一种凝集素。在本研究中，采用MTT法在RPMI - 1640培养基中体外研究了该凝集素对艾氏腹水癌（EAC）细胞的细胞毒性，当在2.5 - 160μg/ml蛋白质浓度下孵育24小时时，观察到细胞生长抑制率在8.0 - 36.0%范围内。通过荧光显微镜和光学显微镜证实了该凝集素诱导EAC细胞凋亡。使用半胱天冬酶抑制剂也证实了凋亡性细胞死亡。在存在半胱天冬酶 - 3和 - 8抑制剂的情况下，凝集素引起的细胞生长抑制（36%）分别显著降低至7.6%和22.3%。采用RT - PCR评估凋亡相关基因Bcl - X、p53和Bax的表达。在未处理的对照EAC细胞中观察到Bcl - X基因的强烈表达，而在经Sheel处理的EAC细胞中该基因消失。同时，Bax基因表达仅出现在经Sheel处理的EAC细胞中，并且在用凝集素处理EAC细胞后，p53基因的表达水平显著增加。该凝集素对EAC细胞表现出强烈的凝集活性。使用流式细胞术研究EAC细胞的细胞周期阶段，观察到该凝集素使细胞停滞在G2/M期。总之，Sheel凝集素通过诱导凋亡抑制EAC细胞生长。
