Blood Transfusion Research Center, High Institute for Research and Education in Transfusion Medicine, Tehran, Iran.
Blood Transfusion Research Center, High Institute for Research and Education in Transfusion Medicine, Tehran, Iran; Tarbiat Modares University, Faculty of Medical Sciences, Department of Immunology, Tehran, Iran.
Life Sci. 2019 May 1;224:249-254. doi: 10.1016/j.lfs.2019.03.072. Epub 2019 Mar 29.
The α-defensins or human neutrophil peptides (HNP 1-3) that exist in azurophilic granules are found to have anticancer activity. The pattern of disulfide bonds in α-defensins is crucial for the functional properties. Therefore, synthesis using the chemical and recombinant approaches is a challenging. A safe source for the production of α-defensins can be the use of leukoreduction filters in blood banks that contain large quantities of neutrophils and are discarded after use. The aim of this study was to purify α-defensins from neutrophils trapped in leukofilters and to investigate its anticancer activity.
Immunoprecipitation was performed to purify α-defensins and the presence of protein was confirmed by Western Blot. The Jurkat T-cell line was incubated with different concentrations (5, 10 and 15 μg/ml) of purified HNP1-3 for 16 h. Cell viability was measured using a WST-1 assay and apoptosis was analyzed for Annexin V/PI markers. Caspase-3/7 activity was determined using fluorescence assay. The effects of purified α-defensins were compared to commercial HNP 1-3.
Purified HNP 1-3 decreased the viability at 10 and 15 μg/ml and commercial HNP 1-3 at 15 μg/ml concentrations. Following to the purified HNP1-3 treatment, the percentage of Annexin V positive population and caspase-3 activity were significantly increased compared to control (p = 0.000 and p = 0.001, respectively) and commercial HNP1-3 (p = 0.034 and p = 0.018, respectively).
Results indicated the anticancer activity of HNP1-3 which can be used as future chemotherapeutic drugs. Furthermore, leukofilters can be considered as economic source for purifying these peptides.
存在于嗜天青颗粒中的 α-防御素或人中性粒细胞肽（HNP1-3）具有抗癌活性。α-防御素中二硫键的模式对于其功能特性至关重要。因此，使用化学和重组方法进行合成具有挑战性。使用血液库中的白细胞减少过滤器作为生产 α-防御素的安全来源是可行的，这些过滤器含有大量的中性粒细胞，使用后会被丢弃。本研究的目的是从白细胞过滤器中捕获的中性粒细胞中纯化 α-防御素，并研究其抗癌活性。
通过免疫沉淀法纯化 α-防御素，并通过 Western Blot 确认蛋白的存在。将 Jurkat T 细胞系与不同浓度（5、10 和 15μg/ml）的纯化 HNP1-3 孵育 16 小时。使用 WST-1 测定法测量细胞活力，并使用 Annexin V/PI 标志物分析细胞凋亡。使用荧光测定法测定 caspase-3/7 活性。将纯化的 α-防御素的作用与商业 HNP1-3 进行比较。
纯化的 HNP1-3 在 10 和 15μg/ml 浓度下降低了细胞活力，而商业 HNP1-3 在 15μg/ml 浓度下降低了细胞活力。与对照组相比，纯化的 HNP1-3 处理后 Annexin V 阳性细胞群和 caspase-3 活性的百分比显著增加（p=0.000 和 p=0.001），与商业 HNP1-3 相比也显著增加（p=0.034 和 p=0.018）。
结果表明 HNP1-3 具有抗癌活性，可作为未来的化疗药物。此外，白细胞过滤器可以被认为是纯化这些肽的经济来源。
