Department of Biotechnology, Mizoram University, Aizawl-796004, Mizoram, India.
Department of Biotechnology, PSG College of Technology, Coimbatore-641004, TN, India.
Anticancer Agents Med Chem. 2019;19(3):337-346. doi: 10.2174/1871520619666181127113848.
To explore the cytotoxic and apoptotic activity of the pierisin-6 protein in HPV HeLa and HepG2 cell lines.
In this study, isolation, and purification of cytotoxic Prierisin-6 from the larvae of Pieris napi by affinity column chromatography techniques. Characterization of full-length mRNA of pierisin-6 gene was performed using 3'/5' RACE PCR. The quantitative RT-PCR used to study the developmental stage-specific expression of pierisin-6 mRNA. The most effective concentration of Pierisin-6 protein was determined by measuring cell proliferation. Apoptosis was assessed using AO/Et-Br, Propidium Iodide, and Rhodamine 123 assays, whereas protein levels of caspase 3, cytochrome C were evaluated by ELISA method. Pierisin-6 induced cell cycle arrest was determined using Propidium iodide by FACS.
In this study, Pierisin-6, a novel apoptotic protein was found to have cytotoxicity against HeLa, HepG2 human cancer cell lines and L-132 human lung epithelial cell line. Among the target cells, HeLa was the most sensitive to Pierisin-6. Flow cytometry analysis confirms an increased percentage of apoptotic cells in sub G1 phase and cell cycle arrest at S phase. Alteration in the transmembrane potential of mitochondria, Cytochrome c released from the mitochondrial membrane, and caspase substrate assay demonstrated the cleavage of Ac- DEVD-pNA signifying the activation of Caspase-3. These findings suggested that Pierisin-6 significantly induce apoptosis in HeLa and HepG2 cells and is attributed mainly through a mitochondrial pathway by activation of caspases. The developmental and stage-specific expression of pierisin-6 mRNA was one thousand-fold increased from second to third instar larvae and gradually declined before pupation.
Pierisin-6 represents a promising therapeutic approach for liver cancer patients.
探讨 Pierisin-6 蛋白在 HPV HeLa 和 HepG2 细胞系中的细胞毒性和凋亡活性。
本研究采用亲和柱层析技术从 Pieris napi 幼虫中分离和纯化细胞毒性 Pierisin-6。通过 3'/5' RACE PCR 对 pierisin-6 基因全长 mRNA 进行特征描述。使用定量 RT-PCR 研究 pierisin-6 mRNA 的发育阶段特异性表达。通过测量细胞增殖来确定 Pierisin-6 蛋白的最有效浓度。使用 AO/Et-Br、碘化丙啶和罗丹明 123 测定法评估细胞凋亡，而通过 ELISA 方法评估 caspase 3、细胞色素 C 的蛋白水平。通过 FACS 用碘化丙啶测定 Pierisin-6 诱导的细胞周期停滞。
本研究发现，一种新型凋亡蛋白 Pierisin-6 对 HeLa、HepG2 人癌细胞系和 L-132 人肺上皮细胞系具有细胞毒性。在靶细胞中，HeLa 对 Pierisin-6 最为敏感。流式细胞术分析证实，亚 G1 期凋亡细胞的百分比增加，S 期细胞周期停滞。线粒体跨膜电位改变、线粒体膜释放的细胞色素 c 和 caspase 底物测定表明 Ac-DEVD-pNA 的切割，表明 caspase-3 的激活。这些发现表明，Pierisin-6 显著诱导 HeLa 和 HepG2 细胞凋亡，主要通过激活 caspase 通过线粒体途径。从第二龄幼虫到第三龄幼虫，pierisin-6 mRNA 的发育和阶段特异性表达增加了一千倍，然后在化蛹前逐渐下降。
Pierisin-6 代表了肝癌患者有希望的治疗方法。
