Department of Biotechnology, Faculty of Science and Humanities, SRM Institute of Science and Technology, Kattankulathur, 603 203 Chennai, Tamil Nadu, India.
Department of Biotechnology, School of Bioengineering, SRM Institute of Science and Technology, Kattankulathur, 603 203 Chennai, Tamil Nadu, India.
Free Radic Biol Med. 2019 May 1;135:198-209. doi: 10.1016/j.freeradbiomed.2019.03.006. Epub 2019 Mar 9.
Glutathione oxido-reductase (GR) is a primary antioxidant enzyme of most living forms which protects the cells from oxidative metabolism by reducing glutathione (GSH) from its oxidized form (GSSG). Although the antioxidant role of the enzyme is well characterized, the specific role of conserved N' peptide sequence in antioxidant mechanism remains unclear. In this study, we have identified an RNA sequence encoding GR enzyme from spirulina, Arthrospira platensis (Ap) and the changes in its gene expression profile was analysed during HO stress. Results showed that HO (10 mM) stimulated the expression of ApGR throughout the timeline of study (0, 5, 10, 15 and 20 days) with highest expression at 5 day post-exposure which confirmed the antioxidant role of ApGR in spirulina during HO induced oxidative stress. A dithiol containing short antioxidant peptide, GGTCVIRGCVPKKLM (GM15) from ApGR was predicted and its radicals (superoxide and hydroxyl radical) scavenging potential was confirmed by in vitro cell-free assays. GM15 (12.5 μM) reduced the intracellular generalized oxidative stress level, as measured using DCFDA assay in HO exposed leucocytes without affecting any of the cellular population. Further, the biomedical application of the radical scavenging property of GM15 was validated in oral carcinoma (KB) cells where GM15 exhibited significant cytotoxicity. Also, GM15 exhibited heterogenous effects on intracellular oxidative stress level in KB cells: at lower concentration (6.25 μM), the peptide reduced oxidative stress whereas, at higher concentration (25 μM) it increased the intensity of oxidative stress. GM15 (25 μM) induced caspase-9 mediated apoptosis in KB cells along with membrane disruption and DNA degradation which are confirmed by propidium iodide (PI) internalization and comet assays, respectively. Overall, the study shows that GM15 peptide i) scavenges superoxide, hydroxyl radicals, and influences intracellular oxidative stress, and ii) has anti-cancer effect in oral cancer cells.
谷胱甘肽氧化还原酶 (GR) 是大多数生命形式的主要抗氧化酶，通过将谷胱甘肽 (GSH) 从其氧化形式 (GSSG) 还原来保护细胞免受氧化代谢的影响。尽管该酶的抗氧化作用已得到很好的描述，但保守的 N' 肽序列在抗氧化机制中的具体作用仍不清楚。在这项研究中，我们从螺旋藻，节旋藻 (Arthrospira platensis) (Ap) 中鉴定出编码 GR 酶的 RNA 序列，并分析了其基因表达谱在 HO 应激期间的变化。结果表明，HO（10mM）在整个研究时间（0、5、10、15 和 20 天）内刺激 ApGR 的表达，暴露后 5 天表达最高，这证实了 ApGR 在螺旋藻中 HO 诱导的氧化应激期间的抗氧化作用。从 ApGR 预测到一种含有二硫键的短抗氧化肽，GGTCVIRGCVPKKLM（GM15），并通过体外无细胞测定证实了其清除自由基（超氧阴离子和羟自由基）的能力。GM15（12.5µM）在不影响任何细胞群体的情况下，使用 DCFDA 测定法降低了 HO 暴露的白细胞中的细胞内普遍氧化应激水平。此外，GM15 的自由基清除特性在口腔癌 (KB) 细胞中的生物医学应用得到了验证，其中 GM15 表现出显著的细胞毒性。此外，GM15 对 KB 细胞内氧化应激水平表现出异质性影响：在较低浓度（6.25µM）下，该肽降低氧化应激，而在较高浓度（25µM）下，它增加氧化应激强度。GM15（25µM）在 KB 细胞中诱导 caspase-9 介导的凋亡，同时伴有膜破裂和 DNA 降解，这分别通过碘化丙啶（PI）内化和彗星试验得到证实。总的来说，该研究表明 GM15 肽 i）清除超氧阴离子、羟自由基并影响细胞内氧化应激，ii）在口腔癌细胞中具有抗癌作用。
