Discipline of Medical Biochemistry, School of Laboratory Medicine and Medical Science, University of KwaZulu-Natal, Durban, South Africa.
Biomedical Resource Unit, School of Laboratory Medicine and Medical Sciences, College of Health Sciences, University of KwaZulu-Natal, Durban, South Africa.
Biotechnol Appl Biochem. 2021 Apr;68(2):257-266. doi: 10.1002/bab.1918. Epub 2020 May 20.
Di-2-picolylamine (DPA) is an organic compound that has been shown to possess antioxidant properties when conjugated to form a metal complex. The basis of this study was to determine the effects of DPA on the proliferation and apoptosis of human hepatocellular carcinoma cells and elucidate the possible mechanisms. The methylthiazol tetrazolium assay served to measure cell viability and generated an IC of 1591 µM. Luminometry was used to investigate caspase activity and ATP concentration. It was observed that the decreased cell viability was associated with reduced ATP levels. Despite increased Bax and caspase 9 activity, cell death was caspase independent as indicated by the reduction in caspase 3/7 activity. This was associated with the downregulation poly(ADP-ribose) polymerase cleavage (Western blotting). However, the Hoescht assay depicted nuclear condensation and apoptotic body formation with elevated DPA levels suggesting DNA damage in HepG2 cells. DNA damage assessed by the comet assay confirmed an increased comet tail formation. The presence of oxidative stress was investigated by quantifying reactive species (malondialdehyde and nitrates concentration) and Western blotting to confirm the expression of antioxidant proteins. The DPA increased lipid peroxidation (RNS), a marker of oxidative stress, consequently causing cell death. The accompanying upregulation of stress-associated proteins superoxide dismutase (SOD2), nuclear factor (erythroid-derived 2)-like 2 (Nrf2), and Hsp70 verifies oxidative stress.
二吡啶胺（DPA）是一种有机化合物，当与金属形成配合物时，表现出抗氧化特性。本研究的基础是确定 DPA 对人肝癌细胞增殖和凋亡的影响，并阐明可能的机制。噻唑蓝（MTT）比色法用于测量细胞活力，得到 IC 为 1591 µM。发光法用于研究半胱天冬酶活性和 ATP 浓度。结果表明，细胞活力的降低与 ATP 水平的降低有关。尽管 Bax 和半胱天冬酶 9 活性增加，但由于半胱天冬酶 3/7 活性降低，细胞死亡与半胱天冬酶无关。这与多聚（ADP-核糖）聚合酶裂解的下调有关（Western blot）。然而，随着 DPA 水平的升高，Hoescht 检测显示核浓缩和凋亡小体形成，提示 HepG2 细胞的 DNA 损伤。彗星试验评估的 DNA 损伤证实了彗星尾形成的增加。通过量化活性氧（丙二醛和硝酸盐浓度）和 Western blot 来研究氧化应激的存在，以证实抗氧化蛋白的表达。DPA 增加了脂质过氧化（RNS），这是氧化应激的一个标志物，从而导致细胞死亡。应激相关蛋白超氧化物歧化酶 2（SOD2）、核因子（红细胞衍生 2）样 2（Nrf2）和热休克蛋白 70 的上调证实了氧化应激的存在。
