Kim Yeong Hoon, Bhatt Lokraj, Ahn Hye-Jin, Yang Zhaoshou, Lee Won-Kyu, Nam Ho-Woo
Department of Ophthalmology, College of Medicine, The Catholic University of Korea, Seoul 06591, Korea.
Department of Parasitology, College of Medicine, The Catholic University of Korea, Seoul 06591, Korea.
Korean J Parasitol. 2017 Oct;55(5):491-503. doi: 10.3347/kjp.2017.55.5.491. Epub 2017 Oct 31.
The effects of tyrosine kinase inhibitors (TKIs) were evaluated on growth inhibition of intracellular Toxoplasma gondii in host ARPE-19 cells. The number of tachyzoites per parasitophorous vacuolar membrane (PVM) was counted after treatment with TKIs. T. gondii protein expression was assessed by western blot. Immunofluorescence assay was performed using Programmed Cell Death 4 (PDCD4) and T. gondii GRA3 antibodies. The TKIs were divided into 3 groups; non-epidermal growth factor receptor (non-EGFR), anti-human EGFR 2 (anti-HER2), and anti-HER2/4 TKIs, respectively. Group I TKIs (nintedanib, AZD9291, and sunitinib) were unable to inhibit proliferation without destroying host cells. Group II TKIs (lapatinib, gefitinib, erlotinib, and AG1478) inhibited proliferation up to 98% equivalent to control pyrimethamine (5 μM) at 20 μM and higher, without affecting host cells. Group III TKIs (neratinib, dacomitinib, afatinib, and pelitinib) inhibited proliferation up to 98% equivalent to pyrimethamine at 1-5 μM, but host cells were destroyed at 10-20 μM. In Group I, TgHSP90 and SAG1 inhibitions were weak, and GRA3 expression was moderately inhibited. In Group II, TgHSP90 and SAG1 expressions seemed to be slightly enhanced, while GRA3 showed none to mild inhibition; however, AG1478 inhibited all proteins moderately. Protein expression was blocked in Group III, comparable to pyrimethamine. PDCD4 and GRA3 were well localized inside the nuclei in Group I, mildly disrupted in Group II, and were completely disrupted in Group III. This study suggests the possibility of a vital T. gondii TK having potential HER2/4 properties, thus anti-HER2/4 TKIs may inhibit intracellular parasite proliferation with minimal adverse effects on host cells.
评估了酪氨酸激酶抑制剂（TKIs）对宿主ARPE-19细胞内刚地弓形虫生长抑制的影响。用TKIs处理后，计算每个寄生泡膜（PVM）内速殖子的数量。通过蛋白质印迹法评估刚地弓形虫的蛋白表达。使用程序性细胞死亡4（PDCD4）抗体和刚地弓形虫GRA3抗体进行免疫荧光测定。将TKIs分为3组；分别为非表皮生长因子受体（non-EGFR）、抗人EGFR 2（抗-HER2）和抗-HER2/4 TKIs。第I组TKIs（尼达尼布、AZD9291和舒尼替尼）在不破坏宿主细胞的情况下无法抑制增殖。第II组TKIs（拉帕替尼、吉非替尼、厄洛替尼和AG1478）在20 μM及更高浓度时抑制增殖高达98%，相当于对照乙胺嘧啶（5 μM），且不影响宿主细胞。第III组TKIs（来那替尼、达可替尼、阿法替尼和培利替尼）在1-5 μM时抑制增殖高达98%，相当于乙胺嘧啶，但在10-20 μM时会破坏宿主细胞。在第I组中，TgHSP90和SAG1的抑制作用较弱，GRA3表达受到中度抑制。在第II组中，TgHSP90和SAG1的表达似乎略有增强，而GRA3表现为无抑制至轻度抑制；然而，AG1478对所有蛋白均有中度抑制作用。第III组中的蛋白表达被阻断，与乙胺嘧啶相当。在第I组中，PDCD4和GRA3很好地定位于细胞核内，在第II组中轻度破坏，在第III组中完全破坏。本研究表明，存在一种具有潜在HER2/4特性的重要刚地弓形虫TK的可能性，因此抗-HER2/4 TKIs可能以对宿主细胞最小的不良反应抑制细胞内寄生虫的增殖。
