Razaghi Ali, Villacrés Carina, Jung Vincent, Mashkour Narges, Butler Michael, Owens Leigh, Heimann Kirsten
Centre for Biodiscovery and Molecular Development of Therapeutics, James Cook University, Townsville QLD 4811, Australia.
Department of Microbiology, University of Manitoba, Winnipeg, MB, Canada R3T 2N2.
Exp Cell Res. 2017 Oct 1;359(1):20-29. doi: 10.1016/j.yexcr.2017.08.014. Epub 2017 Aug 10.
Human interferon gamma (hIFNγ) affects tumour cells and modulates immune responses, showing promise as an anti-cancer biotherapeutic. This study investigated the effect of glycosylation and expression system of recombinant hIFNγ in ovarian carcinoma cell lines, PEO1 and SKOV3. The efficacy of E. coli- and mammalian-expressed hIFNγ (hIFNγ-CHO and HEK293, glycosylated/de-glycosylated) on cytostasis, cell death (MTT, and Guava-ViaCount flow-cytometry) and apoptotic signalling (Western blot of Cdk2, histone H3, procaspase-3, FADD, cleaved PARP, and caspase-3) was examined. Hydrophilic Interaction Liquid Chromatography determined the structure of N-linked glycans present in HEK293-expressed hIFNγ (hIFNγ-HEK). PEO1 was more sensitive to hIFNγ than SKOV3, but responses were dose-dependent and expression platform/glycosylation status-independent, whereas SKOV3 responded to mammalian-expressed hIFNγ in a dose-independent manner, only. Complex-type oligosaccharides dominated the N-glycosylation pattern of hIFNγ-HEK with some terminal sialylation and core fucosylation. Cleaved PARP and cleaved caspase-3 were not detected in either cell line, but FADD was expressed in SKOV3 with levels increased following treatment. In conclusion, hIFNγ did not induce apoptosis in either cell line. Mammalian- expressed hIFNγ increased cell death in the drug-resistant SKOV3. The presence of FADD in SKOV3, which may inhibit apoptosis through activation of NF-κB, could serve as a novel therapeutic target.
人干扰素γ（hIFNγ）可影响肿瘤细胞并调节免疫反应，有望成为一种抗癌生物疗法。本研究调查了重组hIFNγ的糖基化和表达系统对卵巢癌细胞系PEO1和SKOV3的影响。检测了大肠杆菌表达和哺乳动物表达的hIFNγ（hIFNγ-CHO和HEK293，糖基化/去糖基化）对细胞生长抑制、细胞死亡（MTT法和Guava-ViaCount流式细胞术）及凋亡信号传导（Cdk2、组蛋白H3、procaspase-3、FADD、裂解的PARP和caspase-3的蛋白质印迹法）的作用。亲水相互作用液相色谱法测定了HEK293表达的hIFNγ（hIFNγ-HEK）中N-连接聚糖的结构。PEO1对hIFNγ比SKOV3更敏感，但反应呈剂量依赖性且与表达平台/糖基化状态无关，而SKOV3仅以剂量非依赖性方式对哺乳动物表达的hIFNγ有反应。复杂型寡糖在hIFNγ-HEK的N-糖基化模式中占主导，有一些末端唾液酸化和核心岩藻糖基化。在两种细胞系中均未检测到裂解的PARP和裂解的caspase-3，但FADD在SKOV3中表达，处理后水平升高。总之，hIFNγ在两种细胞系中均未诱导凋亡。哺乳动物表达的hIFNγ增加了耐药性SKOV3中的细胞死亡。SKOV3中FADD的存在可能通过激活NF-κB抑制凋亡，可作为一个新的治疗靶点。
