Laboratorio de Biotecnología I, Facultad de Ciencias Químicas, Universidad Autónoma de Chihuahua, Circuito Universitario s/n, Campus II, C. P. 31125, Chihuahua, Chih, México.
The Cellular Characterization and Biorepository (CCB) Core Facility, Border Biomedical Research Center, Department of Biological Sciences, The University of Texas at El Paso, 500 West University Avenue, El Paso, 79968-0519, TX, USA.
Invest New Drugs. 2021 Apr;39(2):400-415. doi: 10.1007/s10637-020-01020-2. Epub 2020 Oct 16.
Lactoferrin has gained extensive attention due to its ample biological properties. In this study, recombinant human lactoferrin carrying humanized glycosylation (rhLf-h-glycan) expressed in the yeast Pichia pastoris SuperMan5, which is genetically glycoengineered to efficiently produce functional humanized glycoproteins inclosing (Man)(GlcNAc) Asn-linked glycans, was analyzed, inspecting its potential toxicity against cancer cells. The live-cell differential nuclear staining assay was used to quantify the rhLf-h-glycan cytotoxicity, which was examined in four human cell lines: acute lymphoblastic leukemia (ALL) CCRF-CEM, T-cell lymphoblastic lymphoma SUP-T1, cervical adenocarcinoma HeLa, and as control, non-cancerous Hs27 cells. The defined CC values of rhLf-h-glycan in CCRF-CEM, SUP-T1, HeLa, and Hs27 cells were 144.45 ± 4.44, 548.47 ± 64.41, 350 ± 14.82, and 3359.07 ± 164 µg/mL, respectively. The rhLf-h-glycan exhibited a favorable selective cytotoxicity index (SCI), preferentially killing cancer cells: 23.25 for CCRF-CEM, 9.59 for HeLa, and 6.12 for SUP-T1, as compared with Hs27 cells. Also, rhLf-h-glycan showed significant antiproliferative activity (P < 0.0001) at 24, 48, and 72 h of incubation on CCRF-CEM cells. Additionally, it was observed via fluorescent staining and confocal microscopy that rhLf-h-glycan elicited apoptosis-associated morphological changes, such as blebbing, nuclear fragmentation, chromatin condensation, and apoptotic bodies in ALL cells. Furthermore, rhLf-h-glycan-treated HeLa cells revealed shrinkage of the microfilament structures, generating a speckled/punctuated pattern and also caused PARP-1 cleavage, a hallmark of apoptosis. Moreover, in ALL cells, rhLf-h-glycan altered cell cycle progression inducing the G2/M phase arrest, and caused apoptotic DNA fragmentation. Overall, our findings revealed that rhLf-h-glycan has potential as an anticancer agent and therefore deserves further in vivo evaluation.
乳铁蛋白因其丰富的生物学特性而受到广泛关注。在这项研究中，我们分析了在酵母毕赤酵母 SuperMan5 中表达的携带人源化糖基化的重组人乳铁蛋白（rhLf-h-glycan），该酵母经过基因糖基工程改造，能够有效地产生包括（Man）（GlcNAc）Asn 连接糖基在内的功能性人源化糖蛋白。我们研究了 rhLf-h-glycan 对癌细胞的潜在毒性。通过活细胞差异核染色分析来定量 rhLf-h-glycan 的细胞毒性，在四种人类细胞系中进行了检测：急性淋巴细胞白血病（ALL）CCRF-CEM、T 细胞淋巴母细胞瘤 SUP-T1、宫颈腺癌 HeLa，以及非癌细胞系 Hs27 作为对照。在 CCRF-CEM、SUP-T1、HeLa 和 Hs27 细胞中，rhLf-h-glycan 的定义 CC 值分别为 144.45±4.44、548.47±64.41、350±14.82 和 3359.07±164μg/mL。rhLf-h-glycan 表现出良好的选择性细胞毒性指数（SCI），优先杀伤癌细胞：与 Hs27 细胞相比，对 CCRF-CEM 的 SCI 为 23.25，对 HeLa 的 SCI 为 9.59，对 SUP-T1 的 SCI 为 6.12。此外，rhLf-h-glycan 在 CCRF-CEM 细胞孵育 24、48 和 72 小时后表现出显著的抗增殖活性（P<0.0001）。此外，通过荧光染色和共聚焦显微镜观察到，rhLf-h-glycan 引起 ALL 细胞中与凋亡相关的形态变化，如起泡、核碎裂、染色质浓缩和凋亡小体。此外，rhLf-h-glycan 处理的 HeLa 细胞显示微丝结构收缩，产生点状/点状图案，并导致 PARP-1 裂解，这是凋亡的标志。此外，在 ALL 细胞中，rhLf-h-glycan 改变细胞周期进程，诱导 G2/M 期阻滞，并导致凋亡性 DNA 片段化。总之，我们的研究结果表明，rhLf-h-glycan 具有作为抗癌剂的潜力，因此值得进一步进行体内评价。
