Recombinant human lactoferrin carrying humanized glycosylation exhibits antileukemia selective cytotoxicity, microfilame
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PRECLINICAL STUDIES
Recombinant human lactoferrin carrying humanized glycosylation exhibits antileukemia selective cytotoxicity, microfilament disruption, cell cycle arrest, and apoptosis activities Sayuri Nakamura-Bencomo 1 & Denisse A. Gutierrez 2 & Elisa Robles-Escajeda 2 & Blanca Iglesias-Figueroa 1 & Tania S. Siqueiros-Cendón 1 & Edward A. Espinoza-Sánchez 1 & Sigifredo Arévalo-Gallegos 1 & Renato J. Aguilera 2 Quintín Rascón-Cruz 1 & Armando Varela-Ramirez 2
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Received: 9 September 2020 / Accepted: 9 October 2020 # Springer Science+Business Media, LLC, part of Springer Nature 2020
Summary 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)5(GlcNAc)2 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 CC50 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 CCRFCEM cells. Additionally, it was observed via fluorescent staining and confocal microscopy that rhLf-h-glycan elicited apoptosisassociated 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-hglycan 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. Keywords Apoptosis . Cancer . Cell cycle . Cytoskeleton . DNA fragmentation . Drug discovery . Humanized proteins
* Renato J. Aguilera [email protected]
Introduction
* Quintín Rascón-Cruz [email protected]
Lactoferrin (Lf) has recently attracted worldwide interest due to its ample range of biological and therapeutic properties [1–4]. Lf is a natural monomeric globular glycoprotein with the iron-binding capability and a member of the transferrin family [5, 6]. Previously known as lactotransf
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