<p class="first" id="d1461530e162">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 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.
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