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Endocytosis and transcytosis of diferric transferrin and apotransferrin in Caco-2 cells
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Endocytosis and transcytosis of diferric transferrin and apotransferrin in Caco-2 cells
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ENDOCYTOSIS AND TRANSCYTOSIS OF DIFERRIC TRANSFERRIN AND
APOTRANSFERRIN IN CACO-2 CELLS
Copyright 2006
by
Mei-Jung Lai
A Thesis Presented to the
FACULTY OF THE GRADUATE SCHOOL
UNIVERSITY OF SOUTHERN CALIFORNIA
In Partial Fulfillment of the
Requirements for the Degree
MASTER OF SCIENCE
(PHARMACEUTICAL SCIENCES)
May 2006
Mei-Jung Lai
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UMI Number: 1437852
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ii
ACKNOWLEGMENTS
I would like to thank Dr. Wei-Chiang Shen for his academic and research guidance
throughout my studies at USC. And I would like to thank Dr. Austin Yang and Dr.
David K. Ann for being my thesis committee members. A special thanks to Daisy
Shen for her support in the lab. Thanks to my labmates, they are helpful and kind
that I fell comfortable in the lab. In addition, I would like to thank my parents and
friends that give me support to complete studies at USC.
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iii
TABLE OF CONTENTS
ACKNOWLEDGEMENTS ii
LIST OF TABLES v
LIST OF FIGURES vi
ABSTRACT viii
I. BACKGROUND
1.1 Introduction 1
1.2 Structure and function of transferrin and apotransferrin 4
1.2.1 Structure 4
1.2.2 Biological function 7
1.3 Transferrin receptor 8
1.3.1 Structure, function and tissue distribution 8
1.3.2 Transferrin receptor 2 1 1
1.3.3 Association with transferrin-receptor protein FIFE 12
1.4 Transferrin receptor-mediated pathway 13
1.5 Application of transferrin receptor for drug delivery 14
1.5.1 Transferrin as a metallodrug carrier 16
1.5.2 Transferrin receptor-mediated transepithelial drug and
gene delivery 18
1.5.3 Transferrin receptor targeting for anticancer drugs 22
1.5.4 Transferrin receptor-mediated delivery across the
blood-brain barrier 24
1.6 Caco-2 cell monolayers 27
1.7 The regulation of absorption and transport by diferric transferrin
and apotransferrin in intestinal epithelia cells 30
II. MATERIALS AND METHODS
2.1 Materials 35
2.2 Preparation of DiFeTf and ApoTf 35
2.3 Iodination of DiFeTf and ApoTf 36
2.4 Cell culture 37
2.4.1 Caco-2 cell monolayers on cluster plates 38
2.4.2 Polarized Caco-2 cell monolayers on Transwell plates 38
2.5 BCA Protein Assay 39
2.6 Pulse-Chase experiments - Determination cell association of 1 2 5 I-DiFeTf
and 1 2 5 I-ApoTf in Caco-2 cell monolayers on cluster plates 40
2.7 Pulse-Chase experiments - Determination cellular uptake of 1 2 5 I-DiFeTf
and 1 2 5 I-ApoTf in Caco-2 cell monolayers on cluster plates 41
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2.8 The competition of different concentration unlabeled DiFeTf and ApoTf on
the cellular uptake of 1 2 5 I-DiFeTf and 1 2 5 I-ApoTf in Caco-2 cell monolayers
2.9 The effect of different chase medium on cellular uptake in
Pulse-Chase studies
2.10 Analysis of uptake and apical-to-basolateral and
basolateral-to-apical transcytosis across polarized Caco-2 cells
III. RESULTS AND DISCUSSION
3.1 Cell association (uptake and surface binding) of DiFeTf and ApoTf in
Caco-2 cells
3.2 Effect of concentration competition on cellular uptake of DiFeTf
and ApoTf
3.3 Effects of different chase medium on cellular uptake of DiFeTf
and ApoTf
3.4 Recycling, uptake, and transcytosis of DiFeTf and ApoTf in polarized
Caco-2 cells
IV. CONCLUSION
REFERENCES
IV
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LIST OF TABLES
Table 1. Proteins involved in intestinal iron absorption
Table 2. Results of percentage of initially endocytosed ligands
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LIST OF FIGURES
Figure 1. X-ray crystal structure of human serum transferrin
Figure 2. X-ray crystal structure of the dimeric ectodomain of the human
transferrin receptor
Figure 3. The pathway of cellular uptake of iron from transferrin via
transferrin receptor mediated endocytosis
Figure 4. Immunoliposomes provide the potential to deliver drugs to specific
target sites
Figure 5. A scheme of a covalent conjugate of a monoclonal antibody to the
TfR using the avidin/biotin technology
Figure 6. Transwell bicameral chamber
Figure 7. Three pathways exist in the small intestinal cell for the uptake of
dietary iron
Figure 8. Percentage of cellular association of DiFeTf and ApoTf in Caco-2
cell monolayers
Figure 9. Measurement of the amount of cell-associated of DiFeTf and ApoTf
Figure 10. Determination of cellular uptake and surface association
Figure 11. Effects of different concentration of unlabeled DiFeTf and ApoTf
on cellular uptake
Figure 12. Cell uptake of DiFeTf and ApoTf at different concentration
Figure 13. Effects of different concentration of DiFeTf and ApoTf in
chase medium on cellular uptake
Figure 14. Effects of exchanged and different concentration chase medium
on cellular uptake
Figure 15. Recycling(A), cell association(B), and transcytosis(C) of DiFeTf
and ApoTf in filter-grown (12-well) polarized Caco-2 cells
Figure 16. Recycling(A), transcytosis(B), and cell association(C) of DiFeTf
and ApoTf in filter-grown (6-well) polarized Caco-2 cells
VI
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49
52
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Figure 17. Apical recycling, transcytosis, and cell association in Caco-2 cells
from apical cell surface dosing
Figure 18. Basal recycling, transcytosis, and cell association in Caco-2 cells
from basal cell surface dosing
Figure 19. The amount of DiFeTf and ApoTf in filter-grown polarized cells
from apical dosing
Figure 20. The amount of DiFeTf and ApoTf in filter-grown polarized cells
from basal dosing
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ABSTRACT
Transferrin (Tf), the major iron transport protein in the blood, has been used
as a potential carrier in oral delivery of protein drugs. To investigate the effect of
iron-binding on the intracellular retention and transcytosis of Tf, diferric transferrin
(DiFeTf) and apotransferrin (ApoTf) were subjected to pulse-chase studies in
Caco-2 cells. The cellular uptake of DiFeTf and ApoTf in nonpolarized Caco-2 cells
were not different from each other. Results suggest that DiFeTf has higher binding
affinity to the receptor. In polarized Caco-2 cells, the recycling of DiFeTf was
higher than that of ApoTf, while the percentage of transcytosis of ApoTf was higher
than that of DiFeTf. No significant difference was found in the cell retention of the
forms of Tf. The data suggest that DiFeTf and ApoTf may have different trafficking
pathways in polarized Caco-2 cells.
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I. BACKGROUND
1.1 Introduction
Most of protein drugs are limited to invasive routes of administration, such as
intravenous or subcutaneous. Oral delivery of protein drugs, the most convenient
noninvasive administration has been studied for decades (Shen, W. C., 2003).
Transferrin (Tf) is the natural transport protein for delivery irons into mammalian
cells, including intestinal epi helCl cells. Cellular iron uptake from the blood is
mediated by T f binding to its specific transferrin receptor (TfR), followed by
TfR-mediated endocytosis, and iron dissociation from Tf in the endosomal
compartment. Iron-depleted apotransferrin (ApoTf) will recycle to the cell surface
and dissociated from TfR (Li, H., et al, 2002; Qian, Z. M., et al, 2002 Windera, A, et
al, 2003). Tf has been considered as a carrier in drug delivery for transcellular
transport, such as crossing small intestinal and alveolar epithelial cells, and the
blood-brain barrier, and for intracellular targeting such as tumor cells (Li, H., et al,
2002; Shah, D., et al, 1995). The delivery mechanism is through TfR expressed on
the cell surface and TfR is expressed abundantly in the human gastrointestinal
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epithelium. Therefore, Tf has been used as a carrier for oral delivery of protein drugs
(Windera, A., et al, 2003; Windera, A, et al, 2004; Xia, C. Q., et al, 2000).
Using Caco-2 cells grown in bicameral inserts, e.g. Transwells, as a model of
intestinal epithelia, it has been shown that apotransferrin and iron-containing Tf
undergo different endocytosis cycles from basolateral membrane (Nunez, M. T., et al,
1997). Diferric transferring (DiFeTf) and apotransferrin (ApoTf) compete to
basolateral TfR-binding which, in turr will determine the iron net exchange
between intestinal cells and the basolateral milieu. However, there were findings
indicating that endocytic cycle of the TfR in intestinal epithelia cell depends on the
nature of the bound ligand (Nunez, M. T., et al, 1997). First, both DiFeTf and ApoTf
recycled to the cell surface after internalization, and ApoTf has a protracted
endocytic cycle. Second, both of the ligands were found in a compartment close to
the basal membrane, but ApoTf reached as well regions closer to the apical
membrane. Confocal microscopy studies revealed a different cellular distribution.
Third, cells incubated with ApoTf presented a more apical distribution of TfRs than
cells incubated with DiFeTf. These results showed that the endocytic cycle of TfRs
in intestinal epithelial cells is determined by the iron content of T f and explained the
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marked inhibition of ApoTf on Tf-mediated iron uptake (Nunez, M. T., et al, 1996),
since ApoTf actually sequesters the TfR in intracellular compartments.
When Caco-2 cells grown in bicameral cambers are a model system for
intestinal iron transport, DiFeTf and ApoTf were internalized from the basal
chamber into distinct vesicles. ApoTf was internalized and routed to a perinuclear
position and DiFeTf to a more basal position below the nuclei (Alvarez-Hemandez,
Z., et al . 2000). These results suggested that Caco-2 cells distinguish ApoTf ffon
DiFeTf and sort them to different compartments early in the processing of me
TfR-bound Tf. Subsequently, ApoTf is routed to a more apical compartment where it
could acquire newly adsorbed iron. Newly adsorbed iron is transported by
DMT1/Nramp2, an iron transport, which is located in the apical membrane of
intestinal epithelium (Canonne-Hergaux, F., et al, 1999). Therefore, a model of iron
transport could include the endocytosis of ApoTf from basolateral membrane to a
compartment where ApoTf could acquire iron. Then iron-transferrin complex would
exocytose and release iron at basolateral surface.
The mechanism involved in ApoTf uptake by different cell types has not been
completed elucidated. Besides, some recent confocal microscopy studies in Caco-2
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cells indicated that ApoTf underwent different basolateral-to-apical from that of
DiFeTf. In order to understand the underlying mechanism in the oral administration
of macromolecular drugs via TfR-mediated transcytosis, the cellular processing of
DiFeTf and ApoTf was investigated in both polarized and non-polarized Caco-2
cells.
1.2 Structure and function of transferrin and apotrrnsfA n
1.2.1 Structure
Tfs are a family of metal-binding glycoproteins composed of a single chain
polypeptide containing 700 amino acids with approximately molecular mass 80 kDa.
The polypeptide chain folded into two structurally similar functionally different
lobes, N and C lobes, connected by a short peptide. Each lobe can be divided into
two domains. There is deep hydrophilic cleft bearing an iron binding site Fe+ 3
coordinated with distorted octahedral geometry to two oxygens from two tyrosines,
one nitrogen from a histidine, one oxygen from an aspartate, and two oxygens from
a bidentate carbonate (Fig. 1) (Li, H., et al, 2002). Lack of this synergistic
bicarbonate anion affects the ability of Tf to release Fe+ 3 once it is bound. The
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ligands are from two domains and two polypeptide strands which cross over
between two domains at the back of iron site. The domains can move apart to form
an open conformation leading iron release (Li, H., et al, 2002; Qian, Z. M„ et al,
2002; Widera, A., et al, 2003). In addition iron, many divalent and trivalent metal
ions can bind to the iron sites. Thus, T f can act as a delivery system for therapeutic
metal ions in the body.
Figure 1. (A) X-ray crystal structure of human serum transferrin. The C-lobe is
shown in a closed form and the apo N-lobe is in an open form. (B) The mental
binding sites of human serum transferrin with residue numbers of the N-lobe in
brackets. (Li, H., Qian, Z. M., 2002, Medicinal Research Reviews, 2002;
22(3):225-250)
Asp302(§3)
Tyr517{1M)
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Tf undergoes conformational changes induced by the decrease in pH during
Fe+ 3 uptake and release. The shape altering affects the binding affinity (Li, H., et al,
2002; Qian, Z. M., et al, 2002; Widera, A., et al, 2003). The iron-loaded Tf binds to
the receptor with greater affinity than ApoTf. The mechanism of Fe+ 3 uptake and
release involve a pH-sensitive interaction. Endocytosis of Fe+ 3-Tf complex into an
acidic endosome (pH 5.5) results in the protonation of residues Lys209 and Lys301
located on opposite domains and provides the drivhr, foice to push two domains
apart (Li, H., et al, 2002; Qian, Z. M., et al, 2002). Fe+ 3 is exposed and facilitated
release to result ApoTf formation.
Deferoxamine (DFO), an iron chelating agent, has been used both in vitro and
in vivo,studies mediated by intracellular pool o f iron (Dayani, P. N., et al, 2004). It is
produced by the bacteria, Streptomyces pilosus and binds iron 1:1 stoichiometrically.
DFO is hydrophilic and does not cross the cell mebrane easily. It is internalized by
fluid-phase endocytosis into pinosomes containingTf. The iron-bound complex,
ferrioxamine, has very high chemical stability. Although transferrin is a bilobular
molecule which binds one Fe+ 3 in each of lobes, previous studies have shown that
apotransferrin and iron-deficient tissue culture medium can be prepared by adding
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DFO (Eby, J. E., et al, 1992). In thisthesis, we used DFO as an affinity matrix to
remove Fe+ 3 from DiFeTf and culture medium.
1.2.2 Biological function
Tfs have been studied intensively few decades. The liver is the major site of
transferrin production and cstabolism. Tfs have been classified into three major
srib-dasses: serum Tf, lacoferrin, and ovotransferrin (Li, I , et al, 2002; Qian, Z. M.,
et al, 2002; Widera, A., et al, 2003). Serum Tf is responsible for binding and
transporting iron into the systemic circulation to the cells and tissues and has been
identified as a potential drug carrier. Lactoferrin is found in breast milk, saliva, and
tear secretions. It acts as a bacteriostatic agent by chelating the iron and also is
involved in modulating immune and inflammatory responses. Ovotransferrin is
found in avian and reptile oviduct secretions and avian egg white. It also exhibits
antimicrobial activity. A new member of the transferrin family, melanotransferrin,
has been identified as an integral membrane protein in human malignant melanoma
cells and in some fetal tissues (Li, H., et al, 2002; Qian, Z. M., et al, 2002; Widera,
A., et al, 2003). Serum Tf has been studied in endocytosis and used as a potential
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drug carrier in drug delivery. In serum, the concentration of Tf is about 2.5 mg/ml.
Although transferrin is primarily an iron-binding protein, only about 30% Tf in
serum is saturated with iron and 70% exists as ApoTf (Du, X. L., et al, 2004).
1.3 Transferrin receptor
1.3.1 Structure, function and tissue distribution
The transferrin receptor (TfR) is a transmembrane h « , mo-;. .mer which consists
two identical monomers with 90 kDa molecular mass. The monomers are covalently
linked by two disulfide bonds. Each TfR monomer binds one moleculae of
transferrin. The ectodomain of the receptor is composed of three distinct domains,
the protease-like, apical, and helical domains (Fig. 2) (Li, H., et al, 2002; Lawrence,
C. M, et al, 1999). The extracellular portion of the receptor is subject to extensive
post-translational modification with three sites of mannose rich N-linked
glycosylation and one O-linked glycosylation. The post-translational modifications
seem to be crucial for its function (Li, H., et al, 2002; Qian, Z. M., et al, 2002;
Widera, A., et al, 2003).
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9
Ap icaJ
Domain ^
H e i i c a l
DomaSn
Proteas e-L.. ue.
Domain
1 1
P lg s m g M e m b r a n e
Figure 2. X-ray crystal structure of the dimeric ectodomain of the hiKian transferrin
receptor. It contains of three distinct domains. (Lowrence C. M., et al, Science, 1999;
286:779-782)
Recent studies showed that both C and N lobe of human serum transferrin are
necessary for receptor recognition. The interaction of Tf and TfR has been a model
of structural characteristics of the two proteins and the available functional
information. Site-directed mutagenesis studies have shown that the central helical
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domains of TfR are keys to the ability of receptor to recognize Tf. Like Tf, TfR also
has conformational changes associated with pH. The motion of apical domain
transduces pH changes into changes in the T f binding cleft. It results the relative
affinity of TfR for DiFeTf and ApoTf (Li, H.,- et al, 2002; Qian, Z. M., et al, 2002;
Widera, A., et al, 2003). Generally, TfRs have a higher affinity to DiFeTf than
ApoTf and different TfRs may have very different affinities toTf. At physiological
pH 7.4, the iron saturation status of Tf plays a large role in the affinity towards TfR.
However, at pH 5.0 in endosomes, Tf is very stable with TfR, which is for the iron
exchange and transport capability of Tf. Crystal structure of TfR complexed with the
hemochromatosis protein HFE showed that HFE binds to the helical domain of Tf.
Thus, HFE and Tf have competitive effect on an overlapping site of TfR (Li, H., et
al, 2002; Qian, Z. M., et al, 2002; Widera, A., et al, 2003).
The TfR assists iron uptake into cells through a cycle of endocytosis and
exocytosis of Tf. TfR is expressed in many tissue types in the body and is
particularly high in erythrocyte precursors, placenta, and the liver. The TfR also has
been found in the epithelium of the small intestine, monocytes, blood-brain barrier,
type II pneumocytes, and neoplastic carcinomas (Li, H., et al, 2002; Qian, Z. M., et
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al, 2002; Widera, A., et al, 2003). In malignant cells, there is TfR expression
attributed to the high level of iron requirement for their growth. The presence of TfR
in different tissues had led to develop targeted delivery therapeutic carriers for
various diseases.
1.3.2 Transferrin receptor 2
A new TfR-b’ke family member, TfR2, has been cloned and sequenced. TfR2
is a tpye II transmembrane glycoprotein, sharing a 45% identity and 66% similarity
in its extracellular domain with TfRl. TfR2 has a similar function to TfRl with
respect to Tf binding and Tf-mediated iron uptake. TfR2 has been thought to be
involved primarily in maintenance of iron homeostasis (Li, H., et al, 2002; Qian, Z.
M., et al, 2002; Widera, A., et al, 2003). Both TfRl and TfR2 interact with T f in a
pH-dependent manner. DiFeTf binds to these receptors at neutral or higher pH and
ApoTf binds prefer at acidic pH. In contrast to TfRl, it is primarily expressed in the
liver. However, unlike TfRl, TfR2 has a much lower affinity for Tf (25 fold lower
than TfRl) (Widera, A., et al, 2003).
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12
1.3.3 Association with transferrin-receptor protein HFE
For iron metabolism, there are two critical proteins: T f and HFE. HFE
mutated in hereditary hemochromatosis to cause excess dietary iron absorption and
iron deposition in several tissues (Li, H., et al, 2002; Qian, Z. M., et al, 2002;
Widera, A., et al, 2003). HFE was found to coprecipitate with TfRl in cultured cells.
Each TfRl is associated with a single HFE molecule (Parkkila, S., et al, 1997). HFE
binds to TfRl with a high affinity at physiological pH pH 7.4 and i competes with
Tf for binding to TfR. Unlike ApoTf, HFE has very low binding affinity for TfR at
the acidic intracellular vesicles pH. It suggests that HFE dissociates from TfRl in
acidic endosomes (Li, H., et al, 2002; Qian, Z. M., et al, 2002;Sharma, N., et al,
2005; Widera, A., et al, 2003). By interactions with T f and TfR, HFE is implicated in
sensmg and regulating iron level, particularly at the level of iron uptake through the
duodental enterocyte.
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1.4 Transferrin receptor-mediated pathway
The mechanism of Fe+ 3 uptake and release involve a pH-sensitive interaction.
This process involves: binding, endocytosis, acidification and dissociation (Fig. 3)
(Andrew, N.C., 1999; Li, H., et al, 2002). It is initiated by the binding of DiFeTf to
TfRs on the outer plasma membrane because TfRs bind to DiFeTf with a high
affinity. Endocytosis of Fe+ 3 -Tf complex via clathrin-coated pits into an acidic
endosome (pH 5.5) allows the release of iron in the endoso ne by decreasing the
affinity. Iron is transported by iron transporter DMT1/Nramp2 into cytoplasm. Then,
the ApoTf-TfR complex is recycled back to the cell surface. At extracellular
physiological pH, apotransferrin dissociates from its receptor due to its low affinity
at pH 7.4 and is released into the circulation and reutilized after reloading with iron.
Iron that enters the cell is used for metabolic functiosn or stored as ferritin.
ATP-mediated energy is necessary for TfR-mediated endocytosis and recycling
(Andrew, N. C., 1999; Li, H., et al, 2002; Qian, Z. M., et al, 2002; Widera, A., et al,
2003).
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14
F erro u s iron ira n tp c rle r
(DM TIt
ClaiiwMi-
coate*i pit
Rsslwsesl
fm
» » • Mrtoctondna
Pi man jsam p
Fonisin
Non«fytli«mi
C « t! s
Cwpiasm
Acidi««S snckHomo
{pH 5.5)
Figure 3. The pathway of cellular uptake of iron from transferrin via transferrin
receptor mediated endocytosis. (Andrew N. C., The New England Journal of
Medicine, 1999; 341: 1986-1995)
1.5 Application of transferrin for drug delivery
Tf and TfR-mediated endocytosis has the potential of sit-specific and
target-oriented drug delivery systems (Li, H., et al, 2002; Qian, Z. M., et al, 2002;
Shah, D., et al, 1995; Widera, A., et al, 2003). The efficient Tf uptake mechanism
has been exploited for drug delivery and targeting, not only for therapeutic agents
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across the intestinal epithelium and blood-brain barrier but also for anticancer drugs.
Because of the abundance of TfR. on the brain endothelium and gastrointestinal
epithelium, Tf appeared to be a carrier for significant transcellular transport. For the
TfR-mediated transport of therapeutics across the blood-brain barrier, anti-TfR
antibodies were chose as delivery carrier to avoid competition with high levels of
endogenous T f present in the serum. Therefore, therapeutics was linked to the carrier
antibody chemically or through genetic engineering to be ie f -ered to the brain
(Qian, Z. M., et al, 2002; Li, H., et al, 2002; Shah, D., et al, 1995). For many
peptides and proteins, the large molecular size and hydrophilicity make them
impermeable to the cellular membranes. T f is a carrier of these proteins in
TfR-mediated transepithelial transport. The Brefeldin A (BFA), a transcytotic
enhancer, has been used overcome the limitation of transcellular transport due to the
low efficiency of transcytosis (Shah, D., et al, 1995; Shah, D., et al, 1996; Wan, J., et
al., 1992; Widera, A., et al, 2003). BFA can change the TfR distribution in the
epithelium to increase the number of TfRs on the mucosal surface of intestinal
epithelium and enhance the mucosal-to-serosal delivery across the intestinal
epithelium. On the other hand, chemotherapy plays a big part in the treatment of
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16
tumors. Tf-mediated pathway has been interested in for cytotoxic drugs targeting
into tumor cells. The density of TfR on the cell surface increases when cells
proliferate highly and become malignant. In addition, the short recycling of TfRs
and high exceeding number of TfRs on the cell surface increase efficiency of
TfR-mediated endocytosis (Shah, D., et al, 1995).
1.5.1 Transferrin as a metallodrug carrier
The transferrins are iron-binding proteins and only 30 % serum Tf saturated
with iron, thus serum Tf has a high capacity for binding to other metal ions of
therapecutic and diagnostic interest, such as Bi+ 3 , Ru+ 3, Ti+ 4 , and Ga+ 3 . These metal
ions have been reported to bind to T f with carbonate, oxalate, or carboxvlate as
synergistic anions (Qian, Z. M., et al, 2002; Sun, H., et al, 1999). Such binding play
a large role in the transport and delivery of medical diagnostic radioisotopes such as
6 7 Ga+ 3 and U 1ln+ 3 and therapeutic metal ions such as Bi+3, Ru+ 3, and Ti+ 4 (Li, H., et
al, 2002; Qian, Z. M., et al, 2002). Ga+ 3 is one of the most useful tumor diagnostic
agents and gallium compounds have been used extensively in the diagnosis and the
treatment of human cancers. Gallium binds to T f in the Fe+ 3 binding sites with
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similar affinity (Harris, W. R., et al, 1983). In vivo studies have shown that Ga in
blood is present in plasma and is tightly bound to Tf (Clausen, J., et al, 1974). So
gallium is transported mainly via TfR mediated mechanism. Similarly, Bi+ 3
complexes are in use in the treatment of ulcers. Bismuth binds strongly to Tf in the
specific iron binding sites with affinities similar as iron and is transported via TfR
mediated endocytosis similar as iron (Li, H., et al, 1996; Sun, H., 1998; Sun, H.,
1999). Bismuth not only interfere with iron uptake by binding to Tf bu. h also be
released within cells and inhibit target enzymes that are for baaierial survival
(Domenico, R, et al, 1996). Ruthenium complexes with amine or dimethylsulfoxide
ligands exhibit high anticancer cancer in vivo and are active against metastases but
not against the primary tumors (Clarke, M. J., et al, 1999; Kraz, R, et al, 1994). It
has been shown that Ru+ 3 is transported in the blood by transferrin and albumin.
Ru+ 3-Tf complex results in high tumor uptake of the metal since TfRs are found in
many tumors than in normal cells. Ru+ 3-Tf complex may provide a less toxic and
more effective antitumor agents by TfR mediated uptake (Ando, A., et al, 1988; Som,
P, et al, 1983). Another example is titanium. Ti+ 4 complexes have been shown to
exhibit high antitumor activities with less toxic side effects (Keppler, B. K., et al,
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18
1993; Kopf-Maier, P., et al, 1987). Cell uptake studies showed that titanium is likely
transported via a similar mechanism as iron, TfR-mediated endocytosis (Guo, M., et
al, 2000; Messori, L., et al. 1999). Therefore, Tf may serve as a carrier to deliver
theses specific metals (Li, H., et al, 2002; Qian, Z. M., et al, 2002 Windera, A, et al,
2003; Shah, D., et al, 1995).
1 :: .2 Transferrin receptor-mediated transepithelial drug and gene delivery
TfR. targeted therapy has emerged as a modem drug-delivery tool. Targeting
of TfR leads to delivery therapeutic agents into tissues or across epithelial barriers
and some advantages of transferrin-drug conjugates includes a preferable tissue
distribution, prolonged half-life in the plasma, and controlled drug release from
conjugates.
Various therapeutic agents have been chemically linked to Tf. The
incorporation of dmgs with Tf by using recombinant protein engineering technique
is a novel approach for cellular delivery of therapeutic agents through TfR uptake
pathway (Li, H., et al, 2002; Qian, Z. M., et al, 2002 Windera, A, et al, 2003). For
example, although doxorubicin is an effective and widely used cancer
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chemotherapeutic agents, cardiotoxicity and drug-resistance limit its utility. The
Tf-doxorubicin conjugate has shown greatly increasing cytotoxicity by accumulation
in tumor cells through transmembrane mechanism, different from doxorubicin, and
overcome drug resistance (Li, H., et al, 2002; Qian, Z. M., et al, 2002). The
Tf-mediated endocytosis also used to delivery insulin in Caco-2 cells and in diabetic
rats using insulin-Tf conjugate linked by a disulfide bond (Li, H., et al, 2002; Xi'a, C.
Q : et al, 2000). This novel approach is potentially useful for developin g "Oral
therapeutic proteins. Liposomes have shown the potential in the introduction of
chemotherapeutic agents with reduced toxicity and extended plasma half-life. They
consist of one or more concentric phospholipids bilayers. A liposomal carrier system
was produced by using small unilameller liposomes made of pure phospholipids
chemically cross-linked to Tf. It was interact specifically with leukemia HL60 cells.
The conjugate was internalized by active receptor-mediated endocytosis (Li, H., et al,
2002; Qian, Z. M., et al, 2002;Sarti, P. et al, 1996). Liposomes conjugated with
anti-TfR also are used for specific drug delivery.
Tf based conjugates have been considered for systemic non-invasive delivery
of therapeutic agents. The small intestine and type II pneumocytes, expressing TfRs,
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provide a potential target for transepithelial delivery of macromolecules when orally
administrated. One problem associated with this approach is that most of surface Tf
lies at the basolateral or serosal membrane in differentiated epithelial cells. This
location tends to prelude the ability of apical-to-basolateral transcytosis of Tf-drug
conjugates. There are three enhancing agents to overcome: lysosomotrophic amines,
lipophilic carboxylic ionophores, and Brefeldin-A (Widera, A., et al, 2003). BFAhas
b . .n extensively used in the study of intracellular trafficking events and it has
dramatic effects on the structure and function of the Golgi complex and the
endosomal sorting pathways (Lippincott-Schuartz, J., et al, 1991; Klausner, R. D., et
al, 1992). In polarized epithelial cells, BFA treatment significantly enhances the
basolateral-to-apical transcytosis of internalized TfR (Prydz, K., et al, 1992). In
addition, the effects of BFA on protein sorting appear to be much more sensitive in
the apical surface (Low, S. H., et al, 1992). In two highly polarized cell systems,
MDCK cells and primary cultured rat type II pneumocytes, BFA has the ability to
enhance TfR-mediated transcytosis in the apical-to-basolateral direction (Windera,
A., et al, 2003). Thus BFA is as an enhancer of TfR-mediated drug delivery. Recent
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21
studies have shown that AG-10, a GTPase inhibitor, also increase the transport of an
insulin-Tf conjugate across Caco-2 cells. (Xia, C. Q., et al, 2001)
The specific delivery of therapeutic genes to defined target cells is a major
goal o f gene therapeutic strategies. The successful introduction of DNA into target
cells could unleash many strategies o f gene therapy that are limited by inefficient
delivery and expression of the therapeutic DNA as a result of low targeting and
transfecuon efficiency and interactions with serum proteins. Non-vial ve ;to ,,
including molecular conjugates and cationic liposomes, have been exploited (Li, H.,
et al, 2002; Qian, Z. M., et al, 2002). Molecular conjugate is composed of nucleic
acids condensed with polycations that can be cross-linked to a ligand for cell
targeting. T f has been used as a general targeting molecule to direct DNA to rapidly
dividing cells. Recently, novel Tf-polylysine and Tf-protamine conjugates have been
shown to be effective carriers for the introduction of genes into cells. It could
enhance cell uptake and transfection activity significantly. Similarly,
Tf-polyethylenimine conjugates also have shown to be as vectors to transfer
therapeutic DNA into cells. Cationic liposomes complexed with DNA have been
used as non-viral vectors for intracellular delivery of receptor or therapeutic genes.
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22
However, poor transfection efficiency is the major drawback and application is
limited by the inhibition of serum. Association of transferrin with cationic
liposome-DNA complexes significantly overcomes the problems. Therefore, this
class of prodrugs is potential for targeted gene delivery (Li, H., et al, 2002; Qian, Z.
M., et al, 2002).
1.5.3 Transferrin receptor targeting for anticancer drugs
The larger numbers of TfRs are expressed on the surface on tumor cells and
TfR is expressed more aboundantly in malignant tissues than their normal
counterparts (Gatter, K. C., et al, 1983; Shindelman, J. E., et al, 1981). TfR is also
more abundant in rapidly dividing cells than quiescent cells (Larrick, L. W., et al,
1979). Therefore, Tf is a good drug carrier and the TfR has been considered as a
suitable target for receptor-mediated endocytosis in delivery therapeutic agents.
Several studies have demonstrated that anticancer drug conjugated with transferrin
through TfR mediated pathway is highly effective for treating cancer in animals and
in humans (Widera, A., et al, 2003). For example, a conjugation of Tf and CRM107
has been used in patients with malignant brain tumors (Li, H., et al, 2002; Widera,
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A., et al, 2003; Qian, Z. M., et al, 2002). Patients received high doses of
Tf-CRM107 exhibit neurological deficits attributed to endothelial damage as a result
of TfRs expressed by capillary endothelial cells in the brain. A new treatment is i.v.
injection of chloroquine during intracerebral infusion of Tf-CRM107; There are dual
benefits: reducing toxicity in undesired tissues and increasing the targeting
efficiency to cancer cells (Hagihara, N., et al, 2000; Laske, D. W., et al, 1997). A
various tovns conjugated to T f has been investigated to increase uptake at the
cellular surface. The incorporation of the toxins in liposomal delivery vehicies,
make them more efficient in cellular uptake (Singh, M., 1999). An alternate
approach to treat cancer cells relies on gene therapy, such as tumor repressor gene
p53. The conjugation with Tf overcomes some limitations of cationic
liposome-based p53 gene delivery vehicles (Seki, M., et al, 2002).
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24
1.5.4 Transferrin receptor-mediated delivery across the blood-brain barrier
The delivery of therapeutic agents to the brain is limited by the low
permeability to the brain capillary endothelial cells that form the blood-brain barrier
(BBB). However, specific receptor-mediated transport mechanism in brain
endothelial cells can be used as means to delivery therapeutic agents (Li, H., et al,
2002; Qian, Z. M., et al, 2002; Widera, A, et al, 2003). Anti-receptor antibodies have
been shown to ta. ge' 8BB endothelium selectively as brain drug transport vectors
(Bickel, U., et ai, 2001). These antibodies are also potential carriers for delivery of
therapeutic agents to the central nerves systems (CNS) and the 0X26 appears to be
the most efficient vector candidate in brain drug delivery. The 0X26 antibody binds
to TfR at a site distinct from the transferrin binding site and does not interfere with
transferrin binding (Qian, Z. M., et al, 2001). Thus, the 0X26 delivery system is not
as sensitive to inhibition by circulation transferrin as the transferrin-drug conjugates
and have applied to antisense, radiopharmaceuticals, therapeutic peptides or protein,
and brain-derived neurotrophic factor (BDNF) (Li, H., et al, 2002; Widera, A, et al,
2003). Therefore, conjugates of therapeutics with the 0X26 antibody undergo
receptor-mediated transcytosis through the BBB via the brain capillary endothelial
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TfR. It has been reported that immunoliposomes (antibody-directed liposomes)
showed potential for both drug and gene delivery to the brain when they conjugated
with the 0X26 monoclonal antibody against the rat transferrin receptor (Fig. 4)
(Huwyler, J., et al, 1996; Li, H., et al, 2002; Qian, Z. M., et al, 2002; Shi, N., et al,
2000; Widera, A, et al, 2003). Small molecule drugs or an exogenous plasmid DNA
has been incorporated into the interior of liposomes, which are pegylated with
polyethylene glycol (PEG) of 2000 Da molecular mass. A thiolated antibody, the
0X26 murine mAh was coupled to the terminal end of PEG 2000 (Huwyler, J., et al,
1996). Besides the chemical conjugation coupling the therapeutics to the 0X26
antibody using the avidin/biotin systems has been made as brain transport vectors
has been made (Fig. 5) (Li, H., et al, 2002; Li, J. Y., et al, 1999; Widera, A, et al,
2003).
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26
0X26
J
DNA
PEG 2000
* * -■D rug
\m •& a » ia
a *, a v a
Liposome * a »
■ * i i
TRENDS in Phaimacofogicai Sciences
Figure 4. Immunoliposomes provide the potential to deliver drugs to specific target
sites. This vector ha;; been shown to be effective in delivery of both small-molecule
drugs and plasmids into the brain. (Li, H., Sun, H., Qian, Z. M., Trends in
Pharmacological Sciences, 2002; 23(5): 206-209)
Blood-Brain
Barrier
TflRMAbi
Brain B l o o d
Figure 5. A scheme of a covalent conjugate of a monoclonal antibody to the TfR
using the avidin/biotin technology. (Brickel et al., Proc Natl Acad Sci USA, 90:
2618-2622,1993)
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27
1.6 Caco-2 cell monolayers
The Caco-2 cell line, derived from a human colon carcinoma, is able to
differentiate spontaneously when grown in standard culture conditions
(Alvarez-Hemandez, X., et al, 1991; Audus, K. L., et al, 1990; Sanchez, L. et al,
1996). Caco-2 cells grown into monolayers on porous membrane in bicameral
chambers are an excellent model system for studies in intestinal epithelial cells.
Because the cells form a-highly polarized monolayer, they exhibit many of the
features of small intestine cells, and have been used to demonstrate vectorial
transport (Alvarez-Hemandez, X., et al, 1991; Audus, K. L., et al, 1990).
For bioavailability studies, cells need to be cultured in Transwell bicameral
chamber until they form fully differentiated monolayers (Fig. 6). The Transwell
bicameral camber has an upper and lower chamber. Upper (apical) chamber stands
for luminal side and lower (basal) chamber stands for serosal side. Transport study
was performed after the transepithelial electrical resistance (TEER) had risen to
more than 250 Q cm2, indicating that the Caco-2 monolayer has formed tight
junctions (Alvarez-Hemandez, X., et al, 1991; Audus, K. L., et al, 1990; Ismail, M.,
1999). TEER is measured with an epithelial voltohmeter. This instrument is a
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chopstick like electrodes with short electrode being inserted in the medium of the
Transwell insert whereas the longer electrode is placed in the medium of the lower
chamber. Electrical resistance values of the cell monolayers in the inserts are
measured daily and values are multiplied by surface area minus the resistance of
blanks of insert without cells.
T & n w d l insert
U p p c a n p r t in e s i
Iwerranprtmerit
Figure 6. Transwell bicameral chamber.
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Caco-2 cells grown on polycarbonate membranes demonstrated a gradual
increase in TEER with maximal TEER being reached at about day 18 of culture.
They have found to exhibit structural and functional differentiation features
normally associated with the characteristics of mature enterocytes. From electron
microscopic analysis of Caco-2 cells, after day 18 cell monolayers have showed
with a highly polarized structure, abundant microvilli in the apical surface, well
developed tight junctions,, glucagons storage grains, and clathrin-coated pits
(Alvarez-Hemandez, X., et al, 1991; Hidalgo, I. J., et al, 1989; Simpson, R. J., et al,
1984). It has been demonstrated that the polarized TfR on the basolateral surface
occurs in the fully differentiated Caco-2 cell monolayers (Shah, D., et al. 1994).Thus
in this study I used differentiated Caco-2 cells grown in bicameral chambers as a
intestinal cell model to study TfR-mediated transport and cell uptake in the
apical-to-basolateral direction.
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30
1.7 The regulation of iron absorption and transport by diferric transferrin and
apotransferrin in intestinal epithelia cells
Iron is vital for living organisms because it is essential for multiple metabolic
processes. Thus, its concentrations in body organs must be regulated carefully.
Intestinal absorption is the primary mechanism for regulating iron concentrations in
the body. Gastrointestinal iron transport has been controlled by several proteins,
such as HFE, associated with hereditary hemochromatosis, and divalent metal
transporter DMT1/Nramp2, a membrane iron transport. These proteins also play an
important role in the transferrin cycle (Tab. 1) (Li, H., et al, 2002; Morgan, E. H., et
al, 2002; Qian, Z. M., et al, 2002; Sharma, N., et al, 2005). There are three pathways
for intestinal iron uptake: the mobilferrin-integrin pathway, the divalent mental
transporter l(DMT-l) pathway, and a separate pathway for uptake of heme by
absorptive cells (Conrad, M. E., et al, 1999). Ferrous iron is taken up by the divalent
metal transporter (DMT1), which is located on the apical villous membrane. DMT1
is a EC/divalent metal symporter that also transports other divalent mentals. The
absorption of dietary inorganic ferric iron is facilitated by the mobilferrin-integrin
pathway. Heme iron is taken up by a DMT 1-independent, protein bound forms such
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31
as acidic isoferrin or hemoglobin (Fig. 7) (Conrad, M. E., et al, 1999; Hentze, M. W.,
et al, 2004; Ma, Y., et al, 2002; Sharma, N., et al, 2005).
Expression
Site
Protein Function Crypt V illus O th er cells
c n e e r o n r o
depletion
DMT1 Fe(ll) transporter
-
+ + (m any) Increase
Ferroportin Fe(tl) tra : no te r
- + 1 Increase
D cytb Ferrireductase
- + -
Increase
H epsestin Ferroxida se - + { N o Change
Ferritin Iron storage + + + (m any) D ecrease
H FE Regulator + - + No Chance
Hepcidin* Regulator
- -
Liver Decrease?
m i Transferrin endocytosis + + I (m any) Increase
TfR2 Transferrin endocytosis
o ■ )
L iver N o Change
Transferrin Iron transport
- -
L iver Increase
IRP1 Regulator + + + (m any) Increase
IRP2 Regulator f + i (m any) Increase
See text for details.
* H epcidiu is a 2 0 -2 5 ainino acid peptide.
+ Iron overload usually produces the opposite effect to iron depletion.
Table 1. Proteins involved in intestinal iron absorption. (Morgan, E. H., Oates, P. S.,
Blood cells, molecules, and diseases. 2002; 29(3):384-399)
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32
Postulated Mechanisms of Iron Absorption
F e +++
F e - M u c in - F e
I
F e
F e A sco rb ate
f= e ++- D C T I-F e” -
Hemoglobin/M yoglobin
'F erritin
F e Fe Fe
■ATR
globin
Luminal Mucosal Corporeal
Figure 7. Three pathways exist in the small intestinal cell for the uptake of food iron.
M = mobilferrin, P = paraferritin. (Conrad, M. E. et al., The American Journal of
The Medical Sciences. 1999; 318(4):213-229)
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The transport of iron across the intestinal epithelium occurs in three phases.
An uptake phase occurs during the transport from the lumen of the intestine into an
apical compartment of the epithelial cells. A transport phase occurs during the
transport across the cell to the serosal or basolateral surface. Finally, a transfer phase
occurs during the transport across the basolateral membrane to the blood. There is a
relationship between apotransferrin and iron flux in intestinal epithelial cells. The
ratio ApoTfrDiFeTf in u e basal medium determines the amount of cellular
absorption of iron (iron absorption) (Nunez, M. T., et al, 1997). Basolateral
apotransferrin increases the apical to basolateral iron transport in iron-deficient
Caco-2 cells. The binding of DiFeTf to TfR was inhibited competitively by ApoTf
and ApoTf inhibited Tf-mediated iron uptake (Nunez, M. T., 1996). This inhibition
was larger than expected solely by receptor competition suggesting that ApoTf
affects other steps in the iron uptake process. Furthermore, densitometric analysis
indicated that DiFeTf distributed mainly in the first half of the basal to apical axis,
while ApoTf distributed into the second third of the axis. Confocal microscopy
revealed differences in the distribution of internalized TfRs when Caco-2 cells were
incubated with ApoTf or DiFeTf. While the incubation with DiFeTf resulted in a
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receptor distribution mostly in the first half of the basal to apical axis, the incubation
with ApoTf produced a more even distribution of receptors in this axis. In addition,
pulse-chase experiments indicated that there is a characteristic protracted endocytic
cycle of ApoTf. It sequesters the TfRs inside the cell making it less available for iron
uptake (Nunez, M. T., et al, 1997).
The role of Tf in the transfer phase has been evaluated in a variety of studies.
Precious studies have shown tha:, tb : iransfer phase is facilitated by the presence of
ApoTf in the basal chamber and ApoTf undergoes a different endocytic cycle than
DifeTf. Besides, the transfer phase of the process of iron absorption is sensitive to
the concentration of ApoTf. This suggests that ApoTf has a unique transport
mechanism, which is different from the traditional TfR pathway, to interact with
basal surface of the Caco-2 cells (Alvarez-Hemandez, X., et al, 1998).
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35
II. MATERIALS AND METHODS
2.1 Materials
Human apotransferrin, deferroxamine, and ferric ammonium citrate were
purchased from Sigma. All cell culture materials including Dulbecco’s Modified
Eagle’s Medium (DMEM), fetal bovine serum, trypsin-EDTA, and
penicillin/streptomycin were products of Gibco BRL (Rockville, MD). Sephadex
G-25 was purchased from Ame.sh,v:it Biosciences (Sweden). Chloramine-T, sodium
metabisulfite, and potassium iodide were purchased from Sigma. BCA Protein Assay
Kit was purchased form Pierce (Rockford, IL).
2.2 Preparation of DiFeTf and ApoTf
Apotransferrin was prepared by dissolving 40 mg human apotransferrin in 1
ml PBS. Differic transferrin was prepared by loading iron to human apotransferrin.
Ferric ammonium citrate (200 pi of 10 mg/ml H2 0 ) was added to 1 ml human
apotransferrin (40 mg/ ml PBS). The mixture was incubated at 37°C for 1 h, then
dialyzed overnight in 2L PBS at 4 °C.
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36
2.3 Iodination of DiFeTf and ApoTf
Differic transferrin was iodinated using the chloramine-T method. 1 2 5 I-labeled
differic transferrin was purified by size exclusion chromatography using a 10-ml
Sephadex G-50 gel column, eluted with phosphate buffer saline (PBS). An aliquot of
1 2 5 I was added to the differic transferrin solution. After mixing, 50 pi Chloramaine-T
(4 mg/0.5 ml PBS), an oxidizing agent, was first added for iodide activation. The
mixture was maintained at room temperature for 5 min, and 50 pi sodium
metabisulfite (2.4 mg/0.5 ml PBS), a reducing agent, was added to terminate the
reaction. Finally, 100 pi potassium iodide (10 mg/ml d.d.F^O) subsequently was
added to complete the nonspecific binding of 1 2 5 I. The reaction mixture was applied
onto the column. Each 1 ml fraction was collected for a total of twelve test tubes. To
determine radioactivity in each fraction, 0.5 pi from each test tube was collected and
measured by using a Packard gamma counter (Packard Instruments, Meriden, CT).
To make sure that transferrin was saturated with iron, 1 2 5 I-diferric transferrin was
incubated with appropriate amount of ferric ammonium citrate for 30 min prior to
the experiments.
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37
The 1 2 5 I-labeled apotransferrin was prepared from 1 2 5 I labeled diferric
transferrin. 2.5 pi deferoxamine (20 mg/ml d.d.H20 ) was added to 0.5 ml
I2 5 I-diferric transferrin (1 mg/ml) and incubated at 37°C for 30 min. Before the
experiments, the appropriate amount of deferoxamine was added to the liquid ensure
the complete conversion of 1 2 5 I -DiFeTf to1 2 5 I-ApoTf.
2.4 Cell culture
Caco-2 cells were obtained from the American Type Culture Collection
(ATCC). They were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM)
supplemented with penicillin/streptomycin and 10 % fetal bovine serum (FBS) in
25cm2 T-25 flasks. When they reached confluence on day 7, the medium was
removed and cells were detached from flask by 0.25ml lOx trypsin-EDTA. The
single cell suspension was formed by gentle crushing with 5 ml fresh medium. The
cell number was counted by a Coulter Counter (Coulter Electronics, Hialeah, FL).
50,000 cells/ cm2 were seeded in 6-well/12-well cluster or Transwell plates for
experiments and appropriate cell numbers were seeded in a new T-25 flask as the
stock.
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38
2.4.1 Caco-2 cell monolayers on cluster plates
Caco-2 cells were cultured and grown into monolayers in 6-well cluster plates
(Costar, Coming, NY). The medium was replaced every 2 to 3 days. The cells
reached confluence for experiment on day 12 to 14. Prior to the experiments, Caco-2
cells were incubated with serum-free medium, containing lmg/ml bovine serum
albumin, at 37°C for 30 min to deplete endogenous transferrin.
2.4.2 Polarized Caco-2 cell monolayers on Transwell plates
Caco-2 cells were cultured and grown into monolayers on 12-well Transwell
plates. The medium was replaced 2 to 3 days. For transport studies, the
transepithelial electrical resistance (TEER) of monolayers as measured with an
epithelial voltohmmeter (EVOM) was >250 ohms*cm2. Prior to the experiments,
Caco-2 cells were incubated with serum-free medium, containing lmg/ml bovine
serum albumin, at 37°C for 30 min to deplete endogenous transferrin. This
procedure did not affect the TEER of the cell monolayers.
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39
2.5 BCA Protein Assay
The BCA Protein assay is based on a detergent-compatible formulation based
on bicinchoninic acid (BCA) for the colorimeric detection and quantitation of total
protein. The purple color reaction product is formed by the chelation of two
molecules of BCA with one cuprous ion. And it exhibits a strong absorbance that is
nearly linear at 562 nm with increasing protein concentrations (20-2000 pg/ml).
Protein concentrations are determined with reference to standards of bovine serum
albumin (BSA).
BCA Protein Assay Kit was used to determine the amount of proteins in the
Caco-2 cells grown on the plates. Half ml of 1 N NaOH was added to each sample
of cell lysate and incubated for 10 min at 37°C. After mixing, 25 pi of each standard
and sample was placed in a 96-well plate. By the Microplate Procedure, first, the
working reagent was prepared by combining 50 parts of BCA Reagent A with 1 part
of BCA Reagent B. Second, 200 pi working reagent was added to each microplate
well which contained 25 pi blank, standard or sample. Third, samples and reagents
were mixed gently for 30 seconds then incubated at 37°C for 1 h. After incubation,
the microplate was cooled down to room temperature. The absorbance at 562 nm of
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40
samples was measured on a plate reader. The concentration of protein in each
sample was calculated by the standard concentration curve prepared from BSA over
a broad range (20-1000 |!g/ml).
2.6 Pulse-Chase experiments - Determination cell association of 1 2 5 I-DiFeTf and
1 2 5 I-ApoTf in Caco-2 cell monolayers on cluster plates
Caco-2 cell monolayers grown on 6-well cluster plates were washed with PBS
twice and preincubated at 37°C for 30 min in serum-free DMEM containing 1
mg/ml bovine serum albumin (BSA) to deplete endogenous transferrin. Caco-2 cells
were pulsed with 1 2 5 I-DiFeTf and 1 2 5 I-ApoTf at 37°C for 30 min. After that, the
medium was removed. Caco-2 cells were rinsed with coid PBS twice quickly and
added 100-fold unlabeled DiFeTf and ApoTf to prevent reintemalization of
dissociated labeled Tf. Cell associated 1 2 5 I-DiFeTf and 1 2 5 I-ApoTf, including cellular
uptake and surface-bound 1 2 5 I-DiFeTf and 1 2 5 I-ApoTf, were chased at 37°C in
serum-free DMEM containing 1 mg/ml BSA and 100-fold unlabeled DiFeTf and
ApoTf for 0 min, 15 min, 30 min, 1 h, and 2 h. At the end of chase period, medium
was collected and the cell monolayers were washed with cold PBS then dissolved in
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41
1 N NaOH. Both of them were assayed in Packard gamma counter. The radioactivity
level in medium was used as a measurement of ligand release. The radioactivity
level measured in the cell monolayers was the amount of cell-associated !25I-DiFeTf
and 1 2 5 I-ApoTf.
2.7 Pulse-Chase experiments - Determination cellular uptake of 1 2 5 I-DiFeTf and
1 2 5 I-ApoTf in Caco-2 cell monolayers on cluster plates
Caco-2 cell monolayers grown on 6-well cluster plates were washed with PBS
twice and preincubated for 30 min in serum-free DMEM with 1 mg/ml bovine
serum albumin (BSA) at 37°C to deplete endogenous transferrin. 1 2 5 I-DiFeTf
(3ug/ml) and 1 2 5 I-ApoTf (3ug/ml) were added in each well for 30 min at 37°C in
serum-free DMEM with 1 mg/ml BSA (pulse). After treatment, the medium was
removed. Caco-2 cells were rinsed with cold PBS twice quickly and added with
100-fold unlabeled DiFeTf and ApoTf in serum-free DMEM, containing 1 mg/ml
BSA, for 0 min, 1 h, and 2 h at 37°C (chase). Caco-2 cell monolayers were washed
with acetate buffer (pH 4) for 5 min at room temperature then detached by
incubation with trypsin. The cells in each well were suspended in 1 ml cold PBS,
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42
and centrifuged at 2500 rpm for 20 min, to remove the supemant. The I25I activity in
the cell pallets was assayed by using a Packard gamma counter.
2.8 The competition on cellular uptake by different concentration of unlabeled
DiFeTf and ApoTf
Caco-2 cell monolayers grown on 6-well cluster plates were washed with PBS
twice and preincubated, at 37°C for 30 min in ? enna-free DMEM containing 1
mg/ml bovine serum albumin (BSA) to deplete endogenous transferrin. The cell
monolayers were treated with 1 2 5 I-DiFeTf (3ug/ml) and I25I-ApoTf (3ug/ml)
together with 0, 10, or 100-fold concentration o f unlabeled DiFeTf and ApoTf,
respectively, in serum-free DMEM containing 1 mg/ml BSA at 37°C for 2 h. After
treatment, the medium in each well was removed. Caco-2 cell monolayers were
washed with cold PBS twice then trypsin was added to detach Caco-2 cells. The cell
suspension from each well was centrifuged at 2500 rpm for 20 min, to remove the
supemant. The radioactivity in Caco-2 cells was determined by using a Packard
gamma counter. The BCA Protein Assay was performed for quantitation of total
protein in Caco-2 cells after the cell pallet was dissolved ini N NaOH.
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
43
2.9 Pulse-Chase studies of different chase medium effect on cellular uptake
Caco-2 cell monolayers grown on 6-well cluster plates were washed with PBS
twice and preincubated for 30 min at 37°C in serum-free DMEM conatining 1
mg/ml bovine serum albumin (BSA) to deplete endogenous transferrin. The cell
monolayers were pulsed with 1 2 5 I-DiFeTf (3pg/ml) and 1 2 5 I-ApoTf (3ug/ml) for 30
min at 37°C. The medium was removed. Caco-2 cells were rinsed with cold PBS
twice quickly and chased with none, 10-fold, and 10Q-fold concentration of
unlabeled DiFeTf and ApoTf, respectively, in serum-free DMEM with 1 mg/ml BSA
for 2 h at 37°C. Caco-2 cell monolayers were washed with cold PBS twice then
trypsin eas added to detach Caco-2 cells. After adding 1 ml PBS in each well, the
content was centrifuged at 2500 rpm for 20 min, to remove the supemant. The cell
pallet was dissolved in 1 N NaOH at 37°C for 10 min, and BCA Protein Assay was
performed to determine cell proteins. In another experiment, Caco-2 cell monolayers
were pulsed in the same conditions and the chase medium was exchanged from each
other. When pulsed with 1 2 5 I-DiFeTf, Caco-2 cells was chased with none, 10-fold,
and 100-fold concentration of unlabeled ApoTf, respectively. When Caco-2 cells
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
44
1 7 S
was pulsed with I-ApoTf, chase medium was exchanged to none, 10-fold, and
100-fold concentration of unlabeled DiFeTf, respectively.
2.10 Analysis of uptake and apical-to-basolateral and basolateral-to-apical
transcytosis of 1 2 5 I-DiFeTf and 1 2 5 I-ApoTf across polarized Caco-2 cells
The polarized Caco-2 cell monolayers grown on 12-well Transwell plates
were washed with. PBS twice and preincubated at 37°C for .30 min in serum-free
DMEM with 1 mg/ml bovine semm albumin (BSA) to deplete endogenous
transferrin. The apical surface of each well was treated with 1 2 5 I-DiFeTf and
1 2 5 I-ApoTf (3ug/ml) in serum-free DMEM with 1 mg/ml BSA for 30 min. After
rinsing with cold PBS twice, the polarized Caco-2 cell monolayers were incubated
for 0 min, 15 min, 30 min, and 2 h in serum-free DMEM with 1 mg/ml BSA and
100-fold weight excess of unlabeled DiFeTf or ApoTf to prevent reintemalization.
The apical and basolateral medium from each well were collected and assayed
radioactivity by using a Packard gamma counter. They were considered as recycling
ligand and transcytosis ligand, respectively. Cell surface association was removed by
mild acid buffer for 5 min at room temperature. The Caco-2 cell monolayers
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45
together with filter membranes were cut and the radioactivity was determined by
using gamma counter as the cellular uptake.
Due to the big standard deviations in results of transcytosis, the experimental
method was altered for measuring the transcytosis specifically. Caco-2 cells were
grown on a 6-well Transwell plate. Before the experiment, they were washed with
PBS twice and preincubated with serum-free DMEM with 1 mg/ml BSA at 37°C for
30 min to deplete endogenous transferrin. The apical surface of each well was pulse
with 1 2 5 I-DiFeTf and 1 2 5 I-ApoTf (3 jig/ml) in serum-free DMEM with 1 mg/ml BSA
for 30 min. After rinsing with cold PBS twice, the Caco-2 cells were chased for 0
min, 15 min, 30 min, and 2 h in serum-free DMEM with 1 mg/ml BSA and 100-fold
weight excess of unlabeled DiFeTf or ApoTf to prevent reintemalizadon. At each
time point, 250ul of the basolateral medium was removed for counting radioactivity.
An aliquot of 250 pi fresh medium was added back to the well in order to maintain
the original volume.
To compare the effects of DiFeTf and ApoTf on trafficking apical-to-basal and
basal-to-apical direction, polarized Caco-2 cells were grown on 12-well Transwell
plates. After preincubated for 30 min in serum-free DMEM with 1 mg/ml BSA, they
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
were pulsed with 1 2 5 I-DiFeTf or 1 2 5 I-ApoTf (3pg/ml) in serum-free DMEM with 1
mg/ml BSA for 30 min on apical surfaces and basal surfaces, respectively. Then,
they were rinsed with cold PBS and chased with serum-free DMEM with 1 mg/ml
BSA and 100-fold weight excess of unlabeled DiFeTf or ApoTf for lmin and 60min.
Apical medium and basolateral medium were collect. Cell membranes were washed
with cold PBS and removed from the Transwells. All of them were subjected to
radioactivity measurement by using a gamma counter.
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47
III. RESULTS AND DISCUSSION
3.1 Cell association (uptake and surface binding) of DiFeTf and ApoTf in Caco-2
cells
Pulse-chase studies were used to investigate the cell association (uptake and
surface binding) of DiFeTf and ApoTf in Caco-2 cells monolayers. After 30 min
pulse, DiFeTf and ApoTf showed similar patterns as the percentage of cell
association (Figure 8). The result of cell association was the sum of jelh. ar uptake
and surface-associated ligand. From Figure 9, the amount of the c i: association of
ApoTf was significantly lower than that of DiFeTf at different chase time. It
suggested that both of DiFeTf and ApoTf had similar TfR-mediated endocytosis, but
ApoTf had lower binding affinity to TfR than that of DiFeTf on Caco-2 cell
monolayers.
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48
90
80
■ * — Diferric-Tf
* — Apo-Tf
70
60
50
40
30
20
10
0
0 0.5 1 1.5 2 2.5
Time (hr)
Figure 8. Percentage o f cellular association of DiFeTf (closed square) and ApoTf
(closed diamond) in Caco-2 cell monolayers. Caco-2 cells were treated with
1 2 5 I-DiFeTf or 1 2 5 I-ApoTf for 30 min pulse, followed by-0 min, 15 min, 30 min, 1 U ,
and 2 h chase at 37°C. The percentage of cell association ligands is the sum of die
percentage of cellular uptake and surface-associated ligands. Each point represents
the mean of three sample wells with error bars representing the standard deviation.
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
49
9.00000
_ 8.00000
> >
■f 7.00000
e
o
| 6.00000
1 3
m 5.00000
c
1 4.00000
2
I 3.00000
<
| 2.00000
1 3
u 1 .00000
0.00000
0 0.5 1 1.5 2 2.5
Time (hr)
Figure 9. Measurement of the amount of cell-associated of DiFeTf (closed circle)
and ApoTf (closed square). 1 2 5 I-DiFeTf and 1 2 5 I-ApoTf were added to Caco-2 cell
monolayers for 30 min pulse, followed by 0 min, 15 min, 30 min, 1 h, and 2 h chase
at 37°C. Caco-2 cells were detached with trypsin and assayed for radioactivity. Each
point represents the mean of three sample wells with error bars representing the
standard deviation.
-♦— Diferric-Tf
■ * — Apo-Tf
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
In order to analyze cellular uptake and surface association separately, Caco-2
cell monolayers were washed with mild acid buffer for 5 min at 25°C to remove
surface associated ligand before being detached by incubation with trypsin. Table 2
showed the percentage of initially endocytosed ligands after the 30-min pulse. The
total cell association, as well as the cellular uptake of DiFeTf and ApoTf was similar.
In pulse-chase studies, the cellular uptake was low (11-15 %) and remained constant
after chase for 1 h and 2 h (Figure 10). The results suggested that both DiFeTf and
ApoTf exist in the similar trafficking route as the endocytosed TfR. It is likely that
and there is a late compartment with a very slow recycling rate that plays a role in
the storage of TfR in Caco-2 cell monolayers.
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51
Pulse chase
studies (in
Caco-2 cells)
Pulse
medium
Total
release
Total cell
association
Cellular
uptake
Surface
association
Chase for 1 h DiFeTf
53.93±3.14 46.07± 3.14 13.54±2.19 32.53dz3.03
ApoTf
55.14±3.24 44.86± 3.24 11.74±3.39 33.12dz5.34
Chase for 2 h DiFeTf
60.25±1.13 39.75±1.13 15.54dzl.90 24.21±3.04
ApoTf
64.30dzl.23 35.70dzl.23 11.14dz 1.11 24.56dz2.17
Table 2. Results of percentage of initially endocytosed ligands (means ± SE, n = 3).
Total cell association is the sum of cellular uptake and surface association.
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52
40.000
35.000
30.000
25.000
20.000
15.000
10.000
5.000
0.000
1 h cell uptake 2 h cell uptake 1 h surface 2h surface
associated associaed
Figure 10. Determination of cellular uptake and surface association. 1 2 5 I-DiFeTf and
1 2 5 I-ApoTf were added to Caco-2 cell monolayers for 30-min pulse, followed by 1 h
and 2 h chase at 37°C. Data were presented as percentage of cellular uptake and
surface association of DiFeTf (gray column) and ApoTf (black column). Each
column represents the mean of three samples with error bars representing the
standard deviation.
■ Difenic-Tf
■ Apo-Tf
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53
3.2 Effect of unlabeled ligands on cellular uptake of 1 2 5 I-DiFeTf and 1 2 5 I-ApoTf
To determine the ability of DiFeTf and ApoTf with different concentration to
compete TfR binding, Caco-2 cell monolayers were incubated with 1 2 5 I-DiFeTf or
1 2 5 I-ApoTf (3ug/ml) in the presence of none, 10- and 100-fold excess of unlabeled
DiFeTf or ApoTf at 37°C for 2 h. Figure 11 shows that only 10-fold concentration of
the corresponding unlabeled ligand exhibited an effective competion with
1 2 5 I-DiFeTf and 1 2 5 I-ApoTf for TfR binding. At 10-fold concentration o f the
unlabeled ligand, the cellular uptake of 1 2 5 I-DiFeTf and I25I-ApoTf reduced to 20.7
% and 14.5 %, respectively. When unlabeled ligand reached 100-fold concentration,
the cellular uptake of both 1 2 5 I-DiFeTf and I2 5 I-ApoTf were almost completely
abolished, i.e., 6.9 % and 3.3 %, respectively. This result suggests that both DiFeTf
and ApoTf are endocytosed into Caco-2 cells predominately by the TfR-mediated
pathway.
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
54
120
J
DiFeTf
ApoTf
50 0 100 150
times of concentration of unlabeled Tf
Figure 11. Effects of different concentration of unlabeled DiFeTf and ApoTf on
cellular uptake. Caco-2 cells were incubated with 3ug/ml 1 2 5 I-DiFeTf (closed circle)
and 1 2 5 I-ApoTf (closed square) in the presence of none, 10-fold (30|j.g/ml) and
100-fold (300|ig/ml) excess of unlabeled DiFeTf and ApoTf, respectively, at 37°C
for 2 h. Each point represents the mean of three samples with error bars representing
the standard deviation.
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
With increasing concentration of unlabeled DiFeTf and ApoTf to compete
with 1 2 5 I-DiFeTf and 1 2 5 I-ApoTf for TfR-mediated endocytosis, the amount of
cellular uptake was reduced significantly. It is known that DiFeTf has higher binding
affinity to TfR than ApoTf. However, the concentration of labeled DiFeTf and
ApoTf that was used in the experiment was much higher than the Kd and therefore
we assumed that there was no receptor saturated problem. Depending on the ratio of
the amount of cellular uptake and the concentration of the total (iodinated and
unlabeled) DiFeTf and ApoTf, the results of amount of T f uptake can be presented
as in Figure 12. With increasing concentration of total concentration of DiFeTf and
ApoTf, the amount of cellular uptake of DiFeTf increased significantly, while that of
ApoTf increased only slightly. The marked difference between these two forms of Tf
indicated that endocytosed DiFeTf may accumulated inside Caco-2 cells.
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56
DiFeTf
ApoTf
100 120
times of concentration of unlabeled Tf
Figure 12. Cell uptake of DiFeTf and ApoTf at different concentration. The data
were derived from the amount of cellular uptake of iodinated and the concentration
of the total (iodinated and unlabeled) DiFeTf and ApoTf.
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57
3.3 Effects of different chase medium on cellular uptake of DiFeTf and ApoTf
In the first study, Caco-2 cells were chased with none, 10-fold, and 100-fold
concentration of unlabeled DiFeTf and ApoTf after 30 min I25I-DiFeTf and
1 2 5 I-ApoTf treatment, The result (Figure 13) showed that the cellular uptake of
DiFeTf was significantly higher than that of ApoTf. The cellular uptake of
1 2 5 I-DiFeTf decreased with increasing concentration of unlabeled DiFeTf in the
chase medium, ana maintain constant at above and of 10-fold concentration of
unlabeled DiFeTf. On the other hand, there was no significant difference in the
cellular uptake of 1 2 5 I-ApoTf at different concentration of unlabeled ApoTf. It is
known that the binding affinity of ApoTf to TfR is lower than that of DiFeTf. This
result suggested that ApoTf may readily dissociate from the surface of Caco-2 cells
in chief without the presence of a high concentration of the ligand in the chase
medium.
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58
30.00
25.00
£ 20.00
< D
| 15.00
*3 10.00
o
5.00
0.00
0 50 100 150
relative concentration of Tf in chase
medium
Figure 13. Effects of different concentration of DiFeTf and ApoTf in chase medium
on cellular uptake. Caco-2 cells were treated with 1 2 5 I-DiFeTf (closed circle) and
1 2 5 I-ApoTf (closed square) for 30 min pulse, and chased with none, 10-fold
(30ug/ml), and 100-fold (300ug/ml) concentration of unlabeled DiFeTf and ApoTf
at 37°C for 2 h, respectively. Each point represents the mean of three samples with
error bars representing the standard deviation.
diferric Tf
Apo Tf
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
In the second study, after the same treatment (pulse), the chase medium was
exchanged from each other. Caco-2 cells pulsed with 1 2 5 I-DiFeTf and 1 2 5 I-ApoTf
were chased with none, 10-fold, and 100-fold concentration of unlabeled ApoTf and
DiFeTf, respectively. The result (Figure 14) showed that the cellular uptake of
DiFeTf was slightly higher than that of ApoTf. The cellular uptake of both of
I2 5 I-DiFeTf and 1 2 5 I-ApoTf decreased significantly with increasing the concentration
of unlabeled ApoTf and DiFeTf in chase medium. Compared with the data in first
study, the cellular uptake of 1 2 5 I-DiFeTf continued decreasing from none to 100-fold
concentration of unlabeled ApoTf and the cellular uptake of I25I-ApoTf decreased
significantly at different concentration of unlabeled DiFeTf in chase medium. This
result suggests that DiFeTf had higher affinity to TfR than ApoTf. The decrease of
cell uptake of 1 2 5 I-DiFeTf by chasing with ApoTf is difficult to explain. However,
one of the possibilities is that ApoTf may decrease the localization of endocytosed
DiFeTf at the storage site in Caco-2 cells is suggested in Figure 10.
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60
6
< D
-2
a
< D
O
25.00
20.00
15.00
10.00
5.00
0.00
100 0 50 150
relative concentration of Tf in chase
medium
• diferric Tf
■ Apo Tf
Figure 14. Effects of different unlabeled ligands in the chase medium on cellular
uptake of 1 2 5 I-DiFeTf and 1 2 5 I-ApoTf. Caco-2 cells were treated with u5I-DiFeTf
(closed circle) and 1 2 5 I-ApoTf (closed square) for 30 min pulse, and chased with
none, 10-fold (3ug/ml), and 100-fold (300ug/ml) concentration of unlabeled ApoTf
and DiFeTf, respectively, at 37°C for 2 h. Each point represents the mean of three
samples with error bars representing the standard deviation.
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
61
3.4 Recycling, uptake, and transcytosis of DiFeTf and ApoTf in polarized Caco-2
cells
The results of recycling, cellular association and transcytosis of DiFeTf and
ApoTf dosed from apical cell surface in polarized Caco-2 cells are shown in Figure
15 A, B, and C. The percentage of recycled and cellular association of ApoTf was
not significantly different from that of DiFeTf. Although the data showed a large
standard deviation in transcytosis, they seemed to show the increasing trend with
time. After improving the experimental method, the results of transcytosis showed
that the percentage of ApoTf was higher than that of DiFeTf (Figure 16B). On the
other hand, the recycling of DiFeTf was higher than that of ApoTf (Figure 16A).
However, there was no difference in the cellular association (Figure 16C). To
compare the effects of DiFeTf and ApoTf on recycling, cellular association, and
transcytosis from different cell surfaces, polarized Caco-2 cells were treated with
I2'l-DiFeTf and 1 2 5 I-ApoTf from both apical and basal side. After pulsed for 3 0 min,
Caco-2 cells were chased for 1 min and 1 h. The results of recycling, transcytosis
and cellular association of DiFeTf and ApoTf were shown in Figure 17 and 18.
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62
DiFeTf
ApoTf
Time(h)
(A)
40.00
20.00
u 10.00
000
0 0.5 1 1.5 2 2.5
Time(%)
—♦— DiFeTf
— » -■ ApoTf
(B)
25.00
g 20.00
| 15.00
| 10.00
5.00
0.00
0 0.5 1 1.5 2 2.5
Time(h)
(C)
Figure 15. Recycling (A), cell association (B), and transcytosis (C) of DiFeTf and
ApoTf in filter-grown (12-well) polarized Caco-2 cells. Apical cell surface was
treated with 1 2 5 I-DiFeTf (closed circle) and 1 2 5 I-ApoTf (closed square) for 30 min
pulse, followed by 0 min, 15 min, 30 min, and 2 h chase at 37°C. Data were
presented as percentage of initially endocytosed ligands. Each point represents the
mean of three measurements with error bars representing the standard deviation.
DiFeTf
ApoTf
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63
70
60
50
40
30
20
10
0 L
0 0.5 i 1.5 2 2.5
Tim e(h)
-D iFeT f
-A p o T f
(A)
DiFeTf
(B)
70 ■ ■ '
60
50
30
10
0
0.5 1.5 2 2.5 0
Tim e(h)
-D iFeT f
“ ApoTf
(C)
Figure 16. Recycling (A), transcytosis (B), and cell association transcytosis (C) of
DiFeTf and ApoTf in filter-grown (6-well) polarized Caco-2 cells. 1 2 5 I-DiFeTf
(closed circle) and 1 2 5 I-ApoTf (closed square) were added to apical cell surface of
Caco-2 cells in individual wells at 37°C for 30 min, followed by 0 min, 15 min, 30
min, and 2 h chase at 37°C. At each time, 10 % of basolateral medium were pulsed
and fresh medium was added to maintain the original volume. Each point represents
the mean of three measurements with error bars representing the standard deviation.
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
When Caco-2 cells were pulsed on the apical surface, and chase for 1 min, the
percentage of apical recycling and transcytosis of ApoTf was higher than those of
DiFeTf. After 1 h chase, those of DiFeTf increased significantly and were similar to
those of ApoTf (Figure 17A,B). In cellular association, there was significantly
different between DiFeTf and ApoTf after 1 min and 1 h chase. In addition, both
forms of T f decreased significantly from 1 min to 1 h chase, especially for DiFeTf
(Figure 17C). The resulted indicated that the percentage of DiFeTf stayed in Caco-2
cells much higr-ej:. than that of ApoTf. After prolonged incubation, cell-associated
DiFeTf went two pathways: apical recycling and transcytosis, with a majority.
However, ApoTf had a short retention in the Caco-2 cells and tended to recycle and
transcytose at a shot time. Besides, in 1 h chase, a significant amount of
cell-assocaited ApoTf was transcytosed.
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
65
120
£ 100
J ? 80
5 60
< U
■ a 4 0
CJ
■ § 2 0
0
is
f t
13 DiFeTf
■ ApoTf
lmin 60min
apical doing, chase for lmin and 60min
(A)
30
~ 25
r 20
c/3
§. 1 5
1 1 0
2
“ 5
0
■
■ DiFeTf
■ ApoTf
lmin 60min
apical dosing, chase for lmin and 60min
(B)
50 i -" "
g 40 -------
lmin 60min
apical dosing, chase for lmin and 60min
B DiFeTf
■ ApoTf
(C)
Figure 17. Apical recycling (A), transcytosis (B), and cell association (C) in Caco-2
cells from apical cell surface dosing. Polarized Caco-2 cells were pulsed with
1 2 5 I-DiFeTf and 1 2 5 I-ApoTf on apical cell surface in individual wells at 37°C for 30
min, followed by 1 min and 60 min chase at 37°C. Each column represents the mean
of three measurements with error bars representing the standard deviation.
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
When pulsed on the basal surface Caco-2 cells, the percentage of ApoTf in
basal recycling was higher than that of DiFeTf after 1 min and 1 h chase, and both of
them increased significantly with time (Figure 18 A). But, in transcytosis and cellular
association, the percentage of DiFeTf was significantly higher than that of ApoTf
after 1 min and 1 h chase. For transcytosis, the percentage of DiFeTf increased
significantly, but not of ApoTf (Figure 18B,C). It indicated that the decrease of
cellular uptake of ApoTf predominately recycled to basolateral cell surface and that
of DiFeTf went through two pathways: recycling and transcytosis.
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
67
■ ■ = 60.00
lmin 60min
basal dosing, chase lmin and 60min
(A)
m DiFeTf
■ ApoTf
12.00
IS s ip
■
■ W i
lmin 60min
basal dosing, chase lmin and 60min
B DiFeTf
■ AooTf
(B)
50.00
£ 40.00
c
I 30.00
| 20.00
=5 10.00
c j
0.00
(C)
Figure 18. Basal recycling (A), transcytosis (B), and cell association (C) in Caco-2
cells from basal cell surface dosing. Polarized Caco-2 cells were pulsed with
l2 5 I-DiFeTf and I2 5 I-ApoTf on basal cell surface in individual wells at 37°C for 30
min, followed by 1 min and 60 min chase at 37°C. Each column represents the mean
of three measurements with error bars representing the standard deviation.
i
S I DiFeTf
■ ApoTf
lmin 60min
basal dosing, chase lmin and 60min
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
Figure 19 and 20 showed the amount of DiFeTf and ApoTf in recycling,
transcytosis, and cell association after 1 min and 1 h chase both from apical and
basal dosing. The total amount of endocytosis of DiFeTf was higher than that of
ApoTf significantly. The results indicated that DifeTf was much easier than ApoTf
to be endocytozed into Caco-2 cells after 30 min pulse.
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
69
3.00
2.50
1.50
1.00
0.50
0.00
.
li
apical recycling transcytosis call association
apical dosing, chase after lmin
il DiFeTf
■ ApoTf
(A)
7.00
6.00
5.00
4.00
Oil
c
3.00
2.00
1.00
0.00
apical recycling transcytosis call association
apical dosing, chase after lh
(B)
1 DiFeTf
I ApoTf
Figure 19. The amount of DiFeTf and ApoTf in filter-grown polarized cells from
apical dosing. Apical cell surface of Caco-2 cells was treated with 1 2 5 I-DiFeTf (gray
column) and 1 2 5 I-ApoTf (blackcolumn) for 30 min, followed by 1 min (A) and 1 h
(B) chase. Each column represents the mean of three measurements with error bars
representing the standard deviation.
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
70
2.00
1.80
1.60
1.40
1.20
1.00
0.80
0.60
0.40
0.20
0.00
X
w m
transcytosis
in
0y'~.
X
ML
S DiFeTf
B ApoTf
basal recycling
basal doing, chase after 1 min
cell association
(A)
transcytosis basal recycling cell association
basal doing, chase after lh
(B)
Figure 20. The amount of DiFeTf and ApoTf in filter-grown polarized cells from
basal dosing. Basal cell surface of Caco-2 cells was treated with l25I-DiFeTf (gray
column) and I25I-ApoTf (black column) for 30 min, followed by 1 min (A) and 1 h
(B) chase. Each column represents the mean of three measurements with error bars
representing the standard deviation.
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
71
IV. CONCLUSION
In order to investigate the endocytosis and transcytosis of DiFeTf and ApoTf
in Caco-2 cells, pulse-chase studies were used in this thesis. In nonpolarized Caco-2
cells, there was no significant difference in the percentage of cell uptake and release
ligands. However, the amount of cell uptake of DiFeTf was higher than that of
ApoTf. By competition studies, it demonstrated that both of DiFeTf and ApoTf were
Internalized into Caco-2 cells through fR- mediated pathway. Besides, DiFeTf has
a higher binding affinity to TfR than ApoTf, and non-specific binding played a
major role in the entering pathway for ApoTf. In polarized Caco-2 cells, there was
little difference between DiFeTf and ApoTf in endocytosis and transcytosis. Form
apical and basal cell surface dosing, recycling and transcysosis were increasing and
cell association was decreasing with time for both of DiFeTf and ApoTf. Comparing
the percentage of each pathway for both apical and basal dosing, recycling was the
major pathway in the trafficking. From apical dosing, the percentage of recycling of
DiFeTf was higher than that of ApoTf, while the percentage of transcytosis of ApoTf
was higher than that of DiFeTf. In addition, the cell association of both ligands was
similar. It indicated that after being endocytozed both ligands went into different
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
intracellular compartments, with DiFeTf favor for the recycling but ApoTf for
transcytosis pathway. From basal dosing, the percentage of recycling of ApoTf was
higher than that of DiFeTf. On the other hand, the percentage of transcytosis of
DiFeTf was higher than that of ApoTf. It also indicated that they went through the
different trafficking pathway after being endocytosed from basolateral cell surface.
For cell association, there was no difference between apical and basal dosing.
Ho wever, the percentage of cell associ rtior of ApoTf was lower than that of DiFeTf
Taken together these results suggested that DiFeTf and ApoTf have oilier ent
trafficking pathway in polarized, but not in non-polarized Caco-2 cells.
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73
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Endocytosis and transcytosis of diferric transferrin and apotransferrin in Caco-2 cells
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