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Introduction
Many toxins of bacteria and plant origins have 2 moieties, with one moiety to bind the cell and help the toxin to enter cell,
and the other to be enzymatically active to exert the
action[1,2]. These toxins have potential in cancer therapy. One promising
approach is to use them in immunotoxins, where the active moiety of toxin is fused with cancer-specific monoclonal
antibodies or other cancer cell-specific
ligands[3,4]. However, this approach may be limited by the lack of highly specific monoclonal
antibodies or other specific ligands for certain types of cancer, and the requirement of large amounts of fusion proteins due
to the lack of the bystander effect.
As a 2-moiety toxin from the seeds of Ricinus
communis, ricin is a ribosome inactivating protein highly toxic to mammalian
cells. It contains 2 peptide chains, A and B, which join together with 2 disulfide
linkages[5]. The ricin A chain (RTA) inhibits
protein synthesis by removing an adenine residue from the exposed loop of 28S ribosomal RNA (eg A4323 in rat 28S RNA),
thus resulting in cell death[6,7]. However, it is generally non
toxic outside the cell due to the low efficiency to enter cells
by itself[8]. Progress has been made to further reduce its side
effect by point mutations[9]. Meanwhile, the ricin B chain
(RTB) is an agglutinin that mediates binding of the toxin to
the cell surface, transporting the molecule to an intracellular
compartment. It is non toxic to cells by
itself[10,11].
In the current study, a novel approach to use these
2-moiety toxins in cancer therapy is proposed and tested
in vitro using ricin as an example. According to the design,
RTB is expressed by an adenovirus vector targeting cancer
tissues while RTA is applied in the form of a purified protein.
When used in vivo, RTA is concentrated in the cancer tissue
where RTB is located due to the strong affinity between RTA
and RTB[3]. It enters cancer cells with the help of RTB, and
exerts its cell-killing function. By-stander effect is also
expected, since the killed cells release more RTB to form a
complex with the external RTA. A prototype of the design,
with no cancer-specificity, was preliminarily tested for its
cytotoxicity in cultured cells and proved to be a potential
alternative for the efficient application of this type of toxin.
Materials and methods
PCR amplification of RTA and RTB genes The leaves of
Ricinus communis were collected from Ningbo, Zhejiang
Province, China. Genomic DNA was extracted using the Plant
Genomic DNA Isolation Kit (Sangon, Shanghai, China).
Primers were designed according to the sequence on Genbank
(No S40336) as follows, with restriction enzyme sites
introduced as indicated: RTA upstream:
5'-GCGGAATTCATGA-TATTCCCCAAACAATACCC-3'
(EcoR I); RTA downstream:
5'-GCGAAGCTTTCAAAACTGTGACGATGGTGGAG-3' (Hind
III); RTB upstream:
5'-TGTCGACATGGCTGATGTTT-GTATGGATCC-3' (Sal
I); and RTB downstream: 5'-TACTCGAGCACACACACTGCAAGAGAGTA-3'
(Xho I). The PCR conditions were: predenaturation at 94
oC for 5 min; 30 amplification cycles of 95
oC for 30 s; 56 oC for 30 s (RTA)
or 62 oC for 30 s (RTB); 72
oC for 1 min; and a final extension of
72 oC for10 min. PCR products were recovered from
agarose gel with the 3S Spin Agarose Gel DNA Purification Kit
(Shenergy Biocolor, Shanghai, China) and cloned into
pMD18-T vector (TaKaRa, Dalian, China) to construct 2
plasmids of pT-RTA and pT-RTB. Both genes were confirmed by
sequence analysis.
Prokaryotic expression of RTA The plasmid pT-RTA
was doubly digested with EcoR I and Hind
III, and the RTA gene was recovered from agarose and ligated with pET32a
(Novagen, San Diego, CA, USA) similarly digested with
EcoR I and Hind III. The resulted plasmid was named
pET32-HisRTA. The RTA production E coli strain was obtained by
transforming BL21 with pET32-HisRTA. The RTA protein
was produced by inducing with
isopropyl-beta-D-thioga-lactoside (IPTG) at 1 mmol/L for 10 h at
20 oC. The expression of RTA was confirmed by SDS-PAGE and Western blotting
with anti-6×His IgG following standard protocols.
Purification of the RTA protein Bacteria cells were
pelleted by centrifugation at 10 000 r/min for 10 min at
4 oC, and lysed with sonication. A 6×His-tagged RTA protein was
purified with Ni2+-nitrilotriacetic acid
(Ni2+-NTA) affinity resin (Shanghai Shenergy Biocolor) under non-denaturing
conditions and eluted with imidazole solution. The fractions with
RTA were pooled and dialyzed in phosphate buffer saline
(PBS) to remove imidazole. RTA was concentrated by
dialysis against polyethylene glycol 8000, quantitated, and stored
at -70 oC for later use.
Construction of RTB-expressing recombinant
adenovirus The recombinant adenovirus was constructed using the
system developed by He et al[13]. The
RTB gene was digested from pT-RTB with Sal
I and Xho I and ligated with the transfer vector
pAdTrackCMV[13] digested with the same enzymes. The resulted plasmid, pAdCMV-RTB, was then
linearized with PmeI and transformed into the
E coli strain BJ5183 (RecA+) harboring the plasmid pAdEasy-1, a
plasmid with an adenovirus genome[13]. Homologous
recombination between linearized pAdCMV-RTB and pAdEasy-1
resulted in the production of pAdEasyCMV-RTB, with the
RTB gene and a kanamycin resistant gene inserted within
the adenovirus genome. The plasmid pAdEasyCMV-RTB
was amplified in E coli DH5a and extracted with the Genomic
DNA Isolation Kit (Shanghai Shenergy Biocolor) from a 200
mL culture. 5 µg of its DNA was linearized with
Pac I and used to transfect human embryonic kidney cell line HEK293
cells with Lipofectamine Reagent (Invitrogen, San Diego,
CA, USA). The recombinant adenovirus AdGFP-RTB was
prepared by the repeated freezing and thawing of transfected
cells 10 d post transfection. Since AdGFP-RTB had an
enhanced green fluorescence protein (GFP) gene, virus
infection could be visualized in infected HEK293 cells 24 h post
infection (pi).
RTB expression and binding with RTA Lysates of the
AdGFP-RTB-infected HEK293 cells were collected at 0, 12,
24, 36, and 48 h pi and dotted on a nitrocellulose membrane.
The membrane was treated with 6×His-tagged RTA in PBS
(10 µg/mL) for 1 h, followed by similar treatment with a rabbit
polyclonal antibody against 6×His (Bioinforbody, Zhuhai,
Guangdong, China, 1:1000) and alkaline
phosphatase-conjugated mice anti-rabbit IgG (Sigma-Aldrich, St Louis, MO,
USA, 1:30000). The blot was then visualized by adding
substrates of nitro blue tetrazolium/5-bromo-4-chloro-3-indolyl
phosphate (NBT/BCIP). The lysates of the cells infected
with AdGFP, an adenovirus without the RTB
gene[14], were used as a negative control. To confirm the release of RTB
from the infected cells, the samples of culture medium from
AdGFP-RTB were concentrated with vacuum freeze-drying,
and assayed with an immuno dot blot as described earlier.
Presence of the RTA protein in AdGFP-RTB-infected
cells To confirm the entry of the RTA protein in
RTB-expressing cells, the RTA protein (40 µg/mL) was added to the
AdGFP-RTB-infected HEK293 cells at 24 h pi, and the cells
were collected 0, 24, and 48 h later. The total cell protein was
analyzed by SDS-PAGE and Western blotting using the
anti-6×His antibody as described earlier. Non-infected HEK293
cells were treated similarly with the RTA protein, collected 24
h later, and used as a control.
Cell-killing effect of the combined application of RTA
and AdGFP-RTB HEK293 and human cervical carcinoma
cell line HeLa were used to seed 24-well plates at a density of
1×105 cells/well. The cells were infected with the
recombinant adenovirus AdGFP-RTB or AdGFP at an MOI (multiplicity
of infection) of 100 pfu/cell, or mock infected. A certain
amount of the RTA protein was added to the culture
supernatant at 24 h pi. Cell mortality was examined with trypan
blue staining 24 h later. The total cells and dead cells were
recorded to calculate the cell mortality. All experiments were
carried out in triplicate and the average mortality and
standard deviation were calculated. MTT assay was also used
to measure the cell viability in treated HeLa and human liver
cancer cell lines Smmc7721 and HL7702 cells using a similar
experimental design as described before, following standard
protocol[14].
By-stander effect The HeLa cells were infected with
AdGFP-RTB or AdGFP at an MOI of 100. RTA 5 µg/mL was
added to the culture at 24 h pi. The supernatant of the
culture was collected 4 d later, diluted, and moved to a 24-well
plate seeded with fresh HeLa cells. The cell viability was
determined by MTT assay 24 h later. The experiment was
carried out in triplicate and the average mortality and
standard deviation were calculated.
Results
Prokaryotic expression and purification of RTA
The expression of the RTA protein in the E
coli BL21 strain was observed in SDS-PAGE, and Western blot analysis was
performed using the specific primary antibody against 6×His
(Figure 1). A strong band with a molecular weight of 47 kDa
was presented in an IPTG-induced sample (Figure 1A), which
was reactive to the anti-6×His antibody (Figure 1B). The
purified RTA protein was obtained from
Ni2+-NTA affinity chromatography eluted with imidazole at the concentration
of 20 and 40 mmol/L (Figure 1C).
Construction of the recombinant adenovirus and
expression of RTB The expression of RTB in AdGFP-RTB-infected
HEK293 cells was confirmed with the dot blot assay using
the RTA protein as an intermediate ligand. The RTB
expression in AdGFP-RTB-infected cells was observed at 24 h pi,
and peaked at 36 h pi, while no RTB expression was
observed in AdGFP-infected cells (Figure 2). Since His-tagged
RTA was used as an intermediate ligand for the detection of
RTB, the result also proved that RTB expressed in cells has
high binding activity to prokaryotically-expressed RTA. The
RTB protein was also detected in the culture medium,
concentrated 20 times, of AdGFP-RTB-infected cells, although
the concentration was very low. This result indicated that
the RTB protein was secreted into the medium, even though
there was not a significant signal peptide in this protein.
Cell-killing effect of RTA and AdGFP-RTB
The cell-killing effect of RTA and AdGFP-RTB was first evaluated
with trypan blue staining in HEK293 and HeLa cells.
AdGFP-RTB infection did not result in significant cell death without
RTA (RTA 0 µg), neither did the treatment of the RTA protein
alone or in combination with AdGFP, even at the highest
dose (Figure 3). However, when AdGFP-RTB and the RTA
protein were used in combination, significant cell mortality
was seen. The mortality increased with the RTA dose, which
reached around 60% with 4.8 µg RTA protein, when the MOI
of AdGFP-RTB was 100 pfu/cell. Similar results were
observed using the MTT assay, where cell viability dropped
by 50% or more when co-treated with AdGFP-RTB and the
RTA protein in HeLa, HL7702, and SMMC-7721 cells (Figure 4).
Entry of the RTA protein into AdGFP-RTB-infected cells
To confirm that the cell death observed above was a result of
the entry of the RTA protein into the cell, HEK293 cells were
infected with AdGFP-RTB, followed by the treatment of the
RTA protein at 24 h pi. The RTA protein was detected in the
cell lysate 24 and 48 h later with Western blotting (Figure 5).
No RTA was detected in the non-infected cells similarly
treated with the RTA protein for 24 h. These results
indicated that infection of AdGFP-RTB prompted the entry of
RTA into the cell.
Bystander effect of the co-application of AdGFP-RTB
and the RTA protein When the RTA protein and
AdGFP-RTB were used in combination, it was expected that RTB
expressed by the adenovirus and the RTA protein would
combine to form the RTA-RTB complex, which would be
highly toxic to cells. To prove the case, the supernatant was
collected from HeLa cells infected with AdGFP-RTB in the
presence of the RTA protein 48 h pi, and used to inoculate
fresh HeLa cells. No green fluorescence was observed in
treated cells 24 h later (data not shown) as expected
indicating that there was no adenovirus in the supernatant.
Meanwhile, cell viability decreased significantly when a high
concentration of supernatant was used (Figure 6). No
significant change of cell viability was seen in the control where
cells were treated with supernatant from AdGFP and
RTA-treated HeLa cells.
Discussion
Ricin is commonly used as a part of immunotoxins for
clinical tumor research and application, although the detailed
mechanism of its function is still under
investigation[15,16]. Previous studies have shown that recombinant RTA
produced in bacteria was highly active to form a complex with
RTB and cause cytotoxicity[8]. Meanwhile, recombinant RTB
expressed in eukaryotic cells has also been shown to be
functional in transporting RTA into the
cell[10,11]. In the current study, a novel approach for ricin-based tumor therapy
was preliminarily tested in vitro to apply RTA as a purified
protein, and RTB as a locally expressed protein in target
cells.
Prokaryotically-expressed RTA was prepared, and an
adenovirus expressing RTB was constructed. RTB expression
and its binding with RTA were confirmed with an indirect dot
blot assay. Further analysis showed, as expected, that
although neither RTA protein nor AdGFP-RTB was cytotoxic
when applied individually, they became highly toxic when
applied simultaneously. Significant cell death or loss of cell
viability was observed in all of the cell lines tested: HEK293,
HeLa, HL7702, and SMMC7721; the cytotoxic effect
correlated with the amount of RTA applied. The RTA protein was
observed in AdGFP-RTB infected cells, but not in
non-infected cells, which was consistent with the cytotoxicity data
and proved that the cell death was due to the entry of RTA
protein into the cells.
The mechanism on how RTA was transported into cells
in this system has not been studied. One possibility is that
some of the RTB produced by cell was leaked to the medium,
formed the RTA-RTB complex with RTA outside the cell, and
helped to move the complex into the cell. The observation of
the RTB protein in the culture medium of AdGFP-RTB
infected cells supported the hypothesis. It is presumed that
the secretive expression of RTB may help to improve the
cell-killing effect further.
The cytotoxic effect caused by the supernatant from
AdGFP-RTB/RTA-treated HeLa cells suggested a potential
bystander effect. It is believed to be due to the presence of
the RTA-RTB complex in the supernatant rather than the
production of more adenoviruses. AdGFP-RTB is a
replication-deficient adenovirus with deletion in the E1A gene. It could
only replicate in HEK293 cells, but not in HeLa cells. This
was consistent with the absence of green fluorescence in
HeLa cells treated by the supernatant.
In the current, we proprosed a novel approach to use
ricin in cancer therapy and preliminarily tested it
in vitro. Experiments in animal models will be necessary to further
confirm the applicability of this approach in
vivo. The method needs to be further developed in terms of specificity and
effectiveness, possibly by using non-fusion native RTA
proteins, cancer cell-specific and replication-competent
recombinant adenoviruses[16], and by the secretive expression
of RTB.
References
1 Pastan I, Hassan R, FitzGerald DJ, Kreitman RJ. Immunotoxin
therapy of cancer. Nat Rev Cancer 2006; 6: 559_65.
2 Johannes L, Decaudin D. Protein toxins: intracellular
trafficking for targeted therapy. Gene Ther 2005; 12: 1360_8.
3 Ng HC, Khoo HE. Cancer-homing toxins. Curr Pharm Des
2002; 8: 1973_85.
4 Schnell R, Borchmann P, Staak JO, Schindler J, Ghetie V, Vitetta
ES, et al. Clinical evaluation of ricin A-chain immunotoxins in
patients with Hodgkin's lymphoma. Ann Oncol 2003; 14:
729_736.
5 Olsnes S, Kozlov JV. Ricin. Toxicon 2001; 39: 1723_8.
6 Robertus JD, Monzingo AF. The structure of ribosome
inactivating proteins. Med Chem 2004; 4: 477_86.
7 Jon R. The structure and action of ricin, a cytotoxic
N-glycosidase. Cell Biol 1991; 2: 23_30.
8 O'Hare M, Roberts LM, Thorpe PE, Watson GJ, Prior B, Lord
JM. Expression of ricin A chain in Escherichia
coli. FEBS Letter 1987; 216: 73_8.
9 Smallshaw JE, Ghetie V, Rizo J, Fulmer JR, Trahan LL, Ghetie
MA, et al. Genetic engineering of an immunotoxin to eliminate
pulmonary vascular leak in mice. Nat Biotechnol 2003; 21:
387_91.
10 Vitetta ES, Yen N. Expression and functional properties of
genetically engineered ricin B chain lacking galactose-binding
activity. Biochim Biophys Acta 1990; 1049: 151_7.
11 Roberts LM, Lord JM. Ribosome-inactivating proteins: entry
into mammalian cells and intracellular routing. Mini Rev Med
Chem 2004; 4: 505_12
12 Ferrini JB, Martin M, Taupiac MP, Beaumelle B. Expression of
functional ricin B chain using the baculovirus sytem. Eur J Biochem
1995; 233: 772_7.
13 He TC, Zhou S, da Costa LT, Yu J, Kinzler KW, Vogelstein B. A
simplified system for generating recombinant adenoviruses. Proc
Natl Acad Sci USA 1998; 95: 2509_14.
14 Chen L, Yin J, Chen Y, Zhong J. Induction of Epstein-Barr virus
lytic replication by recombinant adenoviruses expressing the zebra
gene with EBV specific promoters. Acta Biochim Biophys Sin
2005; 37: 215_20.
15 Stirpe F, Battelli MG. Ribosome-inactivating proteins: progress
and problems. Cell Mol Life Sci 2006; 63: 1850_66.
16 Rao PV, Jayaraj R, Bhaskar AS, Kumar O, Bhattacharya R, Saxena
P, et al. Mechanism of ricin-induced apoptosis in human
cervical cancer cells. Biochem Pharmacol 2005; 69: 855_65.
17 Majhen D, Ambriovic-Ristov A. Adenoviral vectors _ how to use
them in cancer gene therapy? Virus Res 2006; 119: 121_33.
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