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Introduction
Apoptosis, or programmed cell death, is a regulated
process that is essential to metazoan development and tissue
homeostasis[1]. Instructive apoptosis plays a physiological
role in deletion of activated lymphocytes at the end of an
immune response and in elimination of virus-infected cells
and oncogenically transformed cells. Deregulation of
programmed cell death leads to several human diseases,
including cancer, neurodegenerative disorders, and acquired
immunodeficiency syndrome[2]. Since the 1980s, biotherapy
has become an important modality for treating
cancer[3]. Biotherapy is achieved by inducing apoptosis of tumor cells,
which is the ultimate objective of tumor therapy. The
members of the tumor necrosis factor (TNF) family became the
focus of biotherapy of cancer[4]. Despite the potential of
TNF and Fas ligand (FasL; also called CD95L) to induce
apoptosis in many types of tumor cells, severe toxic
side-effects preclude both of these ligand from use in systemic
anticancer therapy[5,6]. TNF infusion causes a lethal
inflammatory response that resembles septic shock mediated by
activation of the proinflammatory transcription factor
NF-kappaB in vascular endothelial cells and macrophages.
Infusion of agonistic anti-Fas antibody causes lethal liver
damage as a result of the induction of Fas-dependent
apoptosis in hepatocytes. Tumor necrosis factor-related
apoptosis-inducing ligand (TRAIL, also called Apo2 ligand)
is a newly discovered TNF superfamily member initially cloned
from human heart and lymphocyte cDNA libraries because
of its sequence homology to TNF and
FasL[7,8]. The TRAIL mRNA is constructively expressed at significant levels in
many tissues in humans (eg lung, liver, spleen, kidney,
thymus, prostate, ovary, intestine, periphero-lymphoid nodes,
heart, placenta, and skeletal muscle). Recent studies have
shown that TRAIL could induce apoptosis in numerous
transformed cell lines of different lineage, but showed
nontoxicity systemically unlike TNF and FasL, ionizing
radiation and chemotherapy[9-11]. The combination of TRAIL
and ionizing radiation or chemotherapy appears to have an
enhancing effect on cancer therapy, and consequently
reduces the dose and side-effects of
chemotherapeutants[12-16]. Therefore, TRAIL is considered to be the most promising
among antitumor agents at present.
TRAIL is expressed as a type II transmembrane protein,
as are other members TNF and FasL[8], and its extracellular
region forms a soluble molecule upon
cleavage[17]. There are several forms of recombinant TRAIL: soluble TRAIL fusion
protein (termed LZ-TRAIL) in which the extracellular region
of the ligand (amino acids 95-281) is linked to an exogenous,
modified leucine zipper that drives
trimerization[11]; soluble
TRAIL containing just the extracellular region (amino acids
114-281); polyhistidine-tagged soluble form (his-TRAIL,
amino acids 114-281)[8,18]; glutathione
S-transferase-TRAIL (GST-TRAIL, amino acids 95-281); and flag epitope-tagged
form (amino acids 95-281)[8,18]. However, these other forms
have deficiencies in solubility, antitumor activity and security.
Therefore, Beijing Sunbio Biotech developed recombinant
mutant human TRAIL (rmh TRAIL). The aim of the present
work was to evaluate the antitumor activity of rmh TRAIL
in vitro and in vivo, and to explore its antitumor mechanism.
Materials and methods
Expression and purification of
protein Escherichia coli BL21 (DE3) strain was used for the expression of rmh TRAIL.
After transformed the plasmid of rmh TRAIL [with a circular
permuted extracellular sequence of native human
Apo2L/TRAIL (amino acid 121-281)], which was built by Beijing
Sunbio Biotech, into the expression vector, expression was
induced by isopropyl-beta-D-thiogalactopyranoside (IPTG)
at 37 °C for 4 h . Optimized IPTG concentration was 1
mmol/L for the protein. Bacteria were harvested and resuspended in
phosphate-buffered saline (PBS, containing 137 mmol/L NaCl,
2.7 mmol/L KCl, 4.3 mmol/L
Na2HPO4, 1.4 mmol/L
KH2PO4, pH 7.3), sonicated on an ice bath, and centrifuged before
further analysis. Purification of rmh TRAIL was performed
with Ni2+-loaded chelating (Amersham Pharmacia Biotech,
Piscataway, USA) and purified protein was subjected to
desalting with S-200 (Amersham Pharmacia Biotech). rmh
TRAIL molecular weight is 19 kDa, and purity is >98%.
Reagents The wild type TRAIL (wt TRAIL) (amino
acids 114-281) used in our experiments was produced by Beijing
Sunbio Biotech, or purchased from Merck (Whitehouse
Station, USA) (used only in vitro). An Annexin V-FITC
apoptosis detection kit was obtained from Oncogene
(Cambridge, USA). In situ cell death detection kit (peroxidase,
POD) and cell death detection enzyme-linked immunosorbent
assay (ELISA)PLUS kit were from Roche (Nutley, USA). DAB
kit was purchased from Beijing Zhongshan Golden Bridge
Biotech. MTT was from Sigma (St Louis, USA). DTT and
proteinase K was from Merck. All the reagents, including
dimethyl sulfoxide (Me2SO) (Tianjin Fuchen Chemicals
Reagent Factory), ethanol, methanol, saturated phenol,
paraformaldehyde (Beijing Chemical Reagents Company)
were of analytical grade.
Animals BALB/c nude mice (female), 6-8 weeks old,
weighing 23-27 g, were supplied by National Institute for
the control of Pharmaceutical and Biological Products.
Animals were kept at room temperature of 18-22 °C, with a rela
tive humidity of 70%.
Cell culture Tumor cell lines: NCI-H460 human
non-small cell lung cancer cells and COLO205 human colon
cancer cells were obtained from American Type Culture
Collection (ATCC); QG-56 human lung squamous cells were
obtained from the cell bank of the Chinese Academy of Sciences;
MDA-MB-231 and MCF-7 human breast cancer cells were
obtained from the Institute of Materia Medica, Chinese
Academy of Medical Sciences and Peking Union Medical College.
Normal cell lines: EC-304 human blood vessel
endothelial cells were purchased from Nanjing KeyGen Biotech.
Human embryonic lung fibroblasts (HELF) were obtained
from the Institute of Materia Medica, Chinese Academy of
Medical Sciences and Peking Union Medical College.
HL-7702 normal human hepatocytes were obtained from the cell
bank of the Chinese Academy of Sciences. CCC-HEH-1
human primary embryo myocardium-derived cells, CCC-HEL-1
human primary embryo liver-derived cells, CCC-HBE-2
human primary embryo trachea-derived cells, MRC-5 human
embryonic lung fibroblasts, CCC-ESF-1 human primary
embryo skin fibroblast cells, and HK-2 human primary nephritic
tubular epithelium cells, were all obtained from cell center of
the Chinese Academy of Medical Sciences and Peking Union
Medical College.
NCI-H460, COLO205, QG-56 , EC-304, HELF and HL-7702
were cultured in RPMI-1640 medium (GIBCO, Carlsbad, USA)
supplemented with 10% fetal bovine serum (FBS) (Lanzhou
National HyClone Bio-engineering, Lanzhou, China).
MCF-7 and MRC-5 were cultured in Minimum Essential Medium
(MEM, GIBCO) supplemented with 10% FBS. MDA-MB-231 cells were cultured in Leibovitz¡¯s L-15 medium (GIBCO)
supplemented with 15% FBS. CCC-HEH-1, CCC-HEL-1,
CCC-HBE-2, and CCC-ESF-1 were cultured in Dulbecco¡¯s
Modified Eagle¡¯s Medium (DMEM) medium (GIBCO)
supplemented with 20% FBS. Insulin 0.2 IU/mL and
hydrocortisone 1 mg/mL were added to CCC-HBE-2 cells. HK-2 cells
were cultured in DF-12 medium (GIBCO) supplemented with
10% FBS.
Cell growth inhibition assay The cytotoxicity of the
rmh TRAIL and wt TRAIL was determined using a
colorimetric MTT assay. Cells in the logarithmic growth phase
were dispersed to prepare a suspension of cell density of
6×104-20×104
mL-1. Then cell suspension was seeded in a
96-well plate in total volume of 100 mL per well and incubated
in a 5% CO2 atmosphere at 37 °C for 24 h. Then, the
supernatant was removed and the cells were added with 100 µL of
different concentrations of the drugs followed by
incubation at 37 °C for another 24 or 72 h. For controls, cells were
placed in medium only. After removal of the supernatant,
the cells were treated with 5.0 g/L MTT for 4 h, the purple
blue sediment was dissolved in 150 µL
Me2SO, and the absorbance at 490 nm was measured using a microplate reader
(BIO-RAD, Hercules, USA, model 550). The survival rate of
cell growth was calculated using the following formula:
Survival rate
(%)=A490(drug)/A490
(control)×100%. Triplicate wells were analyzed for each concentration.
The 50% inhibitory concentration
(IC50) is the concentration required for 50% inhibition, which is calculated from the
linear equation, which was educed by concentration versus
survival rate regression curve. The experiments were
performed in triplicates.
Mouse xenograft tumor model According to the
protocol of transplantation tumor
research[19], NCI-H460 tumor tissues were chopped into 2
mm3, and then transplanted sc into the right flank of nude mice. The diameter of transplantion
tumor was measured with a vernier caliper. When the tumor
size grew to 280 mm3, the 32 tumor-grafted mice were
randomly divided into 4 groups (n=8); that is, negative control
group (treated with normal saline), positive control group
(wt TRAIL, 15 mg/kg) and rmh TRAIL treated groups (3 and
15 mg/kg). All drugs or saline were injected ip once daily for
10 d followed by another 14 d observation.
The tumor volume (TV) was calculated using the
following formula:
TV=1/2×a×b2 (in which a is the length and b is the width
of tumor).
The evaluation index of anti-tumor activity was relative
tumor weight inhibition ratio (%), which was calculated by
the following formula:
Tumor weight inhibition ratio (%)=(1-T/C)×100% (T: test
group¡¯s or positive control group¡¯s mean tumor weight; C:
negative control group¡¯s mean tumor weight).
The data were then subjected to a statistical analysis
(t-test) for actual efficiency of the material tested.
Assessment of apoptosis Various methods, described
below, were used to evaluate NCI-H460 apoptosis.
(a) DNA fragmentation detection by ELISA
A cell death detection ELISA kit was used according to the
manu-facturer¡¯s instructions for DNA fragment
detection[20]. The DNA fragments are discrete multiples of a 180-200 bp
subunit, which can be detected as a DNA ladder on agarose
gel. The enrichment of mono- and oligonucleosomes in the
cytoplasm of the apoptotic cells is a result of DNA
degradation that occurs several hours before plasma membrane
breakdown. The principle on which this test is based is the
detection of mono- and oligonucleosomes in the
cytoplasmic fractions of cell lysates by using biotinylated antihistone-
and peroxidase-coupled anti-DNA antibodies. The enrich
ment of mono- and oligonucleosomes released into the
cytoplasm is calculated as the ratio of absorbance of sample
cells to absorbance of control cells. The enrichment factor
was used as a parameter of apoptosis and is shown on the Y
axis as mean±SD of triplicate experiments performed in
triplicates. An enrichment factor of 1 represents
spontaneous apoptosis. NCI-H460 were grown in 96-well plates and
exposed to rmhTRAIL 10 ng/mL for 0.5 to 4 h or 0.0128
ng/mL to 40 ng/mL for 2 h.
(b) TdT-mediated dUTP nick-end labeling (TUNEL)
assay After incubated with 15 ng/mL rmh TRAIL for 1, 2, and
4 h, NCI-H460 cells were smeared on slides, fixed by 4%
paraformaldehyde in PBS (pH 7.2) for 30 min at room
temperature, washed 3 times with PBS, and blocked by 0.3%
H2O2. Then, cells were permeabilized on ice using 0.3%
Triton X-100 in PBS for 2 min. Subsequent operations were
refered to the manufacturer¡¯s instruction of In
situ cell death detection kit and DAB kit. The samples were dehydrated by
ethanol and cleared in dimethylbenzene, analyzed by
microscopy and photos were taken using a Nikon 4 500 digital
camera.
(c) Flow cytometry For apoptotic cell detection analysis,
NCI-H460 cells were treated with rmh TRAIL or wt TRAIL (1,
10, and 100 ng/mL) for 12 h. All of the attached and the
detached cells were harvested. Detection of the apoptotic
cells was referred to the manufacturer¡¯s instruction of
Annexin V-FITC apoptosis detection kit.
Results evaluation:
Non-apoptotic cells: Annexin V negative and PI negative;
Early apoptotic cells: Annexin V positive and PI negative;
Necrotic cells or late apoptotic cells: Annexin V positive
and PI positive.
Statistical analysis Data were expressed as mean±SD.
Data of the representatives were analyzed for statistical
significance using a t-test. P<0.05 was considered statistically
significant.
Results
Various tumor cell lines are sensitive to rmh TRAIL-induced death in vitro To explore the antitumor activity of
rmh TRAIL against a spectrum of cancer cell lines, we tested
its effect in vitro on the inhibition of 5 different cell lines
derived from cancers of the lung, colon, and breast (2 lung
cancer cell lines, 2 breast cancer cell lines, and 1 colon
cancer cell line) by MTT assay. rmh TRAIL exerted a cytotoxic
effect on these cell lines and inhibited cell growth after cells
being treated for 24 h or 72 h. Furthermore, the antitumor
activity of rmh TRAIL was notably higher than that of wt
TRAIL, and IC50 for rmh TRAIL against all cell lines were
significantly lower than wt TRAIL (Table 1,
P<0.01). Treatment with rmh TRAIL at the dose of 200 ng/mL caused
substantial cell death. The morphology of the cells treated by
rmh TRAIL was characteristic of apoptotic cells: blebbing
and shrinkage of cytoplasm (Figure 1).
Antitumor activity of rmh TRAIL in a mouse xenograft
study To determine whether the tumoricidal activity of
rmh TRAIL observed in vitro could be demonstrated
in vivo, BALB/c nude mice were transplanted sc with NCI-H460
tumor tissue, which showed intermediate sensitivity to
rmh TRAIL in vitro (Table 1), and allowed tumors to establish.
The tumors in the control group grew steadily in an
exponential manner. wt TRAIL treatment gave a 25.7% reduction
in mean tumor volume, which was lower than that of
rmh TRAIL. In contrast, the rmh TRAIL treated groups
showed a marked reduction in tumor size, especially during
the treatment period (Figure 2, Table 2). The tumors in some
mice in the high-dose rmh TRAIL group became invisible
during the treatment period and shortly after withdrawal.
The suppressed tumors recommenced growing 5 d after
withdrawal, but still more slowly than the control group. The
inhibitory effect of rmh TRAIL on transplantation of tumor
NCI-H460 was in a dose-dependent manner. Tumor
suppression in 3 mg/kg rmh TRAIL-treated group was less than
15 mg/kg group, but greater than 15 mg/kg in the wt
TRAIL-treated group (Figure 2, Table 2).
Apoptosis induced by rmh TRAIL
a.rmh TRAIL-induced cell death of NCI-H460 cells
detected by ELISA rmh TRAIL 10 ng/mL was chosen for
DNA fragmentation experiment by ELISA at different time
points. This concentration of rmh TRAIL-induced apoptosis
showed a time-dependent manner. At 2 and 4 h after
treat-ment, rmh TRAIL induced a significant increase of
cytoplasmic nucleosomes (Figure 3A). The result accords with
agarose gel electrophoesis result (data not shown). More-over,
we examined different concentrations (from 0.0128 ng/mL to
40 ng/mL) of rmh TRAIL-induced apoptosis for 2 h. The
result showed a dose-dependent manner (Figure 3B).
b. TUNEL assay The pictures displayed in the Figure
4 were taken after incubation with rmh TRAIL 15 ng/mL for 1,
2, and 4 h. The nucleus condensation and segmentation
were observed by microscope. Lots of apoptotic bodies
were found in rmh TRAIL-treated cells but none in untreated
cells.
c. Flow cytometry To confirm that the percentage of
apoptosis induced by various concentration of rmh TRAIL
in NCI-H460 cells, Annexin V and PI double staining and
flow cytometry were performed. The percentages of apoptosis
induced by rmh TRAIL and wt TRAIL (1 ng/mL, 10 ng/mL,
100 ng/mL) for 12 h were 8.11%, 32.80%, 59.68% and 4.96%,
7.08%, 14.11%, respectively (Figure 5).
Effect of rmh TRAIL on normal cell types
To test whether rmh TRAIL initiates apoptosis in normal cells, 9 lines of
normal cells, were exposed to rmh TRAIL (1 ng/mL-10
µg/mL) or wt TRAIL (1 ng/mL-10 µg/mL) for 24 h. No morphological
evidence of apoptosis was observed as induced by
rmh TRAIL (except for HL-7702); in addition, there was no
decreased cell viability in staining of the cells by MTT (Figure
6), which indicated that rmh TRAIL was not cytotoxic
toward these normal cell types. There was mild injury on
HL-7702 hepatocytes elicited by both rmh TRAIL and wt TRAIL,
and no cytotoxicity on CCC-HEL-1, human primary embryo
liver-derived cells (Figure 6). The
IC50 of rmh TRAIL and wt TRAIL on HL-7702 exceeded 10 µg/mL.
Discussion
Although TNF and FasL have potent cytotoxic activity
against many types of tumor cells, the application of these
death ligands to cancer therapy has been restricted by their
severe toxicity to normal tissues. The discovery of TRAIL
as a death ligand, with its wide tissue-mRNA distribution
and its unique receptor system, suggests that this ligand
might be more suitable than TNF or FasL for systemic cancer
therapy.
rmh TRAIL, developed by Beijing Sunbio Biotech, is a
mutant form of human native TRAIL, but the functional
sequences are conservative. Native TRAIL is expressed as a
type II transmembrane protein that can be cleaved
proteolytically to form a soluble
homotrimer[7,8]. The therapeutic
potential of a recombinant soluble version of human
TRAIL that can be produced in
E coli and purified as a 60-kDa homotrimer has been
evaluated[9,11]. An optimized antineoplastic agent can selectively induce apoptosis of cancer
cells while sparing normal cells. Some scientists have
oppugned the safety of TRAIL on
hepatocytes[22,23]. To obtain a form of TRAIL that has higher antitumor activity and
safety, Beijing Sunbio Biotech produced rmh TRAIL with a
circular permuted extracellular sequence of native human
Apo2L/TRAIL. Our previous work revealed that rmh TRAIL
had a high affinity for its death receptors and
water-solubility (data not shown). This molecule forms more stable
homotrimers. The pharmacokinetics research of rmh TRAIL
indicates that the concentration in tumor tissue was higher
than that in plasma. Furthermore, rmh TRAIL was more
resistant to trypsin proteolysis in vitro than wt TRAIL (data
not shown). All of the above suggests that rmh TRAIL could
contribute to long-standing antitumor activity.
The present study first assesses the antitumor activity
of rmh TRAIL in vitro. A spectrum of cancer cell lines
exhibited sensitivity in vitro to rmh TRAIL. This finding is
consistent with previous reports that native TRAIL is
cytotoxic toward cell lines from cancers of the lung, colon, and
breast[9-11]. rmh TRAIL triggers more cell death
in vitro than wt TRAIL. The study of NCI-H460 xenograft models
indicates that rmh TRAIL can cause tumor regression or
suppress tumor growth, and can initiate tumor cell apoptosis,
which is consistent with its apoptosis-inducing activity
in vitro. The antitumor activity of TRAIL after ip
administration has been demonstrated in several mouse xenograft
models of human cancers, including
colorectal[9], breast[11], and
glioma[24]. rmh TRAIL caused a dose-dependent
suppression of tumor growth, and 15 mg/kg rmh TRAIL resulted in a
86.7% reduction in mean tumor volume, whereas wt TRAIL
only caused a 25.7% reduction using the same dose.
Therefore, in the present study, the antitumor activity of rmh
TRAIL is higher than that of wt TRAIL in the experiments
both in vivo and in vitro. Compared with wt TRAL, rmh
TRAIL can achieve same antitumor activity under lower
dosages.
Second, rmh TRAIL induced apoptosis in NCI-H460 was
confirmed by cell death ELISA assay, TUNEL assay, and
flow cytometry. Nucleus condensation, apoptotic body
appearance, and DNA fragmentation are universal
characteristics in the cells undergoing apoptosis. Furthermore, cell
death ELISA assay and flow cytometry showed the quantity
of apoptotic cells. From the evidence in the present study, it
is elicited that rmh TRAIL could induce NCI-H460 apoptosis,
that and it is time- and concentration-dependent.
Third, MTT assay was used to examine the cytotoxicity
effect of rmh TRAIL on normal cells. Although rmh TRAIL
was cytotoxic toward many tumor cell lines in
vitro,
rmh TRAIL showed no inhibition on normal cells tested in
the experiment from endothelial, fibroblastic, myocardial,
cutaneous, lung, renal, and tracheal origin, supporting the
notion that rmh TRAIL dose did not increase cytotoxicity to
generally nontransformed cells. The results are consistent
with Ashkenazi et al[9,10,25,26]. rmh TRAIL and wt TRAIL
showed mild cytotoxicity to HL-7702 cells, which are
separated from adult liver. The IC50 of rmh TRAIL exceeded 10
µg/mL. The IC50 for non-small cell lung cancer and colonic
cancer, the main clinical indications, are all 0.01 µg/mL, which
is at least 1000 times lower than the
IC50 for HL-7702. Furthermore, the pharmacokinetics research on nonhuman
primates (cynomolgus monkeys) indicated that the maximal
plasma concentration (Cmax) of rmh TRAIL (iv, 5 mg/kg) was
25 µg/mL (data not shown). The dose used in humans would
be no more than 5 mg/kg. Therefore, the
Cmax will be no more than 25 µg/mL, which is less than the
IC50 of HL-7702. The results suggest that the clinical effective dose of rmh TRAIL
might be safety for human.
In conclusion, rmh TRAIL provided potent antitumor
activity in vitro and in vivo, although it did not exert
obvious cytotoxicity on a wide variety of normal cells. Therefore,
rmh TRAIL might prove to be a useful new agent in fighting
cancer cells, leaving normal cells unharmed.
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