Key words PAMAM dendrimer - Suicide gene therapy - Cytosine deaminase - Gene delivery - VEGF promoter - 5-Fluorocytosine
Introduction
Targeted therapy is a type of cancer treatment for precisely identify and attack the
cancer cells. It can be used by itself or in combination with other treatments, such
as standard chemotherapy, surgery or radiation therapy [1 ]
[2 ]
[3 ]. In recent years, several
studies including molecular therapy, antiangiogenesis therapy [4 ]
, immunotherapy [5 ]
, apoptosis regulation [6 ]
, signal-transduction therapy [7 ]
, differentiation therapy [8 ]
, targeted radionuclide therapy [9 ] and gene therapy [10 ] are considered for targeted cancer therapy.
Current gene therapy strategies for the treatment of tumors are restricted by the
lack of targeting the malignant cells. One of the main problems for their in
vivo applications is low transduction (or transfection) efficiency of the
available gene transferring systems [11 ]. The
cytotoxic systems convert a prodrug to an active drug intracellularly. Suicide gene
therapy trials can be more promising than the other available gene therapy
strategies [12 ]
. The safety and
effectiveness are the promising advantages of this approach for cancer treatment
[13 ]. In the suicide gene therapy known as
Gene-Directed Enzyme/Prodrug Therapy (GDEPT), the therapeutic transgenes
have the ability to convert a non-toxic prodrug into cytotoxic drug or to express
the toxic gene expression product [14 ]
[15 ]–[16 ]. Ideally, the gene should be expressed exclusively in the tumor cells
not into the normal cells. The enzyme must reach a sufficient concentration for
catalytic activity to activate the prodrug under physiological conditions for its
clinical use. Because in vivo expression of the foreign enzymes will not
occur in all cells of a targeted tumor, a bystander effect is required, whereby the
prodrug is cleaved to an active drug that kills not only the tumor cells in which it
is formed but also neighboring tumor cells that do not express the foreign enzyme
[17 ].
Among many suicide genes, E. coli Cytosine Deaminase (CD) and Herpes Simplex
Virus thymidine kinase (HSV-tk) are well documented for their strong therapeutic
efficacy in cancer treatment [18 ]
. In
CD/5-fluorocytosine (5-FC) system, a suicide gene, CD, catalyzes the
conversion of the anti-fungal substrate 5-FC into 5-fluorouracil (5- FU), which is a
lethal drug [19 ]
. Suicide gene therapy,
utilizing the CD/5-FC system, is an efficient approach for targeted therapy
in cancer study [20 ]
[21 ]
[22 ].
Most suicide genes under investigation mediate sensitivity by encoding bacterial or
fungal enzymes that convert inactive forms of a drug, into toxic metabolites capable
of inhibiting nucleic acid synthesis [23 ]
[24 ]. The cellular enzymes process the
formulation to three cytotoxic antimetabolites: 1) 5-FdUTP, 2) 5-FUTP and 3) 5-
FdUMP. The cytotoxic effects of CD suicide therapy are based on three properties: a)
formation of (5-FU) RNA, b) 5-FU DNA complexes and c) thymidylate synthase. The
mitochondrial pathways are down-regulated through the control of Bcl-2 [25 ]. Also, heat shock protein 90-beta is
activated, contributing to tumor regression [26 ].
CD/5-FC system has some advantages over other GDEPT systems, including
significant distant bystander effect independent of gap junctions [27 ]
[28 ].
One of the advantages of CD/5-FC system is radiosensitizing ability of 5-FU
to enhance tumor cytotoxicity efficiency along with radiotherapy [25 ]
[29 ].
Some studies have obtained the mutant forms of bacterial CD in order to improve
their kinetics including high affinity and lower IC50 for 5-FC [30 ]
.
The bystander effect can improve the therapeutic consequences which extended beyond
the transfected tumor cells. There are currently five mechanisms investigated for
the regression of untransfected cells due to bystander effects: a) release of
soluble formulations, b) passage through gap junctions, c) passive transportation,
d) stimulation of local microenvironment, and e) endocytosis of apoptotic vesicles.
The CD/5-FU system demonstrates stronger local bystander effect than the
other suicide genes. The 5-FU diffuses efficiently within the tumor cells, and does
not require cell to cell contact [25 ].
There are currently two main vehicles used to deliver the suicide genes: viral
vectors and non-viral vectors [31 ]. Non-viral
vectors are widely used with three different approaches: 1) naked DNA, 2) physical
methods and 3) chemical methods. Non-viral vectors such as cationic liposomes and
cationic polymers are less efficient than viral vectors; they have the advantages of
safety, simplicity of preparation and high gene encapsulation capability. These two
delivery systems have a positive charge, which interacts with the negative charge of
the DNA [32 ]
. PolyAmidoAmine (PAMAM)
dendrimers, is used for drug and gene delivery. The positive charge of PAMAM
dendrimer surfaces decreases their cytotoxicity and has interesting implications for
DNA transfection applications. Depending on its characteristics, such as a high
efficiency in delivery, the capabilities of specific targeting, biodegradability,
non-toxicity, non-immunogenicity, and capability of limiting DNA degradation, PAMAM
dendrimer provides a promising approach for gene delivery [11 ]
[12 ].
Gene therapy for cancer requires vectors that are selectively expressed in tumor
cells. It is achieved by putting the suicide gene under the control of a tumor
specific promoter [33 ]
. A wide number
of cancer tissue specific promoters’ studies have used tissue-specific
promoter elements, such as: human telomerase reverse transcriptase (hTERT) promoter,
carcino-embryonic antigen (CEA) promoter, and osteocalcin (OC) promoter [34 ]
. In the recent years, various new
promising promoters like auxin response factors (ARF) [35 ], glucose-regulated protein (GRP78) [36 ]
, chemokine (C-X-C motif) receptor-4
(CXCR4) [37 ], and osteopontin (OPN) [38 ], have been developed. The human
cytomegalovirus (HCMV) promoter/enhancer is a flexible promoter that is
expressed in many cell types and provides high levels of trans-gene expression [39 ]
. Based on the overexpression of the
vascular endothelial growth factor (VEGF) in breast cancer cells [34 ]
[40 ]
, a recombinant pEGFPN1 harboring the VEGF promoter and the CD
suicide gene (pEGFPN1-VEGFp-CD), was constructed in the present study, for
expression in 4T1 cell line. Plasmid constructs modified with the VEGF promoter
element may provide improvements in gene expression for cancer cells. In the present
study, we designed a tumor-specific promoter system by combining VEGF promoter,
which can target to cancer cells with sufficient transcriptional activity. A
gene-directed enzyme prodrug therapy (GDEPT) system consisting of the bacterial
cytosine deaminase gene (bCD) driven by the VEGF promoter and the prodrug
5-folourosytosine (5FC) was developed and its cytotoxicity effect was evaluated.
Materials and Methods
Cell culture condition, reagents and instruments
4T1 cells, E. coli BL21 and E. coli TOP10, were obtained from
Pasteur institute of Iran and were cultured in LB agar media. 4T1 cells were
cultured in RPMI 1640 medium, supplemented with 10% fetal bovine serum
and %1 penicillin/streptomycin, and incubated at 37°C in
a 5% CO2 incubator.
The restriction enzymes were purchased from Takara Bio, Inc., (Otsu, Japan). T4
DNA ligase, Probest DNA polymerase and were purchased from New England Biolabs,
Inc., (Ipswich, MA, USA). Agarose gel DNA extraction kit, 5-flurocytosine
(5-FC), MTT and dimethyl sulfoxide (DMSO) were purchased from Sigma-Aldrich
(Merck KGaA, Darmstadt, Germany).
The instruments that were used in the present study were: DLS (zetasizer, USA),
ELISA Infinite Tecan (USA), Bio-Rad GenePCR (PerkinElmer, Inc., Waltham, MA,
USA), OLYMPUS Ix53 optical microscope (Olympus Corporation, Tokyo, Japan), an
OLYMPUS BX61 inverted fluorescence microscope and camera system (Olympus
Corporation), BD Calibur Flow Cytometer (BD FACS Calibur, San Jose, CA, USA) and
step -one-plus Real time PCR (ABI, USA).
Molecular cloning of shuttle vector constructions
Amplification of CD gene
E. coli strain BL21 was inoculated into 5 mL liquid LB medium with
shaking, for overnight at 37°C. The genomic DNA of E. coli
BL21 was extracted by lysis method. It used as the template DNA for the
amplification of CD gene by PCR. Specific primers of CD gene were designed
based on published sequence (Genebank, NC-000913). The CD gene was amplified
from the obtained genomic DNA of E . coli BL21 by PCR. The
following primers were used: forward primer, 5′-
TATAAGCTT GTGTCGAATAACGCTTTAC -3′ (Italic part indicates the
recognizing site of Hind III) and the reverse primer, 5′-
ATTAAGCTTT CAACGTTTGTAATCGATGG -3′ (Italic part indicates
the recognizing site of Hind III). The PCR product of CD (1302 bp) and
the expression vector, VEGF-pEGFP-N1 (5349 bp) made in our previous stud
[41 ], were digested by
Hind III and then ligated to obtain the final plasmid,
VEGF-CD-pEGFP-N1 (6650bp). Also, the pEGFP-N1 plasmid was linearized by
EcoR I and Xho I and the CD gene was inserted in its
multiple cloning sites (Hind III). Finally, the recombinant plasmids
sequences were verified by DNA sequencing, and the plasmid was further
amplified and purified by a QIAfilter™ plasmid maxi kit
(Qiagen China Co., Ltd., Shanghai, China), according to the
manufacturer's protocol.
Optimization of dendriplex transfection
Preparation of PAMAM/DNA Dendriplex
G4-PAMAM-D, Sigma-Alderich, (USA), was kindly provided by Dr. Behzad Baradarn
(Tabriz University of Medical Sciences, Tabriz, Iran). Dendriplexes of
DNA/PAMAM complexes were prepared at different N/P ratios.
The N/P ratios were calculated based on PAMAM nitrogen per nucleic
acid phosphate. 2 μg of DNA were mixed in HEPES buffer saline (20
mM) and appropriate amounts of diluted PAMAM by HEPES buffer saline were
added to DNA solutions. Solutions were mixed. The resulting dendriplexes
were incubated for 30 min at room temperature before use.
Gel retardation assay
Gel retardation assay was performed to assess the formation of electrostatic
complexes between PAMAM and plasmid DNA. Briefly, a 0.1% agarose gel was
prepared in TAE buffer (1X) with 0.5 μg/ml safe stain.
Dendriplexes were loaded into the agarose gel wells at N/P ratios of 5,
10, 20, 40, 80, and 160. Samples of dendriplexes were run against free plasmid.
Electrophoresis was conducted at 80 v for 45 min. Gel were photographed under UV
light.
Size and zeta potential measurements
20 μg DNA was incorporated with the different amount of PAMAM in 10 mL
HEPES buffer saline (20 mM) giving N/P =10,20,40,80 and 160
polyplex. Dynamic light scattering was used to measure the mean diameters and
surface Zeta potentials of the polyplexes using Zetasizer-Nano ZS
(Malvern Instruments, Worcestershire, UK). The mean diameters and Zeta
potentials of the dendriplexes were measured three times in triplicate.
Cell culture and cytotoxicity assay of dendriplexes & prodrug on 4T1
cells
4T1 (murine mammary gland tumor), were cultured in RPMI 1640 medium with
10% fetal bovine serum (FBS), and were maintained at 37°C in a
5% CO2 incubator. The potential PAMAM-G4 dendriplex cytotoxicity was
evaluated on 4T1 cells. This study was carried out by MTT assay of cell,
according to the method described by Mosmann [42 ]
. Briefly 5×104 cells were seeded in a
24 well plate and after 24h the cells were treated with different N/P
ratios of dendriplex at 500 μl RPMI1640 media. After incubation at
37°C for 4–6 h, the growth medium was replaced with
fresh FBS+/antibiotics+ media and cultured for one day.
The culture medium was then removed and a total of 20 µl MTT (5
mg/ml) was added to each well and incubated for 4 h at 37°C. The
culture medium was then removed, followed by the addition of 100 µl DMSO
to each well. The absorbance was measured by a plate reader at 570 nm. The cell
viability percentages of different N/P ratios were calculated by
considering the absorbance of control as 100 % viability. Presented
results correspond to n = 3 performed in triplicate and expressed as
mean ± SD.
MTT colorimetric assay was used to evaluate the lethal effect and optimal
concentration of 5FC on 4T1 and transfected 4T1 cells. Cells were cultured in
96-well plates (1×104 cells) in 200 µl RPMI 1640.
After 24 h of culture, the concentration of 5-FC was tested at 0, 50, 100, 200,
400, 600, 800 or 1000 μM. The blank control, experimental and control
groups were set up in five replicate groups. In total, 24, 48 and 72 h after
5-FC treatment, the cell viability was determined using the MTT assay. The
culture medium was then removed and a 20 µl MTT (5 mg/ml) was
added to each well and incubated for 4 h at 37°C. The culture medium was
then removed, followed by the addition of 100 µl DMSO to each well. The
absorbance (optical density; OD) of each well was measured at a wavelength of
570 nm. The cell survival rate (%) was calculated according to the
following formula: (Experimental group OD value/blank control group OD
value)×100. The IC50 value was determined with a Prism 3.03
program for each group.
Cell transfection
One day before transfection, cells were seeded in 6-well plates and cultured in
growth medium without antibiotics. The cell confluency was approximately
70% at the time of transfection. 2 μg plasmid per well was used
for transfection in 6-well plate. PAMAM and plasmid DNA (pDNA) were freshly
diluted to equal volumes with HEPES buffer saline prior to use. The dendriplexes
were prepared by adding PAMAM into pDNA using different N/P ratios. The
dendriplexes were incubated for 30 minutes at RT. Based on standard PAMAM
mediated transfection, the growth medium was replaced by fresh FBS
free/antibiotics free medium (2 mL for 6-well) and then the dendriplexes
diluted by RPMI1640 medium and added to the culture. After incubation at
37°C for 4–6 h, the growth medium was replaced with
fresh FBS+/antibiotics+ media and cultured for one to
two days for detection.
CD and GFP gene expression analysis
Fluorescent microscopy
To qualitatively evaluation of the transfection efficiency, cells transfected
with pEGFP-N1 were observed and imaged using an inverted fluorescent
microscope (Olympus BX61) for GFP expression at days 1 and 2 after
transfection. After 72 h of cell culture (37°C), cells were washed
with PBS three times, trypsinized and collected for observing by
fluorescence microscope. Cells were then centrifuged at 250×g for 2
min, washed with 4°C precooled PBS twice, fixed in 4°C
precooled 3% glutaraldehyde for 4 h and rinsed with PBS for 10 min.
Subsequently, they were fixed on slides at room temperature and observed
under the fluorescent microscope.
Flow cytometry analysis
The transfection efficiency was quantitatively analyzed at cellular level by
detecting and comparing the GFP excitation in the transfected and
non-transfected cells. 2 days after transfection, the cell colonies were
dissociated with trypsin and subjected to flowcytometry analysis (BD
Biosciences, San Jose, CA, USA). The data were processed using FlowJo vX10
software. The transfection efficiency was determined as the percentage of
viable GFP-positive cells among total cells.
Real Time PCR
After 48 h of transfection, total RNA was extracted from transfected
4T1CD+ (CD-transfected 4T1), with TRIzol reagent
(Invitrogen; Thermo Fisher Scientific, Inc.) and then one-step reverse
transcription (RT)-PCR was performed using a One-step RT-PCR kit, according to
the manufacturer's protocol. The forward (5′
CAGCGGCTACCGTGAT-TCA 3′) and reverse (5′ TTTGCACATGGCGTTGG
3′) primers were designed to amplify a 61 bp fragment of the CD gene.
PCR reaction conditions were as follows: 95°C for 5 min, followed by 30
cycles of 94°C for 30 sec, 58°C for 30 sec, 72°C for 1
min, then 72°C for 10 min. Differences in gene expression were
normalized to the β-actin gene expression with the following primers:
forward 5’-aacgagcggttccgatgccctgag-3’; reverse
5’-tgtcgccttcaccgttccagtt3.
Bystander effect
After determining the percentage of transfection, the transfected cells were
mixed with a certain amount of non-transfused cell and the normalized cells were
seeded in 96-well plates 2 days prior prodrug addition. In brief, cells were
washed with RPMI1640 and treated by 5-FC. The number of viable cells after 5-FC
was measured according to the cell cytotoxicity assay mentioned in MTT assay
section [43 ]
[44 ].
Evaluation of the effect of cell soup obtained from transfected cells on
non-transfected cells
According to the transfection instructions, cells in the 6-cell plate were
transfected at N/P ratio 160. 48h after transfection, the supernatant of
the transfected cells was collected. 66% and 33% of the cell
soup were prepared and the cells were treated with 5-FC for 48 hours.
Non-treated cells transfected with cell soup were considered as positive control
and non-transfected cells untreated with cell soup were considered as negative
control.
Statistical analysis
Statistical analyses were performed using GraphPad Prism 8.02
(©1992–2019 GraphPad Software, Inc.). Data were expressed as
mean ± standard deviation of the mean and analyzed by one-way ANOVA. The
means were compared using Duncan’s multiple range tests.
Results
Construction and confirmation of the plasmids
We used in Silico Snap Gene design to obtain the schematic structure of
plasmids and MCS sites ([Fig. 1 ]).
Fig. 1
In Silico SnapGene constructs : a native pEGFP-N1 (with
CMV promoter and total size 4733 bp), b VEGF-pEGFP-N1-
(substitute VEGFpromoter with CMV, size : 5349 bp), c
VEGF-CD-pEGFP-N1-, final construct, size : 6639 bp ), d
CD-pEGFP-N1- ( size : 6023 bp).
VEGF- CD -pEGFP-N1 plasmid
The CD gene was amplified from E. coli BL21 genomic DNA by PCR ([Fig. 2a ]). The VEGF-pEGFP-N1 vector
and CD gene (1302 bp) were digested by Hind III restriction enzyme,
before ligation and transformation into competent cell ([Fig. 2b ]). Colony PCR was performed
for transformation screening [41 ]
([Fig. 3a ]). The recombinant
plasmid was extracted and verified by enzymatic digestion ([Fig. 3b ]).
Fig. 2
a) Gel electrophoresis of PCR product of CD gene; M: 1
Kb Ladder (Fermemtas Co.), 1 : CD PCR product. b Gel
electrophoresis of CD & pEGFPN1-VEGF enzymayic digestion by
Hind III), M: 1 Kb Ladder (Fermemtas Co.),
lane1 : cytosine deaminase, lane 2 : VEGF-
pEGFP-N1.
Fig. 3
a): Gel electrophoresis of colony PCR products after ligation
and transformation, lane M, 1 Kb ladder (Fermentas Co.), lanes 1 to
12, colony PCR products of random selected colonies. Clones of 2,
5,6,7,9 and 12 are positive transformed clones. b ):
Molecular confirmation of correct orientation of inserted CD into
VEGF- pEGFP-N1 by enzymatic digestion by Xho I, Hind III
and electrophoresis: lane M: 1 Kb ladder (Fermentas Co.), lane1:
uncut construct, lane 2: cut VEGF- pEGFP-N1 (5349 bp) and CD (1300
bp).
CD -pEGFP-N1 plasmid
The CD gene was amplified from E. coli BL21 genomic as mentioned
before ([Fig. 2a ]). The pEGFP-N1 and
a 1302 bp fragment of CD sequence were digested, before ligation and
transformation into competent cell ([Fig.
4a ]). Colony PCR was performed for transformation screening ([Fig. 4b ]). The recombinant plasmid was
extracted and verified by enzymatic digestion ([Fig. 4c ]).
Fig. 4
a Gel electrophoresis of PCR product of CD gene; M: 1 Kb
Ladder (Fermemtas Co.), 1 CD PCR product. b Gel
electrophoresis of colony PCR products after ligation and
transformation, lane M, 1 Kb ladder (Fermentas Co.), lanes 1 to 7,
colony PCR products of random selected colonies. Clones of 1, 4 and
5 are positive transformed clones. c Molecular confirmation
of inserted CD into VEGF- pEGFP-N1 by enzymatic digestion and
electrophoresis: lane M: 1 Kb ladder (Fermentas Co), lane1: cut CD-
pEGFP-N1 (4733bp) and CD (1302 bp).
Gel Retardation assay
Gel retardation assay was performed to determine the most appropriate N/P ratio
of dendrimer and plasmid DNA. PAMAM /plasmid complexes were prepared at various
N/P ratios ranging from 5, 10, 20, 40, 80 and 160. After 30 min of incubation
and dendriplex formation, the samples were electrophoresed ([Fig. 5 ]).
Fig. 5 Dendriplex Gel Retardation assay: Lane 1: control
(pEGFP-N1); lanes of 2 to 7: dendriplex (pEGFP-N1+PAMAM) with
different N/P ratios (5, 10, 20, 40, 80, 160).
Transfection optimization
In order to increasing the transfection efficiency, four factors including size,
zeta potential, cell cytotoxicity and N/P ratio of dendriplex were investigated.
As the N/P ratio increases, the particle size and the zeta potential of
dendriplex and also the transfection percentage increase but the cell viability
decreases ([Fig. 6 ]).
Fig. 6 Characterization of the PAMAM /pEGFP-N1 (dendriplex).
a Mean diameter and surface charge of the polyplex versus
various N/P ratios using DLS (data expressed as mean+S.D. of
three-independent measurements). b cellular cytotoxicity and
transfection efficiency of dendriplex versus various N/P ratios;
c and d Polyplex sizes and zeta potentials measured by
Zetasizer for example.
Dendriplexes size and zeta potential
Since PAMAM has a positive charge and DNA has a negative charge in aqueous
solution, PAMAM and DNA can form a complex with electrostatic effects in
physiological conditions. PAMAM/plasmid complexes (dendriplex) at a most
appropriate charge ratio were incubated for transfection of DNA. The particle
size, size distribution and particle charge of PAMAM G4 dendrimer was measured
by DLS (Zetasizer Nano ZS, Malvern Instruments, UK) ([Fig. 6 ]). Data are expressed as mean
± SD. In the following, size and charge of polyplex (DNA+PAMAM)
with various N/P ratios were measured by DLS.
Cellular cytotoxicity and transfection efficiency of dendriplex
Increasing the dendrimer content of the dendriplex increases its cytotoxicity,
but more importantly, increasing the size of the dendriplex may make endocytosis
by cells almost impossible. As shown in [Fig.
6a ], with increasing the dendrimer content of the dendriplex, its size
also increases, so that in the dendriplex with a N/P ratio of 160,
dendriplex size has reached to 400 nm.
Fluorescence microscope and flowcytometry analysis
The constructs expresses a fluorescent reporter (green fluorescent protein) and a
suicide cytosine deaminase gene, under control of VEGF promoter. The GFP
expression was detected in 4T1 cells at different N/P ratios of
dendriplexes ([Fig. 7 ]). These show that
dendriplexes were not cytotoxic in 4T1 tumor cells and were uptaken by these
cells. The increasing N/P ratio may be influencing the total number of
cells was GFP positive and the transfection efficiency. This hypothesis has been
demonstrated qualitatively and quantitatively by fluorescent microscopy ([Fig. 7 ]) and flowcytometry ([Fig. 8 ]), respectively.
Fig. 7 GFP fluorescence imaging: 4T1 cells were transfected by
pEGFP-N1 after 48h at different N/P ratios (Control, and:
N/P=10, N/P=20, N/P=40, N/P = 80, and
N/P= 160).
Fig. 8 Flowcytometry imaging: 4T1 cells were transfected by
pEGFP-N1 after 48 h at different N/P ratios: a Control, b
N/P=10 (% transfection; 7.9), c N/P=20
(%transfect; 17.6), d N/P=40 (%transfect;
25.9), e N/P=80 (%transfect; 35.2), f N/P
= 160, (%transfect; 45.8)
Expression of CD driven by the VEGF promoter or CMV promoter inhibits 4T1
cells growth
To investigate the level of cell proliferation in these enzyme/prodrug systems
(VEGF-pEGFP-N1, VEGF-CD-pEGFP-N1 and CD-pEGFP-N1), 4T1 cells were transfected
with these recombinant plasmids and its sensitivity to the 5-FC was evaluated by
MTT assay. Cell viability % versus 5-FC was drowned using GraphPad Prism
from the dose-cell viability curves. From the IC50 comparisons shown
in [Fig. 9 ], it is clear that the cell
growth inhibition rates in the VEGF-CD-pEGFP-N1 and CD-pEGFP-N1 treated cell
groups was observed while the cytotoxicity in the VEGF-pEGFP-N1 treated group
was observed under the same experimental conditions. Cell growth inhibition
rates were progressively elevated, may be due to ‘bystander
effect’. Therefore, the presence of bystander effect was investigated in
the next experiment.
Fig. 9 4T1 cells transfected with different plasmid shown in [Fig. 10 ]. The MTT assay was
applied for each cell groups at the different concentration of prodrug
(5-FC) at 24 h, 48 h, and 72 h of incubation. Viability percentages and
the error bars are expressed as mean ± SD and represent the
result of five experiments. No cytotoxicity from PAMAM -G4 dendrimer and
5-FC was observed on this cell line.
Bystander effect
For investigation of the bystander effect of each group in response to 5‑FC
treatment, co-cultures consisting of transfected and non-transfected 4T1 cells
were used. First, transfection efficiency was measured by flowcytometry and
normalized cell transfection % was prepared by co-culturing of
transfected and non-transfected 4T1 cells for each group of 4T1 cells. All of
cell groups was treated by 100µM 5‑FC, and cell survival rates were
calculated. Frequency of transfection for VEGF-CD-pEGFP-N1 and pEGFP-N1- CD
treated group cells was 35±3 and 36±4 ([Fig. 10 ]). By increasing in the
transfection rates of VEGF-CD- pEGFP-N1 and CD - pEGFPN1 treated group cells,
cell viability % were progressively decreased, may be due to
‘bystander effect’. However, the most prominent
‘bystander effect’ was observed in the CD-pEGFP-N1 treated
groups.
Fig. 10
a , b Flowcytometry analysis of transfected cells by
VEGF-CD-pEGFP-N1 and CD- pEGFP-N1 respectively c The cell
viability versus normalized cell transfection % diagram, the
blue and red columns represent the cells treated by the VEGF-CD-pEGFP-N1
and pEGFP-N1- CD plasmids, respectively.
Effect of transfected cell supernatant on non-transfected cells
To determine the effect of cell supernatant of transfected cells on prodrug,
concentrations of 33% and 66% of cell supernatant were added on
non-transfected cells and treated with different concentrations of prodrug.
Toxicity in cells treated with cell supernatant was confirmed ([Fig. 11 ]).
Fig. 11 Cell supernatant of transfected cells causes cytotoxicity
on non-transfected cells after addition of prodrug.
RT-PCR of CD gene expression into transfected 4T1 cell
We used RT-PCR to detect the expression of the CD gene 1300-bp fragment and
expression rate comparison between of VEGF-CD- pEGFP-N1 and CD - pEGFPN1 treated
group cells. The transcriptional activity of the CMV promoter (positive control)
in each cell line was considered as 100%. The expression of CD gene by
the VEGF promoter was approximately from 36 to 72% of positive control
(CD - pEGFP-N1) ([Fig. 12 ]).
Fig. 12 Expression of CD gene (mRNA). The high-level of CD
expression was detected in transfected cells by CD-pEGFP-N1.
β -actin was used to normalize the experiment.
Discussion
The purpose of this study was evaluation of the effectiveness of VEGF promoter and
bystander effect on the efficiency of the CD/5-FC enzyme/prodrug system using a
dendriplex-based non-viral gene delivery system. Non-viral gene delivery systems are
highly regarded for their many advantages compared to viral gene delivery systems
[45 ]. However, their low efficiency in
cell transfection has restricted their applications in clinic. Therefore,
researchers are looking for a way to compensate this shortcoming of non-viral
systems and replace non-viral systems with today's high-risk viral systems.
One potential therapeutic strategy for cancer treatment is the technique of
inserting suicide genes that activate prodrugs to produce cytotoxicity in tumor
cells. Suicide gene therapy, utilizing the cytosine
deaminase/5-fluorocytosine (CD/5-FC) system, is an efficient
procedure for targeted therapy in cancer research with promising results in
previously reported studies [46 ]
[47 ]. It is based on the introduction of a viral
or bacterial gene (and or fungal gene) that encodes a metabolic enzyme into target
cells, which allows the conversion of a non-toxic compound into a lethal drug,
causing the death of target cells. The ‘bystander effect’ is a
phenomenon whereby the transfection or transduction of a small fraction of target
cells with the suicide gene may result in widespread target cell death, including
the non-transfected cells [48 ]. Among many
GDEPT, the most frequently used suicide gene therapy for clinical trials approved by
the FDA, are two major systems as: the CD/5-FC system along or with the
(HSV-TK)/GCV system [46 ]. Cytosine
Deaminase, which normally catalyzes the deamination of cytosine to uracil during RNA
biosynthesis, is found in many bacteria and fungi but not in mammalian cells [49 ]. However, the drug has no or low toxicity
for normal mammalian cells. 5-FC is an antifungal drug compound that is
non-cytotoxic but is transformed into 5-FU following deamination by the CD gene,
which is more cytotoxic and may act to inhibit cellular proliferation [48 ]. Because of these many advantages of the
CD/5-FC system and having a suitable bystander effect, we decided to deliver
this system with a dendrimer-based non-viral gene delivery system. We can have a
comparison between the high-risk viral method at literature and the low-risk
non-viral method [50 ], and we hoped that the
low efficiency of the non-viral gene delivery system would be offset by the
bystander effect.
The plasmid (pEGFPN1) contains of the cytosine deaminase (CDase) suicide gene, under
the VEGF promoter (VEGF-CD-pEGFP-N1), was used to transfect 4T1 cells with the
G4-PAMAM dendriplex. The highest efficiency after optimization of the most factors
for our non-viral system was between 40–50%, and we investigated if
cells transfection was 50%, it is enough to kill all of the transfected
cells and nontransfected cells? For this purpose, we organize the bystander effect
test [43 ] performed on on transfected cells,
with non-viral gene delivery systems. The results showed that in 35%
transfection, cells transfected with plasmid VEGF-CD–pEGFP-N1and CD-pEGFP-N1
inhibited the growth of cancer cells by 80 and 90%, respectively.
Another aim of this project was to confirm the expression of CD gene by transfected
cells. For this purpose, three methods were used. First, because we know that both
the CD and GFP genes are under the same promoter, the expression of GFP will also
express the expression of the CD gene in a way that the expression of GFP gene was
quantitatively and qualitatively confirmed. Second, unlike transfected cells with
CD- plasmids, cell transfected with CD+ plasmids
showed toxicity at a certain concentration of prodrug, which could be due to the
function of CD enzyme and conversion of prodrug to a more toxic drug
(5-fluorouracil). Third, the expression of the CD gene at the transcriptional level
was confirmed by real-time PCR method the presence of CD gene mRNA in total RNA of
transfected cells with CD+ plasmids.
In this study, we created a plasmid controlled by a cancer specific promoter (VEGF).
The VEGF promoter was obtained from 4T1 cell lines and inserted into the pEGFP-N1
expression plasmid. Another plasmid was constructed under the control of the CMV
promoter, as a standard promoter [51 ], to make
an appropriate comparison between the function of the VEGF promoter and the standard
CMV promoter. The results of bystander effect test and real-time PCR showed that the
VEGF promoter function can act close to the CMV promoter of the standard pEGFP-N1
plasmid. The mRNA content of the CD gene produced by VEGF-CD - pEGFP-N1 in the 4T1
cells was 70% of mRNA content of the CD gene under control of CMV promoter
(CD - pEGFP-N1) in the 4T1 cells.
In summary, it demonstrated that the G4-PAMAM denderimer-mediated suicide gene
CD/5-FC system can inhibite 4T1 cell proliferation, and exerted a
‘bystander effect’. This approach may be providing a novel framework
for suicide gene therapy to treat breast cancer and or other kinds of cancers
(especially gliomas). 4T1-specific delivery by G4-PAMAM-denderimer-mediated suicide
CD gene with the addition of specific promoters upstream of the suicide gene may be
provide a therapeutic potential for treatment of breast cancer in future.
Conclusion
In this study we have attempted to improve the antitumor efficacy in 4T1
metastasizing breast carcinoma mice by using expressional shuttle vector to transfer
cytosine deaminase gene under control of VEGF promoter in combination
with non-viral PAMAM dendrimer, followed by 5-FC treatment. The results show
inducing apoptosis in 4T1-trasfected cell and a marked inhibition of tumor cell
growth, suggesting CD gene transfer driven by VEGF promoter plus a safe non-viral
vector followed by 5-FC treatment, may be an alternative therapeutic approach for
the breast cancer treatment. Hence; Future studies in animals and clinical trials
remain to follow the in vivo safety and efficiency of this therapeutic
platform.
Authors’ Contributions
M. E. and A.A. contributed to methodology, conceptualization, software, and writing
of original draft; T. S contributed supervision; A.S. and A.B contributed project
administration, supervision, review, and editing.
Informed Consent
All authors have read the manuscript and approved it for submission to your journal.
All authors have agreed to authorship and order of authorship for this manuscript.
Additionally, all authors have the appropriate permissions and rights to the
reported data. Authors declare that they have no competing interests.
