Extract
Note: Please read the
complete full text with Figures and Tables at
Introduction
Suicide gene therapy is a highlight
in tumor gene therapy at present. Therapy using cytosine deaminase/5-fluorocyto-sine
(CD/5-FC) is one of the most widely studied systems, in which CD can
convert relatively nontoxic 5-fluorocytosine (5-FC) into a toxic
metabolite 5-fluorouracil(5-FU) and kill tumor cells[1-4].
Transfer vectors are a key factor in
the tumor gene therapy system, which directly influences tumor
targeting. It is evident that most of the interest and effort in
cancer gene therapy in the coming years should be focused on
transfer vectors. So far viruses are the most widely used suicide
gene vectors. However, viruses are relatively poor in tumor
targeting and safety, which has badly impeded tumor gene therapy[5,6].
It is crucial to search for a novel gene vector that has good tumor
targeting and is relatively safe.
It is evident that the center of a
solid tumor is generally found at a low level of oxygen, and
anaerobic bacteria tend to colonize in a low oxygen environment.
Therefore, anaerobic bacteria is a potential vector for tumor gene
therapy[7-9]. In a previous study, our laboratory
selected a strain of Bifidobacterium Infantis proven to be good for
targeting solid tumors and non-pathogenic[10]. The aim of
the present study was to transfer the CD gene into
Bifidobacterium infantis to construct a Bifidobacterium
infantis/CD targeting gene therapy system and observe the
antitumor efficacy of a CD/5-FC suicide gene therapy system mediated
by Bifidobac-terium infantis on melanoma in vitro and
in vivo.
Materials and methods
Materials
pGEX-1LamdaT plasmid was kindly provided by Research Unit of
Infection & Immunity, West China School of Preclinical and Forensic
Medicine, Sichuan University, China. Strains of E Coli
K12l were purchased from Chengdu Institute of Biological Products,
China. Strains of Bifidobacterium Infantis 2001 were obtained from
West China School of Stomatology, Sichuan University, China.
LA Taq DNA polymerase, T4 DNA
ligase and l-EcoT14 I digest DNA Marker were purchased from
TaKaRa, Japan. 200 bp DNA Ladder Marker was purchased from
Sino-American Biotechnology, China. Purification kit of plasmid,
Wizard PCR Preps DNA Purification Resin, EcoR I and BamH
I were purchased from Promega (Madison, WI, USA).
Mouse melanomacytes B16-F10 were
provided by China Center for Typical Culture Collection. Female
C57BL/6 mice (class 1, aged 6-8 weeks) were provided by the
Experimental Animal Center of Sichuan University.
Methods
Amplification of CD gene
Strains of E Coli K12l were inoculated into 5 mL LB liquid
medium with shaking, overnight at 37 ºC. The next day, genomic DNA
was prepared by phenol/chloroform method and used as template DNA to
perform PCR for the amplification of CD gene. Specific primers of CD
gene were designed based on published sequences (Genebank,
NC-0009131). The upstream primer is 5'-ATG GAT CCG GAG GCT AAC AAT
G-3' and the downstream primer is 5'-GGG GAA TTC TGT AAC CCA
GTC GT-3'. The amplification cycle was repeated 35 times with the
condition of denaturation at 94 ºC for 30 s, annealing at 55 ºC for
30 s and extension at 72 ºC for 2 min. Amplified products were
purified with Wizard PCR Preps DNA Purification Resin and separated
with electrophoresis of 0.8% agarose gel to confirm whether
amplified products had the desired size of 1.3 kb.
Digestion of CD gene and
pGEX-1LamdaT plasmid CD gene 1 µg and pGEX-1 LamdaT plasmid 1 µg
were added into 10 µL 10¡ÁBuffer E reactions, separately. The plasmid
and CD gene were digested with dual restriction endonucleases (1 µL
EcoR I+1 µL BamH I) for 3 h. Once the digestion was
complete, the DNA fragments were separated by electrophoresis with
0.8% agarose gel. The desired bands of 4.9 kb and 1.3 kb were cut
out from the gel. The DNA fragments in the gel were extracted and
recovered with gel extraction kit. Recovered pGEX-1LamdaT plasmid
vector fragment was dephosphorylated with calf intestinal
phosphatase.
Ligation of plasmid vector
fragment and CD gene fragment Recovered CD gene fragment 10 µL,
recovered pGEX-1LamdaT plasmid vector fragment 10 µL, and 1U T4 DNA
ligase were added into the microfuge tube. The reactions were
incubated at 16 ºC overnight. Then the ligation products,
recombinant CD/pGEX-1LamdaT plasmid were separated by
electrophoresis to confirm whether the ligation products had the
desired size.
Transfection of
Bifidobacterium infantis Suspensions of Bifidobacterium
infantis 10 mL were washed with ice-cold pure water and
resuspended in 40 µL ice-cold 10% glycerol. Recombinant
CD/pGEX-1LamdaT plasmid 5 µL was added to the bacterial suspensions,
then mixed and transferred to electroporation cuvette.
Electroporation was carried out to transfect recombinant
CD/pGEX-1LamdaT into Bifidobacterium infantis at 2.0 kV for
10 ms.
Culture of transfected
Bifidobacterium infantis and detection of positively transfected
bacterial colony When electroporation was completed, 1 mL MRS
liquid medium was added into the electroporation cuvette to
resuspend bacteria. Then the bacteria suspensions were transferred
into a 1.5 mL EP tube and incubated in an anaerobic jar at 37 ºC.
Two hours later, bacteria suspensions were plated on MRS solid
medium with ampicillin and incubated in an anaerobic jar at 37 ºC.
After 72 h, a single positively transfected bacterial colony of
Bifidobacterium infantis was picked up and inoculated into 5 mL
MRS liquid medium. Two to three drops of liquid paraffin was added
to the medium and then the cultures were incubated overnight at 37
ºC. The next day, recombinant plasmid was extracted from the
incubated bacteria using Purification kit of plasmid. Extracted
recombinant plasmid was separated with electrophoresis to confirm
whether the recombinant plasmid is the recombinant CD/pGEX-1LamdaT
plasmid. In addition, the extracted recombinant plasmid from
positively transfected bacterial colony was digested with dual
restriction endonucleases of EcoR I and BamH I as
described previously. The size of digested segments were analyzed by
electrophoresis with 0.8% agarose gel.
Sequencing of inserted gene
segment in recombinant plasmid Sequencing of inserted gene
segment in recombinant plasmid extracted from positively transfected
bacteria was performed according to the method of Sanger dideoxy-nucleotide
triphosphate chain termination. Shanghai Genebase Gen-tech
(Shanghai, China) carried out the sequencing.
Processing of positively
transfected Bifidobacterium infantis Suspension of
positively transfected Bifidobac-terium infantis 20 mL was
incubated at 37 ºC anaerobically overnight. The next day, 5-FC with
final concentration 1 mmol/L was added and then the incubation
continued. After 24 h, 10 mL bacteria suspension was taken and
centrifuged for 5 min. The supernatant fluid was collected and 2 mL
ethyl acetate was added and mixed to make the supernatant fluid
suspension in ethyl acetate properly. The mixture was centrifuged
(150¡Ág) at 4 ºC for 10 min. The organic fluid was transferred
to EP pipette and dried in a vacuum. The dried product was resolved
in 1 mL 0.9% saline to be used as the positive treatment fluid.
Suspension of untransfected Bifidobacterium infantis was
processed with the same method and used as the negative treatment
fluid.
Killing effects of treatment
fluid on melanomacyte B16-F10 Melanomacytes B16-F10 were first
digested with 0.125% trypsin and counted. They were then diluted to
a final concentration of 1.5¡Á105 cells/mL and inoculated
into six wells at 1.8 mL/well in 6-well cell culture cluster. The
six wells were randomized into two groups with three wells in each
group. When 50%-60% of the wells were covered by cells, 200 µL
positive treatment fluid and 200 µL negative treatment fluid were
separately added to the two groups with one well from each group
added with saline as the control. The cell culture cluster was
placed in a 5% CO2 incubator at 37 ºC. After 24 h, the
morphology of cells was observed and the number of cells was counted
using trypan blue dye exclusion method to calculate the suppression
rate of each test group, namely; Suppression rate=(Nn-N)/(Nn-N0
)¡Á100% (N0: number of inoculated cells; N: number of
cells in test well after 24 h; Nn: number of cells in
control well after 24 h).
Antitumor effect in vivo
Mouse melanomacytes B16-F10 were inoculated in the muscle of the
right thigh of the female C57BL/6 mice at 5¡Á106
cells/mouse to establish a melanoma model. The experiment was
initiated when the tumor grew to 0.8-1.0 cm in diameter (d 10 after
inoculation). Twenty tumor-bearing mice were divided into test
group(n=10) and control group (n=10) randomly. Diluted
suspension 0.2 mL of positively transfected Bifidobacterium
infantis containing 5¡Á106-6¡Á106 bacteria
was injected into the mice in the test group through the tail vein.
The control group were injected with the same volume and number of
untrans-fected Bifidobacterium infantis. After 7 d, 5-FC was
injected intraperitoneally for each mouse in both groups at a dose
of 500 mg each day, consecutively for 7 d. From the first day of
5-FC injection, the length and width of the tumor were measured
every two days. The volume was calculated using the formula V=1/2¡ÁA¡ÁB2
(A: length; B: width) and the measurement continued for 21 d.
Statistic methods Comparison
of tumor growth suppression rates was examined by chi-square test of
four-fold table. Tumor volume was compared by t-test. The
results were analyzed using statistic software STATA5.0.
Results
Detection of positively transfected
Bifidobacterium infantis colony
Electroporation method was used to transfect recombinant
CD/pGEX-1LamdaT plasmid into Bifidobac-terium infantis. After
72 h, sparse bacterial colonies were observed on MRS solid medium
containing ampicillin. A single positively transfected bacterial
colony was picked up and cultured, then the recombinant plasmid in
the positively transfected Bifidobacterium infantis was
extracted. The size of extracted recombinant plasmid was
approximately 6.2 kb (Figure 1, lane A), which was equal to the size
of recombinant CD/pGEX-1LamdaT plasmid.
Detection of recombinant plasmid
digested with dual restriction endonucleases After the
recombinant plasmid extracted from the positively transfected
bacteria was digested with dual restriction endonucleases of BamH
I and EcoR I, two fragments with sizes of approximately 1.3
kb and 4.9 kb were obtained, which were consistent with the sizes of
CD gene and pGEX-1LamdaT plasmid, respectively (Figure 1,
lane B).
Sequencing of inserted gene
segment in recombinant plasmid extracted from positively transfected
Bifidobac-terium infantis Sequencing results showed that
the size and sequence of nucleotide acid of inserted gene segment
were completely consistent with the size and sequence of nucleotide
acid of CD gene (Genebank: NC-000913). The full length of inserted
gene fragment was 1309 bp. The sequence of two ends of the inserted
gene was also consistent with BamH I site and EcoR I
site. The sequencing identified that the foreign gene, CD gene, was
correctly inserted into pGEX-1LambdaT plasmid and transferred into
Bifidobacterium Infantis. Targeting gene therapy system of
Bifidobacterium Infantis/CD was successfully constructed.
Morphologic changes of B16-F10
cells after treatment After being treated with the positive
treatment fluid for 24 h, most B16-F10 cells were killed and only a
few grew along the wall. Most cells floated and quantities of cell
debris were observed in the medium. The cells left on the wall
underwent significant changes in morphology; the original shape was
gone, cytoplasm became rougher, the nucleus showed pycnosis and the
refraction decreased (Figure 2), demonstrating obvious cellular
damages. In contrast, B16-F10 cells treated with negative treatment
fluid did not appear to have obvious morphologic changes compared
with the control (Figures 3, 4).
Examination of tumor cell growth
suppression rate The negative treatment fluid had no significant
effects on tumor cell growth. The cells growth suppression rate was
4.8%. The B16-F17 cells treated with positive treatment fluid grew
at a critically lower rate. The cells growth suppression rate was
80%, which was significantly higher than that of group treated with
negative treatment fluid (chi-square test:
¦Ö2=7.02,
¦Ô=1,
P<0.01) (Table 1).
Antitumor effects in vivo
There was no significant difference between the tumor volumes in the
test group and control group before treatment. On the d 7 after 5-FC
injection, tumor volumes in the test group were considerably smaller
than those in the control group (P<0.05), and the difference
became greater over time until the end of observation
(Figure 5).
Discussion
It has been proved that a hypoxic
region exists in many human and mouse tumors, especially in solid
tumors, which kill over 90 % of cancer patients. Vaupel[11]
made his study on cancer patients using oxygen electrode
measurement. In his study, Vaupel found the average oxygen partial
pressure in normal tissues read 24-66 mmHg, whereas the readings
dropping to 10-30 mmHg in a tumor tissue with a marked central
region where the readings went below 2.5 mmHg. Based on the presence
of a hypoxic metabolic region in a solid tumor and tendency of
anaerobic bacteria to hypoxic environment, anaerobic bacteria can be
used as transfer vectors for tumor targeting gene therapy.
Compared with viral or other
non-viral vectors, the anaerobic bateria is advantageous for tumor
targeting[13-17]. Yazawa et al[9]
injected non-pathological strains of Bifidobacterium 105-A
and 108-A into mice bearing lung cancer. After 168 h, both strains
of bacteria were observed to have targeted and colonized in the
tumors, whereas no bacteria were found in the liver, spleen, kidney,
normal lung tissues or other normal tissues. When the anaerobic
bacteria were used as the gene transfer vector, it could
specifically proliferate and directly express foreign gene products
in tumor tissues. There is no need to transform cancer cells, which
highly improves the gene expression[9,13,14] .
Most of the transfer vectors used in current studies are strains of
Bifidobacterium. Bifidobac-terium is a non-pathogenic
anaerobic bacteria. It resides in the lower section of the small
intestine and in the large intestine of humans or rodent animals,
and is good for the health of its host and widely used in food
manufacturing and medicine[9,12-15]. It has been proved
in animal experiments that Bifidobacterium has no obvious influence
on body weight, peripheral leukocytes, temperature, or survival time
of mice, hamsters, guinea pigs or rabbits[13]. Moreover,
anaerobic bacteria are highly vulnerable to antibiotics and very low
doses of antibiotics are enough to kill them. Even in the case of
overgrowth, the bacteria can be easily controlled by antibiotics,
which further the safety.
In the present study, we used
Bifidobacterium infantis as a CD gene transfer vector, then
transferred the CD gene into Bifidobacterium infantis and
successfully constructed a Bifidobacterium infantis/CD
targeting gene therapy system.
In the in vitro experiment,
positively transfected Bifido-bacterium infantis was
incubated anaerobically with 5-FC overnight. The supernatant fluid
of the bacteria culture was extracted and resolved in saline. The
final solution was added to the melanomacyte B16-F10 and it was
observed that the growth of melanomacyte B16-F10 was obviously
suppressed at a rate of 80% and the morphology of the cells was also
damaged. It was also observed in our study that the growth of tumors
were greatly inhibited on mice that were injected with 5-FC and
positively transfected Bifidobacterium infantis. These
results indicate that positively transfected with Bifidobacterium
infantis can express CD in vitro and in vivo,
which can convert 5-FC into 5-FU to inhibit tumor growth.
CD/5-FC suicide gene therapy system
mediated by Bifidobacterium infantis has primarily
demonstrated its antitumor effects in vivo and in vitro
in this experiment. This system shows promise as a novel tumor
targeting gene therapy system and is worthy of further study.
References
- 1 Miller CR, Gustin AN,
Buchsbaum DJ, Vickers SM, Manne U, Grizzle WE, et al.
Quantitation of cytosine deaminase mRNA by real-time reverse
transcription polymerase chain reaction: a sensitive method for
assessing 5-fluorocytosine toxicity in vitro. Anal
Biochem 2002; 30: 189-99.
- 2 Miyagi T, Koshida K, Hori O,
Konaka H, Katoh H, Kitagawa Y, et al. Gene therapy for
prostate cancer using the cytosine deaminase/uracil
phosphoribosyltransferase suicide system. J Gene Med 2003; 5:
30-7.
- 3 Plumb JA, Bilsland A, Kakani
R, Zhao J, Glasspool RM, Knox RJ, et al.
Telomerase-specific suicide gene therapy vectors expressing
bacterial nitroreductase sensitize human cancer cells to the
pro-drug CB1954. Oncogene 2001; 20: 7797-803.
- 4 Brown NL, Lemoine NR.
Clinical trials with GDEPT: cytosine deaminase and
5-fluorocytosine. Methods Mol Med 2004; 90: 451-7.
- 5 Xu G, McLeod HL. Strategies
for enzyme/prodrug cancer therapy. Clin Cancer Res 2001; 7:
3314-24.
- 6 Denny WA. Prodrug strategies
in cancer therapy. Eur J Med Chem 2001; 36: 577-95.
- 7 Liu SC, Minton NP, Giaccia
AJ, Brown JM. Anticancer efficacy of systemically delivered
anaerobic bacteria as gene therapy vectors targeting tumor
hypoxia/necrosis. Gene Ther 2002; 9: 291-6.
- 8 Nuyts S, Van Mellaert L,
Theys J, Landuyt W, Lambin P, Anne J. Clostridium spores for
tumor-specific drug delivery. Anticancer Drugs 2002; 13: 115-25.
- 9 Yazawa K, Fujimori M, Amano
J, Kano Y, Taniguchi S. Bifido-bacterium longum as a
delivery system for cancer gene therapy: selective localization
and growth in hypoxic tumors. Cancer Gene Ther 2000; 7: 269-74.
- 10 Wu Y, Yi C, Wang S, Zhang M,
Zhang J. Investigation on tumor-targeting characteristics of
Bifidobacterium infantis toward melanoma in mice. J Sichuan
Univ 2003; 34: 435-8.
- 11 Vaupel PW. Oxygenation of
solid tumors in drug resistance in oncology. In: Teicher BA,
editor. New York: Marcel Dekker; 1993, p53-85.
- 12 Nagy H, Panis Y, Fabre M,
Perrin H, Klatzmann D, Houssin D. Are hepatomas a good
target for suicide gene therapy? An experimental study in rats
using retroviral-mediated transfer of thymidine kinase gene.
Surgery 1998; 123: 19-24.
- 13 Fujimori M, Amano J,
Taniguchi S. The genus Bifidobacterium for cancer gene
therapy. Curr Opin Drug Discov Devel 2002; 5: 200-3.
- 14 Yazawa K, Fujimori M,
Nakamura T, Sasaki T, Amano J, Kano Y, et al.
Bifidobacterium longum as a delivery system for gene therapy
of chemically induced rat mammary tumors. Breast Cancer Res
Treat 2001; 66: 165-70.
- 15 Nakamura T, Sasaki T,
Fujimori M, Yazawa K, Kano Y, Amano J, et al. Cloned
cytosine deaminase gene expression of Bifido-bacterium longum
and application to enzyme/pro-drug therapy of hypoxic solid
tumors. Biosci Biotechnol Biochem 2002; 66: 2362-6.
- 16 Kim TB, Song SH, Kang SC, Oh
DK. Quantitative comparison of lactose and glucose
utilization in Bifidobacterium longum cultures.
Biotechnol Prog 2003; 19: 672-5.
- 17 Makelainen H, Tahvonen R,
Salminen S, Ouwehand AC. In vivo safety assessment of two
Bifidobacterium longum strains. Microbiol Immunol 2003;
47: 911-4.
|
