Combined effects of Cantide and chemotherapeutic drugs on inhibition of tumor cells' growth in vitro and in vivo.

Abstract

To investigate the combination effect of hTERT antisense oligonucleotide "Cantide" and three chemotherapeutic drugs (cisplatin, 5-fluorouracil (5-FU) and adriamycin (ADM)) on inhibiting the proliferation of HepG2, BGC and A549 cell lines in vitro, and to investigate the efficacy of Cantide used in combination with cisplatin (DDP) in vivo.
Cantide was transfected into these tumor cells by Lipofectin, and cell growth activity was calculated by microcytotoxicity assay. In vivo study, cells of HepG2 were implanted in Balb/c nude mice for 4 d. Then Cantide, DDP and Cantide+DDP were given intraperitoneally for 24 d respectively. The body weights of the tumor-bearing animals and their tumor mass were measured later to assess the effect of combination therapy in the nude mice. To evaluate the interaction of Cantide and these chemotherapeutic drugs, SAS software and Jin Zhengjun method were used.
Combination treatments with 0.1 micromol/L Cantide reduced the IC50 of DDP, 5-FU and ADM from 1.07, 4.15 and 0.29 microg/mL to 0.25, 1.52 and 0.12 microg/mL respectively. The inhibition ability of DDP, 5-FU and ADM respectively in combination with Cantide in these tumor cells was higher than that of these drugs alone (P<0.0001). And synergism (Q > or = 1.15) was observed at the lower concentration of DDP (< or = 1 microg/mL), 5-FU (< or = 10 microg/mL) and ADM (< or = 0.1 microg/mL) with combination of Cantide. In vivo, combination treatment with Cantide and DDP produced the greater growth inhibition of human liver carcinoma cells HepG2 in nude mice (0.65+/-0.19 g tumor) compared with that when only one of these drugs was used (Cantide group: 1.05+/-0.16 g tumor, P = 0.0009<0.001; DDP group: 1.13+/-0.09 g tumor, P = 0.0001<0.001).
These findings indicate that Cantide may enhance therapeutic effectiveness of chemotherapeutic drugs over a wide range of tumor cells in vitro, and the combination use of Cantide and DDP can produce much higher inhibition rates, as compared with when either of these drugs was used only in vivo.

Full text

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• BRIEF REPORTS •
Combined effects of Cantide and chemotherapeutic drugs on
inhibition of tumor cells’ growth in vitro and in vivo
Ying Yang, Qiu-Jun Lv, Qing-You Du, Bing-Hu Yang, Ru-Xian Lin, Sheng-Qi Wang
ELSEVIER
PO Box 2345, Beijing 100023, China World J Gastroenterol 2005;11(16):2491-2496
www.wjgnet.com
wjg@wjgnet.com © 2005 The WJG Press and Elsevier Inc. All rights reserved.
World Journal of Gastroenterology ISSN 1007-9327
Ying Yang, Qiu-Jun Lv, Qing-You Du, Bing-Hu Yang, Ru-Xian
Lin, Sheng-Qi Wang, Beijing Institution of Radiation Medicine,
Beijing 100850, China
Ying Yang, Department of Biochemistry, National Institute for
the Control of Pharmaceutical and Biological Product, Beijing
100050, China
Supported by the National Nature Science Foundation of China,
No. 39870879, and Key Technologies R and D Program of China,
No. 2002AA2Z3337
Co-first-authors: Ying Yang and Sheng-Qi Wang
Co-correspondents: Ying Yang and Sheng-Qi Wang
Correspondence to: Professor Sheng-Qi Wang, Beijing Institution
of Radiation Medicine, Beijing 100850, China. sqwang@nic.bmi.ac.cn
Telephone: +86-10-66932211 Fax: +86-10-66932211
Received: 2004-03-31 Accepted: 2004-05-29
Abstract Abstract
Abstract Abstract
Abstract
AIM: To investigate the combination effect of hTERT antisense
oligonucleotide “Cantide” and three chemotherapeutic
drugs (cisplatin, 5-fluorouracil (5-FU) and adriamycin
(ADM)) on inhibiting the proliferation of HepG2, BGC and
A549 cell lines in vitro, and to investigate the efficacy of
Cantide used in combination with cisplatin (DDP) in vivo.
METHODS: Cantide was transfected into these tumor cells
by Lipofectin, and cell growth activity was calculated by
microcytotoxicity assay. In vivo study, cells of HepG2 were
implanted in Balb/c nude mice for 4 d. Then Cantide, DDP
and Cantide+DDP were given intraperitoneally for 24 d
respectively. The body weights of the tumor-bearing
animals and their tumor mass were measured later to
assess the effect of combination therapy in the nude mice.
To evaluate the interaction of Cantide and these
chemotherapeutic drugs, SAS software and Jin Zhengjun
method were used.
RESULTS: Combination treatments with 0.1 µmol/L Cantide
reduced the IC50 of DDP, 5-FU and ADM from 1.07, 4.15
and 0.29 µg/mL to 0.25, 1.52 and 0.12 µg/mL respectively.
The inhibition ability of DDP, 5-FU and ADM respectively
in combination with Cantide in these tumor cells was higher
than that of these drugs alone (P<0.0001). And synergism
(Q
1.15) was observed at the lower concentration of DDP
1 µg/mL), 5-FU (10 µg/mL) and ADM (
(
0.1 µg/mL)
with combination of Cantide. In vivo, combination treatment
with Cantide and DDP produced the greater growth
inhibition of human liver carcinoma cells HepG2 in nude
mice (0.65±0.19 g tumor) compared with that when only
one of these drugs was used (Cantide group: 1.05±0.16 g
tumor, P = 0.0009<0.001; DDP group: 1.13±0.09 g tumor,
P = 0.0001<0.001).
CONCLUSION: These findings indicate that Cantide may
enhance therapeutic effectiveness of chemotherapeutic
drugs over a wide range of tumor cells in vitro, and the
combination use of Cantide and DDP can produce much
higher inhibition rates, as compared with when either of
these drugs was used only in vivo.
© 2005 The WJG Press and Elsevier Inc. All rights reserved.
Key words: hTERT; Antisense oligonucleotide; Tumor;
Chemotherapeutic drugs
Yang Y, Lv QJ, Du QY, Yang BH, Lin RX, Wang SQ. Combined
effects of Cantide and chemotherapeutic drugs on inhibition
of tumor cells’ growth in vitro and in vivo. World J Gastroenterol
2005; 11(16): 2491-2496
http://www.wjgnet.com/1007-9327/11/2491.asp
INTRODUCTIONINTRODUCTION
INTRODUCTIONINTRODUCTION
INTRODUCTION
In some areas of the world, cancer has become or shortly
will become the leading disease-related cause of death in
human beings[1]. In contrast, there are still many inadequate
medical treatments of cancer. The main curative therapies
for cancer surgery and radiation can be only successful in
general if the cancer is found at an early stage and is
localized. Currently conventional chemotherapy for treatment
of the advanced tumors, although quite effective, has been
associated with toxicities to normal tissue and organs, which
is still a major dose limited factor. And chemoresistance is
another major obstacle for successful treatment of cancer[2].
So it is difficult to remove these tumor cell contaminants
with the use of the conventional chemotherapy only. It is
clear that new therapeutic options are necessary.
Recent progresses in identification and characterization of
new molecular targets for cancer and the limited effectiveness
of conventional treatment strategies have attracted considerable
attention on the development of new types of anticancer
drugs. These new drugs would be highly specific for
malignant cells with minimized side effects due to well-defined
mechanisms of action. Antisense oligodeoxynucleotides
(ASODNs) are a new drug they are short, synthetic stretches
of DNA which can hybridize with specific mRNA strands
that correspond to target genes. By binding to the mRNA,
ASODN prevents the sequence of the target gene from
being converted into a protein, thereby blocking the action
of the gene[3]. So ASODNs have been extensively considered

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2492 ISSN 1007-9327 CN 14-1219/ R World J Gastroenterol April 28, 2005 Volume 11 Number 16
for the down regulation of oncogenes in cancer therapy[4].
And a more recent approach was the use of ASODNs in
combination with conventional chemotherapy for potential
anticancer therapy[5-7]. Combination therapy of ASODNs
and cytotoxic drugs potentially has several advantages
including lower doses of chemotherapeutic drugs, less side
effects on normal cells and loss of chemoresistance. Hopefully
the combination therapy will serve as a base for more effective
elimination of tumor cells[8].
In our previous studies it was demonstrated that tumor
cells treated with Cantide, an ASODN targeted to human
telomerase reverse transcriptase (hTERT) mRNA, resulted
in a relatively rapid decrease of tumor cells’ growth in vitro.
Cytotoxic effect of Cantide was also compared with the
sense, random and mismatched ODNs as control sequences.
Only Cantide has potent inhibitory effect on tumor cells
proliferation[9]. In vivo treatment of HepG2 tumor xenografts
with Cantide significantly retarded the growth of the tumors.
In this study, to explore the potential of Cantide used in
combination with chemotherapeutic drugs on inhibition of
human tumor cells’ growth, the cytotoxic interaction between
Cantide and three chemotherapeutic drugs cisplatin (DDP),
5-fluorouracil (5-FU) and adriamycin (ADM) are analyzed
in vitro and investigation of the efficacy of Cantide in
combination with DDP in vivo is presented.
MAMA
MA MA
MATERIALS AND METHODS
TERIALS AND METHODS
TERIALS AND METHODSTERIALS AND METHODS
TERIALS AND METHODS
Cell and culture condition
Human hepatocellular carcinoma cell (HepG2) was
obtained from American Type Culture Collection. Human
lung adenocarcinoma cells (A549) and human gastric cells
(BGC823) were obtained from Chinese National Cancer
Institute, Chinese Academy of Medical Science. HepG2 and
BGC823 cells were cultured in DMEM (GIBCO BRL, Grand
Island, NY, USA), supplemented with 10% FCS (GIBCO
BRL), 100 U/mL penicillin and 100 U/mL streptomycin.
A549 were cultured in RPMI-1640 (GIBCO BRL),
supplemented with 10% FBS (GIBCO BRL), 100 U/mL
penicillin and 100 U/mL streptomycin. Tumor cells were kept
at 37
in a humidified atmosphere containing 50 mL/L
CO2.
Chemotherapeutic drugs
Cisplatin (DDP) was obtained from Qilu Pharmaceutical
General Factory, China. 5-FU was obtained from Tianjin
people’s Pharmaceutical Factory, China. ADM was obtained
from Hisun Pharmaceutical Co. Ltd, China.
Synthesis of Cantide
The antisense phosphorothioate oligodeoxynucleotides
“Cantide” (5’-ACTCACTCAGGCCTCAGACT-3’) was
synthesized on solid supports using Oligo Pilot II DNA
(Amersham-Pharmacia, USA) and purified by HPLC Prep
4000 (Waters Delta, USA) with SOURCE 15Q (Amersham-
Pharmacia, USA).
Evaluation of the effect of Cantide used in combination with
chem otherapeutic drugs on inhibiting proliferation of HepG2 cells
HepG2 cells were seeded at a density of 4×103 cells/well
flat-bottomed plates (100 µL/well). After 24 h, culture
medium was removed and the cells were washed with fresh
FCS-free DMEM. In the above-mentioned medium, Cantide
was delivered into these cells in the form of complex with
Lipofectin (Invitrogen, USA) as described in the direction of
Lipofectin. Using this method, three samples of concentrations
(0.1, 0.2, 0.4 µmol/L) of Cantide were transfected into
HepG2. After incubating for 6 h, 100 µL of cell culture
medium with different chemotherapeutic drugs was replaced
in each well. And five concentrations around the IC50 of
each chemotherapeutic drugs (DDP: 0.25, 0.5, 1, 2, 4 µg/mL;
5-FU: 1.0, 2.5, 5, 10, 20 µg/mL; ADM: 0.025, 0.05, 0.1, 0.2,
0.4 µg/mL) were used. Each anticancer drugs’ different
concentrations was used in combination with Cantide’s three
concentrations (0.1, 0.2, 0.4 µmol/L) respectively, and each
test group was tested thrice. At the same time, treatments
with Cantide alone and either of the anticancer drugs were
assayed, and treatments with cell culture medium without
any drugs were used as control tests. After 72-h incubation,
20 µL of MTS (Promega, USA) was added in each well,
followed by a 90-min incubation at 37
. Inhibition rate (IR)
of tumor cells proliferation was assessed according to
absorption at 490 nm using a Victor 1420 Multilable Counter
(WALLAC, USA).
Analysis of the antitumor profile of combination treatment
with Cantide and chemotherapeutic drugs
Human hepatocellular carcinoma cells (HepG2), human lung
adenocarcinoma cell lines (A549) and human gastric tumor
cells (BGC823) were tested and results obtained are presented
in this paper. A549 and BGC823 cells were treated as what
was done with HepG2 cells (as described above), but the
ranges of concentrations of anticancer drugs were different
according to different cell lines.
Tumors and mice
Female 4-5-wk-old Balb/c nude mice were purchased from
Center for Animals for Experiment, Chinese Academy of
Medical Science. HepG2 tumor cells cultured in vitro and
6×106 cells were injected into the neck of the nude mice.
Four days later, the tumor could be sensed by touch.
Treatment in vivo
The above-mentioned nude mice were divided into four
groups (n = 7-8 mice/group). Cantide only group: Cantide
was dissolved in saline and administered intraperitoneally
(i.p.) 50 mg/kg daily for 24 consecutive days; DDP only
group: DDP was dissolved in saline and administered
intraperitoneally (i.p.) 1 mg/kg every other day for seven
times totally; Combination treatment group: the nude mice
were treated by Cantide plus DDP as described above; and
negative control group (saline, i.p.). Tumor size was measured
in two dimensions by calipers every 3 d, and the volume
was calculated as length×width2×0.52. The nude mice were
then killed and the solid tumors were peeled off on the 27th d
after treatment. Then their tumors were weighed and IR
calculated.
Statistical analysis
The software package for statistical analyses was SAS 6.12,

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Yang Y et al. Combined effects of Cantide and chemotherapeutic drugs 2493
and factorial design was used in the treatments in vitro, while
t test was performed to assess potentially significant
differences between individual groups of observations in
vivo. In all tests, the significance of differences was accepted
at P<0.05.
Analysis of the interaction between Cantide and anticancer
drugs
To analyze the interaction between Cantide and the three
anticancer drugs, Zheng-Jun Jin method[10] was used. This
method provides a “Q” value, according to which the
interaction between two drugs can be classified as antagonistic
effect (Q
0.85), additive effect (0.85
Q<1.15) or
synergistic effect (Q
1.15). And the formula is Q = Ea+b/
(Ea+Eb-Ea×Eb), where Ea+b, Ea and Eb are average effect
of combination treatment, effect of drug A only and effect
of drug B only, respectively.
RESULRESUL
RESULRESUL
RESULTS
Cantide increases the cytotoxicity of DDP, 5-FU and ADM on
HepG2
TS
TSTS
TS
Cantide can decrease proliferation of HepG2 and increase
HepG2 cells’ sensitivity to anticancer treatment. For
each experiment, dose-response curves of each single
chemotherapeutic drug and its combination with Cantide at
different doses were performed. Figure 1A shows that the
drug concentration causing 50% growth inhibition (IC50) of
treatment of HepG2 cells with DDP only is 1.00 µg/mL,
and IC50 of treatment of HepG2 cells with DDP in
combination with Cantide (0.1 µmol/L) is 0.25 µg/mL. On
the other hand, DDP can also increase the efficacy of
Cantide. For example, 0.1 µmol/L Cantide used only (the
dot which corresponds to 0 concentration of DDP on the
dose-response curves) and when it was used in combination
with 0.5 µg/mL DDP reduced the cell viability from 80%
to 35%. Figure 1B indicates that combination treatment
with 0.1 µmol/L Cantide reduced the IC50 of 5-FU from 4.15
to 1.52 µg/mL. And combination treatment with 0.1 µmol/L
Cantide reduced the IC50 of ADM from 0.29 to 0.12 µg/mL
(Figure 1C). The dose-response curves obtained from the
combination experiments indicate that Cantide increased
the cytotoxicity of DDP, 5-FU and ADM on HepG2 cells.
And analysis with SAS software demonstrates statistically
significant differences between any of the various single-
drug treatments and combination treatment as indicated
(P<0.0001).
Cantide synergistically interacts with chemotherapeutic drugs
on HepG2
To investigate the nature of the interaction between Cantide
and the anticancer drugs on HepG2 cells, Zheng-Jun Jin
method[10] was used to analyze the cytotoxicity data for
antagonism, additivity or synergy. Q values in Figure 2
indicate that the synergistic effects appeared for the
combinations of Cantide with lower concentrations of
anticancer drugs. Furthermore, Figure 2A shows that the
combination treatments of DDP plus Cantide obtained
better synergistic effects than those of other anti-drugs’
combinations. The top Q value for combination treatment
of Cantide and DDP was 2.08.
Inhibition effects of combination treatment with Cantide and
chemotherapeutic drugs on different tumor cells
It was confirmed that the reduced cell growth rate in a
variety of carcinoma cells upon treatment with single drug
or the various combination treatments was due to the
induction of cell death and cell viability calculated with
microcytotoxicity assay (MTT) assays. As shown in Table 1,
these values represent IC50 of chemotherapeutic drugs only
and those of various combinations with Cantide on BGC
and A549 cells. Values in first column with concentration
treatments of Cantide are 0.0 (µmol/L) represent treatments
with chemotherapeutic drugs only. And values in the next
three columns represent IC50 of chemotherapeutic drugs
in combination with Cantide. Similar results were obtained
in cultures with BGC and A549 cells that combination
treatments could decrease the IC50 of anticancer drugs.
Antitumor effects of Cantide used in combination with DDP
on human liver tumor xenografts
On the 4th d after injecting HepG2 cell into these nude
mice, the tumor could be sensed by touching and the
volumes of tumors were approximately equal. Then these
Figure 1 HepG2 cells’ growth inhibited by Cantide, DDP(A), 5-FU(B) and ADM
(C) only and at the indicated combinations on HepG2 cells. The dots represent
the concentrations of chem otherapeutic drugs as 0 on the dose-response curves
which means treatments with Cantide only. The cells were treated with Cantide
complexed to Lipofectin for 6 h at 37 . The medium was then replaced with
media containing various concentrations of DDP, 5-FU and ADM. After 72 h of
incubation, MTT assay was performed. Absorbance at 490 nm was normalized
to the control, untreated cells to determine cell viability. Each value represents
the mean±SD from triplicate determination.
Cell viability (%)
110
100
90
80
70
60
50
40
30
20
10
0
DDP
DDP+Cantide (0.1 µmol/L)
DDP+Cantide (0.2 µmol/L)
DDP+Cantide (0.4 µmol/L)
0 1 2 3 4
DDP Concentration (µg/mL)
Cell viability (%)
110
100
90
80
70
60
50
40
30
20
10
0
5-FU
5-FU+Cantide (0.1 µmol/L)
5-FU+Cantide (0.2 µmol/L)
5-FU+Cantide (0.4 µmol/L)
0 5 10 15 20
5-FU Concentration (µg/mL)
Cell viability (%)
110
100
90
80
70
60
50
40
30
20
10
0
ADM
ADM+Cantide (0.1 µmol/L)
ADM+Cantide (0.2 µmol/L)
ADM+Cantide (0.4 µmol/L)
0.0 0.1 0.2 0.3 0.4
ADM Concentration (µg/mL)
ACB

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mice were divided into four treatment groups: DDP only,
Cantide only, DDP+Cantide and control group. Six days
later, the differences between the average tumor volume
of the control group and each of those of the three treatment
groups were statistically significant respectively (all P<0.05).
And the tumor volume of combination therapy group
increased less than that of any other treatment groups.
Twelve days later, the volume of tumor in the combination
treatment group was significantly smaller than those in the
single drug therapy groups (P<0.05). When the test came to
the end, the tumor volume of all groups were as follows: control,
1 995.21±342.77 mm2; DDP 1 mg/kg, 1 107.07±222.66;
Cantide 50 mg/kg, 1 022.88±284.70 mm2; DDP 1 mg/kg+
Cantide 50 mg/kg, 729.86±128.04 mm2 (P<0.01) (Figure 3).
After these nude mice were killed, the solid tumors were
peeled off to measure their weights and calculate the IR.
The IR of combination therapy was 70.0% and it was
significantly greater than the IR of the single drug therapy
groups (P<0.001), where DDP and Cantide were 47.9%
and 51.6% respectively (Table 2). And compared with single
drug groups, the combination therapy had no significant
side effect on nude mice.
DISCUSSIONDISCUSSION
DISCUSSIONDISCUSSION
DISCUSSION
Telomerase is an RNA-dependent DNA polymerase that
synthesizes telomeric DNA sequences and almost universally
provides the molecular basis for unlimited proliferation
potential. Since first discovered in Tetrahymena thermophila in
1985[11], telomerase activity was found to be absent in most
normal human somatic cells but present in over 90% of
cancerous cells and in vitro immortalized cells[12,13]. The
holoenzyme consists of two essential components: one is a
functional RNA component (hTR)[14,15], which serves as a
template for telomeric DNA synthesis; the other is a catalytic
protein (hTERT) with reverse transcriptase activity[16-19]. hTR
is highly expressed in all tissues regardless of telomerase
activity[20], with cancer cells generally having five-fold higher
expression than normal cells. On the contrary, the
expression (mRNA) of hTERT is estimated at less than
1-5 copies per cell and is closely associated with telomerase
activity in cells. hTERT is generally repressed in normal
cells and upregulated in immortal cells, suggesting that
hTERT is the primary determinant for the enzyme activity
[21,22]. Thus, inhibition of hTERT function is expected to
be particularly effective on tumors. And there are some
studies which prove that inhibition of telomerase could
increase the sensitivity of DNA damaging drugs to tumor
cells[23,24]. So, based on our previous studies[9], we investigated
the ability of Cantide, an ASODN against hTERT mRNA,
to sensitize carcinoma cells to hemotherapy and searched
for antisense-drug combinations that could produce
synergistic cytotoxicity.
Figure 2 Q values for the combination treatments of Cantide with DDP (A), 5-
FU (B) and ADM (C) tested on HepG2 cells. Q values were calculated from the
dose-response curves shown in Figure 1 and analyzed with Zheng-Jun Jin
method.
Table 2 Tumor weights after treatment
GroupTumor weight (g) Inhibitory rate (%)
Control
Cantide
DDP
Cantide+DDP
2.17±0.31
1.05±0.16
1.13±0.09
0.65±0.19
0.0
51.6
47.9
70.0
Cantide+DDP group is significantly different from any group treated with a
single drug: P = 0.0009<0.001 vs Cantide group; P = 0.0001<0.001 vs DDP group
(t test). The results are expressed as mean±SD of 7–8 mice per group.
Table 1 IC50 of chemotherapeutic drugs only or that of combination
treatments (µg/mL)
Concentration of Cantide (µmol/ L)
Tumor cell Chemotherapeutic
lines drugs 0.0 0.1 0.2 0.4
DDP
5-FU
ADM
DDP
5-FU
ADM
0.45 0.36 0.27 0.15
10.28 7.84 2.17 1.70
0.60 0.43 0.38 0.11
5.43 3.21 2.04 0.96
9.69 9.06 5.34 0.31
0.76 0.71 0.60 0.43
BGC
A549
Tendency of Q value
DDP+Cantide (0.1 µmol/L)
DDP+Cantide (0.2 µmol/L)
DDP+Cantide (0.4 µmol/L)
Synergistic area
Antagonistic area
Additive area
0 1 2 3 4
DDP Concentration (µg/mL)
Tendency of Q value
5-FU+Cantide (0.1 µmol/L)
5-FU+Cantide (0.2 µmol/L)
5-FU+Cantide (0.4 µmol/L)
0 5 10 15 20
5-FU Concentration (µg/mL)
Tendency of Q value
ADM+Cantide (0.1 µmol/L)
ADM+Cantide (0.2 µmol/L)
ADM+Cantide (0.4 µmol/L)
0 0.1 0.2 0.3 0.4
ADM Concentration (µg/mL)
Synergistic area
Antagonistic area
Additive area
2.08
1.15
0.85
Q value
A
1.25
1.15
0.85
Q value
1.36
1.15
0.85
Q value
CB
2494 ISSN 1007-9327 CN 14-1219/ R World J Gastroenterol April 28, 2005 Volume 11 Number 16
Synergistic area
Antagonistic area
Additive area
Figure 3 Tumor growth curves for Balb/c nude mice after treatment with DDP,
Cantide or DDP+Cantide. Points for these groups represent the average values
for 7-8 mice.
Tumor volume (mm3)
2 500
2 000
1 500
1 000
500
00 6 12 18 24
After treatment (d)
Control
Cantide
DDP
Cantide+DDP

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In this study, the cell lines HepG2, A549 and BGC were
treated with Cantide, DDP, 5-FU or ADM only, or with
different combinations of Cantide and chemotherapeutic
drugs. As the example of HepG2 cells, the results showed
that the inhibition ability of DDP, 5-FU and ADM
respectively when used in combination with Cantide on
HepG2 cells was higher than any of those when only one
of the drugs was used (P<0.0001). And synergism (Q
1.15)
was observed for lower concentration of DDP (
10 µg/mL) and ADM (
1 µg/mL),
5-FU (
0.1 µg/mL) for combination
treatment with Cantide. In order to explore the inhibitory
effects of combination treatments in different tumor cells
further, the cell lines A549 and BGC were tested with the
same methods as that for HepG2 cells. The data also
demonstrated that combination treatment for these tumor
cell lines with Cantide plus anticancer drugs synergistically
induced greater growth inhibition of cancer cells when
compared with treatment in which either one of the drugs
was used. In vivo test, the results of the tumor growth
delay assays were used for Cantide (50 mg/kg) only, DDP
(1 mg/kg) only and Cantide (50 mg/kg)+DDP (1 mg/kg)
in nude mice with intraperitoneal injection (i.p). The IR of
tumor growth of combination therapy was significantly greater
than that of any therapy using only one drug (P<0.001).
These findings could encourage further research in
antisense therapy where Cantide is used in combination with
chemotherapeutic drugs, and our conclusions are also
supported by recent work of others showing the relationship
between telomerase and chemotherapeutic drugs.
Cisplatin (DDP) is frequently prescribed for the treatment
of a wide variety of neoplasms. It causes DNA strand break
especially at guanine residues. Several possibilities exist as
to how cisplatin might interfere with telomerase function[25].
One possibility is that the telomeric repeat sequence (TTAGGG)
n could be cross-linked as G-Pt-G, A-Pt-G or G-Pt-T-Pt-G.
Alternatively, interactions of cisplatin with essential sulfhydryl
groups in the protein part of the enzyme are also possible.
Furthermore, there is evidence that cisplatin might disable
transcription of the telomerase-RNA encoding gene region,
as the expression of human telomerase RNA component,
measured using the hTR-specific TRC3 primers, was
significantly decreased[26]. Since cisplatin’s effect on telomerase
activity is distinct from other cytotoxic drugs as described
above, one might propose that inhibition of telomerase
activity could, in part, contribute to cisplatin’s remarkable
efficacy against tumors and thus inhibition of telomerase
activity might have therapeutic potential.
ADM promotes apoptotic cell death in a variety of
experimental tumor cell lines[27]. It is found that ADM-
induced DNA damage appears to preferentially target
chromosome ends resulting in substantial telomere-related
cytogenetic abnormalities, indicating that the observed
senescence is due to telomere dysfunction[28].
5-FU, an inhibitor of thymidylate synthase, was shown
to be more cytotoxic when used in combination with ASODN
specific for thymidylate synthase mRNA. And Mitsui et al.,
found that some cancer cells exposed to 5-FU showed a
diminished telomerase activity preceded by a time-dependent
decrease in the mRNA expression of hTERT[29]. Thus,
therapeutic strategies involving applications of ASODN in
combination with conventional cytotoxics appear promising
for potential cancer therapy[30].
Although a critical and careful evaluation of telomere
inhibitors in cancer chemotherapy in vivo is certainly required,
and further studies on the mechanism of combination
therapy are needed, our present data indicate that Cantide,
the inhibitor of telomerase maintenance, may act to make
cancers chemosensitize to DDP, 5-FU and ADM. Thus, it
can encourage the development and evaluation of this
therapeutic combination of drug applications.
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