Antisense phosphorothioate oligodeoxynucleotides enhance the cytotoxic activity of in vitro anti-breast cancer
【Abstract】 Objective To study the HER2 mRNA as the target of the antisense oligodeoxynucleotide (S ODNs) HA6722 alone
and in combination with paclitaxel over-expression of HER 2
breast cancer cell line MDA MB 453 in vitro
proliferation in vivo. Methods HER2 over-expression of MDA
MB 453 cells with low HER2 expression in MDA MB 231
cells, MTT were observed HA6722 alone and in combination with paclitaxel on the two kinds of tumor cell proliferation; to terminal transferase-mediated The dUTP nick end labeling
(TUNEL) detection of apoptosis. Results HA6722 and taxol alone may dose-dependent manner inhibited MDA MB 453 cells in
vitro, IC50 values were 47.6 ? 7.9nmol * L 1 (n = 3, mean
? s), and 19.4 ? 4.1nmol * L -1 (n = 3, mean ? s). Plus
low-dose of HA6722 can enhance paclitaxel on MDA MB 453
cell line, IC50 values from combined pre-19.4 ? 4.1nmol * L-1
fell to 13.6 ? 5.2nmol * L-1 (n = 3, P "0.05). The joint
application, at the IC50 concentration, the joint between the two indices (CI) of 0.81 ? 0.43 (n = 3), its MDA MB 453
cells showed positive interaction between synergies. Conclusion antisense oligonucleotide HA6722 paclitaxel combined with cytotoxic drugs for HER 2 overexpressing
breast cancer cells in vitro synergistic inhibitory effect.
Key words antisense oligonucleotide Taxol Breast Cancer HER2 mRNA
The chemical treatment of breast cancer has been significant progress in recent years, however, the multi-line
treatment for late recurrence and metastasis is still the treatment of patients is still very difficult. Including biological targeting therapy to explore new therapies,
including recent research has become one of the hot.
The preliminary laboratory studies have shown that in order for the target specificity of HER2 mRNA antisense oligonucleotides, can inhibit HER2 overexpressing breast
cancer cell line proliferation . This study intended to right past the synthesis of antisense oligonucleotide HA6722 alone or with paclitaxel, when combined with the anti-breast
cancer activity of research, trying to explore new methods of
breast cancer treatment.
1 Materials and methods
1.1 cell lines and culture
In this study, the cell lines: MDA MB 453 (HER2
overexpression), cell culture medium required for the L15 (Gibco, BRL), containing 10% fetal calf serum (FCS, Hyclone), at 37 ?, non-CO2 in incubator; MDA MB 231 (HER2 low
expression) in vitro using RPMI 1640 (Gibco, BRL) medium
containing 10% fetal calf serum (FCS, Hyclone), Purchase 37 ?, with the volume fraction of 5% CO2 incubator passage. When the cell growth to 80% ~ 85% confluence with 0.25%
trypsin during digestion, passage and collection of cells.
1.2 Antisense oligonucleotide synthesis and in vitro lipofection
Antisense oligonucleotide targeting HER2 mRNA antisense oligonucleotide HA6722 sequence containing 20 nucleotides of the oligonucleotide, its nomenclature and synthesis see
reported in the literature . Full thio modified by gene technology Co., Ltd. Beijing Parkson Cup synthesis. Sequence is as follows: HA6722: 5'CAG CAG CCG AGC CAG CCC GA3 '
Agarose gel electrophoresis results showed that the
antisense oligonucleotide purity to meet the requirements. Antisense oligonucleotide with serum-free antibiotic-free L
15 or RPMI 1640 medium dissolved concentration of 50μmol *
L-1 solution, filtration sterilization, -20 ? storage,
temporary use, when diluted to the required concentration.
Antisense oligonucleotides in vitro lipofection by Invitrogen, Gibco BRL Inc. lipofection method described in the instructions. When the cultured cells reached 65% ~ 70% confluence, the transfection, specific methods to see the
1.3 paclitaxel drug companies from Hainan Hai, 30mg / support, temporary use, when diluted with sterile saline solution into the required concentration.
1.4 MTT (methyl thiazolyl tetrazolium, MTT) assay cell
activity logarithmic growth phase of the cells inoculated with
different number of 96-well culture plates (Costar, USA), the number of vaccination were: MDA MB 453 5 × 104 / Kong,
MDA MB 231 2 × 104 holes. Experimental group and
control group are equipped with three parallel holes cell culture. When cell growth to 65% ~ 70% confluence, the 2000 will be using liposomes with different concentrations of HA6722 transfected cells 4 ~ 6h, then add the same molar concentration of taxol, and then continue to build 24 ~ 72h, tilting to the culture medium, Add fresh configuration MTT
solution (0.5mg/ml) 200μl, were incubated for 4h, suction to the MTT solution, each well add 200μl DMSO, gently shock 20 ~
30min, enzyme-linked measure the light absorption values (OD value).
1.5 of apoptosis (apoptosis) of apoptosis kit purchased from Beijing Zhongshan Biotechnology Co., Ltd.. In order to 100nmol * L-1 of the HA6722 and taxol were treated separately MDA MB 453 cells in 12h, and using 50nmol * L-1 of the
HA6722 and the same concentrations of taxol has the role of
MDA MB 453 breast cancer cells 6h, collected by taxol and HA6722 after single or combined effects of MDA MB
453 breast cancer cells, centrifugal smears, according to the instructions detection of apoptosis. Brown precipitate appeared in the nucleus as positive, otherwise negative. Apoptotic index (AI) = (number of apoptotic cells / total cells counted) × 100%.
1.6 antisense drug and evaluation of the efficacy of Taxol
Logarithmic growth phase of the MDA MB 453 cells
and MDA MB 231 cells were treated with series of concentrations of HA6722 and the combined effects of paclitaxel alone or after, MTT determination of OD values. Paclitaxel alone, when a series of final concentration of 1,4,16,64,250 and 1 000nmol * L-1, while HA6722 alone, when a
series of final concentration of 12.8,32,80,200,500,1 250nmol * L -1; a joint application between the two mixed in equal molar concentration, the series followed by the concentration of 6.4,16,40,100,250 and 625nmol * L-1, the final reaction
volume of 200μl. The survival fraction f according to the following formula: f = ODtreat / ODcontrol (1)
Clinical evaluation of a joint program carried out according to the literature [4,5], that is, log (1 / f 1)
of the log (drug concentration) mapping, parallel linear fit, may x-axis intercept of the curve shall log (IC50), and linear slope of m, using the following formula f generated when the effects of single drugs and drug concentration in the joint when: Dosef = DoseIC50 [(1 / f) 1] 1 / m (2)
As the joint program with a fixed molar concentration ratio, resulting in an effect of the drug concentration f can be decomposed into a joint drug concentration (D) 1 and (D) 2, for producing any one effect of f, when the combined index of the drug (combine index, CI) can be calculated the following formula:
CI = (D) 1 (Df) 1 + (D) 2 (Df) 2 + α (D) 1 (D) 2 (Df) 1
(Df) 2 (3)
Where (D) 1 and (D) 2 represent the effect of combination
therapy produced when the concentration of f, and (Df) 1 and (Df) 2 represent the effects of drugs alone generated when the
concentration of the time f; hypothesis that the two drugs does not rule out the possibility, or mutually exclusive, α
values 1 or 0; CI reflects the interaction of two-drug
combination, greater than 1 indicates antagonism, said the sum is equal to 1, less than 1 indicates synergy. All tests were carried out three more times to repeat.
1.7 Statistical analysis
SPSS10.0 statistical analysis software used for statistical analysis, experimental data to ? s that are used
to compare the number of Student's t test and analysis of variance F test (One Way Anova).
2.1 HA6722 and paclitaxel alone when two kinds of breast
cancer cells in vitro
The 1,4,16,64,250 and 1 000nmol * L-1 the final
concentration and taxol on the growth of two kinds of breast cancer cells show a dose-dependent inhibitory effect, its
inhibition IC50 values were 19.4 ? 4.1nmol * L-1 and 23.6 ?
5.7nmol * L-1, Figure 1A; while 12.8,32,80,200,500,1 250nmol * L-1 under the action of a series of concentrations, HA6722 on two different levels of HER2 expression breast cancer cells in vitro is very different, for HER2 over-expression of MDA MB
453 cells showed a significant dose-dependent inhibition,
while low expression of HER2 in MDA MB 231 cells have
almost no inhibition, IC50, respectively to 47.6 ? 7.9nmol *
L-1 and 731.8 ? 14.3nmol * L-1 (n = 3, P <0.01, Figure 1B.
2.2 HA6722 and taxol emulsion in combination on MDA MB
453 breast cancer cells in vitro
Preliminary results from the above you can see, 12.8nmol * L-1 concentration of HA6722 on the MDA MB 453 cells,
the inhibition rate was only 4.7%, and therefore the concentration used as a non-therapeutic concentration used to enhance the pharmacological effects of Taxol study, results showed that the , as the concentration of HA6722 to join and taxol on MDA MB 453 cells in vitro enhanced, IC50 values from 19.4 ? 4.1nmol * L-1 fell to 13.6 ? 5.2nmol * L-1 (n =
3, P <0.05 ).
Equimolar concentrations of HA6722 combination with paclitaxel studies have shown that by 6.4,16,40,100,250, and 625nmol * L-1 of the two drugs together, MDA MB 453
cells showed more obvious inhibition, see Figure 2 A, B, C. IC50 concentrations in the combined index CI = 0.85 ? 0.16 (n
= 3), in research involving the full range of drug concentration, the two drugs combined index of fluctuations in
the (0.85 ? 0.11) ~ (0.87 ? 0.27), between Figure 2D.
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In order to clarify HA6722 synergistic anti-tumor effect
with paclitaxel whether the cells in selective low expression
of HER2 breast cancer cell line MDA MB 231 cells to the
same study, results show that equimolar concentrations of two kinds of drug combination on the The joint index of cell lines detected drug concentrations, fluctuations in the (1.13 ?
0.21) ~ (1.19 ? 0.24) between the Figure 3.
2.3 HA6722 and taxol emulsion in combination on MDA MB
453 breast cancer cell apoptosis
The 100nmol * L-1 of the HA6722 and taxol separately dealing with or 50nmol * L-1 of the HA6722 and the same
concentrations of taxol has the role of MDA MB 453
cells, TUNEL method for apoptosis detection showed, HA6722 and paclitaxel Combined enhanced cell apoptosis (n = 3, P <0.05), in Table 1.
Have shown that, proto-oncogene HER2 over-expression is
an independent, poor prognostic indicators, will lead to breast cancer patients resistant to certain chemotherapy regimens , shortened survival .
Figure 1 Taxol and anti-sense oligonucleotide HA6722
alone when two kinds of breast cancer cell lines, MDA MB
453, and MDA MB 231 cells in vitro (omitted)
Figure 3 equimolar concentrations of HA6722 in combination with paclitaxel on MDA MB 231 association
index with the change in the survival fraction curve
Figure 2 paclitaxel and HA6722 alone or in combination when MDA MB 453 cell proliferation (omitted)
Table 1 HA6722 with paclitaxel alone or combined application of MDA MB 453 cell apoptosis (omitted)
MDA MB 453 Group * P