Daidzein on neuroblastoma cell proliferation
Author: Xu Ying Zhu Heng-ying, Li shume, Lai Ri-yong,
[Abstract] Objective To investigate the daidzein on neuroblastoma SH-SY5Y cell
apoptosis and proliferation. Methods Cell growth inhibition assay (MTT), flow cytometry methods such as observation of SH-SY5Y cells, morphological changes,
apoptosis rate and cell cycle. Results of 10.0 ~ 80.0 μ g / ml of daidzein significantly inhibited cell proliferation, and there is dose-dependent; with the control group
compared with daidzein treatment 24 h after SH-SY5Y cells in each group suffered
from sub - diploid peak (Sub-G1), cell apoptosis rate and daidzein in a dose-dependent
manner. Conclusion of daidzein can induce neuroblastoma SH-SY5Y cell apoptosis and
proliferation of SH-SY5Y cells significantly inhibited.
[Key words] neuroblastoma proliferation Daidzein
Abstract: ObjectiveTo observe the effect of Daidzein on neuroblastoma. MethodsThe proliferation was tested by MTT assay.Cell cycle and apoptosis rate were determined by flow cytometry. Results10.0 ~ 80.0 μ g / ml of Daidzein could significantly in hibit proliferation of SH-SY5Y cell, Daidzein extract could induce
apoptosis of SH-SY5Y cell and the rate of apoptosis was in creased with the increase of the drug concentration. ConclusionDaidzein extract can induce apoptosis and inhibit proliferation of SH-SY5Y cell.
Key words: Neuroblastoma; Daidzein; Proliferation
Neuroblastoma (neuroblastoma, NB) is the more common malignant tumor of children, the incidence rate of malignant tumors accounted for the first three children, and other common childhood cancer compared to chemotherapy is poor, a serious threat to children's lives. Therefore, the search for new therapies become an issue of great concern. In recent years, various studies found that soy isoflavones can inhibit many types of cancer and the therapeutic effect , but the anti-cancer effect of soy
isoflavones study focused on breast and prostate cancer and other hormone-dependent
tumors, with regard to their Children's Neuroblastoma Study on the Effect the growth of few reports. Therefore, we observed in vitro daidzein on NB cell line SH-SY5Y
growth with a view to further study its mechanism and clinical application of laboratory basis.
1 Materials and methods
1.1 Material SH-SY5Y cell line as a Key Laboratory of Molecular Biology, Guiyang Medical College presented. Daidzein (Daidzein) Saide Hi-Tech Biology Co.,
Ltd. Shaanxi gifts, purity of 98%. RPMI -1640 produced for the Gibco, fetal calf serum
for the Hangzhou Sijiqing company, dimethyl sulfone dumb (DMSO) and methyl thiazolyl tetrazolium (MTT) for the Sigma company. BD FACSCalibur flow cytometer
BD produced for the United States; MK3-based microplate reader and 3111 were the
United States-based CO2 Incubators Thermo company.
1.2.1 Cell culture and inoculation of cultured SH-SY5Y cell line containing 5% calf
serum RPMI -1640 medium, CO2 Incubators, within (5%), constant temperature 37 ? . When the cells when covered with 0.25% trypsin digestion, cells were collected, 1 000 r / min, centrifuge 5 min, PBS washed two times to a concentration of ~ 2 * 104 in 96-well plates were inoculated into each hole volume of 180 ml.
1.2.2 Drug treatment of subjects will be daidzein dissolved in DMSO, the dubbed 100mg/ml storage solution, -20 ? to save. When used with the complete medium be
diluted to 10 mg / ml of the working fluid, with a diameter of 0.22 μ m micropore
membrane filter placed on sterilization stored at 4 ? , and further diluted with
complete medium were required to The different concentrations until 24 h after the cells were inoculated with different concentrations of the subjects were medicine, each
well 20 μ l, respectively, to a final concentration of 0.1,1.0, 10.0,20.0,40.0, 60.0, 80.0,100.0,150.0 mg / ml. Each concentration of four holes, while the complete medium containing DMSO as control and blank together. MTT with PBS (pH 8.2) dubbed in
5.0mg/ml solution, magnetic stirrer stirring 30 min, a diameter of 0.22 μ m micropore membrane filter stored at 4 ? after sterilization, is valid for 2 weeks.
1.2.3 Effect of cell growth inhibition was determined by MTT assay . Cells were
seeded 24 h after being drug subjects, followed by every 24 h using MTT colorimetric assay optical density values, which correspond to the number of viable cells. To do the following: Canadian subjects a certain time intervals after drug MTT solution plus 10 μ
l, 37 ? to continue to foster 4 h, after the termination of culture supernatant carefully absorb holes, each hole by adding 150 μ l DMSO, oscillator oscillation 5 ~ 10 min, so that fully dissolve crystals. Select wavelength 490 nm as measured by enzyme-linked
immunosorbent assay instrument light absorption value of each hole and record Daidzein acting on the SH-SY5Y cells, the concentration effect data and time-effect
data, using the following formula to calculate drugs on cell growth inhibition rate, and
Inhibition rate (%) = measured against the average OD value of hole - plus drug
group, the average OD value is measured according to the average OD value of measured holes * 100%
Table 1 of daidzein on SH-SY5Y cell proliferation inhibition rate of (omitted)
Compared with control group, ? P <0.05; n = 3
1.2.4 Detection of apoptosis by flow cytometry and cell cycle analysis of cell culture in each group 48 h after the collection of each group cell suspension 1 ml, the concentration of about 106 / ml, 1 000 r / min centrifugation 5 min and abandon the culture medium, washed twice with PBS centrifugation, with 70% cold ethanol-fixed,
4 ? to save 1 ~ 24 h, 1 000 r / min after 5 min centrifugation, PBS wash rinse to ethanol, two times; adding RNase A (final concentration of 60 μ g / ml), shaking, 37 ?
incubated 30 min; accession PI (final concentration 50μ g/ml) dye 1 ml, 4 ? dark
stained 30min. By flow cytometry, each sample measurement (4 ~ 8) * 103 cells, using MoufitLT software content of each group, DNA samples and cell cycle analysis.
1.3 Experimental statistical methods were repeated three more times. All the measured indicators are used ? s, said, using SPSS 14.0 statistical package for analysis and apoptosis analysis of variance experiment, cell proliferation assay using t test. With P <0.05 indicated significant difference. Reposted elsewhere in the paper for free download http://
2.1 Daidzein on SH-SY5Y cell growth and inhibition of cell proliferation inhibition test results showed that compared with normal control group, the concentrations of daidzein handle 24 ~ 72 h of cell inhibition rate compared with the normal control
group were There was a significant increase (P <0.05) (Table 1, Figure 1). Daidzein treatment SH-SY5Y cells, 24,48,72 h the IC50 were 65.0,47.0,40.0 μ g / ml. These results suggest that daidzein on SH-SY5Y cells in vitro has a strong inhibitory effect, its
inhibitory effect within a certain range of dose and time-dependent, and showed a
certain degree of saturation.
Figure 1 of daidzein on SH-SY5Y cells in vitro inhibitory effect of (omitted)
2.2 Daidzein on SH-SY5Y cell cycle and apoptosis after 48 h of drug treatment, various concentrations of daidzein can induce H-SY5Y cell apoptosis, G1 hypodiploid
peak before the peak occurs (Sub -G1), that is characteristic of apoptotic cells apoptosis peak (Figure 2). Daidzein-induced apoptosis in SH-SY5Y cells showed a definite dose-
dependent, apoptosis was significantly higher than the normal control group (P <0.05). With the increase of the concentration of daidzein, G0 / G1 and G2 / M phase cells decreased significantly, DNA synthesis phase (S phase) cells were significantly increased, indicating that daidzein to SH-SY5Y cells to S phase arrest , inhibit cell
Figure 2 Daidzein on SH-SY5Y cell cycle and apoptosis (omitted)
NB treatment is difficult, it has been actively looking for new treatments. China's traditional Chinese medicine for the treatment of cancer has a long history with the curative effect, advantages of low toxicity. Soy isoflavones are important
phytochemicals, from 12 kinds of monomers, which include genistein, daidzein, soybean Flavin three kinds of these three kinds of daidzein and daidzein glycosides derived from the nine kinds of ingredients. A large number of studies have shown that
[3,4] soy isoflavones have a high concentration of anti-cancer tumor suppressor role. In
recent years, from the perspective of apoptosis mechanism of Chinese herbal anti-
tumor effect and from the screening of natural drugs, anti-cancer drugs has become a
research hotspot. In this study, MTT method and flow cytometry to detect daidzein on SH-SY5Y cell proliferation, cell cycle and cell apoptosis.
Cell proliferation, cell cycle and cell apoptosis. MT results also showed that daidzein on SH-SY5Y cells significantly inhibit the in vitro, in the 10 ~ 80 μ g / ml within the framework of its inhibitory effect with the increase of drug concentration and effect time, an extension of its inhibitory effect is more obvious; When the concentration is higher than 80 μ g / ml, its inhibitory effect is no longer with the drug concentration increased and further enhance the role of time-dependent, showing a
certain degree of saturation. Flow cytometry results showed that daidzein can induce apoptosis in SH-SY5Y cells, and with the increase of the concentration of daidzein, G0 / G1 HEG2 / M phase significantly reduced cell number, DNA synthesis phase (S phase) was significantly increased, suggesting that daidzein can SH-SY5Y cells to S phase
arrest, thereby inhibiting cell proliferation. At present, for soy isoflavones inhibit cell proliferation mechanism of inhibiting cell proliferation in that it includes sex hormone-
like effects, anti-oxidation, induce apoptosis, inhibit tyrosine kinase activity and
inhibition of topoisomerase activity, etc. [5 ~ 8]. Our findings suggest that daidzein through the SH-SY5Y cells to S phase arrest to suppress the proliferation of SH-SY5Y
cells. Whether there are other ways to inhibit the proliferation of SH-SY5Y cells in this
experiment is not involved, do not know.
In summary, this study shows that daidzein in vitro on SH-SY5Y cells significantly
inhibited the proliferation and can induce apoptosis, although there is by activating cells to which the pathways to induce apoptosis in SH-SY5Y cells, wither death is still
unknown, pending further study, but this study or the treatment of NB could provide a new idea.
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