Berbamine-induced apoptosis in human leukemia Jurkat
Abstract [Objective] Berbamine-induced apoptosis in human
leukemia Jurkat mechanism. [Methods] MTT Measuring IC50 values of human leukemia Jurkat cells and cell toxicity, Hoechst33258
fluorescent staining Berbamine apoptotic body formation, flow cytometry Annexin-? FITC-PI to detect the incidence of
apoptosis, PI staining for detection of apoptosis and cell cycle peak, while flow cytometry caspase-3 protein expression.
[Results] Berbamine selectively inhibit the growth of human leukemia Jurkat cells and the concentration-dependent
relationship, the role of 48h Jurkat cells, IC50 value of 6.6 ? 0.5μg/ml; Berbamine can induce Jurkat cell apoptosis, the cell cycle arrest in the S phase; the same time, cells increased caspase-3 protein expression. [Conclusion] Berbamine
can activate caspase-3 protein expression, induced apoptosis in human leukemia Jurkat cells and was time-effect
【Key Words】 Jurkat cells
Experimental Study of Berbamine Inducing Apoptosis in Human Leukemia Jurkat Cells
Abstract: [Purpose] To explore the mechanism of berbamine inducing apoptosis in human leukemia Jurkat cells. [Methods] IC50 value and cytotoxity of human Jurkat cells were detected by MTT method; apoptotic body, by fluorescent staining with Hoechst33258; cell apoptotic rate, by FCM with Annexin-? FITC-PI staining method; apoptotic peak, cell cycle and protein expression of caspase-3, by FCM. [Results]
Berbamine could significantly inhibit the proliferation of Jurkat cells in a dose-dependent manner and the IC50 value was
6.6 ? 0.5μg/ml at 48 hours. Berbamine also selectively induced apoptosis of Jurkat cells which were arrested in the S phase. In addition, expression of caspase-3 protein of Jurkat
cells increased after exposure to berbamine. [Conclusions]
Berbamine can selectively induce apoptosis of human leukemia Jurkat cells in the manner of dose and time dependence through up-regulation expression level of caspase-3 protein.
Key words: berbamine; Jurkat cells; apoptosis; caspase-3
Berbamine is a two-benzyl-isoquinoline alkaloid, has
anti-oxidant, immune suppression, elevated white blood cells and other clinical effects . Recent studies have shown that Berbamine can regulate intracellular free calcium concentration [2,3], reduced expression of multidrug
resistance gene in order to reverse multidrug resistance , a small number of studies have shown that its inhibitory effect on tumor cell proliferation [5,6], but its mechanism is still unclear. The experimental observation Berbamine on human
leukemia Jurkat cells induced apoptosis, and its mechanism of action were discussed.
1 Materials and methods
1.1 Drugs and reagents
MTT, propidium iodide (PI), Berbamine, Hoechst33258 for the Sigma company's products, RPMI 1640 medium for the United
States Hyclone products, Annexin-? FITC-PI-reagent Immunotech
company's products, Cytofix / Cytoperm, Caspase3-PE and the
same type of negative control mice IgG1-FITC/PE U.S.
Pharmingen company's products.
1.2 Experimental materials and major equipment
Cell culture bottles and the culture plates Nunclon Danish company's products; Clean Benches and CO2 Incubators for Germany Heraeus products; Wallac microplate reader for Finnish products; FACSCan flow cytometry BD products for the
United States; Axioskop 2 fluorescence microscope for the Zeiss company's products.
1.3 cell lines and culture
Jurkat cells were purchased from Shanghai Cell Biology, 10% fetal calf serum, 5% CO2, 37 ? incubation box routine
1.4 MTT method Jurkat cells measured IC50 value and the growth inhibition curve of
Logarithmic growth phase cells were seeded in 96-well
culture plate, 6 re-holes. Each hole 3 × 104A/ ml cells with
different concentrations of Berbamine, the total volume of 200μl, cultured 48h, each hole by adding 1mg/ml MTT working solution 50μl, 37 ? placed 4h, centrifuged 2 000r/min, 5min,
supernatant of each hole by adding 150μl DMSO, shock
dissolved crystal at a wavelength of 570nm compared with color, find the half inhibitory concentration IC50 and growth inhibition curves. Adoption of healthy human peripheral blood normal separation of liquid separation Ficol mononuclear blood cells.
1.5 Detection of apoptosis
Jurkat cells treated with different drug treatment, take 1 × 105/ml cell suspension 1ml phosphate buffer wash two times, by adding buffer 490μl, Annexin-? 5μl and propidium
iodide (PI) 5μl, dark place ice response to 10min, the machine testing, CellQuest software analysis. Another take 2 × 106 cells, 70% ice ethanol fixed, 24h after the PBS washing two times, by adding 1mg/ml RNase A 200μl, 37 ? water bath,
30min, coupled with PI Staining dark reaction of 30 min, the machine detected in 10 000 cells, Multicycle software analysis sub-diploid peak and the cell cycle.
1.6 Hoechst33258 staining apoptotic morphology
100μl incubated with 1mg/ml Hoechst33258 1μl cells (8
× 105/ml) 10min, samples for cell rejection film, air-dried
coverslip added, Zeiss fluorescence microscope, CCD camera.
1.7 caspase-3 expression in flow detection
Take 2 × 106 cells were treated with phosphate buffered saline wash two times, cells fixed and rupture of membrane, fluorescent antibody staining reagents according to the instructions at the same time using the same type of mouse
IgG1-FITC/PE the establishment of negative control. Detected on the machine. CellQuest software analysis.
1.8 Statistical analysis
All data in this experiment are used x ? s said that
between the two groups analyzed by the t test.
2.1 Berbamine cytotoxicity
Berbamine significantly inhibited the growth of Jurkat
cells, and there is a concentration-dependent relationship. As
the concentration of drug action from 0.5μg/ml to 10μg/ml,
cell viability was reduced to 93.69% from 14.85%, in this role of the concentration range Berbamine on normal human peripheral blood leukocytes had no significant cytotoxic effect (Figure 1 ). Berbamine role in 48h, Jurkat cells, IC50 value of 6.6 ? 0.5μg/ml. Reposted elsewhere in the paper for free download http://
2.2 Berbamine-induced apoptosis in Jurkat cells
Fluorescence microscopy observation of normal Jurkat cells, the nucleus can be seen as a circular, stained uniform. Using 5μg/ml Berbamine 48h after treatment, some cells underwent apoptosis, nuclear chromatin condensation and marginalization, apoptotic body (Figure 2).
With Annexin-? / PI double staining Quantitative
analysis showed that the normal Jurkat cells, the low rate of spontaneous apoptosis, about 3.4%. 5μg/ml Berbamine role in
12h after, Jurkat cell plasma membrane phosphatidylserine (PS)
valgus increased, the apoptotic rate increased to 5.97%, 24h apoptosis rate was 20.5%, 48h apoptosis rate increased to 44.72% (Figure 3). 24h and 48h apoptotic rates, the difference was significant (P <0.01).
2.3 Berbamine on Jurkat cell cycle
Jurkat cells were 5μg/ml Berbamine Berbamine 48h after
treatment, apoptotic peak (sub-G1 phase), peak of 28.5%, while
changes in the cell cycle, G0/G1 phase cells without drug
treatment from the percentage of the time 53.8% decreased to 35.7%, S phase cells marked by the percentage of time without medication 41.0% to 59.5%, G2 / M phase cell percentage changed little, respectively, 5.2% and 4.8%.
2.4 caspase-3 protein expression in
Without the drug treatment of Jurkat cells, caspase-3
protein expression rate was 1.62%, after 12h the role of 5μ
g/ml Berbamine caspase-3 protein expression after a slight
increase, to 2.23%; the role of caspase-3 after 24h rate of
protein expression 9.83%, after 48h the role of caspase-3
protein expression rate was 20.9%.
At present the view that the incidence of cancer and the imbalance between cell proliferation and apoptosis related . Apoptosis in the tumor growth, plays a negative regulatory role in the process, you can curb the rapid growth
of tumors. People in the long-term cancer therapy practice,
come to realize that you can induce apoptosis of tumor cells to achieve the purpose of treatment of cancer.
Berbamine around in 1980 and elevated white blood cell as a drug subject to research, early research focused on immune function and its clinical efficacy on the , was later found Berbamine with anti-tumor, anti - oxidation [4 ~ 7], is a
calmodulin antagonist [2,3].
Berbamine anti-tumor effect is mainly manifested in
inhibition of tumor cell proliferation, but little research in this area [5,6]. XU Rong-zhen, etc. The study found Berbamine can reduce the K562 cells, bcr / abl fusion gene expression, induce apoptosis of K562 cells. This study shows that, Jurkat
cells were treated Berbamine role, we could make a dose-
dependent relationship between cell proliferation was inhibited, and cells to form apoptotic bodies and apoptotic peak eversion membrane phosphatidylserine (Annexin-? cells)
that Berbamine anti-cell proliferation by inducing apoptosis
to achieve, this role has obvious time-effect relationship. At
the same time, the same concentration of Berbamine on normal hematopoietic cells, no obvious toxicity.
Many anticancer drugs make tumor cell cycle arrest at a
certain time period, thereby inhibiting tumor cell proliferation . Our research found that the role of Berbamine Jurkat cells after the G2 / M phase was significantly reduced, S-phase cells increased, indicating
that Berbamine on Jurkat cell proliferation inhibition by
inducing apoptosis, so cells were arrested in S phase .
Caspase enzyme in apoptosis play a critical important role in the pro-apoptotic signals under the action of a series of proteins such as cytochrome C, etc., combined with the
caspase enzymes to start the caspase cascade, eventually leading to cell apoptosis [ 9]. Studies have shown that with the increase in the concentration Berbamine role, activated caspase-3 protein expression increased, suggesting that the activation of caspase Berbamine enzyme to induce cell apoptosis.
Berbamine pairs of normal human lymphocytes without apparent toxicity, but it can induce apoptosis of leukemia cells may be a promising drug for the treatment of leukemia, it is worth further study.
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