DOC

Berbamine-induced apoptosis in human leukemia Jurkat experimental study_1906

By Phyllis Reynolds,2014-10-30 11:21
11 views 0
Berbamine-induced apoptosis in human leukemia Jurkat experimental study_1906

Berbamine-induced apoptosis in human leukemia Jurkat

    experimental study

     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

    relationship.

     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 [1]. 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 [4], 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

    training.

     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 Results

     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%.

     3 Discussion

     At present the view that the incidence of cancer and the imbalance between cell proliferation and apoptosis related [7]. 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 [1], 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 [8]. 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.

     References

     [1] Luo Chong Nian, Lin Xin, Wang Li as, et al. Berbamine on mouse spleen cytotoxic T lymphocyte activity in inhibition of [J]. Chinese Journal of Pharmacology and Toxicology, 1995, 9 (2): 159 -160.

     [2] Po-Yan, FU Bing, Yan-Ling Zhao, et al. Berbamine in

    cultured Hela cells, the role of intracellular free calcium concentration [J]. Acta Pharmacol Sin, 1999, 20 (11) :1011-

    1014.

     [3] Po-Yan, Guo-Fen Qiao, Yan-Ling Zhao, et al. Berbamine on

    ATP-induced cultured smooth muscle and cardiac intracellular

    calcium mobilization of [J]. Acta Pharmacol Sin, 1999, 20

(8) :705-708.

     [4] Qing-Hua Dong, Zheng Shu, XU Rong-zhen, et al. Berbamine

    K562/Adr multi-drug resistant cells in vitro [J]. Chinese Journal of Medicine, 2004, 24 (9) :820-822.

     [5] Xu R, Dong Q, Yu Y, et al. Berbamine: A novel inhibitor of bcr / abl fusion gene with potent anti-leukemia activity

    [J]. Leuk Res, 2006, 30 (1) :17-23.

     [6] Wang GY, Zhang JW, Lu QH, et al. Berbamine induces apoptosis in human hepatoma cell line SMMC7721 by loss in

    mitochondrial transmembrane potential and caspase activation [J]. J Zhejiang Univ Sci B, 2007, 8 (4): 248-255.

     [7] Evan G, Littlewood T. A matter of life and cell death [J]. Science, 1998, 281 (5381) :1317-1322.

     [8] Carnero A. Targeting the cell cycle for cancer therapy [J]. Br J Cancer, 2002, 87 (2) :129-133.

     [9] Wolf BB, Schuler M, Echeverri F, et al. Caspase-3 is the

    primary activator of apoptotic DNA fragmentation via DNA fragmentation factor-45/inhibitor of caspase-activated DNase

    inactivation [J]. J Biol Chem, 1999, 274 (43) :30651-30656.

    reposted elsewhere in the paper for free download http://

Report this document

For any questions or suggestions please email
cust-service@docsford.com