Cyclosporin A, tetrandrine reversed K562/A02 elements and combination of the two multi-drug resistant cell
【Abstract】 Objective: To explore the cyclosporin A (CsA), Su-tetrandrine (Tet) and
the combined use of both drug-resistant human leukemia cell line K562/A02 the role of
multi-drug resistance reversal. Methods: MTT determination of daunorubicin (DNR) on cell volume of half inhibition (IC50), flow cytometry cell apoptosis rate and the concentration of intracellular DNR. Results: DNR on the K562 cells and K562/A02
cells, IC50 were 0.27 and 21.22 mg * L-1; CsA (1 mg * L-1) and Tet (0.1 mg * L-1) after
individual and joint processing K562/A02 1) The role of K562/A02 cell apoptosis rate was 2.70%, CsA, and Tet alone or in combination treatment apoptosis rates were
10.27%, 17.64% and 52.79%; 2-treated cells after chemotherapy has significantly
increased the concentration of DNR. Conclusion: CsA and Tet resistance can be reversed, and the combined effects of a stronger reversal effect.
Key words Cyclosporine A; tetrandrine hormone; K562/A02 cells; multi-drug
Chemotherapy is an important means to treat cancer, one of the multi-drug
resistance (multidrug resistance, MDR) is the main reason leading to the failure of chemotherapy. P-glycoprotein (P-gp)-mediated MDR is the most studied tumor MDR,
one of the main mechanism, studies have shown that its expression and cellular capacity of the powerful drug efflux on . Cyclosporin A (CsA) is an immunosuppressive agent, adjustable P-gp function, inhibit the overexpression of P-gp
and reverse the tumor cell MDR ; tetrandrine Su (Tetrandrine, Tet) is a traditional Chinese medicine Stephania tetrandra root of the main component is a non-specific
Ca2 channel blockers, studies have shown that it can adjust a variety of P-gp-mediated
MDR cell lines resistant . Two drugs alone in vitro experiments, reversal of drug resistance has been a certain effect, but reversed MDR in combination at home and abroad there is no relevant reports. In order to find an effective low toxicity co-
resistance reversal agent, for providing laboratory basis for clinical treatment of leukemia, we leukemia cell line K562 and its resistant cell line K562/A02 experimental study in vitro experiments to study the combined application of these two kinds of of
drugs on the role of MDR reversal.
1 Materials and methods
1.1 Cell lines and culture conditions
Sensitive strains K562 and MDR strains of Hematology K562/A02 by the Chinese Academy of Medical Sciences Institute, sensitive cells were cultured in 1640 containing 10% FBS culture medium, K562/A02 were cultured with 2 μmol * L-1 doxorubicin,
10% fetal bovine serum in 1640, was suspended 1 week before the experiment of adriamycin. Two kinds of cells were placed in 37 ?, 5% CO2 incubator, 2 ~ 3 d
subcultured one time.
1.2 Experimental equipment, drugs and reagents
LDZ522 centrifuge (Beijing Medical Centrifuge Factory), SartoriusBS110S balance (Beijing Sartorius Balance Co., Ltd.), HZ28201K constant temperature oscillator
(Taicang Science and Education Equipment Factory), Model 550 microplate reader (Japan BIO2RAD company), FACSCAL BUR-flow cytometry (FCM, the U.S. BD
company), daunorubicin (DNR, Italy Farmitalia company), CsA (Sandoz, Switzerland),
Tet (Jiangxi Silver Tao Pharmaceutical Co., Ltd.), tetrazolium salt ( MTT, Sigma Company, with PBS preparation of 5 g * L-1), fetal bovine serum (Hyclone Inc.), RPMI
1640 medium (GIBCO Company).
1.3.1 Experimental grouping experiments logarithmic growth phase cells, cell density adjusted according to different experimental requirements: (1) control group, K562, or K562/A02 cell suspension; (2) DNR group, cell suspension by adding a certain dose of DNR; (3) DNR plus Tet group, cell suspension by adding a certain dose of DNR and Tet; (4) DNR plus CsA group, cell suspension by adding a certain dose of DNR and CsA; (5) DNR plus Tet plus CsA group, cell suspension by adding a certain dose of DNR, Tet, and CsA.
1.3.2 MTT Determination of reversal agents on DNR cytotoxicity impact reference documentation and kit instructions. Logarithmic growth phase of the drug-resistant
strains and sensitive cell lines by adding reversal agent (alone or in combination, CsA final concentration of 1 mg * L-1, Tet final concentration of 2.5 mg * L-1) the role of 1
h, according to the set gradient with different concentrations of DNR, each of the concentration of complex is located three holes. Serum-free culture solution for the
zero-hole group, cell suspension without the drug for the blank control group, inoculated in 96-well plates, each well containing 2 × 105 cells suspension of 200 μl. Placed in 37 ?, 5% CO2 incubator removed after 48 h incubation, add MTT reaction solution 20 μl, to continue to build 4 h after the plate centrifuge 3 000 r * min-1
centrifuge 10 min, discard supernatant, each well add 150 μl of dimethyl sulfoxide dissolved, micro-mixing oscillator, measured on microplate reader wavelength of 540 nm absorbance (A). Calculating cell growth inhibition rate:
Growth inhibition rate (%)=( 1 - Hole A value of experimental / control hole A
value) × 100%.
IC50 = cell growth inhibition rate of 50% of the drug concentration
Resistance to multiple drug-resistant cell = IC50 / sensitive cell IC50
The experiment was repeated three times, each time re-established three holes,
taking the average of the final result.
1.3.3 FCM detection of apoptosis rate to take K562/A02 cells, a final concentration of 2 × 105 ml-1, according to the experimental group and each drug (alone or in combination, DNR final concentration of 1 mg * L-1, CsA final concentration of 1 mg *
L-1, Tet final concentration of 0.1 mg * L-1), a separate control group and only
K562/A02 plus DNR (1 mg * L-1) in the control group K562/A02 cells, respectively, after 48 h incubation cells were collected, with 4 ? pre-cooling of the PBS washed
twice with 1 ml blot buffer re-suspended cells, each adding Annexin ?-FITC 10 μl,
another blank control and FITC-based contrast dark at room temperature 15 min,
after washing cells, FCM on the detection of cell apoptosis.
1.3.4 FCM detection of the concentration of intracellular DNR take K562/A02 cell
line, a final concentration of 2 × 105 ml-1, group dosing were incubated 48 h, separate
K562 cells and cells without reversal agents K562/A02 control were added to DNR (final concentration of 1 mg * L-1), placed in 37 ?, 5% CO2 incubator were incubated
2 h, washed three times cold PBS solution, 1 500 r * min-1 centrifuge 5 min, discard
supernatant using FCM measured relative fluorescence intensity of intracellular DNR, this value is proportional to the concentration with the DNR.
1.4 Statistical analysis
The use of SPSS 13.0 statistical software for analysis and significance test using single-factor analysis of variance, P