[Abstract] Objective To investigate mifepristone in JEG choriocarcinoma cell 3 in vitro proliferation of non-classical human leukocyte class I antigen HLA G, HLA E expression in. Methods Cultured high expression of HLA G HLA E choriocarcinoma cell lines JEG 3, using the MTT assay mifepristone on cell proliferation, respectively by RT PCR technology and flow cytometry analysis techniques to observe its effect on HLA G cells, the expression of the mRNA and protein levels of HLA E. Results mifepristone showed concentrations trust inhibition of cell proliferation in JEG 3, high concentrations of mifepristone significantly lower in JEG 3 cells HLA G HLA E mRNA and protein levels. Conclusion Mifepristone anti-tumor mechanism may be one of the body can break immune tolerance to tumors, thereby curbing the growth of the tumor.
Key words mifepristone; human leukocyte antigen; immune tolerance
Effect of Mifepristone on Proliferation and Expression of Non classical Human Leucocyte Antigen Class Ⅰ Molecules in Choriocarcinoma Cell Line
WANG Jian ying1, LI Yong2, CHENG Jian xin1, FAN Li qiao2, WU Xiao hua1, ZHAO Qun2, WANG Shi jie2
1.Department of Gynecolory and Obstetric, The Forth Hospital of Heibei Medical University, Shijiazhuang 050011, China, 2.Department of SurgeryAbstract: Objective To explore the effect of mifepristone on the proliferation and the expression of nonclassical HLA I molecules (HLA G and HLA E) in choriocarcinoma cell line JEG 3.Methods The HLA G highly positive cell line of choriocarcinoma (JEG 3) was cultured in vitro, and MTT assay was used to examine antiproliferative effect of mifepristone on the JEG 3 cells , the mRNA expression of HLA G and HLA E were detected by RT PCR, and protein level by flow cytometry.Results Mifesitone produced concentration dependent and time dependent antiproliferative effect on JEG 3 cell at all experimental concentrations.High concentration mifesitone could significantly down regulate both mRNA and protein expression of HLA G and HLA E.Conclusion One of mifesitone’s antineoplastic mechanisms is surmounting immune tolerance to the neoplasm and inhibit its growth.Key words Mifesitone Human Leucocyte Antigen immune tolerance.
Key words: Mifestitone; Human Leucocyte antigen; Immune tolerance
Studies have shown that the human leukocyte antigen G (human leukocyte antigen G, HAL G) and human leukocyte antigen E (human leukocyte antigen E, HLA E) by the immunosuppressive pathways play in the process of tumor immune escape 1].
Many in vitro and in vivo experiments show that mifepristone for sex hormone-dependent gynecological tumors have significant anti-tumor effect, but the mechanism of action is not clear, its role lowered HLA G, HLA E, has not been reported. This study will to mifepristone direct role in the high expression of of HLA G and HLA E of choriocarcinoma cell lines JEG 3, observe the killing activity of mifepristone JEG 3 cells and the cells of HLA G HLA E levels, and to explore the anti-tumor mechanisms of mifepristone, to provide a theoretical basis for clinical application.
1 Materials and methods
1.1.1 cell lines choriocarcinoma cell line JEG 3, was purchased from the Chinese Academy of Sciences, Institute of Zoology, family planning and reproductive biology National Key Lab.
1.1.2 Reagents mifepristone (RU486), pure by Beijing Zizhu Pharmaceutical Co., Ltd.; methyl thiazolyl tetrazolium blue (MTT) was purchased from Sigma; reverse transcription-polymerase chain reaction (RT PCR) kit purchased from Fermentas; primers were synthesized by Shanghai Sangon Biological Engineering Technology & Services Co., Ltd.; HLA G, HLA E monoclonal antibody was purchased from British Abcam.
1.2.1 JEG 3 cells cultured with 10% fetal bovine serum, 100u/ml penicillin, 100g/ml streptomycin 2mmmol / L DMEM/F12 culture medium, at 37 ° C, 5% CO2 thermostat incubator culture, digested with 0.25% trypsin, 2 to 3 days passaged once.
1.2.2 MTT assay mifepristone mifepristone JEG 3 cell proliferation dissolved in anhydrous ethanol, dubbed 10mg/ml RPMI1640 medium to the desired concentration, the experimental group m non-Division ketone final concentration were 1.25μg/ml, 2.5μg/ml, 5μg/ml, 10μg/ml, 20μg/ml, 40μg/ml, 80μg/ml. In order to avoid the impact of ethanol on the test, set the ethanol (7) the control group, so that the final concentration of ethanol and the ethanol content of the various experimental groups. Cell proliferation was measured by MTT assay, take the logarithmic growth phase cells, adjusting the concentration of 2 × 105 / ml, were seeded in 96-well culture plates, each well 100μl, adherent culture different concentrations for 24h drugs, each concentration of six wells per plate of three blank control well. The cell culture after a set period of time, each well was added 10μl MTT (concentration of 5mg/ml), culture was continued for 4h, discard the culture liquid, each well was added dimethyl sulfoxide (DMSO) 150μl oscillation 10min, a microplate reader at 600nm absorbance (A) was measured at the value, and the following equations to calculate the rate of cell growth inhibition in each group. The experiment was repeated five times.
Cell growth inhibition rate = (hole A value of control wells A Value – the experimental hole A value) / control × 100%.
1.2.3 RT PCR detection of mifepristone mifepristone JEG 3 cells HLA G, HLA E mRNA levels of the test group a final concentration of 40μg/ml, 80μg/ml, a drug control group. Drugs that act on the JEG 3 cells 72h, total cellular RNA was extracted using Trizol kit with UV spectral photometric method for quantitative RNA. The same amount of RNA was reverse transcribed to cDNA strand, to obtain the reverse transcription product as a template for PCR, amplification of HLA G gene, HLA E gene and β actin gene. Using the gel analysis system density analysis of the amplified bands, and the resultant value as the average gray scale value represents the amount of expression of the corresponding gene, and dividing by the average gray value of the β actin mRNA relative expression of each amplification product amount. The amplification conditions: HLA G 94 ℃ for 3min, 94 ° C denaturation 45s, 55 ° C annealing 45s, extension at 72 ° C for 1 min, 30 cycle, 72 ℃ for 7min; HLA E to 94 ° C denaturation 3min, 94 ℃ for 30s, 55 ° C annealing at 55s was 72 ° C extending 2min, 40 cycles, 72 ℃ for 7min. 1% agarose gel electrophoresis 80V 30min, to observe changes in gene expression. Primer sequences : of HLA G upstream primer material to 5 ‘ AGA CGC CAA GGA TGG TGG the TCA 3’, downstream primer material to 5 ‘ AGG AAA GGT GAT TGG GGA AGG 3’; HLA E upstream primer material for 5 ‘ TCC GAG CAA AAG TCA AAT 3’, downstream primer for the 5 ‘ AGA TCC AAG GAG AAC CAG 3’; β actin upstream primer 5 ‘ AAG AGA GGC the ATC the CTC the ACC CT 3’, downstream primer material is 5 ‘ GGA AGG AAG GCT GGA AG 3’. Amplification products: HLA G 770 bp was HLA E 447 bp – length of β actin 619bp. The experiment was repeated five times.
1.2.4 measured by flow cytometry impact test group mifepristone mifepristone on the protein expression of HLA G, HLA E JEG 3 cells at a final concentration of 40μg/ml and 80μg/ml, drug action JEG the 3 cells 72h, taking a single cell suspension of 1 × 106/ml were added 1:100 mouse anti-human monoclonal antibody to HLA G HLA E 100μl incubated 30min PBS washed supernatant was added 1:20 sheep anti mouse FITC IgG100μl, and incubated for 30min, unbound antibody and washed with PBS, and then PBS was added, and filtered through 500 mesh copper mesh on the machine after detection, provided the background and negative controls. The fluorescence index (FI) indicates the relative content of the protein. The experiment was repeated five times.
1.2.5 statistical methods of data to ± s, using SPSS statistical software for data analysis, F-test.
2.1 mifepristone JEG 3 cells proliferation
As can be seen from Figure 1, With the prolonged duration of action, the same concentration of mifepristone on inhibition rates gradually increased 40μg/ml and 80μg/ml concentration role in cell inhibition rate after 72h more than 50%, so the choice 72h the mifepristone best experimental duration of action.
Compared with blank control group, after 72h of drug action, the killing effect of various concentrations of mifepristone on JEG 3 cells in varying degrees, in a concentration-dependent manner (P <0.05). When mifepristone concentration lower than 10μg/ml weak inhibition, but the concentration of 20μg/ml more inhibition showing a rising trend, the inhibition rate reached almost 100% when the concentration of 80μg/ml. For mifepristone dissolved ethanol has no significant influence on cell growth, as shown in Figure 1.
2.2 mifepristone the JEG 3 cells of HLA G mRNA and protein level
Mifepristone the role in JEG 3 cells after 72h, pairwise comparisons between the control group, 40μg/ml group and 80μg/ml groups, HLA G mRNA and protein expression changes were statistically significant. Wherein the control group and 80μg/ml group compared, P <0.01; compared to the control group and 40μg/ml, 40μg/ml group and 80μg/ml group, all P <0.05. Table 1, Figure 2,4. Table 1 mifepristone in JEG-3 cells HLA G
2.3 mifepristone JEG 3 cells HLA E mRNA levels and protein levels.
80μg/ml mifepristone in JEG 3 cells after 72 h compared with the control group, HLA E mRNA and protein expression were significantly lower; 40μg/ml group compared with the control group, HLA E mRNA and protein levels No significant changes were (respectively, P = 0.164, P = 0.2); compared with 80μg/ml 40μg/ml group, mRNA expression was not changed significantly (P = 0.098), reduced protein expression was statistically significant (P = 0.018), see Table 1, Figure 3,5.
The main effector cells of the immune surveillance role CTL (cytotoxic T cell and CTL) and NK cell recognition of CTL for tumor cells to be dependent on the tumor cell surface major histocompatibility complex Ⅰ (major histocompatibility complex, MHC identification of I) class I molecules. The tumor cells do not express MHC Ⅰ molecules, NK cells play its cytotoxicity to attack the tumor cells.
At the same time, the tumor cells through a variety of ways to evade the body’s immune surveillance role, including classical MHC Ⅰ molecules lost or allosteric, resulting in the loss of CTL killing; another tumor cells in the loss of MHC Ⅰ molecules high expression of non-classical MHC Ⅰ molecules (such as HLA G, HLA E), to pass a strong anti-inhibitory signals, causing tumor cells to escape and then escape CTL killing of NK cells.
NK cell receptors include two categories, namely, the immunoglobulin superfamily, and C-type lectin superfamily, each category including inhibitory receptors and activation receptor. Immunoglobulin superfamily inhibitory receptors KIRs (killer cell inhibitory receptor) ILT 2 (Ig like transcript 2) and ILT identify HLA G. C-type lectin superfamily inhibitory receptor CD94/NKG2A identify HLA E . HLA G and HLA E has a similar NK inhibitory function polymorphism, extensive tissue distribution. HLA G with KIR binding and activation of the KIR cytoplasmic part contains the immunoreceptor tyrosine-based inhibitory motif, the motif MHC Ⅰ molecules combined, can turn on negative regulation signal, inhibiting the activation of NK cells . HLA E combined allele specificity of the leader sequence peptides derived from other HLA I molecules, this peptide must be able to stabilize the expression of HLA E, CD94/NKG2A recognition combined with the HLA E by body, inhibit the cytotoxicity of NK cells and T lymphocytes . HLA G and HLA E have a complementary or synergistic effects, the former can promote and stable expression of the latter.
High expression of HLA G depends on activation in the tumor microenvironment irritants, such as IFN γ, GM CSF, IL 2, IL 10, etc., can enhance the expression of HLA G protein on the expression of HLA E also have some role [5,6]. Expression of a large number of its cells in the placental maternal surface chorionic trophoblast CHEN Chun-ling et al  found that play an important role in the regulation of expression of HLA G, HCG, high expression of HCG in choriocarcinoma may raise HLA G involved in immune escape has important significance. 2006 Yie etc.  studies have reported progesterone, human chorionic gonadotropin, and progesterone by raised levels of HLA G to achieve the purpose of miscarriage, which indicated that the clinical labor induction drugs such as mifepristone caused abortion may be the mechanism can cut their HLA G level of HLA E, thereby suggesting that the anti-tumor mechanism of immune escape.
To terminal differentiation, mifepristone main mechanism of anti-tumor activity; mifepristone also by lowered bcl 2, induction of TGF β1 expression to induce apoptosis ; study also found that the rice non mifepristone is a specific cell cycle inhibitory drugs, the tumor cells by inhibiting DNA synthesis arrest in the G0/G1 phase, can not enter the S and G2 / M phase, thereby inhibiting the proliferation of tumor cells . This study shows, has a strong inhibitory effect of mifepristone on JEG 3 cells in a time-and dose-dependent manner. High concentrations of mifepristone role in JEG 3 cells 72h HLA G, HLA E gene mRNA and protein levels can be adjusted downwards, 80μg/ml group lowered HLA G, the degree of HLA E above 40μg/ml group especially HLA G performance is more obvious, other mechanisms may prompt of mifepristone anti tumor can break immune tolerance of the body’s tumor and restore the role of the effector cells to kill tumor, thereby curbing the growth of the tumor, which also tumor treatment provides a new direction of development. Especially for highly expressed in cancer tissues HLA G the patients HLA E or a conventional drug-resistant patients, during tumor immunotherapy research process, it should give full consideration to the tumor cells express HLA G, HLA E antigen.