Abstract Objective To investigate bortezomib induced high expression of the ABCG2 drug nasopharyngeal carcinoma cell Allo  NK cell killing sensitivity mechanism. Using immunomagnetic beads to separation ABCG2High CNE2/DDP cells and Allo  NK cells, after the separation of flow cytometry to detect cell purity and by bortezomib treated NKG2D ligand expression rate of the target cells, LDH release assay detected by boron behalf of the rank and file meters before and after the the sensitivity of the Allo  NK cells, cells ABCG2HighCNE2/DDP destruction. Results ABCG2HighCNE2/DDP cell separation ABCG2 expression was (91.40 ± 2.32)%, after NK cell sorting CD3-CD16 + CD56 + cells of 90% purity. After bortezomib treatment target cells of MICA, MICB, of ULBP1 ULBP2 ULBP3 expression rate before drug treatment (2.92 ± 0.33)% and (4.27 ± 0.33)% and (5.80 ± 0.62)%, (11.10 ± 3.15 )% and (7.75 ± 1.14)%, respectively, rising to (17.52 ± 2.04)%, (12.53 ± 3.68)%, (15.24 ± 2.91)%, (62.02 ± 6.85)%, (35.69 ± 3.23)%. Effector to target ratio 10:1,20:1 Allo  NK cell killing rate of the bortezomib treated ABCG2HighCNE2/DDP cells (15.32 ± 13.86)%, (27.26 ± 6.81)% and (35.06 ± 5.10 )%, (52.34 ± 4.78)%. The killing rate before and after treatment the difference was statistically significant (F = 26.03, P = 0.000). Conclusion bortezomib by tumor cells induced by high expression of NKG2D ligands (of MICA / B of ULBP1  3), so that the the destruction increased sensitivity of tumor cells Allo  NK cells.

Key words of ABCG2; allogeneic natural killer cells; of NKG2D; destruction sensitivity to bortezomib;

Induction of Expression of Ligands for NKG2D Receptor in ABCG2High Nasopharyngeal Carcinoma by Bortezomib

    HUANG Yu  xian, WANG Yang, HU Liang  shan, SONG Chao  yang, GUO Kun  yuan

    Department of Hematology, Zhujiang Hospital, Southern Medical University, Guangzhou 510282, China

    Corresponding Author: GUO Kun  yuan, E  mail: gzyuan@pub.guangzhou.gd.cnAbstract: Objective To investigate the mechanism on the effects of improving cytotoxic sensitivity of ABCG2High CNE2/DDP cells to Allo  NK cells which exerted by bortezomib. Methods ABCG2HighCNE2/DDP cells and Allo  NK cells were isolated by magnetic activated cell sorting (MACS). Flow cytometry was used to evaluate the purity of isolated cells and the expression of NKG2D  ligands on target cells before and after incubation with bortezomib.Subsequently , the cytotoxic sensitivity of treated and un  treated ABCG2High CNE2/DDP cells to Allo  NK cells were measured by LDH releasing assay.Results The expressions of ABCG2 in ABCG2High CNE2/DDP cells were (91.40 ± 2.32)%. More than 90% of isolated NK cells showed tobe CD3-CD16 + CD56 + cells which would definitely meet the needs of experiments.The expressions of MICA, MICB, ULBP1, ULBP2, ULBP3 on target cells incubated with bortezomib have respectively increased from (2.92 ± 0.33)%, (4.27 ± 0.33)%, (5.80 ± 0.62)%, (11.10 ± 3.15)%, (7.75 ± 1.14)% to (17.52 ± 2.04)%, (12.53 ± 3.68)%, (15.24 ± 2.91)%, ( 62.02 ± 6.85)%, (35.69 ± 3.23)%. At the E: T ratio of 10:1 and 20:1, the cytotoxic sensitivity of ABCG2HighCNE2/DDP cells to Allo  NK cells increased from (15.32 ± 13.86)% and (27.26 ± 6.81)% in un  treated groups to (35.06 ± 5.10)% and (52.34 ± 4.78)% in bortezomib treated groups.Data above showed that cytotoxic sensitivity of target cells in each group before and after bortezomib treatment had significant differences (F = 26.03 P = 0.000). Conclusion Bortezomib can up  regulate expressions of NKG2D  ligands (MICA / B, ULBP1  3) in ABCG2High nasopharyngeal carcinoma cells, which resulted in higher cytotoxic sensitivity to Allo  NK cells.

    Key words: Bortezomib; ABCG2; Allo  NK cell; NKG2D; Sensitivity cytotoxicity

Boron Bortezomib (Bortezomib is administered, PS  341, VELCADE), the trade name Velcade, is a dose-dependent and reversible proteasome inhibitor, is the first proteasome inhibitor used in clinical treatment. It is mainly by the ubiquitin-proteasome pathway works by specifically blocking effectively inhibit cell growth, angiogenesis, and the rapid induction of apoptosis, not only for multiple myeloma, lymphoma have a good effect, and can for the treatment of a variety of solid tumors, such as thyroid cancer, breast cancer, lung cancer, pancreatic cancer, prostate cancer [1  5]. Recent research data show that bortezomib can increase the sensitivity of tumor cells for immunotherapy, but the exact mechanism is unclear. This article using immunomagnetic beads separation ABCG2HighCNE2/DDP cells and Allo  NK cells by flow cytometry before treatment with bortezomib, changes in the rate of target cells after expression of NKG2D ligands, LDH release assay target cells Allo  NK the cytotoxicity sensitivity changes, resistant nasopharyngeal carcinoma cell killing Allo  NK cells sensitivity to changes in its mechanism of bortezomib induced Allo  NK cell adoptive immune chemotherapy laid the theoretical basis.

    1 Materials and methods

    1.1 Materials and Equipment

    Reagents: boron bortezomib (Xianyangsen), anti-NKG2D ligand (anti  MICA / B monoclonal antibody, and anti  of ULBP1  3 monoclonal antibody, BD Company), PE-conjugated goat anti-mouse IgG1 (eBioscience Company), PE-labeled mouse anti-human ABCG2 monoclonal antibody (eBioscience), anti-CD56 immunomagnetic beads, anti-PE immunomagnetic beads (Miltenyi Biotec, Germany), the killing activity detection kit (Cytotox96 non  radioactive cytotoxocity assay, Promega Corporation), fetal bovine serum ( Hangzhou Evergreen), RPMI 1640 (Gibco Company). Instrument: 5810R Fast the low temperature the centrifuge (Eppendorff Company) 2010 microplate reader (Zhengzhou Cyber ​​Instruments, Inc.), Olympus inverted fluorescence microscope (Olympus, Japan), carbon dioxide incubator (Thermo, USA), Beckman Coulter of the EPICS AITRA type flow cytometer (Beckman Coulter Inc.), electronic scales (CE Company, Germany).

    1.2 Methods

    1.2.1 highly expressed in the of ABCG2 CNE2/DDP cells and Allo  NK cells isolated and expression of detection (1) ABCG2HighCNE2/DDP cell separation and purification: to to collect CNE2/DDP cells PBS washed twice, counted cells were resuspended in PBS . Cell density of 1 × 106/ml plus 0.5μg PE-labeled mouse anti-human ABCG2 monoclonal antibody, 4 ℃ incubated for 30min. Centrifuged, and the supernatant was removed, resuspended to a cell density of 1 × 107l/ml adding 20μl anti  PE immunomagnetic beads for cell positive selection, flow cytometry analysis ABCG2HighCNE2/DDP purity (high expression of ABCG2 CNE2/DDP cells denoted ABCG2HighCNE2/DDP). (2) Allo  NK cells isolated and purified: conventional density gradient centrifugation from human peripheral blood mononuclear cells were washed twice with PBS and count cells, CD56 MicroBeads for the cells positive selection, CD3-CD56 + cells, streaming cytometric detection of CD3-CD16 + CD56 + cell purity.

    1.2.2 Cell culture containing 10% fetal bovine serum, 100U/ml penicillin, 100μg/ml streptomycin, 3 μmol / L DDP RPMI 1640 medium, 37 ° C, 5% CO2 conventional culture experimental cells used are in the right logarithmic growth phase. The rhIL of 1000u/ml cultured NK cells  2.

    1.2.3 Flow cytometric analysis of NKG2D ligand expression rates of target cells were collected by bortezomib treated without bortezomib treatment and the boron bortezomib 100ng/ml of incubation ABCG2HighCNE2/DDP cells in, washed 2 times with PBS, counting cells, the cell density of 1 × 106/100μl each added the 2μg mouse anti-human NKG2D ligands (MICA, MICB, ULBP1, ULBP2, ULBP3) monoclonal antibody, 4 ℃ for 30 min, the PBS washed twice, adding PE-conjugated goat anti- mouse monoclonal antibody, the same type of IgG1 (Pharmingen company) as a negative control antibody, 4 ℃ incubated for 20min, PBS washing, using flow cytometry analysis of samples of 1 × 104 cells positive cells, calculate the percentage of fluorescence labeled cells, experiments Repeat three times.

    1.2.4 LDH release assay detection Allo  NK cells to target cell killing activity collect ABCG2HighCNE2/DDP cells, divided into three groups before and after treatment with bortezomib. Treatment group: boron bortezomib 100ng/ml of incubation 4h in ABCG2HighCNE2/DDP cells; untreated group: without the bortezomib the pretreatment ABCG2HighCNE2/DDP cells; control group: K562 cells, containing 5% fetal calf serum RPMI 1640 complete culture medium to adjust the cell density of 1 × 105/ml, added to 96-well plates, each well 50μl. Allo  NK cells freshly isolated effector cells at different effector to target different amounts of effector cells were added to each 50 μl of ratio (10:1,20:1). Each group are located three wells at 37 ° C under 5% CO2 in co-incubated for 4h, centrifugal, draw 50μl supernatant was added to 96-well flat-bottomed microtiter plates, adding 50μl LDH substrate reaction solution at room temperature for 30min added to each well 50μl reaction-terminated liquid. Microplate reader wavelength 490nm punishable blank group measured OD values. Calculate the activity of NK cells (%) = (experimental group OD value – target cells the natural release the group OD value – effector cells the natural release group OD value) ÷ (target cells release group OD value  target cells the natural release the group OD value) × 100%.

    1.3 statistical methods

    SPSS13.0 software for data processing, data to ± s NKG2D ligand expression rate of target cells before and after drug treatment using paired t test, target cells Allo  NK cytotoxicity sensitivity of single-factor analysis of variance was used to compare the difference was statistically significance (P <0.05).

    2 Results

    Of 2.1 ABCG2HighCNE2/DDP cells, Allo  NK cell separation purity and expression rate detection

    The ABCG2 expression display ABCG2HighCNE2/DDP cells was (91.40 ± 2.32)%, its obvious cell atypia, flow cytometry results shown in Figure 1. NK cells microscope observation after sorting cells small, rounded cell morphology, flow cytometry sorting CD3-CD16 + CD56 + cell purity of over 90%, in line with the experimental requirements, see Fig. 2.

    2.2 Flow cytometric analysis of expression of NKG2D ligands on target cells before and after treatment with bortezomib

    Flow cytometry analysis showed that: without bortezomib treatment ABCG2HighCNE2/DDP of NKG2D ligands MICA, MICB, of ULBP1 ULBP2 ULBP3 expression rates were (2.92 ± 0.33)%, (4.27 ± 0.33)%, ( 5.80 ± 0.62)%, (11.10 ± 3.15)% and (7.75 ± 1.14)%, five ligands are weak expression or non-expression; after bortezomib treatment, NKG2D ligands MICA, MICB, of ULBP1, ULBP2 ULBP3 expression was significantly elevated, were (17.52 ± 2.04)%, (12.53 ± 3.68)%, (15.24 ± 2.91)%, (62.02 ± 6.85)%, (35.69 ± 3.23)%, NKG2D ligands on target cells before and after treatment expression rates of ligand t-test difference was statistically significant (t = 11.38, P = 0.008; t = 8.39, P = 0.05; t = 9.03, P = 0.05; t = 13.85, P = 0.005; t = 12.17, P = 0.006), as shown in Figure 3.

    2.3 LDH release assay to detect a target cell killing activity of the Allo  NK cells to bortezomib before and after processing

    LDH release assay results: Allo  NK cell killing activity ABCG2HighCNE2/DDP cells and K562 cells with enhanced effector to target ratio increased, the pretreatment of the target cells, effector to target ratio of 10:1, the killing rate (15.32 ± 13.86)%, (36.41 ± 4.01)%; effector to target ratio of 20:1, the destruction rate (27.26 ± 6.81)%, (52.33 ± 5.49)%; description Allo  NK cells have normal destruction capacity. The target cells after bortezomib treatment, effector to target ratio 10:1,20:1 destruction rate before treatment (15.32 ± 13.86)%, (27.26 ± 6.81)% and (35.06 ± 5.10)% rose to , (52.34 ± 4.78)%, In addition, pretreatment of the target cells, Allo  NK cytotoxicity sensitivity K562 control cell destruction sensitivity flat. In both effector to target ratio, the the groups target cells sensitivity difference was statistically significant (F = 26.03, P = 0.000), as shown in Figure 4.

    3 Discussion

    The ABCG2 resistance-associated protein is a newly discovered, belonging to the ATP-binding cassette (ATP  binding cassette, ABC) transporter family G subfamily members. ABCG2-mediated multidrug resistance is atypical multidrug resistance (multi  drug resistance, MDR), mitoxantrone, daunorubicin, topotecan, SN  38, according to Lee irinotecan drugs such as cross-resistance medicine, and P  gp, MRP-mediated MDR substrates of cisplatin, vincristine, paclitaxel and does not produce cross-resistance [6]. In addition, some scholars believe that the specific ABCG2 expression in SP cells may be potential markers of stem cells [7]. The traditional treatment for this type of multi-drug-resistant tumor cells, unable to obtain satisfactory results, which may be related to the incidence of tumor resistance mechanisms complexity, and therefore need to combine high expression of the treatment of of ABCG2 drug nasopharyngeal auxiliary other treatments or open up their diameter.

    Recent studies show that NK cells have a wide range of anti-tumor effect, including in vitro killing activity of a variety of tumor cell lines (acute and chronic leukemia, lymphoma, myeloma, melanoma, prostate cancer, breast cancer), and in animal xenograft model showed good antitumor activity [8-9]. NK cell functions rely mainly on the NK cell surface receptor and the expression of MHC and non-MHC class cell binding ligand, transfer inhibition or activation signal, to adjust the activity of NK cells; NK cell activation signal path (NKG2D  the DAP  10) mediated cytotoxic activity of killer cell immunoglobulin-like receptors (Killer cell immunoglobulin  like receptor, KIR) and MHC Class I binding inhibitory signal [10  11]. In addition, in vitro intervention factors such as cytokines (IL  2 and IL  15, IL  18), molecular targeted anti-tumor drugs (Iressa, Glivec) can regulate the activation of NK cells and target cells with receptor expression, which improve the activity of NK cells to improve the sensitivity of tumor cells to NK cells [12  14]. The present study, the target cells without boron before alternate zolmitriptan processing, and target cells five NKG2D ligands the infirm expression or no expression of the lethal sensitivity lower. Five ABCG2HighCNE2/DDP cell expression of NKG2D ligands rate than before drug treatment significantly increased after bortezomib treatment, indicating that bortezomib can induce NKG2D ligands, with the target cells after drug treatment, expression of NKG2D ligands liter Allo  NK cell killing activity than the target cells before drug treatment significantly enhanced. In the analysis of the cytotoxic activity of of Allo  NK cells to target cells, scholars habits K562 cells as NK cell killing activity detection control cells, mainly because it is sensitive cell lines of NK cells did not express the inhibitory signal, and NK cell activation signal high expression of NKG2D ligands [8], the author is the same as the control cells to detect the activity of NK cells with K562 cells.

    Bortezomib is a highly specific 26S proteasome inhibiting ability Dipeptidyl boric acid compound, by inhibiting the activity of the catalytic center of the proteasome 20S, which selectively inhibit the degradation of some important regulatory role of the protein in the body, further cell division arrest at the G2 / M phase, leading to cell apoptosis [15  16]. Recent studies have also confirmed bortezomib can effectively reduce the threshold value of chemotherapy drugs induce apoptosis and reversal of drug resistance, while eliminating the resistance caused by the cell adhesion-mediated, so that the tumor cells to chemotherapy, radiation therapy is more sensitive; Further , Valés  Gómez M bortezomib can induce tumor cell expression of immune effector cell activation ligands (of MICA / B, ULBPS), increased the immunotherapy sensitivity of tumor cells [17]. From the above experimental results verify bortezomib selectively induced the expression of the tumor cells NKG2D ligands, so that the enhanced sensitivity of the tumor cells, NK cells, but specifically through what mechanism bortezomib induced target cells highly expressed NKG2D ligand remains to be further studied.