[Abstract] Objective create resistance to melphalan in multiple myeloma cell line MOLP  2 / R, and a preliminary study of its biological characteristics and resistance mechanisms. The melphalan concentration gradient increments law establishing multiple myeloma cell line resistant to melphalan. Detect morphological differences of the two cells, the growth curve and doubling time; drug sensitivity test detects two cells of melphalan and a variety of chemotherapy drugs half inhibitory concentration (IC50) and resistance index (RI); using Western blot comparing two cells of P  gp, MRP and FANCD2 expression differences to investigate the possible mechanisms of resistance. Results established resistance index of 6.03 MOLP  2 / R resistant cell lines compared with MOLP  2 cells, MOLP  2 / R resistant cell Alien obvious; resistant cell doubling time was significantly prolonged (P <0.05 ); MOLP  2 / R and ADM, CTX, DDP, VP  16 cross-resistance; of MOLP  2 / R in FANCD2 the monoubiquitinated expression than MOLP  2 significantly increased P  gp, MRP in no higher expression in the resistant cell line. Conclusion MOLP  2 / R has a typical multi-drug resistant characteristics, provided the experimental basis for further study of resistance reversal ways. The FANCD2 protein monoubiquitinated expression enhancements may be MOLP  2 / R cells one of the major mechanisms of acquired resistance.

Key words multiple myeloma; melphalan; FA / BRCA (Fanconi anemia / BRCA) pathway; resistance; multidrug

Establishment of Melphalan  resistant Multiple Myeloma Cell Line MOLP  2 / R and Its Multidrug Resistant Mechanisms

    XIAO Hui, ZHANG Ke  jian, ZUO Xue  lan

    Department of Hematology, Zhongnan Hospital of Wuhan University, Wuhan 430071, ChinaAbstract: Objective To establish melphalan  resistant cell line of human multiple myeloma MOLP  2 / R and to investigate its biological characteristics and possible mechanisms of acquired resistance.Methods Melphalan  resistant cell line of multiple myeloma MOLP  2 / R was established by continuous stepwise selection in increasing concent ration of melphalan. Cell morphology, growth curve and population doubling time, protein level of P  gp, MRP and FANCD2 monoubiquitination were investigated to determine the biological features of MOLP  2 / R cell line. The IC50 and resistance index (RI) were measured by MTT assay.Results A melphalan  resistant cell line MOLP  2 / R was successfully established. The resistance index (RI) of MOLP  2 / R cells was up to 6.03.Besides melphalan it was cross resistant to many other chemotherapeutic agents, such as ADM, CTX, DDP and VP  16. Comparing with its parent cell line, the multiplication time was postponed (P <0.05), but 0.05). Western blot studies showed that the levels of Pgp, MRP expression in the MOLP2/R cells were similar with the sensitive cells, but">the proportion of S phase wasn’t significantly higher than parent cell line (P> 0.05). Western blot studies showed that the levels of P  gp, MRP expression in the MOLP  2 / R cells were similar with the sensitive cells, but enhanced FANCD2 protein monoubiquitination.Conclusion MOLP  2 / R cell line with stable melphalan  resistance shows typical multidrug resistance phenotypes and may serve as an ideal model for exploring the mechanism of MDR and the new route of reversing multidrug resistance. Over  expression of FANCD2 protein monoubiquitination might contribute to acquired drug resistance in MOLP  2 / R.

    Key words: Multiple myeloma; Melphalan; FA / BRCA (Fanconi anemia / BRCA) pathway; Drug resistance; Multiple

Multiple myeloma (multiple myeloma, MM) is a plasma cell malignancy, clinical will melphalan as first-line treatment of MM has been in use ever since, most MM patients early in the treatment of drug better response, but will eventually acquired drug resistance and relapse. The MM cell multidrug resistance is the main reason for the MM treatment failure. Recent studies have found that MM cells resistant to repair DNA damage is closely related to [1  2]. But the specific mechanisms and pathways of resistance is still not clear. Establishment of the ideal MM resistant cell model is in-depth study of MM cell multidrug resistance mechanisms premise. Increasing concentration gradient method, MM melphalan resistant cell lines MOLP  2 / R, and the detection and identification of biological traits, preliminary exploration FA / BRCA pathway key protein monoubiquitinated FANCD2 expression levels in significance in the resistant cell line.

    1 Materials and methods

    Of 1.1 medicines and reagents

    RPMI 1640 medium and fetal bovine serum from Gibco. Anti-FANCD2 and β  actin antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Melphalan, cisplatin (Cisplatin, DDP), vincristine sulfate (Vincristine, VCR), MTT were purchased from Sigma. Doxorubicin (Adriamycin, ADM), cytarabine (Ara  c) purchased from Zhejiang Hisun Pharmaceutical Co., Ltd.. Cyclophosphamide (cyclophosphamide, CTX), etoposide (VP  16) were purchased from Jiangsu Hengrui Pharmaceutical Co., Ltd.

    1.2 Experimental Methods

    1.2.1 induced by drug-resistant cell lines

    Human multiple myeloma cell line MOLP  2 was purchased from Germany microbial and cell cultures (DSMZ), containing 10% fetal bovine serum at 37 ℃, 5% CO2, 4nmol / L L-glutamic acid, 1% penicillin and streptomycin RPMI1640 medium subculture. A gradual increase in melphalan concentration induced by drug-resistant cell lines MOLP  2 cell culture medium. The initial concentration of melphalan in the resistant cell culture medium to 0.25 μmol / L, every two weeks to increase the concentration of 0.25 μmol / L, the melphalan resistant cell lines obtained in vitro after 40 weeks of continuous culture cells, melphalan resistant cell culture medium in a final concentration of 5μmol / L. This resistant cell lines named MOLP  2 / R. Lymphoblastoid cell lines HSC93 and from the known Fanconi anemia  F-type (Fanconi anemia  F, FA  F) patients to the normal person the original raw lymphocytes VU698 as experimental control [3], the above two cell lines by Dutch Free University professor Joenje kindly train the same way MOLP  2 cell lines.

    1.2.2 Morphological observation

    Take the logarithmic growth phase cells, to a concentration of 5 × 105/ml cell smears after Giemsa staining, cell morphology was observed structure. Take Exponentially growing parental cells MOLP  resistant cells MOLP  2 / R ultrastructure of the cells were observed under a transmission electron microscope.

    1.2.3 Cell growth curve and population doubling time (TD) Determination

    Inoculation cell density of 2 × 104 / ml in 24-well cell culture plates, each well 1 ml at 37 ° C, 5% CO2 incubator culture consecutive days, daily count 3 hole cell averaging. The cell growth curve, according to Patterson formula to calculate the cells in the logarithmic growth phase TD = Tlg2 / lg (N / N0), and T is the incubation time, the cell number N for the moment, N0 as the starting cell number.

    1.2.4 susceptibility test

    MTT assay MOLP  2 and MOLP  2 / R cell of melphalan, ADM, VCR, CTX, Ara  c, VP  16 and DDP’s sensitivity. Two weeks MOLP  2 / R before the experiment cells cultured in the culture fluid without melphalan after the backup. Take the logarithmic growth phase MOLP  2 and MOLP  2 / R cells, RPMI1640 culture liquid into single cell suspension inoculated into 96-well plates (cell concentration of 2 × 104 per well) were added 48 h after different concentration of chemotherapy drugs, parallel to each concentration of 3 holes. The supernatant was discarded after 48 h of incubation, each well 5 mg / ml solution of MTT 20 μl continue to cultivate carefully discard the supernatant after 4 h, each well was added 200 μl DMSO, dark and shaken for 10 min crystals fully dissolved. The OD value of the absorbance of each well with the the microplate measured wavelength of 570 nm in control wells for the serum-free RPMI 1640 culture medium. The concentration of the drug for the horizontal axis, and cell viability as the vertical axis, draw concentration – effect curve to determine the median inhibitory concentration (IC50) calculated resistance index (RI) = resistant cells IC50 / parental cell IC50 of.

    The 1.2.5 cell cycle distribution of DNA content

    Take parental cells and resistant cells in log phase 1 × 106 PBS washing. Preparation of single cell suspension, 322 g (1 200 r / min) centrifugation 5 min, the supernatant was discarded. Resuspended in 2 ml staining solution (0.15 mol / L NaCI; 0.1 mol / L Tris / HCI; 0.5 mmol / L MgCl2; 1.0 mmol / L CaCl2; 0.1% Igepal; 0.002% BSA) and incubated at room temperature for 15 min. Add 20 μl of Hoechst stock (1 mg / ml) 4 ℃ incubated for at least 15 min after cell cycle analysis on a flow cytometer (Becton Dickinson, San Jose, CA).

    1.2.6 P  gp, MRP and of FANCD2 expression of the determination

    Using the Western blot method. To take logarithmic phase cells, adding RIPA (Radio  Immunoprecipitation Assay, U.S. Pierce) lysate. Ice for 10min at 4 ° C, 20 800 g (14 000 r / min) centrifugation for 10 min the supernatant. The BCA method (BCA protein assay reagent kit, U.S. Pierce) for protein quantitation, protein sample is added to an equal volume of 2 × SDS gel sample buffer and mixed, placed in a boiling water bath for 10min. Protein samples (sample volume are each well of 20 μg of total protein) in SDS  PAGE gel electrophoresis (3% ~ 8% Tris  acetate gradient gel, Invitrogen Inc.) separation, separation of protein bands transferred to PVD membrane with anti-human P  gp, MRP, of FANCD2 and β  actin antibody was incubated overnight at 4 ℃, reaction with horseradish peroxidase-labeled secondary antibody and ECL Western blot analysis system (Amersham Pharmacia Biotech Products) to detect and developing.

    1.3 statistical methods

    SPSS10.0 software package, data to ± s statistical methods using two sample t-test, P <0.05 considered significant difference.

    2 Results

    Determination of 2.1 cell growth curves and population doubling time

    Cell growth curve The MOLP  2 and MOLP  2 / R peak in 6-7 days, TD, respectively (32.9 ± 2.6) (48.7 ± 3.1) h, the drug-resistant cell doubling time 44.5% longer than the parental cells , MOLP  2 / R growth the proliferation significantly slowed (P <0.05). The MOLP  2 and MOLP  2 / R of the growth curve is shown in Figure 1.

    2.2 Cell Morphology

    Resistant cell volume size is inconsistent, irregular shape, cytoplasm, nuclear small and irregular, visible phenomenon of megakaryocytes the pseudopodia and black particles in the cytoplasm increased significantly; parental cells are round, the same size, nuclear round clear cytoplasm, evenly distributed.

    2.3 P  gp, MRP and FANCD2 expression

    P  gp, MRP in parental cells and resistant cells were negative expression, suggesting that the resistant cell resistance mechanism has nothing to do with the high expression of P  gp, MRP. FANCD2 monoubiquitinated expression was significantly increased in the resistant cells, suggesting that this resistance may occur with the FA / BRCA pathway activity enhancements related, as shown in Figure 2.

    2.4 cell cycle detection

    Occurs mainly because the a FANCD2 single ubiquitination in G1 ~ S phase, we detected by flow cytometry MOLP  2 / R cells arrested at the G1  S period to seek FANCD2 single expression enhanced ubiquitination. Flow cytometry cell cycle G1, S and G2 / M phase cells, found MOLP  2 / R MOLP  2 cells, the G1 to S phase proportion of resistant cells than parental cells, but the difference was not statistically significant 0.05),见表1。">(P> 0.05), as shown in Table 1. Table 1 MOLP  2 and MOLP  2 / R cell cycle comparison (%)

    2.5 MOLP  2 / R of RI and its cross-resistance

    Of melphalan resistance to ADM, CTX, DDP, VP  16 cross-resistance, while Ara  c, VCR no cross-resistance remains similar to the parental cell sensitivity, as shown in Table 2. The MOLP  2 / R in the culture medium without melphalan repeatedly subcultured after 10 weeks of resistance and stability, RI 6.01 12 weeks after the the measured resistance index dropped 5.35 5 μmol / L melphalan culture need to re-join, and still get the same resistance, the measured resistance index of 6.07. Table 2 MOLP  2 / R cells resistant spectrum

    3 Discussion

    Induced resistance is actually a process of screening cells due to cell heterogeneity, the most sensitive cells are killed, a small number of sensitive cells survive and be amplified. This experiment using a progressive administration of law, in the culture medium in vitro using human multiple myeloma cell line MOLP  melphalan induced culture. MOLP  2 resistant cell lines, melphalan relative tolerance than the parental cells increased 6.03-fold compared with parental cells resistant cell growth slowed, no significant changes in cell volume, is a secondary sex-resistant cell lines with multidrug resistance characteristics, the structure and mechanism of action of CTX, ADM, DDP variety of chemotherapy drugs exist to varying degrees of cross-resistance, no cross-resistance Ara  c, VCR. Is generally believed that the resistance index of less than 5 low resistance, resistance index of 5 to 15 moderate resistance, resistance index of greater than 15 highly resistant [4]. This shows that the study established a moderate resistance MOLP  2 / R multi-drug resistant cell lines to study the of melphalan resistance mechanisms and screened reversal agents ideal model.

    Studies have shown that a variety of ways to participate in the occurrence of the MDR, including some intracellular factors, such as of TNF, IL  2 enhanced DNA repair [5  6] and aberrant DNA repair caused by MDR [7]. Have been found to FA / BRCA (Fanconi anemia / BRCA) pathway plays an important role in DNA damage repair process [8-9] multiple the FA gene encoding the protein through a common mechanism of FA / BRCA (Fanconi anemia / BRCA) pathway to maintain cell stability of the genome, at least eight FA proteins (A, B, C, E, F, G, L and M) interacting to form a FA protein core composite, the composite is activated by promoting FANCD2 monoubiquitinated of FANCD2, The FANCD2 activated FA complex Preparation role. A variety of DNA cross-linking agent to exert its effect through the channels, has confirmed that acquired drug resistance in ovarian cancer DDP related to the re-activation of the FA / BRCA pathway [9]. Even in combination with chemotherapy in breast line CTX and doxorubicin FA / BRCA pathway a protein FANCG upregulation [10]. A variety of drugs to DNA cross-linking agents in clinical commonly used chemotherapy drugs, such as mitomycin C (Mitomycin C (MMC), MMC), DDP, CTX, melphalan, etc., are widely used in a variety of solid tumors and hematological malignancies treatment . FA / BRCA pathway adjustable cells to DDP and other DNA cross-linking material generated reaction [11]. The experimental results confirmed that FANCD2 monoubiquitinated in resistant multiple myeloma cells MOLP  2 / R expression was significantly increased, suggesting that this resistance may occur related to the FA / BRCA pathway activity enhancement.

    In summary, a class of DNA cross-linking agent used in cancer chemotherapy drugs is likely to play a role in its secondary resistance may be related to the strength and the activity of the pathway through the role of the FA / BRCA pathway. This prompted us to achieve enhanced chemotherapy drugs and lifting resistance by inhibiting the activity of the pathway.