| BackgroundMultiple myeloma(MM) is a presently incurable B-cell cancer characterized by accumulation of malignant plasma cells in the bone marrow and painful bone destruction that accounts for approximately 1% to 2% of all human cancers, High-dose chemotherapy with stem cell support has achieved higher, But few patients remain in long-term remission. In recent years, the role of cytokines in the development of MM has been the focus of many studies. Although interleukin 6 (IL-6) is the best documented growth factor in this malignancy, the role of a second potential growth factor, insulin-like growth factor 1(IGF-1), has been less clearly defined. The object of the study is to investigate the effect of IGF-1 on MM cell lines RPMI8226, and provide a new method of the aimed therary of MM.Methods1. Cell culture of RPMI8226RPMI8226, a cell line derived from human multiple myeloma, were cultured in RPMI1640 medium, supplemented with 10% heated-inactivated fetal bovine serum, 100U/ml penicillin G and 100μg/ml streptomycin, at 37℃in a humidified of 5% CO2 in-air atmosphere. The cells were passaged every two days. Cells at logarithmically growing state with viability≥95% as assayed by trypan blue exclusion staining were used for the studies described below.2. Cell culture of BMSCsHeparinized bone marrow was obtained after informed consent from healthy donors. All of the bone marrow cells were cultured in LG-DMEM medium, supplemented with 10% heated-inactivated fetal bovine serum, 100U/ml penicillin G and 100μg/ml streptomycin, at 37℃in a atmosphere of 5% CO2. After incubated for 48 hours, non-adherent cells were removed, and the remaining adherent cells were considered as"adherent BMSCs"and were used for the present studies.3. Growth effects of IGF-1 on RPMI8226 cellsRPMI8226 cells in exponential growth state were exposed to IGF-1 at different concentration for the designed time period(up to three days). Four hours before the ending point, 20μl of MTT solution(5mg/ml)was added to the cells in each well. The supernatants were discarded after centrifugation, then each well added 150μl DMSO and dissolved the precipitate, the amount of converted MTT was quantified as value of absorbance at 570nm test wavelength in an ELISA Reader.4. Detection of RPMI 8226 cells cell cycle and apoptosis before and after IGF-1 treatment by FCM.RPMI8226 cells in exponential growth state were exposed to IGF-1 at different concentration for the designed time period(48 hours). 1×106 cells were harvested, washed in cold PBS, Following addition of 1ml PI, Cells were incubated at 4℃temperature for 30 minutes, then analyzed by FCM and apoptosis rate was calculated by Muticycle AV software. To detect the apoptosis of RPMI8226 cells, After exposed to IGF-1, bortezomib and dexamethasone for 72 hours, the same method as before.5. The expression level of adhesion molecule CD44 on RPMI8226 cells before and after IGF-1 treatment, measured by FCM.RPMI8226 cells in exponential growth state were cultured with IGF-1 for 72 hours, harvested the cells and washed in cold PBS, After addition of anti-CD44, Cells were incubated at room temperature for 30 minutes, the expression level of CD44 was analyzed by FCM.6. The effect of IGF-1 on the adhesion of RPMI8226 cells and BMSCs.BMSCs were planted in 96-well plates at a density of 1×105 cells/ml in LG-DMEM medium. After 24 hours incubation of BMSCs, each well added 5×104 RPMI8226 cells and different concentration IGF-1, and incubation 24 hours. Discarded the supernatants and PBS washed the non-adherent cells, 20μl of MTT solution(5mg/ml)was added to the cells in each well. The supernatants were discarded, each well added 150μl DMSO and dissolved the precipitate, the amount of converted MTT was quantified as value of absorbance at 570nm test wavelength in an ELISA Reader.7. The effect of IGF-1 on the migration of RPMI8226 cells.The effect of IGF-1 on migration of RPMI8226 cells under coculture system was demonstrated in 24-well transwell cell culture chambers with the upper chamber containing filters of 8.0μm pore size. The upper surface of the filters was coated with a mixture of basement membrane components (Matrigel matrix) and dried overnight. RPMI8226 cells (1×105) were plated in the upper chambers, followed by cocultivation with RPMI1640 medium and IGF-1 in the lower chamber. After 24 hours incubation take out the chamber, washed in PBS and cells on the upper surface of the filters were removed with a cotton swab. Cells on the lower surface of the filters were fixed, stained with HE's staining and counted. Ten fields (×400) per chamber were counted under a microscope and average of three chambers determined.Results1. IGF-1 caused a dose-and time-dependent growth promotion of RPMI8226 cells. The MTT assay result showed that the proliferation ratio was (14±6)%,(41±9)%,(61±7)%,(77±14)%,(92±14)% respectively, after the cells treated with IGF-1 at the concentration of 12.5,25,50,100,200 ng/ml, for 24 hours. The proliferation ratio was (20±8)%,(42±9)%,(65±11)%,(77±9)%,(89±9)% , at the same concentration for 48 hours. The proliferation ratio was (23±6) %,(44±7)%,(67±11)%,(80±12)%,(90±10)%, at the same concentration for 72 hours.2. The IGF-1 converted the cell cycle from G1 period into S peroid, When RPMI8226 cells had been treated with IGF-1 at the concentration of 50ng/ml,100ng/ml,200ng/ml, the percentage of S period cells were 44%,55.8%,55.1%.3. The FCM assay results showed IGF-1 inhibited the apoptosis in RPMI8226 cells, the percentage of apoptosis cells decreased from 32.81% in the culture of bortezomib to 20.73% after exposure to IGF-1 for 72 hours, in the case of dexamethasone, similar results were obtained.4. The expression level of CD44 on RPMI8226 cells treated with IGF-1 at the concentration of 50ng/ml for 72 hours was higher (4.6%) than that on RPMI8226 cells without IGF-1 treatment (3.7%). The results showed that IGF-1 could increase the adhesion of RPM I8226 cells to BMSCs. When RPMI8226 cells had been treated with IGF-1 at the concentration of 25ng/ml,50ng/ml,200ng/ml, the adhesion rates were 23.8%,30%,38.2% respectively.5. Pretreated with IGF-1 for 24h, the invasive number of RPMI8226 cells under microscope (×400) were 68 and 147 respectively obviously increased in contrast with control (20).ConclusionsGrowth effect and antiapoptosis of IGF-1 on RPMI8226 cells was in a dose-and time-dependent maner, IGF-1 can inhibite the apoptosis induced by bortezomib and dexamethasone. IGF-1 up-regulated the expression level of adherent molecule CD44 on RPMI8226 cells. IGF-1 can also promote the adhesion and migration of RPMI8226 cells.
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