| ObjectivesMultiple myeloma(MM)is a plasma-cell neoplasm. The annual incidence rate of MM is 1/100000 in our country and about 4/100000 in the western industrialized countries. The number of yearly newly-diagnosed MM is about 14,000 in the United States. Until now, MM is an incurable malignancy, with a 3-year median survival time (MST), accounting for 20% of mortality rate among all hematopoietic malignancies. The main methods for treating MM include combination chemotherapy and haemopoietic stem-cell transplantation. Because the median age at diagnosis is about 60 year-old and 50% to 60% patients with renal dysfunction, most patients have no chance for autologous stem-cell transplantation.In the past decades, MP (melphalan-prednisone),VAD (vincristine-doxorubicin-dexamethasone) and M2 (vincristine, BCNU, melphalan, cyclo- phosphamide, and prednisone) were the common multidrug regimens for the treatment of MM and the response rates were 42% to 50%. Thalidomide is effective in treating relapsed and refractory MM patients and the response rate was 25%. When thalidomide is combined with dexamethasone, the response rate is 50% for relapsed patients and 64% to 74% for newly-diagnosed MM patients. In 2001, a phaseⅡclinical trial of dexamethasone combined with Lenalidomide, the analog of thalidomide, showed that the response rate was 91%. With the usage of Bortezomib alone, the first clinical applied proteasomes inhibitor, A 33% and a 50% response rate was achieved for relapsed and refractory MM patients, respectively. Despite these advances in the treatment, multiple myeloma remains incurable, and the need to develop effective treatments remains urgent.TRAIL (Tumor Necrosis Factor-related apoptosis inducing ligand), is a member of the Tumor Necrosis Factor (TNF) superfamily. TRAIL can induce apoptosis of tumor cells but not normal cells in in vitro studies or in experimental animals. Therefore, it is reasonable that TRAIL can be considered as an ideal candidate cancer treatment.The present study is thus designed to examine the growth inhibition and adhesion effect of TRAIL on RPMI8226, a multiple myeloma cell line and on bone marrow stroma cells (BMSCs), and investigate TRAIL's functioning mechanism. Methods1 Cell culture1.1 Cell culture of RPMI8226RPMI8226, a cell line derived from human multiple myeloma, were cultured in RPMI 1640 medium, supplemented with 20% (v:v) heat-inactivated fetal bovine serum (Hyclone), 100U/ml penicillin G and 100μg/ml streptomycin sulfate, at 37℃in an atmosphere of 5% CO2. The cells were seeded at a density of 3×105 cells/ml and passaged every 5 days. Cells at Logarithmically growing state with viability≥95% as assayed by trypan blue exclusion staining were collected and used for the studies described below.1.2 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% (v:v) heat-inactivated fetal bovine serum (Hyclone), 100U/ml penicillin G and 100μg/ml streptomycin sulfate, at 37℃in an 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.2 The effect of TRAIL on the adhesion of RPMI8226 and growth inhibition effect of TRAIL on BMSCs2.1 Growth inhibition effect of TRAIL on BMSCs assayed by MTT methodBMSCs were planted in 96-well plates at a density of 2×105 cells/ml in LG-DMEM medium. After 24 hours incubation of BMSCs, cells were exposed to TRAIL at different concentrations for different time period (up to 4 days). Six hours before the ending of culture, 20μl of MTT solution (5mg/ml) was added to the cells in each well. The supernatants were discarded after centrifugation and the precipitant was dissolved in DMSO. The amount of converted MTT was quantified as value of absorbance (A) at 550nm test wavelength in an ELISA Reader. The effect of TRAIL was shown in the dose-effect curve.2.2 The expression level of adhesion molecule CXCR4 on RPMI8226 cells before and after TRAIL treatment, measured by FCM.RPMI8226 cells in exponential growth were planted in 24-well plates at a density of 2×105 cells/ml in RPMI1640 medium, with 1 ml cell suspension in each well. After cultured with different concentrations of TRAIL for 24 hours, RPMI8226 cells were harvested, washed in cold PBS. After addition of 6μl anti-CD184 (that is anti-CXCR4), cells were incubated at room temperature for 30 min, then, the expression level of CXCR4 was analyzed by FCM.3 Growth inhibition effects of TRAIL on RPMI8226 cells RPMI8226 cells in exponential growth state were exposed to TRAIL at different concentrations for the designed time period (up to 4 days). Six hours before the culture ending point, 20μl of MTT solution (5mg/ml) was added to the cells in each well. The supernatants were discarded after centrifugation and the precipitate was dissolved in DMSO. The amount of converted MTT was quantified as value of absorbance (A) at 570nm test wavelength in an ELISA Reader. The growth inhibition effects of TRAIL on RPMI8226 cells were shown in the dose-effect curve.4 Detection of RPMI8226 cells apoptosis induced by TRAIL-treatment4.1 Detection of RPMI8226 cell Apoptosis before and after TRAIL-treatment by FCM. The early apoptotic cells was detected with annexin V-PI detection kit.Cells in exponential growth state were planted in 24-well plates at a density of 2×105 cells/ml in RPMI1640 medium, with 2ml cell suspension in each well. After cultured with TRAIL at different concentrations, RPMI8226 cells were harvested, washed in cold PBS, and re-suspended in 200μl of binding buffer. Following addition of 10μl AnnexinⅤand 5μl PI, cells were incubated at room temperature for 30 min, then analyzed by FCM and apoptotic rate was calculated by CELLQuest software.4.2 Measurement of the expression level of Bax,Bcl-2,Mcl-1,CARP1,CARP2,XIAP and cFLIP mRNA in RPMI8226 cells before and after TRAIL-treatment by RT-PCR.4.3 Analysis of the expression level of nuclear factorΚB(NF-ΚB) phosphor-P65,caspase-3 and Mcl-1 proteins levels in RPMI8226 cells before and after TRAIL-treatment by Western blot analysis.For measuring the expression level of NF-κB P65,caspase-3 and Mcl-1 protein by western blot, RPMI8226 cells were lysed in RIPA buffer (containing Tris-Hcl 20mM, Nacl 150mM, PMSF 1mM, Nonidet P-40 1% v:v , SDS 0.1%, EDTA 1mM, Leupeptin 2mg/L, Sodium deoxycholate 0.5%) at 4℃for 1h. After centrifugation, the supernatants were collected and the protein concentrations were detected by Lowry method. 150μg protein samples were separated by electrophoresis on a 8% SDS-PAGE gel and transferred to nitrocellulose membrane. Then the membrane was blocked overnight at 4℃in TTBS containing 1% BSA and 5% (v:v) no-fat powdered milk. For immunoblot analysis, blot was incubated with mouse anti-human NF-κBP65,caspase-3 and rabbit anti-human Mcl-1 for 1h at room temperature on shaking, then washed in TTBS, finally incubated in secondary horse-radish peroxidase-labeled goat anti-mouse antibody and goat anti-rabbit antibody for 1h. After three further washes with TTBS, bound secondary antibody was detected using ECL.Results1 TRAIL affected the expression level of adherent molecules on RPMI8226 cells. No significant growth inhibition effect of TRAIL on BMSCs was observed.1.1 TRAIL had almost no effect on the growth of BMSCs. Our result showed that the RPMI8226 cells grow better when the cells were co-cultured with BMSCs than the cells were cultured alone.1.2 The expression level of CXCR4 on RPMI8226 cells treated with TRAIL at the concentration of 5μg/ml for 24 hours was much higher (72.58%) than that on RPMI8226 cells without TRAIL-treatment (47.82%).2 TRAIL exerted a dose- and time-dependent growth inhibition on RPMI8226 cells. The MTT assay result showed that the growth inhibition rate of RPMI8226 cell was 46%, 54%, 59%, respectively, after the cells treated with TRAIL at the concentrations of 2.0μg/ml, 5.0μg/ml and 10μg/ml, for 24 hours. There is a statistically significant difference in the growth inhibition rate among different group of cells which were treated with TRAIL for different times.3 The aopoptosis-inducing effect of TRAIL on RPMI8226 cells3.1 The morphological changes of RPMI8226 cells treated with TRAIL were as following: concentration of chromatin, vacuolization, altitude coacervation of chromatin, marginalization, cell nucleus pyknosis and chip and apoptosis body appearing. There was no apoptotic morphology change was observed in the control RPMI8226 cells cultured in medium without TRAIL.3.2 The FCM assay result showed that the ratio of apopototic cells to non- apopototic cells increased after TRAIL-treatment. The difference in the percentage of apopotosis cells was statistically significant before TRAIL and after TRAIL treatment.3.3 The RT-PCR measurement showed that the relative expression level of Bcl-2,CARP1 and CARP2 mRNA in RPMI8226 cells decreased significantly after TRAIL treatment. The relative expression level of Mcl-1,cFLIP and XIAP mRNA decreased either, but without statistic significant. No expression of Bax in RPMI8226 cells was measurable before TRAIL treatment, while it increased obviously after TRAIL-treatment for 24 hours. 3.4 Western blot assays showed that TRAIL down-regulated the expression levels of caspase-3 and NF-κB P65 protein in RPMI8226 cells. TRAIL-treatment showed no obvious effect on the expression level of Mcl-1 protein in RPMI8226 cells.Conclusions1 TRAIL had almost no effect on the growth of BMSCs. Our result showed that the RPMI8226 cells grow better when co-cultured with BMSCs than cultured alone.2 TRAIL up-regulated the expression level of adherent molecule CXCR4 on RPMI8226 cells significantly.3 Growth inhibition effect of TRAIL on RPMI8226 cells acted in a dose-and time-dependent maner.4 TRAIL induced the apoptosis of RPMI8226 cells. The expression level of apoptosis related genes and proteins in RPMI8226 changed after TRAIL-treatment.5 The results of the present study indicated that TRAIL induced apoptosis of RPMII8226 cells via inhibiting the expression of anti-apoptosis genes and proteins while increased the expression of apoptosis-inducing genes and proteins.
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