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OBJECTIVE@#To investigate the effects of down-regulating of c-Met expression to the proliferation, invasiveness and apoptosis of human multiple myeloma RPMI 8226 cells.@*METHODS@#According to transfection the RPMI8226 cells were dividide into RPMI 8226 (untreated RPMI 8226), RPMI 8226 /shRNA-Met and RPMI8226/shRNA-control group, respectively. Protein expression level of c-Met was detected by Western blot so as to evaluate transfection condition; the proliferation of the cells was detected by MTT; apoptosis and cycle of the cells were detected by flow cytometry; effect of c-Met/shRNA on RPMI 8226 cell adhesion was detected by RPMI 8226 cell adherence to ECM (Fn and Matrigel) and ECV304 cells. Invasiveness of RPMI 8226 cell was detected by Transwell assay.@*RESULTS@#The c-Met short hairpin RNA (shRNA) was successfully transfected into RPMI 8226 cells, and could inhibit the expression of c-Met significantly. The down-regulation of c-Met could inhibit the proliferation of RPMI 8226 cells significantly. The percentage of cells in the G/G phase and apoptotic rate (sub-G) in the RPMI 8226/shRNA-Met group were higher than those in the control group, the adhesion rate and the number of migrated RPMI 8226/shRNA-Met cells were decreased significantly as compared with control group. There were no significant differences in each indexes between RPMI 8226/shRNA-control and control group.@*CONCLUSION@#Knockdown of c-Met can affect the proliferation, adherence, invasiveness and apoptosis of human multiple myeloma RPMI 8226 cells.
Subject(s)
Humans , Apoptosis , Cell Line, Tumor , Cell Proliferation , Multiple Myeloma , RNA, Small InterferingABSTRACT
<p><b>OBJECTIVE</b>To investigate if NS-398 could enhance the chemosensitivity of Bortezomib (BOR) on human multiple myeloma RPMI 8226 cells.</p><p><b>METHODS</b>After the treatment of NS-398 combined with BOR, MTT assay was used to detect the proliferation inhibition effect on human multiply myeloma RPMI 8226 cells in vitro, Flow cytometry was used to analyze their effect of apoptosis and cell cycle; the caspase-3 activity of different treatment group was detected by using ELISA and the activity of Cox-2 was measured by using Cox-2 activity assay kit.</p><p><b>RESULTS</b>The inhibitory rate of NS-398 combined with BOR was higher than that of NS-398 or BOR alone(Q>0.85). After treatment of NS-398 combined with BOR, the percentage of cells arrested in the G/Gphase and apoptotic rate were both higher than that of treatment with each drug alone(Q>1.15). The caspase-3 activity in cells treated with combined of NS-398 and BOR was significantly higher than that of treatment of each drug alone(Q>1.15).</p><p><b>CONCLUSION</b>NS-398 combined with BOR shows a synergistic effect on the growth inhibition of RPMI 8226 cells in vitro.</p>
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<p><b>OBJECTIVE</b>To establish bortezomib (BOR)-resistant human multiple myeloma U266 cell line U266/BOR and to detect its biological characteristics.</p><p><b>METHODS</b>U266 cells were constantly exposed at low dose and progressively increasing dose of BOR to establish U266/BOR, the cell morphology was observed by inverted microscopy, ICand resistant index were determined by MTT assay, cell growth curve was drawed and the doubling time was calculated; cell cycle distribution were analyzed by flow cytometry, and RT-PCR was used to detect the mRNA expression of resistance-related genes.</p><p><b>RESULTS</b>The MM U266/BOR cell line was successfully constructed and its resistance index was up to 19.8. The both cell morphologies were not different. Compared with U266 cells, the multiplication time was postponed with the increase of G/Gcell ratio, and S phase was reduced. The mRNA expression of PTPROt, Beclin 1 and PTEN were reduced, and the mRNA expression of c-Maf was enhanced in U266/BOR cells; as compared with U266 cells, but the MDR1 mRNA expression was not different between U266 cells and U266 BOR cells.</p><p><b>CONCLUSION</b>The BOR-resistant U266 cell line has been establiseed successfully. It provides an ideal cell model for further exploration of the mechanism for BOR resistance.</p>
ABSTRACT
<p><b>OBJECTIVE</b>To investigate if Ad-NK4 can enhance the chemosensitivity of human multiple myeloma RPMI 8226 cells to bortezomib(BOR).</p><p><b>METHODS</b>The cell-matrix adhesion test and PRMI 8226 cell-ECV 304 cell adhesion test were used to analyze the effect of Ad-NK4 on adhesion of RPMI 8226 cells; Western blot was used to detect the expression changes of adhesion and invasion-associated proteins MMP2, MMP3, MMP7 and VEGF; MTT assay was used to detect the effect of Ad-NK4 on proliferation of RPMI 8226 cells; the flow cytometry with PI staining was used to detect the effect of Ad-NK4 on cell apoptosis; the expression of cleaved caspase-3, BAX and BCL-2 was assayed by Western blot.</p><p><b>RESULTS</b>These 2 adhesion assays indicated that Ad-NK4 significantly inhibited the adhesion of human multiple myeloma RPMI 8226 cells. In addition, Erk and JAK/STAT pathway may be involved in the process. The expression level of MMP-2, MMP-3 and VEGF were decreased in Ad-NK4 group, compared with untreated or Ad-GFP group (P<0.05). However, the expression of MMP-7 protein in Ad-NK4 group was not significantly different from untreated or Ad-GFP group (P>0.05). The inhibitory rates of the proliferation in cells treatedly Ad-NK4 combined with BOR was significantly higher than that with BOR or Ad-NK4 alone. Similarly, Western blot indicated that the level of cleaved caspase-3 and BAX in cells treated with Ad-NK4 combined with BOR was significantly higher than BOR or Ad-NK4 alone, but BCL-2 protein expression was significantly lower. Meanwhile, the ratio of BAX/BCL-2 was increased.</p><p><b>CONCLUSION</b>Ad-NK4 can enhance the chemosensitivity of human multiple myeloma RPMI 8226 cells to BOR,which is associated with increasing of both BAX/BCL-2 ratio and Caspase-3 activation.</p>