[Effect of Met Knockdown on Biological Behavior of Multiple Myeloma RPMI 8226 Cells].

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 G0/G1 phase and apoptotic rate (sub-G1) 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.

[Establishment of BOR-Resistant U266 Cell Line and the Detection of Its Biological Activities].

OBJECTIVE: To establish bortezomib (BOR)-resistant human multiple myeloma U266 cell line U266/BOR and to detect its biological characteristics. METHODS: 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, IC50 and 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. RESULTS: 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 G0/G1 cell 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. CONCLUSION: 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.

[Anti-proliferation Effect of NS-398 in Combination with Bortezomib on Multiple Myeloma RPMI 8226 Cells In Vitro].

OBJECTIVE: To investigate if NS-398 could enhance the chemosensitivity of Bortezomib (BOR) on human multiple myeloma RPMI 8226 cells. METHODS: 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. RESULTS: 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 G0/G1 phase 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). CONCLUSION: NS-398 combined with BOR shows a synergistic effect on the growth inhibition of RPMI 8226 cells in vitro.

[Ad-NK4 Enhances the Chemosensitivity of Human Multiple Myeloma RPMI 8226 Cells to Bortezomib].

OBJECTIVE: To investigate if Ad-NK4 can enhance the chemosensitivity of human multiple myeloma RPMI 8226 cells to bortezomib(BOR). METHODS: 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. RESULTS: 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. CONCLUSION: 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.

Knockdown of DEPTOR inhibits cell proliferation and increases chemosensitivity to melphalan in human multiple myeloma RPMI-8226 cells via inhibiting PI3K/AKT activity.

OBJECTIVE: The present study determined the role of DEP domain containing mTOR-interacting protein (DEPTOR) in the proliferation, apoptosis and chemosensitivity of RPMI-8226 multiple myeloma cells, using small hairpin RNA (shRNA) to knock down DEPTOR gene expression in vitro. METHODS: DEPTOR mRNA and protein levels in RPMI-8226 cells treated with DEPTOR-specific shRNA were evaluated by reverse transcription-polymerase chain reaction and Western blotting. Expression of apoptosis-associated proteins (including cleaved caspase-3 and cleaved poly-ADP ribose polymerase [PARP]) and activation of the phosphatidylinositol 3-kinase (PI3K)/v-akt murine thymoma viral oncogene homologue 1 (AKT) signalling pathway were detected by Western blotting. RESULTS: Transfection of DEPTOR-specific shRNA successfully knocked down DEPTOR gene expression in transfected RPMI-8226 cells. These transfected cells, together with control RPMI-8226 cells, were treated with 20 µmol/l melphalan for 24 h. Knockdown of DEPTOR exacerbated melphalan-induced growth inhibition and apoptosis, increased levels of cleaved caspase-3 and cleaved PARP, and reduced levels of phosphor-AKT. CONCLUSION: Downregulation of DEPTOR inhibited proliferation and increased chemosensitivity to melphalan in human multiple myeloma RPMI-8226 cells via inhibiting the PI3K/AKT pathway.