Anti-tumor Efficacy And Mechanisms Via COX-2 And 12-LOX Pathways In Multiple Myeloma

PART 1Expression of COX-2 in Multiple Myeloma and Efficacy of Meloxicam on Proliferation and Apoptosis of Myeloma CellsObject: To investigate the expression of cooxygenase-2 (COX-2) in multiple myeloma (MM) and the efficacy of Meloxicam on the proliferation and apoptosis of myeloma cells.Methods: Human myeloma cell lines, RPMI8226, KM3 and U266 were culutured and maintained in RPMI1640 medium containing 10% fetal bovine serum. After treatment of various concentrations of Meloxicam, cell proliferation and viability were checked by MTT and typan blue staining, cell apoptosis was assayed by Annexin-V/PI double-staining, TUNEL, and AO/EB staining. Expression of COX-2 protein and mRNA was checked by Western blot and RT-PCR, respectively. Result: (1) 10 to 50μM of Meloxicam inhibited proliferation of RPMI8226 and KM3 cells in a time- and dose-dependent manner, whereas had no effect on proliferation of U266 cells and normal human peripheral monocytes. (2) Apotosis rates, checked by Annexin-V/PI TUNEL, increased after treatment of Meloxicam, and typical apoptosis changes could be observed by AO/EB staining with fluorescence microscopy. (3) Expression of COX-2 on mRNA and protein levels of the cell lines was inhibited by various concentrations of Meloxicam.Conclusion: Human myeloma cell lines of RPMI8226, KM3 and U266 express COX-2. Meloxicam, a COX-2 specific inhibitor, can inhibit proliferation and induce apoptosis in RPMI8226 and KM3 cells, in which inhibited expression of COX-2 may be involved.PART 2Small interfering RNA of cyclooxygenase-2 induces growth inhibition and apoptosis independently of Bcl-2 in human myeloma RPMI8226 cellsObject: To investigate the effects of small interfering RNA of cyclooxygenase 2 (COX-2) on the proliferation and apoptosis of human multiple myeloma RPMI8226 cells and its relation with the Bcl-2 family in vitro.Method: The COX-2 siRNA fragment targeting exon 5 of COX-2 gene was transfected into the cells with the Amaxa nucleofection technique. The cells were divided into three groups, transfected cells (RPMI8226siRNA), untreated cells (RPMI8226Untreated) and cells in uncleofector solution, without siRNA and with the application of the electroporation program (RPMI8226Blank). Transcription and expression of COX-2 were checked by RT-PCR and Western blot analysis, respectively. The inhibition of cell growth was detected by 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium (MTT) assay. Apoptosis was estimated by Annexin-V/propidium iodide double-labeled cytometry and confirmed by terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling assay. Bcl-2 and Bax expression was evaluated by Western blot analysis.Result: The COX-2 siRNA fragment could be successfully (>78%) transfected into RPMI8226 cells with the nucleofection technique, which resulted in the significant inhibition of transcription and protein expression of COX-2 in the myeloma cells. Proliferation of the transfected cells was inhibited and apoptosis was induced (6.52%±0.32%, 12.53%±2.52%, 24.39%±3.51% and 36.48%±4.96% for 0, 12, 24 and 48h, P<0.01). However, the expression of Bcl-2 and Bax in the RPMI8226 cells had no significant changes after nucleofection.Conlusion: COX-2 siRNA transfection can suppress COX-2 expression in human myeloma RPMI8226 cells, which leads to growth inhibition and apoptosis independently of Bcl-2. PART 3Expression of 12-lipoxygenase in Multiple Myeloma RPMI8226 Cells and Role of Baicalein on the Cell Proliferation and ApoptosisObject: To investigate the expression and role of 12-lipoxygenase in human multiple myeloma RPMI8226 cells and efficacy and mechanisms of baicalein on RPMI8226 cells.Method: Human myeloma cell lines, RPMI8226, KM3 and U266 were culutured and maintained in RPMI1640 medium containing 10% fetal bovine serum. After treatment of various concentrations (20μM, 40μM and 60μM) of baicalein, cell proliferation and viability at different time points were checked by MTT and typan blue staining, cell apoptosis was assayed by Annexin-V/PI with flow cytometry, TUNEL and Wright-Gimesa staining, and cell cycle distribution was checked by flow cytometry. Expression of COX-2 protein and mRNA was checked by Western blot and RT-PCR, respectively.Result: Human myeloma RPMI8226 cells express 12-LOX. Various concentrations from 20 to 60μM of baicalein inhibited the proliferation of RPMI8226 cells, and IC50s were 80, 46 and 30μM for 24, 48 and 72 h, respectively. And metabolite of 12-LOX could reverse inhibitory effect of baicalein on RPMI8226 cells. Significant apoptosis rates happened and typical morphology changes appeared after the treatment of baicalein. Compared with the control (0μM), 20μM, 40μM and 60μM of baicalein could lead to significant G0/G1 phase arrest with a decrease of S phase from 21.5%±5.0% to 5.9%±2.2%, while no changes for G2/M phase cells. The expression of COX-2 in protein was inhibited in a time- and dose-dependent manner significantly (P<0.01, compared with the control) and was nearly completely inhibited after treatment of 60μM of baicalein. 20μM of baicalein could inhibit activation of Erk1/2 in RPMI8226 cells in a time-dependent manner.Result: 12-LOX is expressed by human MM RPMI8226 cells and plays a important role in cell proliferation and apoptosis. Baicalein can lead to inhibited proliferation and induced apoptosis in RPMI8226 cells, which inhibited expression of 12-LOX and activation of Erk1/2 were involved in.