| Objective:Multiple myeloma(MM)is a neoplasm derived from plasma cell. The remission rate and median survival time of patients treated with conventional chemotherapy are low. Although hematopoietic stem cell Transplantation and new chemotherapy drugs such as proteasome inhibitor are effective in prolonging survival, MM is still an incurable hematological malignancy.Norcantharidin (NCTD) is an important derivant of cantharidin which extracted from blister bug. It has been confirmed that NCTD could inhibit the proliferation of several tumor cell lines in vitro. In the present study we investigated the effects of NCTD on human MM cell line U266 and the possible mechanisms of NCTD. We also studied the synergistic anti-tumor effect of NCTD with adriamycin (ADM) on human MM cell line U266. The purpose of this study is to provide experimental evidence for clinical application of NCTD in patients with MM.Methods1. The growing inhibition of NCTD and/or ADM on U266 cells at different concentrations was detected by MTT assay.Human MM U266 cells were cultured and treated by NCTD with different concentrations (0, 2.5, 5, 10, 20, 40 and 80μmol/L) for 48h. U266 cells were treated by NCTD(0,2.5,5 and 10μmol/L)combined with ADM (0, 0.25, 0.5, 1, and 2μmol/L) for 48h. Cell growth inhibition was detected by MTT assay and then calculated using half inhibition concentration (IC50). 2. After treating U266 cell with different concentrations of NCTD and/or ADM, the change of the cell density cell morphology, and structure were observed by inverted phase contrast microscope.3. Detection of cells apoptosis by AnnexinⅤ/ propidium iodide (PI) flow cytometry.Human MM U266 cells were cultured and treated with NCTD 5μmol/L and ADM 0.25μmol/L alone or in combination for 48h then collected. AnnexinⅤand PI were added into culture plate. Apoptosis was determined quantitatively using flow cytometry4. Determination of caspase-3, caspase-8 and caspase-9 in U266 cells by spectrophotometry.Human MM U266 cells were treated with NCTD using IC50 for different times (12h, 24h, 36h, and 48h), The expression of caspase-3, caspase-8, and caspase -9 in these cells was detected by spectrophotometry.5. Detection of the expression of survivin protein using immunohistochemical staining.Human MM U266 cells were treated with NCTD using IC50 for different times (12h, 24h, and 48h). Immunohistochemical staining was used to detect the expression of survivin protein in these cells.Results:1. The effects of NCTD on U266 cells.Our results showed that NCTD could inhibit the growth of U266 cells in vitro with a dose-dependent manner. The IC50 of NCTD to U266 cells at 48h was 22.046μmol/L.2. The synergistic effect of NCTD and/or ADM on U266 cells.(1) The changes of IC50 in different groups: The IC50 of ADM to U266 cells at 48h was 1.23±0.06μmol/L. When cultured with NCTD at different concentrations (2.5, 5 and 10μmol/L), it was declined to 1.02±0.11 (P<0.05), 0.74±0.07 (P<0.05) and 0.46±0.04μmol/L (p<0.05), respectively.(2) The changes of apoptosis in different groups: The apoptosis rate in combined group (44.3±2.20%) was significantly higher than that of NCTD group (12±1.21%), ADM group (14.2±3.51%) and control groups (2.8±0.43%) (p<0.05).(3) Morphology of apoptosis: The apoptosis ratio was significantly higher in combined group than that of ADM group, there was no changes of apoptosis ratio in control group for a better cell vigor.3. The effect of NCTD on the expression of apoptosis related proteins in U266 cells.NCTD could increase the activity of Caspase-3, -8 and -9 proteins under limit active time. The activity of caspase protein was positively correlated with action time of NCTD. The changes of activity of Caspase-3, -8, and -9 proteins showed obvious time-effect relationship. The results also showed that the expression of apoptosis related proteins in U266 cells changed obviously in the IC50 of NCTD treated cells for different times (12h, 24h, and 48h). Survivin expression rate in treatment group were lower than that in control group (P<0.05), the expressions rate of survivin decreased obviously when treated time was adequate.Conclusions1.NCTD showed a growth inhibition effect on U266 cells with a dose-dependent manner. The inhibition effect could be related to the apoptosis effect of NCTD.2.Our results suggest NCTD can enhance the anti-myeloma effect of ADM.3.The apoptosis of U266 cells could be related to up-regulation of caspase-3,-8,-9 expression, and down-regulation of survivin expression. |
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