| Multiple myeloma (MM) is a B-cell malignancy characterized by the latent accumulation in bone marrow of secretory plasma cells with a low proliferative index and an extented life span. The median age of MM patients is 50 to 60 years with a natural course of half to one year. The routine treatment is combination chemotherapy. But patients often couldn' t tolerate such intensive treatment because of the age and the primary disease.Curcumin (diferuloylmethane) is a potent antioxidant and chemopreventive agent derived from the plant Curcuma longa. The antioxidant and chemopreventive effects were remarkably confirmed to be safety. So curcumin has been considered by oncologists as a potential third generation cancer chemopreventive agent. More and more researchers paid close attention to it. We have domestrated that curcumin could induce apoptosis in human acute, chronic leukemia cell lines and lymphoma cell line (HL-60, K562 and CA46 cells). At the same time we have domestrated that the curcumin-induced K562 cells apoptosis correlated with down-regulation of the abundance of P210bcr/ebl, which might ultimately lead to retardation of the Ras signal transduction pathway. On the basis of that previous study we chose U266 cells as objective and tried to reveal the effect of curcumin on it. There are few reports about the effect of curcumin on MM cells, apart from the effect of curcumin on nuclear factor(NF), Iκ Bα kinase and IL-6 inducible STAT3 Phosphorylation of MM cells internationally. The related report of curcumin on MM cells is not found domestically.Major researches we took: The growth inhibition of U266 cells by curcuminwas analyzed using cell growth curve, MTT assay and colony forming assay. Apoptotic cells were detected by staining with AO-EB ( acridine orange-ethidium bromide), and confirmed by transmission electron microscopy (TEM), flow cytometry analysis, DNA fragmentation analysis and TdT-mediated dUTP nick end labeling(TUNEL) assay. The mitochondrial transmembrane potential was detected by flow cytometric analysis. The expression of mRNA was determined by reverse transcript-polymerase chain reaction (RT-PCR) assay. The expression of protein was detected by ELISA assay, flow cytometric analysis and Western blot technique. The activity of telomerase in U266 cells was detected by the Telo TAGGG Telomerase PCR ELISA assay.Major results we achieved: 1. Curcumin could inhibite U266 cells proliferation significantly in time and dose dependent manner. 2. Curcumin can induce apoptosis in U266 cells as follows: ①Staining of cells with AO-EB revealed that curcumin induced nuclear chromatin condensation and nuclear fragmentation. ②Apoptotic body and swollen bioblast were observed by TEM. ③Sub-G1 fraction was detected by flow cytometry analysis. ④Agarose gel (1.5%) electrophoresis also showed that cells treated with curcumin induced internucleosomal DNA fragmentation in the form of a laddering pattern. ⑤ TUNEL detection showed that curcumin induced apoptosis of U266 cells in a time-depended manner, and the percentage of apoptosis was enhanced from 13. 4±0. 64% to 46. 2 ±1. 31%. ⑥Flow cytometry analysis showed that the ratio of changed mitochondrial transmembrane potential in U266 cells treared with curcumin was higher than that of control group. 3. Curcumin could regulate the expression of apoptosis-related genes: RT-PCR analysis showed that the expression of c-myc, bcl-2, bcl-xl, mcl-1, c-fos, c-Jun, VEGF and hTERT was down-regulated in U266 cells treared with curcumin, whereas the expression of bax, WTp53, TGF-β1 and fas was up-regulated. 4. Curcumin could regulate the expression of apoptosis-related proteins: ①ELISA analysis showed that the level of autocrine TGF-β1 raised dramatically in U266 cells treared with curcumin and the level of autocrine IL-6 descended slightly. ②Western blotanalysis showed that the protein expression of C-myc and C-fos was down-regulated in U266 cells treared with curcumin but the protein expressions of Bax and Caspase3 were advanced. ③Flow cytometry analysis showed that the masculine rate of bcl-2 in U266 cells treared with curcumin was decreased evidently, however, the masculine rate of fas in U266 cells treared with curcumin was conspicuously. 5. Curcumin could influence the quant of telomerase activity in U266 cells: PCR ELISA assay showed that the quant of telomerase activity in U266 cells treated with curcumin was decreased sharply.Conclutions: Curcumin could inhibite proliferation of U266 cells and induce its apoptosis. The mechanism by which curcumin induced the apoptosis of U266 cells may involve modulation of the expression of c-myc, bcl-2, bcl-xl, mcl-1, c-fos, c-Jun, VEGF, hTERT, bax, WTp53, TGF-β1 and fas. |
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