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• W151907173 abstract "Multiple myeloma (MM) is a uniformly fatal disease characterized by a clonal proliferation of neoplastic plasma cells in the bone marrow and is the second most commol hematological malignancy.1 At present, a cure for multiple myeloma has not been achieved with chemotherapeutic regimen, and many patients die of drug-resistant disease. Thus, the development of new effective anticancer agents to treat both early and advanced MM seems to be an attractive perspective. Chinese herbal medicines are treasures inherited from our ancestor, and many constituents of herbs had been shown great effects on many kinds of diseases. Among them, many herbal medicines have been used historically in anti-inflammatory therapy. As an important cytokine in the development of inflammatory, IL-6 has been confirmed to be one of the most essential mediators during MM pathogenesis,2 and it is believed that both inflammatory and MM may be induced partly in the same way. Thus, we have kept trying to find some anti-inflammatory Chinese herbs that could treat MM. Baicalein (5,6,7-trihydroxyflavone) is a flavonoid derived from the root of Scutellaria baicalensis or Huang Qin, a widely used Chinese herbal medicine, which has used historically in anti-inflammatory therapy, and has been confirmed to inhibit the activity of 12-lipoxygenase (12-LOX) that is overexpressed in tumor tissues such as prostate cancer, breast cancer and so on. 12-LOX inhibition can induce apoptosis and inhibit proliferation of many human cancer cells.3–5 However the expression 12-LOX in the myeloma cells has not been reported. In this study we try to examine whether Baicalein has effect on apoptosis and proliferation of myeloma cells, if 12-LOX is expressed by them and whether Baicalein can inhibit the expression of 12-LOX in MM cells. METHODS Cells and groups Human myeloma cell line (RPMI8226), obtained from America Type Culture Collection (ATCC, USA), was cultured in RPMI-1640 medium (Gibico, USA) supplemented with 10% fetal bovine serum (Gibico, USA). Cells were grown at 37°C in a humidified 5% CO2 atmosphere, and then were treated with various concentrations of Baicalein at 70% — 80% confluence. Cells were divided into four groups with different concentrations of Baicalein at 0, 20, 40 and 60 μmol/L, respectively. Baicalein, purchased from Sigma (USA), first dissolved in DMSO and then serially diluted in RPMI-1640 medium immediately prior to experiments. Proliferation assay Cells were seeded onto 96-well plates (200 μl/well) and treated with 0, 20, 40 or 60 μmol/L of Baicalein for 24, 48 and 72 hours, respectively. Subsequently, 20 μl of MTT at a concentration of 5 mg/ml was added to each well, and cells were incubated with for an additional 4 hours at 37°C. After a brief centrifugation, supernatants were carefully removed and 200 μl DMSO was added to the cells. After insoluble crystals were completely dissolved, absorbance at 490 nm was measured using an ELx800 reader (Bio-Tek Instruments, USA), and optical density (OD) value was obtained to determine viable cells. For trypan blue exclusion assay, cell suspension was mixed with the same volume of 0.4% trypan blue solution; cells were counted using light microscopy and cell viability was estimated by the percentage of the number of the unstained in comparison with the untreated control. All experiments were repeated in triplicate. Apoptosis assay At first, apoptosis of cells treated with various concentrations of Baicalein at different time points was estimated using an Annexin-Vapoptosis assay kit (Bender Medsystems, USA) by flow cytometry (FACScan, Becton Dickinson, USA). Then, apoptosis morphological changes were determined by acridine orange (AO) and EB staining. One μl AO/EB (AO: 100 μg/ml; EB: 100 μg/ml) was mixed with 25 μl treated or untreated cells of 1×106/ml, and then 10 μl was tested by fluorescence microscope (Olympus, Japan) immediately. For quantification, three different fields were counted under the microscope and at least 300 cells were enumerated in each field. All experiments were repeated in triplicate. Cell cycle analysis by flow cytometry Cell cycle analysis was performed on the cells incubated for 48 hours. The cells were fixed in chilled 70% ethanol for at least 24 hours before staining with 50 μg/ml propidium iodide and 100 μg/ml RNase A (Sigma, USA) and were analyzed using FACScan (Becton Dickinson, USA) 30 minutes after staining. All experiments were repeated in triplicate. Western blotting Treated cells (1 × 106) were washed twice with PBS and lysed in ice-cold cell lysis buffer [50 mmol/L Tris (pH 7.2), 150 mmol/L NaC1, 1 mmol/L ethylene-diaminetetraacetic acid, 1% Triton X-100, 2.5 mmol/L sodium pyrophosphate, 1 mmol/L sodium orthovanadate, and 1 μg/ml leupeptin] containing 1 mmol/L phenylmethylsulfonyl fluoride. Equivalent amounts of proteins (20 μg) were boiled in Laemmli sample buffers and fractionated on 10% polyacrylamide gel electrophoresis (PAGE) with sodium dodecyl sulfate (SDS). Seperated proteins in gel were transferred onto PVDF membranes by an electroblot apparatus (Bio-Rad, USA). Filters were blocked for 2 hours in PBS, containing 0.1% Tween 20 (PBS-T) with 5% low fat milk and were then incubated with goat anti-rabbit antibody for platelet-type 12-LOX and goat anti-actin (Santa Cruz, USA) at 4°C for 12 hours. Membranes were then washed with PBS-T and incubated with HRP-conjugated anti-goat antibody or anti-rabbit antibody for 1 hour. Specific signals were detected on X-ray films using an enhanced chemiluminescene detection system (ECL, Super signal Pierce; USA). Blots were then stripped and reblotted with anti-β-actin antibody to ensure that equivalent levels of proteins were present in each lane. Human platelet-type antigen acted as a positive control. Statistical analysis Data were expressed as mean ± standard deviation (SD). Statistical analysis was performed using F test and q test by SPSS10.0, and statistical significance was defined as P<0.05. RESULTS Growth inhibition of Baicalein on RPMI8226 cells The effects of the specific 12-LOX inhibitor Baicalein on proliferation of myeloma cells was determined by MTT colorimetric survival assay (Fig.1A). Inhibitory effects on proliferation were also confirmed by the trypan blue dye exclusion method (Fig.1B). Treatment with Bacalein inhibited growth of RPMI8226 cells in a time and dose-dependent manner. IC50 were 80 μmol/L for 24 hours, 46 μmol/L for 48 hours, and 30 μmmol/L for 72 hours, respectively.Fig.1.: Inhibition of cell proliferation by Baicalein, group of without Baicalein-treated as control. A: After culture in medium with Baicalein for 24, 48, or 72 hours, cell growth was measured by MTT assay. B: Viable cell number was determined by trypan blue dye exclusion, and cell viability is shown as the percent of control. Compared with the control, AP<0.01.Baicalein induced apoptosis of RPMI8226 cell line To clarify the mechanism of growth inhibition induced by Baicalein, apoptosis of cells were studied by flow cytometry using Annexin-V and PI staining. Baicalein caused cell apoptosis in a dose-dependent manner (Fig.2). Baicalein-induced apoptosis was also evaluated by AO/EB staining, which showed that RPMI8226 cells underwent typical apoptotic morphological changes of nuclear and cytoplasmic condensation, loss of cell volume, and nuclear fragmentation upon treatment with 40 μmol/L Baicalein for 48 hours.Fig.2.: Baicalein induces significantly apoptosis in RPMI8226 cells in a dose-dependent manner. *P<0.01 compared with the control group.Cell cycle arrest Compared with the control of (71.5 ± 12.1)%, 20 to 60 μmol/L of Baicalein caused a significant G0/G1 phase arrest of (79.4 ± 15.2)% to (83.4 ± 15.6)% (P<0.05) and concurrent decrease of S phase from (21.5 ± 5.0)% to (5.9±2.3)% (P<0.01) in a dose-dependent manner. No significant change for G2/M phase cells was observed. Western blot of 12-LOX protein expression A decrease of 12-LOX expression occurred after incubation with Baicalein, and this decrease was caused by Baicalein in a time- and dose-dependent manner (P<0.01 compared with the control). The expression of 12-LOX protein was nearly inhibited completely in 60 μmol/L Baicalein group compared with the control. DISCUSSION In this study, we found that Baicalein inhibited proliferation and induced apoptosis of RPMI8226 cells in a time- and dose-dependent manner and caused G0/G1phase arrest, which show that cell arrest is involved in inhibiting growth of myeloma cells. This is the first assessment of 12-LOX expression in MM. The molecular study of the well-known relationship between polyunsaturated fatty acid metabolism and carcinogenesis is revealing novel molecular targets for cancer chemoprevention research. The cyclooxygenase (COX)s and LOXs are two important enzyme classes that metabolize polyunsaturated fatty acids and affect carcinogenesis. 12-LOX is expressed in a variety of human tumor cells including leukemia cells6 and contributes substantially to such tumorigenic events as carcinogenesis, invasion, metastasis, and angiogenesis.7–11 However, it is unclear whether 12-LOX is expressed in human MM cells and what role 12-LOX plays in myeloma cells. We confirmed the expression of 12-LOX protein in RPMI8226 cell line and Baicalein was able to inhibit 12-LOX protein expression in a time- and dose-dependent manner. Since Baicalein has been confirmed to be a specific inhibitor of 12-LOX, and 12-LOX plays a very important role in many tumors,7–9,12 additionally, Baicalein was able to inhibit proliferation and induce apoptosis of human myeloma RPMI8226 cells while inhibited expression of 12-LOX protein in a time- and dose-dependent manner, it is of possibility that the effects of Baicalein on myeloma cells are related with its inhibiting 12-LOX protein expression as a specific 12-LOX inhibitor. Baicalein induces apoptosis through the activation of caspase-3 in human leukemia HL-60 cells in which 12-LOX has been identified.13 Caspase-3 is involved in the apoptosis mechanisms of Baicalein-treated myeloma cells in our research (data not shown). Another report suggests that 12-LOX activates NF-kappaB in prostate cancer cells,14 and Baicalein-induced downregulation of 12-LOX expression possibly attenuates transcription factor NF-kappa B activity in myeloma cells, and this can reduce IL-6 in MM as a result. This is in agreement with the present reports.15,16 COX-2 has been reported to be frequently expressed in MM and is an independent predictor of poor outcome,17 and Baicalein suppressed activity of COX-2 followed by the inhibition of prostaglandin E2 and cell proliferation in cancer cells,18 so we remain to investigate if Baicalein could influence COX-2 expression in myeloma cells. LOX family includes 5-LOX, 8-LOX and 15-LOX besides 12-LOX, we keep trying to find out the exact role of 12-LOX and if other LOXs have been involved in. After all, results presented in this report suggest clearly that Baicalein can directly inhibit proliferation of myeloma cells, and down-regulate 12-LOX protein expression which is related with the effects of Baicalein on myeloma cells. Huang Qin has been historically used in China without any obvious side-effect, and our findings have confirmed Baicalein has no toxic effect on normal bone mononuclearcell (data not shown), Baicalein is suitable for clinical trial in MM and 12-LOX is worthy of further investigation as a new important cure target for MM." @default.
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• W151907173 title "Role of Baicalein in the regulation of proliferation and apoptosis in human myeloma RPMI8226 cells" @default.
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