Abstract
Purpose: To investigate the effect of bryostatin on the proliferation of ependymoma cells, and the underlying mechanism(s).Methods: Ependymoma cell lines (SC-EPN1 and SC-EPN2) were cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10 % fetal bovine serum (FBS) and streptomycin (10 mg/ml) in a humidified incubator at 37 °C and 5 % CO2 atmosphere. Rhe cells were randomly assigned to six groups: control group and five bryostatin groups treated with increasing concentrations of bryostatin (10 - 50 μM). Cell proliferation was determined by MTT assay, while real-time quantitative polymerase chain reaction (qRT-PCR) was used to determine the levels of expressions of apoptosisrelated genes. Expressions of cyclooxygenase-2 (COX-2), interleukin-8 (IL-8), Bcl-2, Bax and Pglycoprotein were determined using Western blotting.Results: Treatment with bryostatin significantly and concentration-dependently down-regulated COX-2 and IL-8 mRNAs expressions (p < 0.05). On the other hand, proliferation of SC-EPN1 and SC-EPN2 cells were significantly and concentration-dependently inhibited by bryostatin, relative to control group (p < 0.05). After 72 h of treatment with bryostatin (50 μM), the extent of apoptosis was significantly higher in SC-EPN1 (57.43 %) and SC-EPN2 cells (52.29 %) than in control group (2.37 %, p < 0.05). The results of Western blotting showed that treatment with bryostatin significantly reduced the expressions of Bcl-2 in ependymoma cells, relative to the control group (p < 0.05). However, there were no significant differences in the expression of Bax among the groups (p > 0.05). P-glycoprotein expression was significantly higher in bryostatin groups than in control group (p < 0.05). The results of flow cytometric analysis of rhodamine-123 (rh123) fluorescence showed that after 72 h of treatment with bryostatin (50 μM), rhl23 fluorescence significantly decreased in SC-EPN1 (8.10 %) and SC-EPN2 cells (10.11 %), relative to control group (20.83 %, p < 0.05).Conclusion: Bryostatin exerts anti-proliferative and apoptotic effects on ependymoma cells by suppressing COX-2 and IL-8 expressions. Thus, the inhibition of COX-2 expression may constitute an effective chemotherapeutic strategy for ependymoma treatment.Keywords: Bryostatin, Ependymoma cells, Proliferation, Apoptosis, Expression
Highlights
Ependymoma is the third most common childhood tumor, and it accounts for 15 % of brain tumors in children [1,2]
The RNAs were maintained in diethyl pyrocarbonate (DEPC) water, and their concentrations determined spectrophotometrically
Results from Western blotting showed that treatment with bryostatin significantly reduced the expressions of Bcl-2 in SC-EPN1 and SCEPN2 cells when compared with the control group (p < 0.05)
Summary
Ependymoma is the third most common childhood tumor, and it accounts for 15 % of brain tumors in children [1,2]. After 24 h of incubation, the cells were treated with varied concentrations of bryostatin (10 - 50 μM) and cultured for 72 h. This was followed by the addition of 20 ml of 0.5 % MTT solution within 4 h, after which the culture medium was changed. The cells were incubated for 72 h, harvested and subsequently treated with HEPES binding buffer containing Annexin V-FITC and PI. The expressions of COX-2, Bcl-2, Bax, and Pglycoprotein in SC-EPN1 and SC-EPN2 cells treated with bryostatin (40 and 50 μM) were determined using Western blotting. The cells were further incubated in HEPES-buffered medium supplemented with 1 μM rh123 They were thereafter treated with bryostatin (40 and 50 μM) or DMSO (control group) for 72 h, and incubated with calcium ion-free phosphate buffer containing trypsin. Values of p < 0.05 were considered statistically significant
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