| Brazilin is a bioactive molecle isolated from Caesalpinia sappan wood. It has shown promising anti-tumor effects in certain cancer cell types. However, the molecular mechanisms of the inhibitory effect of brazilin on tumor cell have not been fully undertstood. In this study, the inhibitory effect of brazilin in human bladder cancer T24cells was investigated using MTT assy and flow cytometry. The digital gene expression profiling was used for investigate the inhibitory mechanism of brazilin on T24cells. The study will provide theoretical foundation for identification of new drug targets and development of noval anti-cancer drugs.1. Cytotoxicity assay of brazilin in human bladder cancer T24cellsMTT assay was used to measure cell growth and cytotoxicity. Cells were treated with different concentrations of brazilin for6hours, resulted in a dose-dependent antiproliferative effect. The antiproliferative effect was reaching the plateau at64μg/mL. The calculated LC50was32μg/mL. Treatment of T24cells with brazilin dosage of32μg/mL for different time resulted in a time-dependent antiproliferative effect. The result indicated that the inhibitory effect of brazilin on T24cells is dose-dependent and time-dependent.Apoptosis and cell cycle progress was evaluated by flow cytometry. The results demonstrated that brazilin has inhibitory effect on T24cells and numbers of live cells were significantly decreased with brazilin treatment. The cell cycle was arrested at G2period.2. Analysis of gene expression by digital gene expression profilingWith the development of next-generation sequencing technology, the Digital Gene Expression (DGE) tag profiling allows us to analyze gene expression more efficient, with accurate level changes and in full scale. We analyzed differentially expressed genes with brazilin treatment at dosage of32μg/mL. Cells were treated with32μg/mL brazilin for6hours, with saline treatment as a control. The total RNA was isolated from control and treated cells. Thhe quality of RNA was confirmed and then used for Digital Gene Expression Profiling analysis. The result showed that521genes were up and968genes were down regulated. Classification analysis of differentially expressed genes has identified that17genes are associated with apoptosis and21genes with cell cycle. The combined studies of GO and pathway analysis results have helped to select17differentially expressed genes for further validation using Real-time PCR.3. Validation of differentially expressed genes by Real-time PCRBased on the results from DGE Profiling,17genes were found strongly associated with the inhibitory effect of brazilin and were selected for further validation by Real-time PCR. The results indicated that there are no significant changes in mRNA expression levels of Caspase3, Caspase8, Caspase9, CycD, CycE, CDK2, CDK4and CDK6. The gene in death receptor-mediated apoptosis pathway TRADD was highly upregulated with brazilin treatment, while no significant changes in FADD and FAS. Transcription levels of HSP40and HSP70A were upregulated with3h and6h brazilin treatment, and HSP70B with3h,6h and12h brazilin treatment. HSP47mRNA levels did not change significantly. c-Fos were also upregulated after3h,6h and12h brazilin treatment. c-Fos was dramatically increased32folds with6h brazilin treatment and therefore, selected for further investigation.4. Functional analysis of the differentially expressed gene c-FosReal-time PCR analysis showed that c-Fos was highly expressed with6h brazilin treatment. Similar result was observed when analyzed by western blot. The c-Fos over-expression plasmid was co-transfected with GFP expression vector into T24cells. Analysis by Real-time PCR and western blot showed that transfection of c-Fos led to a significant upregulation of both c-Fos mRNA and protein levels in T24cells. Apoptosis event was investigated afer the co-transfection. The result demonstrated that cell morphology changed dramatically with the c-Fos over-expression, which includes cells shrinkage and rounding, adherent deterioration, and at the same time, the number of the cell were decreased significantly. To evaluate the cytotoxic effect of c-Fos overexpression on T24cells, viability of the cells was measured by a MTT assay. The results indicated that T24cell activity was significantly reduced to33.8%. Our preliminary results showed that overexpression of c-Fos led to decrease of cell viability and cell death. These results may suggest that c-Fos may be a important mediator and plays critical roles in T24cell survival under brazilin treatment. |
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