Toxicity And Molecular Mechanism Of Brazilin On Bladder Cancer Cell

Bladder cancer is one of the major diseases, characterized with high incidence and high recurrence rates after operation. Brazilin, which is one of the major ingredient of Caesalpinia sappan. L, has been demonstrated to effectively inhibit the growth of bladder cancer cells by MTT assays. Furthermore, Digital Gene Expression (DGE) profiling was performed to analyze differentially expressed genes of T24 cell before and after brazilin treatment in our previous study. In this research, we first selected (the differently expressed) GADD45β gene based on the DGEs and studied its function by overexpessing GADD45β in T24 cell. Then, the toxicity effect of brazilin on different tumor cells was detected by MTT assay and the expression of c-Fos and GADD45β in different tumor cells after brazilin treatment was also verified. Finally, we isolated mouse embryonic fibroblast cell (MEF) as the normal cell and investigated the safety of brazilin. The main contents were listed as follows:1. Function analysis of GADD45β gene expressionBased on the results of Digital Gene Expression profiling,18 differentially expressed genes involved in apoptosis and growth arrest were selected for quantitative Real-time PCR (qPCR) verification. The nucleotide sequences of these genes were obtained from the NCBI. The results showed that no significant change for mRNA expression of Caspase family and cycle regulatory genes. Growth arrest and DNA damage gene GADD45β were increased 2.7-fold after brazilin treatment for 6h. Therefore, we selected GADD45β for further research. In order to investigate the role of GADD45β in T24 cells, the eukaryotic expression vector with GADD45β gene was constructed. The full-length cDNA of GADD45β was amplified by PCR from T24 cells and cloned into vector pcDNA3.1, named as pcDNA3.1-GADD45β. Then the empty vector and pcDNA3.1-GADD45β was transfected into T24 cells with Liprofectamine 2000. Significant up-regulation of GADD45β mRNA was detected by qPCR(, and the results suggested that the eukaryotic expression vector had entered into T24 cells successfully). Meanwhile, the cell morphology of T24 cells was changed, including cell shrinkage, rounding and adherent deterioration. The viability was reduced to 15.45%. These results suggested that GADD45β may play an important role in T24 cells death.2. Brazilin effect on different cancer cell lines and mRNA expression of c-Fos and GADD45βWe studied the toxic effect of brazilin on human lung cancer cell line Spc-A-1, hepatoma cell line Smmc-7721, cervical cancer cell line Hela229 and mouse melanoma cell line B16 by MTT. The results showed that brazilin effectively inhibits the growth of these cancer cells as a broad-spectrum drug. qPCR analysis showed that c-Fos and GADD45β were highly expressed after brazilin treated 6h in T24 cell. Cell viability was decreased by 66.22% and 85.45% when c-Fos and GADD45β was over expressed in T24 cells respectively. These results suggested that c-Fos and GADD45β may play an important role in T24 cells death. (In order to study whether the pathway of brazilin killing other tumor cells is same to T24 cell,) the mRNA expressions of c-Fos and GADD45βin different tumor cells after brazilin treatment were tested by qPCR. And no significant change of c-Fos was detected for mRNA expression in different tumor cells after brazilin treatment for 6h, while GADD45P in T24, Spc-A-1 and Smmc-7721 were also significantly increased.3. The effect of brazilin on normal cellsIn order to study the effect of brazilin on normal cells, we successfully isolated mouse embryonic fibroblasts (MEF) cell. MTT assay was used to detect the effects of different concentration and different exposure time of brazilin on cell viability in the MEF cell line. The results showed a dose-dependent anti-proliferative effect on MEF. However, the viability of MEF were significantly higher than T24 cells at each concentration of brazilin and the relative activity of MEF was 60% higher than that of T24 cells when the treated with 32μg/mL for 6h, indicating that the toxicity of brazilin is less significant to normal cells than T24 cells. Brazilin is potentially a safe drug.