The Study Of Brazilin Induced Bladder Cancer T24 Cells Necroptosis

Bladder cancer was the major cancer in the urinary system,with the characteristics of high incidence and recurrence.Irrigation was the main prevention methods of postoperative recurrence for bladder cancer patient.Brazilin,the effective ingredient of Su Funing lotion could effectively prevent postoperative recurrence of bladder cancer and kill bladder cancer T24 cells.However,the broad-spectrum and safety of Brazilin and the death mechanism of bladder cancer cells treated by Brazilin remain unclear.In this research,the different kinds of bladder cell lines were used to test the broad-spectrum and safety of Brazilin.We detected the overlay proliferation properties,lactate dehydrogenase,calcium,mitochondrial membrane potential of T24 cells to further clarify the pharmacological toxicity of bladder cancer treated by Brazilin.We found that Brazilin killed T24 cells by necroptosis based on the treatment of different death inhibitor,combing with the enzyme activity assay and transmission electron microscopy.Finally,we proposed that Brazilin caused T24 cell necroptosis mediated by RIP1/RIP3/MLKL complex by using gene expression profiles analysis and rear-time qPCR assay of T24 cells treated by brazilin.There are four research contents:Firstly,the low serum test showed that Brazilin could kill bladder cancer cells T24,5637 and J82 cell and proved that the potential of Brazilin as a broad-spectrum therapy for bladder cancer.We used normal bladder epithelial SV-HUC-1 cells and bladder cancer T24,5637 cells to carry out the cytotoxicity test.Compared with T24 and 5637 cells,the cytotoxicity of Brazilin on SV-HUC-1 cells was lower.The results showed that Brazilin was relatively safe as a bladder cancer drug.Secondly,we detected some physiological and biochemical properties of T24 cells after Brazilin treatment,and found that Brazilin could inhibit proliferation and superposition of T24 cells,change the cellmembrane permeability to make the stable lactate dehydrogenase release into the solution,increase the concentration of Ca2+,and decrease the mitochondrial membrane potential Involved in cell metabolism significantly.Thirdly,we utilized inhibitors of different death modes,cysteine protease activity assay and morphological observation to screen the death mode of Brazilin killing T24 cells.Apoptosis inhibitor z-VAD-fmk could not inhibit the T24 cell death leading by Brazilin,and Caspase enzyme could not be activated by Brazilin.These results indicated that Brazilin killing T24 cells not through the Caspases induced apoptosis.Meanwhile,both autophagy inhibitor 3-MA and necroptosis inhibitor Nec-1 could inhibit Brazilin killing T24 cells,combined with the morphological characteristics of necroptosis in T24 cells observed by transmission electron microscopy,indicating that Brazilin killed T24 cells by necroptosis.Fourthly,we found the expression of RIP1/MLKL were up-regulated in Digital gene expression data after the half lethal concentration of Brazilin treatment.Moreover,the real-time quantitative PCR also showed the necroptosis related gene RIP1/RIP3/MLKL were over-expression.These results indicated that Brazilin killed T24 cells through RIP1/RIP3/MLKL compex mediated necroptosis.