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The Interaction of Cathepsin D and Bcl-X_L in Apoptosis of Chronic Myelogenous Leukemia K562 Cell Induced by Glucosamine Sulfate

Author: ZuoDanDan
Tutor: ZuoQingGuo
School: Fourth Military Medical University
Course: Pathology and Pathophysiology
Keywords: Glucosamine sulfate Chronic myeloid leukemia Apoptosis Cathepsin D Bcl-XL
CLC: R363
Type: Master's thesis
Year: 2010
Downloads: 27
Quote: 0
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Abstract


Background cell is the structural and functional units of the basic body, coordinated with each other in the process of metabolism, wherein the apoptosis (apoptosis) has an important role for the regulation of tissue development and homeostasis. Apoptosis is a form of cell death, a basic phenomenon of life, a variety of physiological processes involved in the body's growth and development, and aging, is also involved in the pathogenesis of many diseases, including tumors, including important factors. Apoptotic signal transduction and molecular regulation mechanism is a very important problem in the field of life science research in many diseases, including tumors, including the development and progression. Apoptotic signal transduction pathways have been clear, lysosomal-mediated apoptosis vital structures, and different induction factors trigger apoptosis, apoptosis signal transduction pathways differ, but in a multi- kinds of incentives between lysosomal and mitochondrial apoptotic signal transduction mechanisms are not yet clear. This experiment was carried out on the basis of preliminary studies, previous we found that glucosamine sulfate (Glucosamine Sulfate, GS) can induce apoptosis of K562 cells and apoptosis through the release of lysosomal pathway of Cathepsin D (Cat D) mediated, and is associated with down-regulation of Bcl-XL, but Cat D signal from lysosomes to mitochondria, inhibition of apoptosis and mitochondrial surface protein Bcl-XL has what kind of relationship is still unknown. Whether the interaction on the basis of clear apoptosis lysosomal release of Cat D and the outer mitochondrial membrane expression of Bcl-XL, Bcl-XL is for lysosomal Cat D with mitochondrial apoptotic signal transduction new path downstream substrate molecules, we carried out a study of their mechanism of action. The purpose of the establishment of glucosamine sulfate (Glucosamine Sulfate, GS) K562 cell apoptosis induced by chronic myelogenous leukemia (Chronic Myeloid Leukemia CML) model, containing 5.0 mmol.L-1GS K562 cells induced by immunofluorescence and Western Blot Cat D RNA interference method detection lysosomal release mitochondrial outer membrane Bcl-XL, and explore the model of apoptosis of K562 cells, Cat D and mechanism of action of Bcl-XL. Method 1. Apoptosis of chronic myelogenous leukemia K562 cells before and after the MTT assay cell activity before and after apoptosis; apoptosis of K562 cells before and after HE staining the, Hoechst33342 detect changes in cell morphology; applications the DNA ladder detect cell apoptosis; application flow cytometry detection induced cell state. The application of laser confocal microscopy double labeling immunofluorescence and Western blot detection of apoptosis before and after the application Pepstatin A suppression mechanism of interaction of the cells before and after the Cat D Cat D and Bcl-XL. SANTA company purchased specificity Cat D and Bcl-XL small interfering RNA and negative RNA. Cationic liposomes siRNAs were transfected into K562 cells, inverted cell morphology was observed under a microscope, double immunofluorescence staining of laser confocal imaging, reverse transcription PCR to detect the transfection and then use the GS treatment before and after 48h after Cat D Bcl-XL mRNA expression; immunoblotting techniques Cat D cells transfected with siRNAs, GS before and after treatment and Bcl-XL protein expression was measured; 1.0.5 mmol.L-1, 1.0 mmol.L-1, 5.0 mmol.L-1 GS-induced showed cytostatic most 5.0 mmol.L-1 GS of inhibition. HE staining control group K562 cells were round, smooth border, membrane shrinkage, while 5.0 mmol.L-1 GS after 72 h of treatment, cell shrinkage, membrane is not smooth, and cell vacuoles phenomenon budding phenomenon . 5.0 mmol.L-1 GS for 72 h after Hoechst33342 staining showed the presence of apoptotic cells. 5.0 mmol.L-1 GS 72 h after treatment on the DNA ladder electrophoresis picture clear bands of 180-200bp; control K562 cells in flow cytometry, little apoptosis, spontaneous apoptosis of the tumor cells, After 5.0 mmol.L-1 GS-induced K562 cells after 72 h, apoptosis increased significantly. K562 cells compared with untreated 5.0 mmol.L -1 GS 72 h after induction of apoptosis rate 3.8% becomes 12.4%, P lt; 0.05. Induced K562 cells after 72 h after 5.0 mmol.L-1 GS Cat D and Bcl-XL confocal laser signal is significantly enhanced, Cathepsin D protein expression increased, decreased expression of Bcl-XL protein. K562 cells were treated with Pepstatin A, then 5.0 mmol.L-1 GS induced 72 h than GS treated Cat D expression was decreased significantly enhanced co-localization signal of Cat D and Bcl-XL signal than before, Cat The D protein decreased expression of Bcl-XL protein expression did not show a significant difference. 3 liposomal transfection interfering RNA immunofluorescence after double labeled laser confocal display: K562 cells were transfected Cat D siRNA and Bcl-XL siRNA group the signal suppression transfection significantly decreased in the co-localization signal. 5.0 mmol.L-1 GS induced 72 h were transfected with the Cat D siRNA and Bcl-XL siRNA group, Cat D and Bcl-XL common the positioning signal CatD relative enhancement. Compared with transfection of K562 cells, GS before and after induction of apoptosis Cat D protein expression increased while no significant differences in protein expression of Bcl-XL. Conclusion 1. Successfully constructed Glucosamine sulfate-induced apoptosis of chronic myelogenous leukemia model. The 5 mmol.L-1, GS-induced K562 cells; 2.5 mmol.L-1, GS-induced apoptotic cells Cat D colocalization of Bcl-XL signal is significantly enhanced. Cat D protein expression of Bcl-XL protein expression decreased. Pepstatin A inhibits Cat D, then 5 mmol.L-1 GS induction with a simple 5 mmol.L-1, GS K562 cell can be seen Pepstatin A suppressed, Cat D and Bcl-XL co-localization of the signal appeared Cat D signal enhancement. Cat D protein decreased expression of Bcl-XL protein expression did not show a significant difference. 3. Liposomal transfection interfering RNA Cat D and Bcl-XL after both co-localization signal weakened. Cat D after interference, the colocalization signal performance of the Cat D and Bcl-XL Cat D decreased expression of Bcl-XL relatively enhanced; interfere with Bcl-XL, Cat D expression relative enhancement. Interfere with the expression of the Cat D both before and after the Bcl-XL protein is governed by inhibition of 5 mmol.L-1, GS-induced enhanced expression of the Cat D, and Bcl-XL did not change significantly.

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