Experimental Study On The Signal Transduction During Leukemic Cell Apoptosis Induced By Iron Chelator

Iron is essential element for life. It plays key roles in many actions such as cellular DNA synthesis, electron delivery, oxygen transportation. Iron is also a required cofactor in many basic metabolic enzymes, including respiratory enzymes and ribonucleotide reductase. So, iron deficiency can result in numerous physiological dysfunction. But, on the other hand, iron overload also does harm to human body and even increase the fatalness of tumor's growth.Apoptosis is closely related to tumors. With the further studies on the mechanisms and gene regulation of tumor cell apoptosis, as well as on the separation and clone of apoptosis- related genes, it may hopefully come trueto artificially regulate tumor cell's apoptosis at molecular level in order to promote the treatment effect of tumors and achieve the purpose to treat and prevent neoplasitc diseases. Our study focuses on exploring the signal transduction pathway during leukemic cell apoptosis induced by iron chelator, and providing the useful experimental data for developing new drugs to treat tumors.Objective: To explore the signal transduction pathway during iron chelator-mediated apoptosis by detecting the expression and activation of associated caspase(caspase-9,-3).Methods: 1.Observe the effect of DFO in different concentrations on the proliferation of K562 cell by MTT assay.2. Observe the effect of DFO on K562 cell's cycle and the action of DFO's inducing apoptosis by FCM and fluorescence microscope.3.Detect the effect of DFO on the expressions of caspase-9 and -3 mRNA by TaqMan Real-time RT-PCR.4. Detect the activation of caspase-9 and -3 induced by DFO among the caspase cascade by Western Blot Analysis.Results: When K562 cell was treated with DFO, the following events occurred: 1 .The cell's proliferation of treatment group is less than that of control group, the higher concentration of DFO, the lower proliferation of K562 cell. 2.Cell's cycle was arrested. Cell without iron is unable to enter S phase from Go/Gi phase. The amount of cells during Gi phase increased and cells in S phase decreased, and cells in G2 phase not too change. The proliferation index of cell decreased along with the increased dosage and prolonged treatment time.3. K562 cell treated with DFO appeared in classical morphological hallmarks of apoptosis after 24 hours. 4. The expressions ofcaspase-9 and -3 mRNA were upregulated by DFO. The peaks of caspase-9 and -3 mRNA expression curve overlap at the 16th hour point. 5.Caspase-9 and -3 were sequentially activated.The non-active procaspase-9,-3 decreased and the active subunits of caspase-9,-3 gradually increased and then the caspase cascade is triggered.Conclusions: 1 .Iron chelator can inhibit cell's proliferation in dosage-dependent.2. Iron chelator can induce both cell cycle arrest and apoptosis by iron deprivation.3.Iron chelator can upregulate caspase-9 and -3 mRNA expression. This may also be a manner for iron chelators to regulate apoptosisAIron chelator can sequentially activate caspase -9 and -3 and then trigger caspase cascade to regulate apoptosis by mitochondria! pathway.