Molecular Mechanisms Of Betulin-induced Apoptosis In A549 Cells

Malignant tumor is a leading killer of human health in the world, and its morbidity is markedly increasing in a long peried. Among which,Lung cancer is the first cause of human death. The standard treatment for most tumors continues to be surgery,radiation and chemotherapy. Chemotherapy - the use of medications to treat cancer - has played a major role in cancer treatment for half a century. There is several medication for chemotherapy, for example, etoposide, taxinol and G-Rh2. Years of research and testing have proved chemotherapy to be an effective method for cancer therapy. However, chemoresistance and the damage for normal cells are the major causes of cancer treatment failure. To investigate new medications has become to be hot spots to overcome tumor resistant and enhance sensitivity of anticancer reagents to tumor cells .Betulin is an abundant, naturally occurring triterpene that is predominantly found in bushes and trees, it forms the principal extract (up to 30% of dry weight) of the bark of birch trees. Here is its structure: Betulin can be used as a starting compound for other useful materials and compounds with various interesting pharmacological properties. Its structurally-related derivatives, particularly betulinic acid, have been demonstrated to be cytotoxic drugs and exhibit antitumor activity against many types of tumors. However, the cytotoxicity of betulin has rarely been reported. The aims of the our study were, therefore, to elucidate the effect of betulin on proliferation of tumor cells and investigate the mechanisms responsible for the apoptosis-inducing activity of betulin in A549 cells.In this thesis we draw some conclusions as follows:1. Betulin obviously reduces the proliferation/viability of A549 cell lines in a dose-dependent manner and the value of IC50 is 12 ug/ml.2. We further study on the Mechanisms of Betulin in A549 cells. Our results showed the loss of viability in A549 cells by betulin is due to the induction of apoptosis.3. Apoptosis of betulin-induced A549 cells is mediated through depolarization of mitochondrial membrane potential and cytochrome c, Smac release. While caspase-8/Bid-mediated mitochondrial pathway may also be involved in the apoptosis of A549 treated with betulin.The inhibition of apoptosis is the key cause of tumorigenesis, so to activate an apoptotic program is an effective approache for cancer therapy. Most of chemotherapeutics has been showned to be the inducer of apoptosis in cancer cells. Recent studies suggest that mitochondria play a critical role in apoptosis induced by chemotherapeutic drugs, because mitochondria-initiated apoptosis involved in multiple factors function in conjunction and in parallel to trigger cell death. And the pathway is able to feed-forward and amplify the apoptotic signal. Even when caspase-dependent and caspase-independent pathways cannot function properly, mitochondrial dysfunction caused by apoptotic stimuli may lead passively to cell death, owing to compromised energy production.We designed a series of methods to test the effect of betulin on A549 cells and further invastigate the signal transduction pathway of betulin–induced apoptosis in A549 cells. The MTT assay showed that betulin effectively inhibits cell proliferation in A549 cells in a dose dependent manner with an IC50 value of 12μg/ml. In the presence of 10μg/ml betulin, A549 cells undergo an apoptosis program, as evidenced by apoptotic morphology such as, cell shrinkage, membrane blebbing, nuclear condensation and fragmentation. Flow cytometry analysis showed that the subG1 fraction was significantly upregulated in betulin treated A549 cells. Kinetics analysis showed that depolarization of the mitochondrial membrane potential and release of the mitochondrial apoptotic proteins, cytochrome c and Smac, occurred as early as 2 hours after treatment with 10μg/ml of betulin. Sequential activation of caspases-9, -3 and cleavage of PARP occurred after those mitochondrial events. In addition, caspase-8 activation and Bid cleavage may also be involved in this apoptosis program.Taken together, betulin triggers an extensive apoptotic cell death in human lung cancer A549 cells via mitochondrial cytochrome c release and caspase-9 prosessing. Caspase-8/Bid-mediated mitochondrial pathway may also be involved in the apoptosis of A549 cells treated with betulin via a positive feedback loop to amplify the mitochondrial apoptotic pathway.