Triptolide-induced Apoptosis In Myelodysplastic Syndromes MUTZ-1 Cells And Its Mechanism

The myelodysplastic syndromes (MDS) are a group of clonal hematopoietic stem cell disorders characterized by ineffective hematopoiesis and dysplasia. It can be regarded as a pre-malignant condition that typically has one of three outcomes. Patients can survive for an extended period of time with the disease, patients can die due to complications of severe cytopenia, or the disease can transform to acute leukemia. Now it is commonly accepted that the carcinogenesis of MDS is a multistep process involving many etiologic alterations such as chromosome aberrations, gene mutations, immune abnormalities, environmental changes, and so on. Based on its pathogenesis, our therapy should focus on eliminating malignant clone, reconstructing hematopoiesis and regulating immunity.Triptolide (TPL) is a purified component extracted from Tripterygium wilfordii Hook. F (TWHF) and has been demonstrated to be effective in patients with a variety of inflammatory and autoimmune diseases, such as rheumatoid arthritis, nephritis and lupus erythematosus. Recent studies showed that TPL possessed potential anti-tumor properties. In the present study we investigated the therapeutic effects and mechanisms of TPL against human MDS cell line MUTZ-1.First of all, we used MTT assay to examine the effects of different concentrations of TPL on the growth of MUTZ-1 cells. The results indicated that cell viability in the presence of TPL decreased in a dose-dependent manner. The inhibitory rates of cell growth were positively correlated with TPL concentrations (P< 0.01). The growth- inhibitory IC50 value were 55.06ng/ml; 48.60ng/ml; 22.57ng/ml after the treatment of tiptolide for 24,48,72 hours, respectively.Then, in order to investigate whether apoptosis is associated with the antitumor activity of TPL in human MUTZ-1 cells, we analyzed the fragmentation of genomic DNA and cell morphology using transmission electron microscope. We observed the typical ladder pattern of internucleosomal fragments and apoptotic morphology such as pyknosis and apoptotic body after MUTZ-1 cells were exposed to TPL for 24hr.As we all know, the translocation of phosphatidylserine (PS) is considered to be one of the hallmarks of apoptosis. We used flowcytometry to evaluate the exposure of PS on MUTZ-1 cells after double staining with fluorescein isothiocyanate (FITC)-labeled annexin V and propidium iodide (PI). Exposure to 0,20,40,60ng/ml TPL for 24 hours resulted in 2.71%, 9.99%, 31.25%, 52.3% cells undergoing apoptosis, respectively. The apoptotic rate was positively correlated with the concentrations of TPL (r=0.985; P=0.015).To further confirm apoptosis and determine the mechanism for its induction, we used western blot to assay the protein expression of caspase-3, -8, -9 and PARP.Caspase-3 is a member of the cysteine protease family, which plays a crucial role in apoptotic pathways by cleaving a variety of key cellular proteins including poly (ADP-ribose) polymerase (PARP). Considering the central role caspase-3 plays in executing apoptosis, we examined the expression of caspase-3 in MUTZ-1 cells. When the concentrations of TPL were added to 40ng/ml and 60ng/ml, the cleaved caspase-3 subunits were clear, indicating caspase-3 was activated and proteolytic processed in TPL induced cell apoptosis.Our western blot analysis also established concentration-dependent PARP cleavage in response to TPL. Caspase activation caused degradation of 113-kDa holoenzyme to a 89-kDa fragment and a 24kDa fragment which are recognized by the same antibody. Low concentration of TPL (20ng/ml)achieved PARP degradation, which became more pronounced at higher doses.Because activation of mitochondrial pathways has been observed in most cases of drug-induced apoptosis, we first sought to determine whether TPL treatment was associated with the activation of caspase-9, an active component of the apoptosome. MUTZ-1 cells were treated using TPL at various doses for 12h and the result showed that with the increased doses of TPL, the produced minor accumulation of its cleavage product, a supposedly activ...