Plumbagin Enhanced TRAIL-induced Apoptosis Of Kasumi-1Cell In NOD/SCID Mice

Background:The translocation (8;21)(q22;q22) is one of the most common chromosomal abnormality in acute myeloid leukemia(AML). The incidence of AML with t(8;21) is6%-10%in primary AML and18%-40%in AML-M2.AML with t(8;21) is generally considered as a good prognostic type according to WHO classification. However, the recurrence and drug resistance of AML with t(8;21) still threat the life of patients. Therefore, it’s important for finding new drugs to overcome the drug resistance of leukemic cells, elevate the rate of complete remission and improve the long-term survival of patients in clinical practice.Tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) belongs to TNF superfamily, a type Ⅱ membrane protein,which can trigger apoptosis specifically by binding with its death receptors DR4and DR5in tumor cells including human leukemia cells, but it can spare against normal host cells. However, the resistance to TRAIL remains a therapeutic challenge in practice. Exploring new agents to enhance TRAIL-induced apoptosis would increase the potential clinical utility for therapy of leukemia.Plumbagin (5-hydroxy-2-methyl-1,4-naphthoquinone), a Chinese traditional herb extracted from the root of plumbago zeylanica L has been shown to have diverse pharmacological effects including anticancer, anti-bacterial, anti-reproduction, and anti-coagulant, the property of anticancer is the most promising among them. Previous research has confirmed that plumbagin can enhance TRAIL-induced apoptosis of Kasumi-1in vitro. It means that plumbagin has potential action to inversing the resistance of TRAIL. Therefore, it’s necessary to verify the finding in animal experimental leukemic mode in vivo before clinical usage.Non-obese diabetic/severe combined immunodeficient mouse not only have inherent immunodeficiency but also have lack of T and B lymphocytes. A variety of tumor cells can be implanted in NOD/SCID mouse, and it will have fewer rejection reactions and graft-versus-host disease (GVHD) at the same time. On the basis of in vitro studies, the effect of TRAIL, plumbagin and their combination on Kasumi-1cells were observed in NOD/SCID mouse, and the possible mechanisms were also investigated in the study.Part Ⅰ Plumbagin could enhance rsTRAIL induced apoptosis on Kasumi-1cells in vivoAIM:To investigate the effect of plumbagin alone, rsTRAIL alone and their combination on leukemic Kasumi-1cells in vivo. METHODS:NOD/SCID mouse bearing xenograft tumor of Kasumi-1cells were randomly divided into normal saline group, Ara-C group, rsTRAIL alone group, plumbagin alone group and rsTRAIL combined plumbagin group. The tumor volume and weight changes of mouse were observed.The pathological slices of tumor and organs of mouse were stained with HE method and observed under microscope.RESULTS:(1) Compared with the control group, both rsTRAIL and plumbagin could inhibit the growth of the xenograft tumor(P<0.01).They had a synergistic effect (P<0.05).The inhibitory rates in rsTRAIL, plumbagin and rsTRAIL combined plumbagin were56.74%,65.17%and90.07%respectively.(2)The NOD/SCID mouse mode of AML with t(8;21) were successfully established, and the main marker is the formation of subcutaneous mass.(3)The main toxicity of plumbagin6mg/kg was diaeehea in tumor-bearing mouse. CONCLUSIONS:Plumbagin could enhance TRAIL induced apoptosis of Kasumi-1cells in vivo without obvious damaging of the vital organs.Part II Mechanisms of the enhancement effect of plumbagin on the apoptosis of Kasumi-1cells induced by rsTRAIL in vivoAIM:To investigate the mechenisms of plumbagin alone, TRAIL alone and their combination on leukemic Kasumi-1cells in vivo. METHODS:The morphology of tumor cells was observed by microscope. The apoptotic rate of single cell suspension of xenograft tumor was calculated by flow cytometry. The expression of DR4and DR5at protein level on the surface of cells was detected by flow cytometry. RESULTS:(1)T he ratios of annexin V positive cells were (47.39±9.99)%,(51.35±3.58)%and (68.23+6.74)%in rsTRAIL alone groups, plumbagin alone and the combination of the both group respectively, and was significantly higher in the combination group than in the group of rsTRAIL alone or plumbagin alone.(2)Plumbagin could increase the expression of DR4and DR5in cells of xenograft tumor which were demonstrated by flow cytometry.CONCLUSIONS: Plumbagin can enhance TRAIL induced apoptosis of Kasumi-1cells by upregulation of DR5expression.