| Background: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 DR4 and DR5 in 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 leukemia therapy.Plumbain (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. There were lots of reports about its anti-proliferation in diverse cancer cell lines, such as leukemia, prostate cancer, breast cancer, ovarian cancer, lung cancer, cervical cancer, and so on. However, there are no papers about its effect in combination with TRAIL in leukemia.Kasumi-1 cell line was cultured from a patient with t (8,21) positive acute myelogenous leukemia. AML with t (8,21) which approximately accounted for 10%-15% of acute myeloid leukemia is generally considered as a good prognosis subtype, but recurrent and resistence to drug are still a tough problem in clinical. Then, it is important to explore new reagents and methods to conquer this problem. The effect of TRAIL, plumbagin and their combination on Kasumi-1 cells were observed, and the possible mechanisms were investigated in the study.Part I Plumbagin enhanced rsTRAIL induced apoptosis on Kasumi-1 cells AIM:To investigate the effect of plumbagin alone, rsTRAIL alone and their combination on leukemic Kasumi-1 cells. METHODS:Kasumi-1 cells were treated with plumbagin alone, rsTRAIL alone at different concentration, and their combination at variant concentrations. The changes of morphology of Kasumi-1 cells were observed under microscope after Wrighting staining, the proliferation of cells were measured by CCK-8 assay and the apoptosis were analyzed independently through Annexin V/PI double staining by flow cytometry and TUNEL staining. RESULTS:Both rsTRAIL and plumbagin could induce the apoptosis of Kasumi-1, and rsTRAIL-induced apopotosis of Kasumi-1 could be enhanced by plumbagin. The ratios of annexin V positive Kasumi-1 cells were (27.7±2.9)%, (25.6±3.1)% and (52.1±3.3)% in 100 ng/mL rsTRAIL groups,2μmol/L plumbagin and the combination of the two agents group respectively, and the combination group was significantly higher than the group of rsTRAIL or plumbagin alone. TUNEL assay demonstrated that the number of apoptotic cells in groups of plumbagin combining with rsTRAIL were higher than the groups of rsTRAIL or plumbagin alone. CONCLUSIONS:Plumbagin can enhance TRAIL induced apoptosis of Kasumi-1 cells.Part II Mechanisms of the enhancement effect of plumbagin on the apoptosis of Kasumi-1 cells induced by rsTRAILAIM:To investigate the mechenisms of plumbagin alone, TRAIL alone and their combination on leukemic Kasumi-1 cells. METHODS The contents of GSH in Kasumi-1 cells treated with plumbagin were measured by a microplate reader. The expression of DR4 and DR5 at mRNA lever was detected by real-time PCR. The expression of DR4 and DR5 at protein lever in cytomembrane was detected by flow cytometry. The expression of signal transduction proteins, such as DR5, caspase-3, caspase-8, caspasep-9, Bid, Bax, Bcl-2 and c-FLIP was detected by Western blotting. Effect of anti-DR5 neutralizing antibodies on Kasumi-1 apoptosis was detected by flow cytometry. The Effect of NAC on the apoptosis Kasumi-1 cells was detected by flow cytometry. RESULTS:Plumbagin decreased the content of GSH on Kasumi-1 cells. Plumbagin could up-regulate the expression of DR5 at mRNA levels in Kasumi-1 cells by real-time PCR assay. The up-regulation of DR5 and Bax, the activation of caspase-8 as well as the down-regulation of c-FLIP at protein level could be detected in plumbagin alone and the combination with rsTRAIL groups. Anti-DR5 neutralizing antibodies could inhibit the apoptosis in TRAIL treated group and abolish the enhancement effect of TRAIL induced apoptosis by plumbagain. ROS scavenger NAC could inhibit the apoptosis of Kasumi-1 cells induced by plumbagin alone and its combination with rsTRAIL; meanwhile, the effects of up-regulation of DR5, Bax, and down-regulation of c-FLIP at protein level were also abrogated by the ROS scavenger NAC. CONCLUSIONS:Plumbagin can enhance TRAIL induced apoptosis of Kasumi-1 cells and the mechanisms include the upregulation of DR5, Bax, activation of caspase-8 and the degradation of c-FLIP which is ROS dependented. |
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