Study On The Effection Of TRAIL On The Leukemic Cell Lines

Identifying targeted agengts capable of selectively inducing death of cancer cells,while sparing normal host cells,has cells,has been the ultimate goal of cancer therapeutic drug development. One promising and potentially important novel approach is the induction of programmed cell death through proapoptotic receptors. An additional TNF family member that also could promote apoptosis of tumor cells, called TNF-related apoptosis-inducing ligand (TRAIL), was subsequently identified. As a novel biological agent, TRAIL has good clinical application perspective because of selectively kill malignant cells whereas normal cells are largely unaffected by this treatment.It had been confirmed that in vitro trail can suppress or induce most common human tumor cell death, such as colon carcinoma, lung cancer, breast cancer, glioblastoma, renal carcinoma, malignant melanoma, and so on. In athymic mice with established subcutaneous xenografes from the human colon or breast cancer cell line. TRAIL induced substantial tumor regression followed by either a marked delay in tumor regrowth or complete tumor ablation.Many abord reportes have certified that targeting death receptors with tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) has the remarkable potential to selectively kill malignant cells whereas normal cells are largely unaffected by this treatment. However, some tumor cells, including leukemia cells, exhibit resistance to this molecule. To investigate the basis for resistance of leukemia cells to TRAIL is the focus of clinical tumor reseach. Several different mechanisms have been identified by researchers that could block TRAIL-mediated apoptosis, such as : defects or mutations at the level of the TRAIL receptors, including loss of DR4 expression through homozygous deletion or lack of expression of death receptor on the cell surface, and competitive binding of TRAIL by the decoy receptors TRAIL-R3 (DcR1) and TRAIL-R4 (DcR2) or the soluble receptor OPG; compromised function of the death-inducing signaling complex as a result of overexpression of cellular FLICE-inhibitory protein or loss of caspase-8 expression by gene methylation; lack of activation of the mitochondrial pathway by virtue of overexpression of antiapoptotic (e.g., Bcl-2 and Bcl-XL) or mutation of pro-apoptotic (e.g., Bax) Bcl-2 family members; caspase inhibition by up-regulation of the inhibitor of apoptosis protein family members.Many abord papers have reported that the mechanisms of resistance to TRAIL paradoxically activates pro-survival pathways .Jurkat T leukemic cells respond to Etoposide and TRAIL, not only disclosing the occurrence of apoptosis but also a kind of resistance. A similar rate of viability upon the exposure to these two drugs up to 24 h has been evidenced, followed by the occurrence of a rescue process against TRAIL, not performed against Etoposide, along with an highernumber of dead cells upon Etoposide exposure, in comparison with TRAIL treatment. These preliminary results let us to speculate on the possible involvement of PI-3-kinase in TRAIL resistance disclosed by surviving cells (20%), may be phosphorylating Akt-1.To evaluate the involvement of survival pathways in the response of Jurkat T leukaemic cells sensitive to the cytotoxic action of TRAIL, Co-treatment of the cells with inhibitors of PI-3 kinase (LY294002) and nuclear factor kappaB (NF-kB) (SN50) pathways.leads to an earlier significantly increased cytotoxicity, respectively in the form of apoptosis and necrosis. Consistently with the data obtained with the pharmacological inhibitors, the activation and nuclear translocation of both PI-3K and NF-kB were observed. The results provide evidence that even in sensitive neoplastic cells TRAIL paradoxically activates pro-survival pathways,which protect against TRAIL-mediated death since their inhibition leads to an earlier and increased cytotoxicity.In this study, different leukemia cell lines treated with TRAIL, we use the evaluation of cell cycle and apoptosis, western blot analysis to anlyse some aspects of the PI-3K/Akt pathway,notably survival pathways, in order to add new evidences to explain the resistance of leukemia cells to TRAIL.We used different leukemia cell lines,such as Jurkat, K562, NB4 and MCF-7 for the objects of the experiment,cells were treated with different concentrations (100-1000ng/ml) TRAIL for 6, 12, 24, 48 hours,and assayed cell activity and inhibition rate by MTT. The results showed that the TRAIL inhibited cell growth in a dose, time-dependent manner, and the dose larger,the time longer, the rate of inhibition was higher. Different cell line has different sensitivity to TRAIL, MCF-7 is the most sensitive. The inhibition of leukemia cell is 18.07%,15.94%,15.65%. TRAIL is more than 300ng/ml, the rate of inhibition grow slowly;at the same dose, the rate of inhibition has no significant deviation.So we choose the best dose and time is 300ng/ml,24h.We used different leukemia cell lines treated with 300ng/ml TRAIL for 24 hours, analysis of PI fluorescence was performed with an EPICS Coulter Flow Cytometer ,multicycle software was used for cell cycle phase analysis.The result showed that G0/G1 phase increase, S phase degrade, G2/M phase degrade. But no statistics significance.TRAIL is synergistic with 10uM LY294002 (a selective pharmacologic inhibitor of PI-3k activity) to treat leukemia cell lines, 24 hours later, Applicating PI/annexin V labeled apoptosis cell and detected apoptosis rate by flow cytometry. The results showed that the combined treatment can improve leukemia cell line apoptosis rate, P<0.05.We used different leukemia cell lines treated with 300ng/ml TRAIL, half of 1 hour and 1 hour later, cell were harvested in lysis buffer differently, protein setermination was performed,Equal amounts of protein(30ug) for each sample were run .Signalling pathways activated by TRAIL were assayed in Western blottong.total homogenates showed an increased expression and activation of AKT-1.In this experiment, We think that the parallel activation of survival and death pathways even by death inducers should be taken into account to improve therapeutic responses and sensiticities of leukemic cells to chemotherapeutic agents. TRAIL used in combination with pharmacological inhibitors of PI-3K pathway could be considered as an alrernativw strategy for leukemia treatment.