| Tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) also known as apoptosis ligand2(Apo2L) is a member of TNF family and discovered by Wiley et al. in1995by searching homologues of the TNF family proteins in EST(expressed sequence tag) database. TRAIL is a type II membrane protein. TRAIL possesses five receptors, dis-symmetrically expressed on normal and cancer cells. Of the five receptors, TRAIL-R1(death receptor4, DR4) and TRAIL-R2(death receptor5, DR5), comprise a cytoplasmic death domain (DD), which could transmit an apoptotic signal upon TRAIL stimulation. TRAIL-R3(decoy receptor1, DcRl) devoiding of any transmembrane and cytoplasmatic domains is linked to the cell surface via a glycosylphosphatidylinositol (GPI). TRAIL-R4(decoy receptor2, DcR2) consists of a partial death domain. OPG (osteoprotegerin) is a soluble receptor which completely lacks transmembrane and cytoplasmatic domains. Therefore, DR4and DR5are two functional receptors which could lead to the recruitment of Fas-associated death domain (FADD), and form the death-inducing signaling complex (DISC) and activate the caspase cascade. DcRl, DcR2and OPG could bind with TRAIL, but couldn’t result in cell death.TRAIL has been known to induce apoptosis in a variety of tumor cells and some virally infected cells, but not in most normal cells. Thus TRAIL have attracted considerable attention for the potential use in cancer therapy since it was found. According to report, although TRAIL was an effective therapy to some types of leukemia in clinical trial, there are exceptions. The mechanism that chromic myelogenous leukemia (CML)-derived lymphoblast K562cells were resistant to TRAIL-induced apoptosis was unclear.Chemotherapeutic drugs Harringtonine, Daunorubicin, Cytarabine, Hydroxyurea and Cisplatin which were used as the treatment of CML and other cancers were used to treat K562cells alone or with rsTRAIL in our reserach. Among the drugs, hydroxyurea had synergy with rsTRAIL in the induction of apoptosis. Flow cytometry analysis death receptors DR4,DR5,DcR1and DcR2on the surface of K562cells which were treated with rsTRAIL or(and) HU revealed change in the level of DR5. It is suggested that DR5may play important role in deciding K562cell sensitivity to rsTRAIL and chemotherapeutic drugs-induced apoptosis. The results of gene chip before revealed rsTRAIL decreased expression of STAT5in THP-1cells, suggesting that STAT5involved in rsTRAIL-induced apoptosis. It was found that co-treatment of K562with rsTRAIL and HU can trigger the inhibition of STAT5. Detection of active forms of kinases which were upstream of STAT5, and the expression level of its target genes, found that the phosphorylation of direct upstream kinase JAK2and MEK were inhibited, the phosphorylation of another kinase JNK was elevated. The target gene bcl-xl and c-myc were inhibited at protein and mRNA levels respectively. Reduced phosphorylation of IkB occurred, suggest NF-κB was involved in the regulation of sensitivity changes in K562. Detection of apoptotic effector molecular revealed enhanced cleavage of Caspase8,3and9in rsTRAIL and HU combined treatment group. Apoptosis-inhibited molecule FLIP and cIAP1were significantly reduced. AG490, a JAK2-specific inhibitor, can inhibit the activation of STAT5at the molecular level. The combination of AG490with rsTRAIL and(or) HU can induce increased sensitivity, proving that STAT5in K562cells determined their sensitivity to drugs-induced apoptosis.In summary, we demonstrated in the present study that the synergy between rsTRAIL and HU in the cytotoxic effect to K562was caused by the inhibition of STAT5activity. The effect was mediated through JAK2/STAT5/NF-κB pathway, activation of Caspase Cascade and expression inhibiton of cFLIP and cIAP1. Since CML-derived lymphoblast K562cells had the characteristics of cancer stem cells, it played an important role in cancer recurrence and metastasis. Therefore, this article had a certain value in providing a theoretical support for CML treatment. |
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