The Effect Of α2,8-sialyltransferase On Multidrug Resistance In Human Chronic Myeloid Leukemia

Objective:Human chronic myelogenous leukemia (CML) is a one of the malignant tumor of the bone marrow, which accounts for about 20% in the adult leukemia, but its pathogenesis is still not clear. Because of the shortage of the hemotype hematopoietic stem cells, the chemotherapy had become the preferred method to cure CML. The clinical data shows, most of the patients with CML have certain resistance to chemotherapy, hindered the treatment of CML. With the development of glycoprotein and glycomics, more and more researchers draw attention to the relations between tumors and glycosylation. In recent years, researchers have shown that sialic glycosylation modification are associated with tumors and their drug resistance, but the mechanisms are still not yet uncovered. Alpha 2,8-sialytransferases as a menber of the sialytransferases family are closely related to the drug resistance in leukemia, but the cause of CML resistant mechanism has not been reported. In this study, to investigate the differential expression of 6 a2,8sialyltransferases in the both three pairs of chronic myeloid leukemia cell lines and peripheral blood mononuclear cells (PBMC) of CML patients, and to find out the potential correlation of a2,8sialyltransferase and multidrug resistance (MDR) of CML. For further diagnose and cure CML multidrug resistance to provide a new therapeutic strategy and target point of CML.Methods:(1) Using real-time PCR(RT-PCR) for quantitative detection of the expression of a2,8sialyltransferase genes in three pairs of CML cell lines and peripheral blood mononuclear cells (PBMC) of CML patients.(2) Manipulating the differentially expressed a2,8sialyltransferase genes (i.e., >3-fold higher) in three pairs of CML cells, to detect the changes of gene levels by RT-PCR, the sialyltransferase and main PI3K/Akt signaling molecules protein levels by Western blot, the chemosensitivity to anti-tumor drugs in vitro by MTT, the anti-tumor experiment and IHC analyze for in vivo, and the flow cytometry analysis for the expression of P-gp.(3) Using LY294002 or Akt shRNA to inhibit the expression of PI3K/Akt molecules in K562/ADR cells, with the above methods to detect the chemosensitivity of K562/ADR cells in vitro and vivo, and analyze the expression of P-gp.Results:(1) In the three pairs of CML cell lines, only ST8SIA4 and ST8SIA6 (i.e., >3-fold higher) were differentially expressed, and had a significant difference (P<0.05), the others have no significant difference or were not detected. In PBMC of CML patients, there also have the same result.(2) The specific silencing of ST8SIA4 and overexpression of ST8SIA6 in K562/ADR cells resulted in increased chemosensitivity to anti-tumor drugs (P<0.05), while decreased chemosensitivity to anti-tumor drugs (P<0.05) in K562/ST8SIA4 cells and K562-ST8SIA6 shRNA1 cells.(3) The specific silencing of ST8SIA4 in K562/ADR cells resulted in decreased expression of the main molecules of PI3K/Akt signaling pathway and P-gp (P<0.05), while increased in K562/ST8SIA4 cells.(4) Blocking the PI3K/Akt pathway by its inhibitor LY294002 or Akt shRNA in K562/ADR cells resulted in the decreased expression of the main molecules of PI3K/Akt signaling pathway and P-gp, and increased the chemosensitivity to anti-tumor drugs (P<0.05).Conclusion:(1) The expression of a2,8sialyltransferase genes was significant difference in three pairs of CML cell lines and PBMC of CML patients. The differential expression are associated with multidrug resistance in CML(2) The mechanism of α2,8sialyltransferase involved in the development of MDR of CML cells is likely to be through ST8SIA4 regulate the activity of PI3K/Akt signaling pathway and then cause the change of P-gp.