Inhibition Of Notch Signaling Induces Apoptosis And Influences The NF-?B Signaling In Multiple Myeloma Cells

Objective: To explore Notch signaling pathway in human multiple myeloma cell lines-RPMI8226 and U266; and to investigate the effects on proliferation, apoptosis, as well as cell cycle after the DAPT treatment in multipie myeloma cells; and to evaluate the regulation of NF-?B signaling by Notch signaling; and to reveal the possible mechanisms of cross-talk between Notch and NF-?B signaling in multiple myeloma.Methods: The expression of Notch1 transmembrane subunit(NTM) protein and Hes1 m RNA were respectively examined by Western blot and q RT-PCR assay, with PBMC of health donors as negative controls and T-ALL cell line Jurkat cell as a positive control. RPMI8226 and U266 cells were cultured with various concentrations of DAPT for 24 h, 48 h and 72 h, then CCK-8 test was used to measure cell proliferation and calculate the half maximal inhibitory concentration(IC50). The apoptosis and cell cycle of multiple myeloma cell lines after DAPT treatment were examined by Flow cytometry. NTM, c-Myc,p-IKK?,p-I?B? proteins as well as the p65 and p50 proteins in nucleus and cytoplasm were detected by Western blot after 48 h treatment with DAPT. At the same time, Notch1, Hes1, c-Myc, IKK?, I?B?, p65 and p50 m RNA expression were detected by q RT-PCR after 48 h of DAPT treatment.Results: 1. Comparing with the PMBC, RPMI8226 and U266 cells express more NTM protein.In addition,the expression of the NTM protein in RPMI8226 cells is also more than that in Jurkat cells. According to q RT-PCR results, both RPMI8226 and Jurkat cells express significantly amounts of Hes1 m RNA except that the U266 cells express the same m RNA for the low level,but still higher than that in PMBC(p<0.01).2. The proliferation of RPMI8226 and U266 cells are inhibited by DAPT with time and concentration dependent manner to some extent.3.The apoptosis of RPMI8226 and U266 cells is significantly induced by DAPT(p<0.01). 4. DAPT induces S phase arrest in RPMI8226 cells and G0/G1 phase arrest in U266 cells.5. DAPT mediates a significantly decrease on NTM and c-Myc proteins, as well as on Notch1, Hes1 and c-Myc m RNA expression in multiple myeloma cell lines. 6. For NF-?B signaling pathway, the expression of p50 and p65 protein in nucleus as well as the m RNA decrease along with the p50 and p65 protein increase in cytoplasm after DAPT treatment for 48 h in human multiple myeloma cell lines. At the same time, the positive regulator p-IKK? protein and m RNA decrease and the negative regulator protein p-I?B? increase after DAPT treatment for 48 h in human multiple myeloma cell lines.But the standard of the negative regulator I?B? m RNA increase in RPMI8226 cells and decease in U266 cells after DAPT treatment for 48 h.Conclusion: 1. Multiple myeloma cell lines: RPMI8226 and U266 cells express high amounts of NTM protein and Hes1 m RNA. 2. DAPT inhibits the proliferation, induced the apoptosis and cycle arrest of RPMI8226 and U266 cells. 3. The possible mechanism of the phenomenon mentioned above may be due to the decrease of c-Myc protein and Hes1 m RNA expression through inhibiting the proteolytic cleavage of NTM, and the decrease the activity of NF-?B signaling pathway.