Mechanism Activities Of NF-κB Pathway Inhibition By Parthenolide Via Ubiquitination In Multiple Myeloma

Objective Parthenolide(PTL) is an active component of sesquiterpene lactones, which has an anticancer activity to various humor, and its ability of anticancer relates to inhibition of NF-κB. Multiple myeloma(MM) is a malignant proliferative disease, we had already confirmed that PTL could kill myeloma cells, and the mechanism might be due to inhibit NF-κB in MM, but molecule mechanism had still not been completely clear yet. The lately study shows that PTL can induce apoptosis though controlling ubiquition course of key proteins, which may be important parts in a signal pathway. Our research will study the molecule mechanisms how PTL inhibit NF-κB, and discuss the possible potential targets in NF-κB signal pathway.Methods We use RPMI8226 cells as research object in vitro. Supply different doses of PTL to the cells for different time, then use MTT to detect generation of RPMI8226 cells, FACS tested cell period and apoptosis, to investigate the effect of key proteins we use immunoprecipitation to test expression of ubiquition proteins, include the TRAF6 and Nemo, the level of IκB-αin cytoplasm and p65 in nucleus expression were measured by Western-Blot, to discovery condition of p65 translocated from cytoplasm to nucleus, we use Immunofluorescence to identify.Results The cell growth was reduced after treated with PTL, also in a dose-depended way(P<0.01), The apoptosis of cells reflected by AnnexinⅤ/PI assay, showed a rapidly increased when treated by 40μmol/L of PTL, under a fluorescence wo could see the nucleus revealed rupture,pycnosis, and apoptotic body existed; We had discovered PTL could increase the proportion of cells in G2 phase significantly; Treated with different doses of PTL after 24 hours, p65 in nucleus expression showed remarkably decreased, in a dose-depended way (p<0.05), detected by a fluorescence microscope, we found p65 which translocated from cytoplasm to nucleus had reduced; We revealed that ubiquition TRAF6 and Nemo both showed obviously reduce, in a dose-depended way; The expression of IκB-αin cytoplasm showed evidently increase, in a dose-depended way; ubiquition total protein also manifested the same result, decreased evidently, all the changes had a positive correlation with dose of PTL; With computer software Autodock we predicted that PTL could bond to TRAF6 steadily, then induced decreased of ubiquition TRAF6.Conclusions Our research confirmed PTL had an affect to NF-κB activity in MM, and TRAF6, an E3 ligase, could be bonded directly to PTL, as a target protein. Though this kind of effect, the activation of NF-κB signal pathway had been influenced, then MM cells had been induced apoptosis.