The Effects Of Cucurmosin And The Mechanism Of Notch-Signaling Pathway In Multiple Myeloma Cell RPMI8226

[Objective] To study the proliferation, apoptosis mechanism with CUS on RPMI8226.Thenused the vitro proliferation, apoptosis detection experiment and Notch signal pathway related toresearch with the application of high efficiency, low toxicity of natural active drugCucurmosin(CUS) on multiple myeloma cell line RPMI8226.[Methods] The MTT method for the detection of CUS on PRMI8226cell proliferationinhibition (volume efficiency and effectiveness); Growth curve of PRMI8226cell treatedwith CUS was measured for the effects of growth Using trypan blue exclusion test; Proliferationinhibition of PRMI8226cell was detected by Colony proliferation inhibition test after CUStreatment in vitro; Transmission electron microscope were used to observe morphologicalchage and study Ultrastructural changes of PRMI8226cell exposed to CUS; Apoptosis rate ofPRMI8226cell was examined by Annexin V/PI double staining flow cytometry analysis.6.PIsingle staining flow cytometry DNA ploidy analysis; Propidium iodide (PI) staining associatedwith flow cytometry was used to study the DNA ploidy analysis on PRMI8226cell; Westernblot was used to analyze the expressions of apoptosis related proteins including bcl-2、caspase-3、caspase-8、caspase-9、Notch1、Jagged-2、P-Akt and Nf-kB of PRMI8226cell afterCUS treatment.[Results]1.The results of MTT assay showed that the rate of proliferation inhibition in atime-dependent and dose-dependent manner increased from7%to86.4%when RPMI8226cellexposed to0,0.0078,0.0156,0.0625,0.125,2.0umol/L CUS for24,48and72hours.The50％inhibitory concentrations(IC50)of CUS for24,48and72h were80umol/L，0.65umol/L，0.2umol/L.There was a significant difference between the treatment group and the control groupat each period (P<0.01).2.Growth curve through Trypan blue staining test counting viablecell indieated that, compared with control group， cell growth of CUS treated group wasinhibitied, latent period was prolonged, curve tends to declined gently and cells doubled timeextended. with the increase of the concentration。There was a significant difference between the treatment group and the control group at each dose (P<0.05).3.colony proliferation inhibitionassay showed that: the colony formation rate from50.5%down to5%, colony inhibition ratewas raised from18.5%to91.9%. with different dosage of CUS(0.0078,0.0156,0.0312,0.0625,0.125,0.25umol/L) in RPMI8226cell after7days. There was asignificant difference between the treatment group and the control group at each dose (P<0.05).4. Morphological analysis through transmission electron microscopy presents the shrinking ofcell volume, irregular morphology of nuclear, increase of nuclear heterochromatin and typicalmorphologic changes of apoptosis such as chromatin condensation, shrunken, fragmentationnucleus as well as “apoptotic body”after RPMI8226cells exposured to cucurmosin.5.Theresults of annexinV/PI stainin detected cells apoptosis indicated that the rate of apoptosisimproved(7.44%,12.49%,17.48％and26.41%)following the augmentation of the CUSconcentratio((0,0.125,0.5and2umol/L for72h). PI single staining flow cytometry results alsoshowed a concentration dependent, DNA ploidy showed typical massive hypodiploid cellpopulation(sub-G1) and the percentage of apoptotic cells was23.1％,31.9％,4.2％and59.8％,respectively after NB4cell exposure to0.0156,0.0312,0.0625,0.25umol/L CUS for72hshowing a significant dose-dependent manner. There was a significant difference between thetreatment group and the control group at each dose (P<0.05).6.Western blot analysis showedthat bcl-2proteins sharply reduced, meanwhile expression of cleaved caspase-3, cleavedcaspase-8and cleaved caspase-9protein strongly upregulate in a time-dependent anddose-dependent way, At the same time, the Notch1、Jagged-2、P-Akt and Nf-kB also reduced inthe Notch signal pathway.[Conclusion]1.The proliferation of RPMI8226cell was remarkbly inhibited by CUS in aconcentrationand time-dependent manner in vitro.2.Cucurmosin can effectively inducesapoptosis of multiple myeloma RPMI8226cell in a concentration-and time-dependent manner.3.It is likely one of anti-multiple myeloma mechanism in RPMI8226cell treated with CUS thatNotch1, Jagged-2、P-Akt and Nf-kB was highly down-regulated.