Effects Of Regulating Autophagic Activity On Proliferation And Apoptosis Of Multiple Myeloma Cells RPMI 8226

Objective:The study was aimed to investigate the effects of autophagic medications including autophagy activator RAPA(rapamycin),autophagy inhibitor HCQ(hydroxychloroquine)and 3-MA(3-methyladenine)on proliferation and apoptosis of human multiple myelomacells RPMI 8226.Method:RPMI 8226 cells which were cultured under the normal serum condition were treated with RAPA,HCQ and 3-MA,respectively,with normal serum cultured cells as control group.Cells were stained with MDC(Monodansylcadaverine)and autophagesomes were counted under the fluorescencemicroscope;CCK-8(Cell Counting Kit-8)was used to detect the cell proliferation andFCM(flow cytometry)was used to quantify the proportion of apoptotic cells.Western blot was used to evaluate the expression level of LC-3b(microtubule-associated protein 1 light chain 3b),caspase-3(cysteinyl aspartate specific proteinase-3),Bcl-2(B-cell lymphoma-2)and PARP(poly ADP-ribose polymerase).Results:Fluorescence microscope showed moreautophagosomes in RPMI 8226 cells treated with RAPA for 24 h than those in the control group,while autophagsomes in RPMI 8226 cells treated with HCQ and 3-MA was significantly fewer than control group;RAPA and HCQ inhibited the cell proliferation in a timeand dose-dependent manner while 3-MA had no significant effect on cellproliferation.After 24 h treatment with different concentration(0,100,500 and 1000 nmol/L)of RAPA,the proportions of apoptotic cells(%)(X±SD)were 0.9 ± 0.3,9.1 ± 1.2,28.6 ± 0.8 and 43.9 ± 2.1(P <0.05)respectively.The proportion of apoptotic cellswere 0.9±0.3,8.3 ± 1.8,15.9 ± 1.5 and 21.3 ± 2.0(P <0.05)respectively in different concentrations of HCQ(0,4,8and 16 ?mol/L)groups(P<0.05).No statistical differencewas found among different concentrations(0,5,10 and 20 ?mol/L)3-MA groups(P>0.05).After 24 h treatment of RAPA,theLC-3protein expression levelof RPMI 8226 cells was higher,and Bcl-2protein expression levelof RPMI 8226 cells waslower,while caspase-3and PARPwere higher in higher concentration RAPA groups than in lower concentration groups.After HCQ treatment,the LC-3 and Bcl-2protein expression levels were lower,while caspase-3andPARPprotein expression were higherin higher concentration HCQ groups than in lower concentration groups.LC-3 protein expression levelof RPMI 8226 cells was lower in higher concentration 3-MA groups than in lower concentration groups.However,caspase-3,PARP,Bcl-2protein expression levels were similar and no statistically significant difference was found among different concentration 3-MA groups.Conclusion:RAPA could induce autophagy in RPMI 8226 cells while HCQ and 3-MA inhibit autophagy.RAPA and HCQ could inhibit the proliferation and promote apoptosis of RPMI 8226 cells and 3-MA had no significant effect on cell proliferation and apoptosis.