Fucoidan Prevents Multiple Myeloma Cell Escape From Chemotherapy-induced Drug Cytotoxicity

Objective: To observe the preventive effect of Fucoidan on reducingmultiple myeloma (MM) cells escape from cytarabine and its mechanismby constructing vitro model, which provides a theoretical basis forpreventing MRD formation in clinical.Methods:(1)ELISA was used to detect the SDF-1content in Serumfrom12healthy volunteers and53MM patients (8patients (15.1%) atDurie-Salmon stage I,17patients (32.1%) at stage II, and28patients (52.8%)at stage III).(2) We used serum from healthy volunteers and MM patients indifferent disease stages, a Boyden chamber and matrigel glue to construct themigration and invasion model in order to detect the influence of cytarabine(apeak of the drug concentration) on RPMI8226and U266cells escape, thechemotaxis effects of serum from MM patients in different disease stageson MM cells and the best observation time point.(3) The MTT assay wasused to evaluate the effect of Fucoidan (25,50,100,200and400μg/ml) onthe growth of RPMI8226and U266cells at24,48and72h. Thehalf-maximal Fucoidan inhibitory concentration (IC50) values for the RPMI8226and U266cells were calculated.(4) The Annexin V-FITC and PIwere respectively used to detect the effect of12.5μg/ml Fucoidan on theapoptotic rate of RPMI8226cells and U266cells.(5) To further observe theeffect of fucoidan on MM cells escape from cytarabine cytotoxicity in vitromodel. The experiment was divided into a control group, a cytarabine group,a fucoidan+cytarabine group and a fucoidan group.(6) Flow cytometry wasused to assess the effects of cytarabine and Fucoidan on RPMI8226andU266cells surface CXCR4expression.(7) Western blotting was used toassess the protein expression of MMP9and RhoC induced or un-induced bySDF-1α.Results：(1) The SDF-1serum concentration was1.71±0.08ng/ml inhealthy volunteers,1.77±0.05ng/ml in stage I,2.22±0.18ng/ml in stage IIand2.80±0.13ng/ml in stage III. The SDF-1serum concentration increaseswith the increasing of the clinical stages.(2) The results demonstrate thatcytarabine can promote MM cell escape, and that the number of cells in thelower chamber increased with increasing clinical disease stage in vitromodel and the best observation time point is at6h.(3) The result of MTTdemonstrated that the preventive effect of Fucoidan on RPMI8226andU266cells growth in a time dose dependent manner and the IC50wererespectively48μg/ml and53μg/ml.(4) The RPMI8226and U266cellswere treated with12.5μg/ml Fucoidan for72hours. The apoptosis rate ofRPMI8226and U266cells doesn’t have significant difference compared with the control group.(5) The RPMI8226and U266cells were treated with12.5μg/ml Fucoidan for72hours and the result shows that Fucoidan canpreventatively decrease RPMI8226and U266cells escape from cytarabineand the number of transmembrane cells reduces obviously compared withthe cytarabine group.(6) Flow cytometry was used to measure CXCR4expression in all groups. The result shows that the expression in cytarabinegroup is higher than that in the control group, fucoidan group andfucoidan+cytarabine group and that shows significant difference(P<0.05).Compared with the control group, the expression in fucoidan group andfucoidan+cytarabine group decreases obviously. There is no significantdifference between fucoidan group and fucoidan+cytarabine group (P>0.05).(7) Compared with the groups un-induced by SDF-1α, MMP-9, RhoCprotein expression increase significantly in the groups induced bySDF-1α.And the expression in cytarabine group is higher than that in thecontrol group, fucoidan group and fucoidan+cytarabine group and thatshows significant difference (P<0.05).Compared with the control group, theexpression in fucoidan group and fucoidan+cytarabine group decreases andthat shows significant differences (P<0.05). There is no significantdifference between fucoidan group and fucoidan+cytarabine group(P>0.05).Conclusions:(1) Cytarabine can promote RPMI8226and U266cellsescape.(2) Fucoidan can preventatively decrease RPMI8226and U266cells escape from cytarabine cytotoxicity.(3) Up-regulating RPMI8226and U266cells surface CXCR4expression may be an important mechanism ofcytarabine promoting MM cells escape.(4) Down-regulating RPMI8226and U266cells surface CXCR4expression, MMP-9and RhoC proteinexpression may be an important mechanism of fucoidan decreasing MMcells escape from cytarabine cytotoxicity.(5) RhoC, MMP-9protein andSDF-1/CXCR4axis play a key role in the escape.