The Effects And Mechanisms Of Disulfiram/copper Inhibiting ALDH~+ Stem Cell-like Population In Multiple Myeloma

Part?Identification of stem cell-like properties of ALDH~+cells isolated from multiple myelomaObjective:To isolate the ALDH~+cells from multiple myeloma(MM)cells,and identify the stem cell-like properties of ALDH~+cells.Methods:Aldefluor assay was used to detect the percentage of ALDH~+population in MM cell lines NCI-H929 and RPMI-8226,and ALDH~+and ALDH~-population were isolated from NCI-H929 cells.Colony formation assay and western blotting were performed to identify the stem cell-like characteristics of ALDH~+cells.Moreover,the percentage of CD138~-cells in ALDH~+population compared with ALDH~-population was examined.In vivo,we developed the NOD/SCID mouse model to identify the tumorigenesis capacity of ALDH~+population.Results:We found that both NCI-H929 and RPMI-8226 cells contain a small subset of ALDH~+cells with the percentage of 1.21%(1.21±0.05)?0.67%(0.67±0.05),respectively.Compared to ALDH~-cells,ALDH~+cell endowed with more enhanced colony formation capacities(66.6±6.8 and 11.3±4.1,P<0.01)and more increased expression of stem cell transcription factors NANOG and OCT4.ALDH~+cells had more increased percentage of CD138~-cells compared to ALDH~-cells,with the percentage of 7.62%(7.62±0.55)and 1.19%(1.19±0.42)respectively(p<0.001).In vivo assay showed that ALDH~+population had the stem cell-like characteristics of tumorigenicity.Conclusion:MM cell lines contained a very small population of ALDH~+cells which had tumorigenicity capacities both in vitro and in vivo endowed with the properties of stem cell-like cells.Moreover,ALDH~+populaton and CD138~-cells had overlap at some extent.Part ? The influence of disulfiram/copper on cells and stemness of multiple myelomaObjective: To examine the effects of DS with or without Cu on the cells and stemness of MM,providing the theoretical basis for the safety and effectiveness of clinical application.Methods: CCK-8 assay was used to detect the inhibition effects of DS with or withour Cu on both NCI-H929 and RPMI-8226 cell lines.Aldefluor assay was performed to examine the effects of the percentage of ALDH+ population.The expression levels of NANOG and OCT4 after treating by DS or in combination with Cu detected by western blotting.The efficacy of DS or with Cu on the clonogenic capacities of MM was evaluated by colony formation assay.Annexin V/PI assay was used to detect the pro-apoptosis effects of DS with or without Cu on both MM cell lines.To investigate the inhibition effects of DS/Cu in vivo,a xenograft mouse model was developed.Results: Cu alone was not toxic to both NCI-H929 and RPMI-8226 MM cells.The inhibition effects of DS displayed a biphasic effect in two cell lines.The effects of DS increased with the concentration from 0.05 ?M to 0.5 ?M,and then the action reduced with increasing concentration.On the whole,the DS alone did not display obvious effects.However,DS/Cu had more significant inhibition effects than DS alone,and the effects were dependent on the concentration.In both NCI-H929 and RPMI-8226 cells,Cu alone was unable to reduce the proportion of ALDH+ cells.However,DS/Cu decreased the proportion of ALDH+ cells more significantly than DS alone.DS administrated alone did not obviously decreased the expression of NANOG and OCT4,however,DS with Cu remarkably reduced the levels of both proteins in two cell lines.DS/Cu had stronger inhibition effects on colony formation than DS alone.Compared to DMSO control,Cu administrated alone did not have obvious action.Compared to DS administrated alone,the pro-apoptosis effects of DS/Cu were significantly increased.In vivo assay,xenograft tomors of DS/Cu group significantly reduced in tumor growth,and the tumor volumes were 23.2% of the size of those in the control group(p < 0.001).Ki-67,ALDH1A1,NANOG,and OCT4 detected by immunohistochemistry showed that DS/Cu reduced the expression of all proteins evidently compared to control group.Conclusion: DS/Cu inhibits the cells and stemness of MM,and the effects of DS are in a Cu-dependent manner.Part ? The mechanisms of disulfiram/copper inhibiting ALDH+ stem cell-like population of multiple myelomaObjective: ALDH isozymes have 19 subtypes,and DS as an ALDH inhibitor,we in order to investigate which subtype of ALDH was inhibited by DS/Cu suppressing the stemness of MM and its downstream pathway.Methods: Recent studies determined that Aldefluor assay can detect ALDH1A1,ALDH1A2,ALDH1A3,and ALDH2.So we examined the expression of ALDH1A1,ALDH1A2,ALDH1A3,and ALDH2 of MM cells after treating by DS or in combination with Cu by western blotting.We further overexpressed ALDH1A1 or ALDH2 in NCI-H929 cell line by lentivirus.Aldefluor assay was used to examine the percentage of ALDH+ population in ALDH1A1 or ALDH2 overexpression cells.Western blotting was used to detect the expression of NANOG and OCT4 in ALDH1A1 or ALDH2 overexpression cells.In order to evaluate the clonogenic capacities in ALDH1A1 or ALDH2 overexpression cells and the effects of DS or with Cu in ALDH1A1 overexpression cells,a colony formation assay was performed.In addition,we used western blotting and lentivirus-mediated ALDH1A1 overexpression technology to explore the downstream mechanism.Results: In NCI-H929 cells,DS/Cu significantly inhibited the expression of ALDH1A1 and ALDH2,and DS alone did not have evident effect on ALDH2.In RPMI-8226 cells,the inhibition effects of DS/Cu on ALDH1A1 and ALDH2 were more efficient than DS alone.However,the expression of ALDH1A2 and ALDH1A3 were not suppressed by DS with or without Cu in both cell lines.We found that both ALDH1A1 and ALDH2 contribute to the functional ALDH activity and can be detected by Aldefluor assay.The levels of NANOG and OCT4 and the clonogenic capacities were increased obviously in ALDH1A1 overexpression cells,but not in ALDH2 overexpression cells.Compared to control,DS/Cu showed significantly inhibition effects on clonogenic capacities in ALDH1A1 overexpression cells(p < 0.01).These results showed that ALDH1A1 is associated with the stemness of MM.In addition,DS/Cu inhibited the levels of Hedgehog(Hh)pathway transcription factors Gli1 and Gli2,and the expression of Gli1 and Gli2 were increased in ALDH1A1 overexpression cells.Conclusion: Although DS/Cu inhibited both ALDH1A1 and ALDH2 subtypes,only ALDH1A1 plays the pivotal role in maintaining the stemness of MM.DS/Cu suppressed ALDH+ stem cell-like population via ALDH1A1 and Hh pathway regulated by ALDH1A1 at least in part.DS/Cu may be a promising agent in inhibiting the stemness of MM,and ALDH1A1 is a potential therapeutic target to treat MM.