| The cancer stem ceils(CSCs) play a critical role in both cancer initiation and relapse as they are resistant to most of cytotoxic agents and able to proliferate indefinitely.The plant alkaloid oxymatrine(OM) has many biological activities including the ability to induce cell cycle arrest and apoptosis,which makes it a potentially useful agent for targeting cancer cells.So in order to determine whether it has beneficial pharmacological properties to eradicate CSCs,we have analyzed the effects of OM on MCF-7 breast cancer cells.Cytotoxicity and cancer stem-like cells (side population,SP) identification and sorting were performed.Inhibitory effect was evaluated on the sorted SP and non-SP cells.SP cells were identified with percentage (4.02±0.18%) in MCF-7 cells.OM caused a dose-dependent reduction in the proliferation of MCF-7 cells and a decrease in SP cells relative to untreated controls. As conon-SPared to non-SP cells,inhibitory effect in the SP cells was higher.Wnt/β-catenin signaling pathway was also examined by analyzing the expression of total P-catenin and phosphorylated p-catenin in cytoplasm,OM treatment was associated with an inhibition of Wnt/p-catenin signaling pathway relative to untreated controls.These results indicate that the growth inhibitory effects of OM treatment on MCF-7 cells may be partly via effects on SP and Wnt/p-catenin signaling pathway. Further work is warranted to explore whether OM may be a useful novel therapeutic drug for targeting breast CSCs.1.Isolating and characterizing SP cells from MCF-7 cells1.1 METHODS:Cells were trpsinized and re-suspended at 10~6cells/ml in conon-SPlete RPMI1640.Hoechst33342 was added to a final concentration of 5μg/ml with or without 100uM verapamil hydrochloride and cells were incubated for 90 minutes in a 37℃water bath.The MCF-7 and the SP/non-SP cells from MCF-7 cultured 7 days were analyzed and sorted by FACS.β-catenin,phosphorylatedp-catenin,c-myc and cyclinDl were also detected by fluorescence,FACS,Western blot and anon-SPlisensor quantitative PCR among SP and non-SP,MCF-7 cells.1.2 RESULTS:SP is contained in MCF-7 for about 4.02±0.18%.The SP cells perform a higher speed proliferation rate and higher clone formation rate than non-SP cells,and SP cells can be separated into SP and non-SP cells after being cultured 7 days.There is no difference between the percentages of SP in the parental cells and the descendant cells.As we speculated,the expressions of p-catenin,c-myc and cyclinD1 were higher,and phosphorylated p-catenin was lower,as compared with non-SP cells.1.3 CONCLUSION:The percentage of SP in MCF-7 cells is 4.01±0.18%.The ability of SP cells developing into SP and non-SP means that SP is in the higher hierarchy.And the activity of Wnt signaling pathway is over-regulated in SP cells.2.The effects of OM on MCF-7 cells2.1 METHODS:MTT experiments were performed to determine whether OM can inhibit the viability of MCF-7 cells.In order to determine whether OM can induce the apoptosis of MCF-7 cells,the cells treated with different concentrations OM for 48h were stained with AV-PI,and analyzed by FACS,at the same time,the fluorescence microscope detection were performed,β-catenin,phosphorylated p-catenin,c-myc and cyclinD1 were also detected by fluorescence microscope,FACS,Western blot and anon-SPlisensor quantitative PCR among OM treated MCF-7 and the control one. Cisplatin was the positive control drug of these experiments.2.2 RESULTS:There was a dose and time depended anti-proliferative activity of OM ranging from 62.5ug/ml to 4mg /ml(Fig.4A) and 0.5mg/ml from 6 to 48h.The percentages of Phosphorylatedβ-catenin positive cells by FACS.The percentages were 26.26%,41.50%,and 62.77%in 0.25mg/ml,0.5 mg/ml and 1mg/ml OM treated cells and 18.52%of untreated cells,respectively.The expressions ofβ-catenin,c-myc and cyclinD1 were all decreased in the OM treated cells,as compared with untreated cells.2.3 CONCLUSION:OM could decrease MCF-7 cell viability,induce MCF-7 cells apoptosis,decrease the expression of totalβ-catenin and its nuclear location,decrease the mRNA of P-catenin,c-myc and cyclinD1 and increase the phosphorylatedβ-catenin in cytoplasm.Most of these effects were mediated in a dose-dependent manner.3 The effects of OM on cancer stem like cells(SP cells) from MCF-7 cells3.1 METHODS:MTT experiments were performed to determine whether OM can inhibit the viability of SP cells from MCF-7cells.In order to determine whether OM can decrease the percentage of SP cells in the total of MCF-7 cells,the cells treated with different concentrations OM for 48h were restained with Hochest33342 and analysis by FACS.At the same time,β-catenin,phosphorylatedβ-catenin,c-myc and cyclinD1 were also detected by fluorescence microscope,FACS,Western blot and anon-SPlisensor quantitative PCR among OM treated MCF-7 and the control one. Cisplatin was the positive control drug of these experiments.3.2 RESULTS:The inhibitory effects of OM on SP cells from MCF-7 cells were significant difference among different concentrations of OM.Conon-SPared with cisplatin,OM showed a higher inhibitory effect in breast CSCs(SP),and a lower inhibitory effect in breast cancer cells(non-SP).In OM-treated cells,the SP population was 3.1%,1.7%,and 0.2%,at 0.25mg/ ml,0.5mg/ml,1mg/ml OM, respectively.The staining intensity of cytoplasm and nuclearβ-catenin of the re-cultured SP cells treated with OM was significiantly decreased,andβ-catenin was typically found at the cell membrane only conon-SPared with controls.The expression of phosphorylatedβ-catenin of re-cultured SP cell treated with OM positive cells were increased and the expression of c-myc and cyclinD1 were all decreased,as compared with untreated cells,but this effect was not observed in cisplatin treated group. 3.3 CONCLUSION:This result showed that the breast cancer stem-like cells(SP) were more sensitive to OM than cisplatin,it leads to a dramatic decrease in the SP population and down regulating the activity of Wnt/β-catenin signaling pathway...
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