| Botanical Medicines has great application value in the tumor therapy and adjuvant therapy, many antineoplastic agents such as taxol, vincristine that originated from botanical medicines have been widely used in clinical. The main reason for the failure of chemotherapy are due to the occurrence of multidrug resistance (MDR) of cancer, it is estimated that about90%of the failure are ascribed for MDR. Many MDR reverse agents had been stopped in clinical trials due to their toxicity, and the therapeutic effect is less than their toxicity effect. Plenty of botanical medicines has long medicinal history and its side effects are also be relatively clear, therefore it has great advantages in looking for MDR reverse agents from botanical medicines. In this research, the accumulation of Rho-123in1Caco-2cells was used as the drug screening model for MDR reverse agents extracts from Botanical Medicines, which including GanodermaLucidum Karst, Rabdosia rubescens (Hemsl.) and Hara Sophora tonkinensis Gapnep. We determined the effective component group of Ganoderma Lucidum Karst and Rabdosia rubescens (Hemsl.),and the mechanism of these extracts on MDR reverse effects is also discussed. The main results are as follows:1. The accumulation of Rho-123in Caco-2cells was used as the drug screening model, the intracellular mean fluorescence intensity associated with Rh123by the the positive drug verapamil was used as criterion,20kinds of botanical medicines extracts were screened,and the results showed:extracts from Ephedra sinica Stapf, Sophora flavescens Ait, Zanthoxylum schinifolium Sieb. et Zucc, Sinomenium acutum (Thunb.), Chrysanthemum indicum L., GanodermaLucidum Karst, Rabdosia rubescens (Hemsl.) and Hara Sophora tonkinensis Gapnep could increase the intracellular accumulation of Rh123, as for that extracts from the Ephedra sinica Stapf, Sophora flavescens Ait, Zanthoxylum schinifolium Sieb. et Zucc, Sinomenium acutum (Thunb.), Chrysanthemum indicum L.had been verified to be positive in MDR reverse effects, it is also confirmed that this screening model is suitable for screening MDR reverse agents from the complex extracts; When extracts from GanodermaLucidum Karst,Rabdosia rubescens (Hemsl.) or Hara Sophora tonkinensis Gapnep were used in conjunction with ADM,the cytotoxicity of ADM were increased, and the intracellular accumulation of ADM are also increased,which suggested that these three extracts has potential MDR reverse effects.2. Extracts of GanodermaLucidum Karst by different solvent were prepared as GP,GC,GE,GB and GW, each extracts was screened by aforementioned cell model, the results shows:when treated at non-toxicity dose, except for the GP, other extrats GC,GE,GB and GW could increase the intracellular accumulation of Rh123, when combination ADM with GC,GE,GB and GW, GE, GB and GW could enhance the cytotoxicity of ADM and increase the intracellular accumulation of ADM. The fluorescence accumulation multiple associated with ADM are2.27(GE,20μg/mL)>3.51(GE,100μg/mL),4.48(GB,4μg/mL),4.22(GB,20μg/mL),1.94(GW,4μg/mL)and2.63(GW,20μg/mL), respectively, and the effect are concentration dependently.3. The effects of GC,GE,GB and GW are also concentration-dependently in enhancing the ADM cytotoxicity and its intracellular accumulation. GC,GE,GB and GW from Ganoderma Lucidum Karst could reverse the MDR of SGC-7901/ADR cells to ADM at non-toxicity dose, and the revers folds are3.71(GE,20μg/mL),4.53(GE,100μg/mL),2.12(GB,4μg/mL),2.64(GB,20μg/mL),2.22(GW,4ug/mL)and2.16(GW,20μg/mL), respectively.4. Studies on the mechanism of MDR reverse effects shows:GE and GB could enhance the intracellular accumulation of ADM in SGC-7901/ADR cells, and the fluorescence accumulation multiple associated with ADM are3.35(GE,20μg/mL)>2.52(GB,20μg/mL) and3.26(GW,20μg/mL),which indicated that the MDR reverse effects of GE, GB and GW may be mediated by modulating the activities of p-gp. The effects of GE and GB on the transcription of MDR1were determined by RT-PCR. The results shows:when treated with GE and GB, the transcription of MDR1are decreased by32.16%(GE,20μg/mL),50.27%(GE,100μg/mL)>25.34%(GB,4μg/mL) and41.53(GB,20μg/mL), respectively; the abundance of p-gp is determined by immunofluorescence method, the p-gp relevant mean fluorescence intensity was decreased by21.5%(GE,20μg/mL),32.7%(GE,100μg/mL),20.5%(GB,4μg/mL) and27.8%(GB,20μg/mL), indicated that the MDR reverse effects of extracts from Ganoderma Lucidum Karst are mediated by modulating the activities of p-gp.5. GE and GB could also reverse the MDR of MCF-7/ADM cells to ADM, when treated with non-toxicity dose, the reverse folds are1.78(GE,8μg/mL),3.56(GE,40 μg/mL),2.11(GB,8μg/mL)and3.47(GB,40μg/mL), respectively. As in the ADM accumulation assay, the fluorescence accumulation multiple associated with ADM are3.31(GE,8μg/mL),4.48(GE,40μg/mL),1.94(GB,8μg/mL) and2.57(GB,8μg/mL), and the effect is also concentration-dependently. GE and GB also has the same effects in the inhibition of the transcription of MDR1and the decrease of p-gp expression, these results indicated that the MDR reverse effects of GE and GB on MCF-7/ADM cells is also mediated by modulating the activities of p-gp, and the MDR reverse effects of GE and GB is also wide-spectrum, in all the items detected, the effect of GE is better than that of GB, indicated that GE is the major effective component group in MDR reverse effects.6. Extracts of Herba Rabdosiae Rubescentis by different solvent were prepared as HP,HC,HE,HB and HW, each extracts was screened by aforementioned cell model, the results show:when treated with non-toxicity dose, except for the HP and HB, HC,HE and HW could increase the intracellular accumulation of Rh123and ADM, and enhance the cytotoxicity of ADM, indicated that HC,HE and HW are the potential effective component groups in MDR reverse effects.7. HC、HE and HW could reverse the MDR of SGC-7901/ADR cells to ADM at non-toxicity dose. The reverse folds are2.51(HC,20μg/mL)、2.80(HC,100μg/mL),2.29(HE,20μg/mL)、3.15(HE,100μg/mL),1.47(HW,20μg/mL) and1.64(HW,100μg/mL), respectively. The fluorescence accumulation multiple associated with ADM are3.05(HC,20μg/mL)、2.74(HE,20μg/mL)、2.62(HW,20μg/mL), respectively.8. The effect of HC and HE on the transcription of MDR1are determined by RT-PCR. The results shows:when treated with HC and HE, the transcription of MDR1are decreased, and the abundance of p-gp is also decreased by17.10%((HC,10μg/mL),14.7%(HE,10μg/mL)when determined by immunofluorescence method. These results indicated that the MDR reverse effects of HC and HE on SGC-7901/ADR cells is also mediated by modulating the activities of p-gp, and the potential effective component group are in HC and HE.9. HC and HE could also reverse the MDR of MCF-7/ADM cells to ADM, when treated with non-toxicity dose, the reverse folds are1.87(HE,4μg/mL),4.02(HE,20μg/mL)、2.16(HC,4μg/mL)and4.60(HC,20μg/mL), respectively. As in the ADM accumulation assay, the fluorescence accumulation multiple associated with ADM are3.97(HE,4μg/mL)、4.21(HE,20μg/mL)、61(HC,4μg/mL)and3.83(HC,20μg/mL), it is in line with the MDR reverse experiments on SGC-7901/ADR cells, The difference is that their MDR reverse effect on MCF-7/ADM cells is more efficient than that on SGC-7901/ADR cells, indicate that extracts from Rabdosia rubescens (Hemsl.) may have difference efficiency on different origin of MDR cells.10. HC and HE could also inhibite the transcription of MDR1,and decrease the expression of p-gp, these results suggested that the MDR reverse effects of HC and HE on MCF-7/ADM cells is also mediated by modulating the activities of p-gp, and HC and HE are the major effective component group in Rabdosia rubescens for MDR reverse effects. |
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