植物药中肿瘤多药耐药逆转剂的筛选及其逆转机制研究

【作者】 李发荣；

【导师】 王喆之； 董忠民；

【作者基本信息】 陕西师范大学 ， 植物学， 2013， 博士

【摘要】 植物药在肿瘤的治疗和辅助治疗中有重要的应用价值,许多植物药来源的抗肿瘤药物已经广泛在临床使用,如紫杉醇、长春新碱等。肿瘤多药耐药性的发生是肿瘤化疗失败的重要原因,据估计约有90%的转移性肿瘤的化疗失败应归因于肿瘤细胞的多药耐药性。多数肿瘤多药耐药逆转剂临床实验失败的主要原因是由于逆转剂本身的毒副作用过大,从而导致其治疗效应小于相应的副作用。由于很多植物药有悠久的药用历史,其毒副作用相对较为清楚,所以从植物药中寻找肿瘤多药耐药逆转剂是值得期待的研究方向。本课题以Caco-2细胞中Rho-123的蓄积为筛选模型,筛选出了灵芝、山豆根和冬凌草三种具有潜在逆转作用的植物药提取物,并确定了灵芝、冬凌草的具有逆转MDR作用的活性组分群,初步探讨了其逆转MDR的作用机制。主要研究结果如下：1、以Caco-2细胞中Rho-123的蓄积为筛选模型,以阳性药维拉帕米处理组中Rho-123相关的荧光强度为筛选标准,对20种植物药进行筛选,结果显示：麻黄、苦参、花椒、青风藤、野菊花、山豆根、冬凌草以及灵芝提取物均显示了促进Caco-2细胞中Rho-123蓄积的作用。其中麻黄、苦参、花椒、青风藤提取物是已经证实的具有逆转MDR的作用植物药,也印证了本筛选模型适用于成分复杂的植物提取物的筛选：山豆根、冬凌草以及灵芝提取物与阿霉素联用时可增加ADM对Caco-2细胞的细胞毒作用,可以促进ADM在Caco-2细胞中的蓄积,说明冬凌草、灵芝、山豆根提取物具有潜在逆转p-gp介导MDR的作用。2、制备了灵芝的GP、GC、GE、GB和GW提取部位,以Caco-2细胞中Rho-123的蓄积筛选模型对灵芝不同提取部位进行筛选,结果显示：在非毒性剂量下(<IC20),除GP之外,其他几个提取部位均可以增加Caco-2细胞中Rho-123蓄积。各活性提取部位与ADM联合使用,GE、GB和GW可以增强ADM对Caco-2细胞的细胞毒性,促进Caco-2细胞中ADM的蓄积,其荧光蓄积倍数分别为2.27(GE,20Hg/mL)、3.51(GE,100μg/mL)、4.48(GB,4μg/mL)、4.22(GB,20μg/mL)、1.94(GW,4μg/mL)、2.63(GW,20μg/mL),显示了较为明确的剂量依赖关系。3、灵芝活性提取部位可以有效逆转SGC-7901/ADR耐药细胞对ADM的耐药性,在非毒性剂量下(<IC20),其逆转倍数分别为3.71(GE,20μg/mL)、4.53(GE,100μg/mL).2.12(GB,4μg/mL)、2.64(GB,20μg/mL)、2.22(GW,4μg/mL)、2.16(GW,20μg/mL)。4、对其逆转机制的研究中揭示：灵芝活性提取部位可以促进SGC-7901/ADR细胞内阿霉素的蓄积,其阿霉素相关的荧光蓄积倍数分别为3.35(GE,20μg/mL)、2.52(GB,20μg/mL)、3.26(GW,20μg/mL),说明灵芝提取物逆转SGC-7901/ADR的MDR与其促进细胞内阿霉素的蓄积相关,有可能是通过调节p-gp活性实现的。灵芝乙酸乙酯部位和正丁醇部位对SGC-7901/ADR细胞MDR1基因转录影响的RT-PCR结果显示,不同活性部位处理后,MDR1基因的转录下降率分别为：32.16%(GE,20ng/mL)、50.27%(GE,100μg/mL)、25.34%(GB,4μg/mL)、41.53(GB,20μg/mL)。对细胞总P-gp的免疫荧光检测显示：GE (20μg/mL,100μ/mL)、GB(4μg/mL、20μg/mL)处理后,细胞荧光强度分别下降了21.5%、32.7%、20.5%、27.8%。呈明显的剂量效应,说明灵芝提取物逆转MDR的效应是通过抑制P-gp的作用实现的。5、GE和GB也可以有效逆转耐药细胞MCF-7/ADM细胞的耐药性,在非毒性剂量下(<IC20),其逆转倍数分别为1.78(GE,8μg/mL)、3.56(GE,40μg/mL)、2.11(GB,8μg/mL)、3.47(GB,40μg/mL),呈现较明显的剂量效应,在阿霉素的蓄积实验中,其阿霉素相关的荧光蓄积倍数分别为3.31(GE,8μg/mL).4.48(GE,40μg/mL),1.94(GB,8μg/mL)、2.57(GB,40μg/mL),与在SGC-7901/ADR细胞的相关实验结果相一致。GE和GB对MCF-7/ADM细胞MDR1基因的转录及也有相似的抑制效应,也可以有效地降低MCF-7/ADM细胞中P-gp的表达,说明灵芝提取物逆转MCF-7/ADM细胞的耐药性同样与抑制P-gp的活性相关。也说明灵芝提取物用于逆转肿瘤的MDR具有一定广谱性,其活性组分群位于GE和GB,在阿霉素蓄积、逆转倍数及P-gp表达量等测试指标中,GE较GB稍好,说明GE是灵芝具逆转MDR作用的最主要的活性组分群,对其成分的深入分析有可能发现新的逆转MDR作用的活性组分。6、制备了HP、HC、HE、HB和HW提取部位,以Caco-2细胞中Rho-123的蓄积筛选模型对冬凌草不同提取部位进行筛选和初步验证的的结果显示：在非毒性剂量下(<IC20),HC、HE和HW均可以有效增加细胞内Rho-123及阿霉素的蓄积,促进阿霉素的细胞毒性,提示HC、HE和HW是冬凌草具有潜在的逆转MDR活性的活性组分群。7, HC、HE和HW可以有效逆转SGC-7901/ADR细胞的阿霉素耐药性,逆转倍数分别为其逆转倍数分别为2.51(HC,20μg/mL)、2.80(HC,100jig/mL)、2.29(HE,20μg/mL)、3.15(HE,100μg/mL)、1.47(HW,20μg/mL)、1.64(HW,100μg/mL),对SGC-7901/ADR细胞中阿霉素相关的荧光蓄积倍数分别为：3.05(HC,20μg/mL).2.74(HE,20μg/mL)、2.62(HW,20μg/mL)。8、利用RT-PCRJI技术对HE、 HC的SGC-7901/ADR细胞MDR1基因转录的半定量检测显示,HC和HE可以抑制MDR1基因的转录；对细胞总P-gp的免疫荧光检测显示：药物处理后,P-gp表达量分别降低约17.10%(HC,10μg/mL)和14.17(HE,10μg/mL),说明冬凌草提取物逆转MDR的效应是通过抑制p-gp的表达实现的。冬凌草逆转作用的主要活性组分群位于HC和HE。9、HC和HE也可以有效逆转耐药细胞MCF-7/ADM细胞的耐药性,在非毒性剂量下(<IC20),其逆转倍数分别为1.87(HE,4μg/mL)、4.02(HE,20μg/mL)、2.16(HC,4μg/mL)、4.60(HC,20μg/mL),呈现较明显的剂量效应。在阿霉素的蓄积实验中,其阿霉素相关的荧光蓄积倍数分别为3.97(HE,4μg/mL)、4.21(HE,20μg/mL)、3.61(HC,4jig/mL)、3.83(HC,20μgmL),与对SGC-7901/ADR细胞的逆转效果相比较,结果基本一致,但是对于MCF-7/ADM细胞的耐药逆转效应要优于SGC-7901/ADR细胞,提示冬凌草提取物在逆转肿瘤MDR时,对于不同肿瘤细胞的逆转效应可能有所不同。1O、HC和HE对MCF-7/ADM细胞MDR1基因及也有相似的抑制效应,也可以有效地降低MCF-7/ADM细胞中P-gp的表达,说明冬凌草提取物逆转MCF-7/ADM细胞的耐药性同样与抑制P-gp的活性相关。也说明冬凌草提取物逆转肿瘤的MDR的活性组分群位于HC和HE。更多还原

【Abstract】 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.更多还原