甘草提取物及其主要成分对Caco-2细胞膜上P-gp功能和表达的影响

 目的 初步考察甘草提取物及其主要成分（甘草甜素、甘草次酸和甘草苷）对Caco-2细胞膜上P-糖蛋白（P-gp）功能和表达的影响,以备进一步探讨甘草新的解毒机制。方法 利用流式细胞术检测Caco-2细胞内罗丹明123（Rho-123）的荧光强度和细胞膜上P-gp的表达,以考察P-gp外排功能和表达水平的变化。结果 经过60 min的干预,甘草提取物(1, 10, 100 μg·mL-1)、甘草甜素(1, 10, 100 μg·mL-1)和甘草苷(1, 10, 100 μg·mL-1)Caco-2细胞内Rho-123的荧光强度较阴性对照组分别降低了34.66%、28.90%、24.56%、27.89%、26.29%、37.33%、19.51%、21.86%和19.03%（P＜0.05）。经过72 h的干预,甘草提取物中质量浓度(10 μg·mL-1)组,甘草甜素低、中、高质量浓度(1, 10, 100 μg·mL-1)组,甘草次酸中、高质量浓度(1, 10 μg·mL-1)组Caco-2细胞膜上P-gp表达的阳性率较阴性组分别增加了31.18%、61.67%、70.32%、77.43%、37.58%和49.14%（P＜0.05）。结论 甘草提取物、甘草甜素和甘草苷可能增强Caco-2细胞膜上P-gp的功能,而中质量浓度的甘草提取物,低、中、高质量浓度的甘草甜素和中、高质量浓度的甘草次酸则可能上调P-gp的表达。甘草甜素既能增强Caco-2细胞膜上P-gp的功能,又能上调其表达,可能是甘草影响P-gp的有效成分。     

治疗剂量下4种抗结核药物与Caco-2细胞上P-gp相互作用研究

 目的 研究治疗剂量下4种抗结核药物(异烟肼、左氧氟沙星、乙胺丁醇、吡嗪酰胺对Caco-2细胞上P-糖蛋白功能、表达及MDR1 mRNA表达的影响,从而解释联合抗痨治疗中不同组合的合理性。方法 采用流式细胞仪测定细胞内罗丹明-123的浓度,考察药物对P-糖蛋白功能的影响,流式细胞术分析药物对Caco-2细胞上P-糖蛋白表达的影响,实时荧光定量PCR技术分析药物对Caco-2细胞MDR1基因mRNA水平表达的影响。结果 含药培养20 d后,异烟肼和乙胺丁醇减少了罗丹明-123在Caco-2细胞内的蓄积(P＜0.05,为诱导作用;异烟肼、乙胺丁醇均上调了Caco-2细胞上P-糖蛋白的表达(P＜0.05,其P-糖蛋白表达量分别为阴性对照组的3.5和3.8倍;同时上调了Caco-2细胞上MDR1 mRNA的表达(P＜0.05,其MDR1 mRNA的表达量分别为阴性对照组的11.5和11倍。左氧氟沙星增加了罗丹明-123在Caco-2细胞内的蓄积(P＜0.05,为抑制作用;下调了Caco-2细胞上P-糖蛋白和MDR1 mRNA的表达(P＜0.05,其P-糖蛋白和MDR1 mRNA表达量分别为阴性对照组的50%和32%。而吡嗪酰胺与P-糖蛋白无明显相互作用。结论 治疗剂量的异烟肼和乙胺丁醇为P-糖蛋白的诱导剂,左氧氟沙星为P-糖蛋白的抑制剂,而吡嗪酰胺对P-糖蛋白功能和表达无明显影响。     

解毒祛瘀方对乳腺癌细胞MCF-7/ADM的耐药逆转作用

目的:研究解毒祛瘀方对人乳腺癌耐药细胞MCF-7/ADM耐药逆转的作用及机制。方法:以MCF-7/ADM细胞为研究对象,利用噻唑蓝(MTT)比色法检测解毒祛瘀方对人乳腺癌耐药细胞MCF-7/ADM生长的影响;应用流式细胞术检测肿瘤细胞内罗丹明123(Rh-123)的含量;分别利用实时荧光定量聚合酶链式反应(Real-time PCR)及蛋白免疫印迹法(Western blot)检测肿瘤细胞内多药耐药蛋白1(MDR1),乳腺癌耐药相关蛋白(BCRP)mRNA及蛋白表达变化。结果:与空白组比较,经1.25,2.5 g·L~(-1)解毒祛瘀方作用后,阿霉素对人乳腺癌耐药细胞MCF-7/ADM的逆转倍数(RF)分别提升1.7倍和3.0倍(P0.05);人乳腺癌耐药细胞MCF-7/ADM中Rh-123含量分别提高了1.8倍和2.5倍(P0.05),MDR1和BCRP蛋白和mRNA表达水平明显下降(P0.05),1.25,2.5 g·L~(-1)解毒祛瘀方MDR1 mRNA表达分别降低35.5%和56.0%(P0.05),BCRP mRNA表达分别降低41.6%和49.5%(P0.05)。结论:解毒祛瘀方可提高人乳腺癌耐药细胞MCF-7/ADM对阿霉素的敏感性,逆转该细胞对阿霉素的耐药性,其机制可能与降低MDR1和BCRP蛋白和mRNA的表达,抑制细胞药物外排作用相关。     

18α-甘草酸和18β-甘草酸对Caco-2细胞P-gp功能和表达的影响

目的:研究甘草酸18位差向异构体18α-甘草酸、18β-甘草酸对Caco-2细胞上P-糖蛋白功能和表达的影响。方法:建立Caco-2细胞模型,采用罗丹明-123摄取法评价P-糖蛋白的功能;利用流式细胞术和荧光定量PCR分析Caco-2细胞膜上P-糖蛋白的表达。结果:中、高浓度(10,60μmol.L-1)α-GL使细胞内Rho-123摄取增加,对P-gp表现出抑制作用,但没有呈现出剂量依赖性;β-GL各浓度组使细胞内Rho-123摄取减少,对P-gp表现出诱导作用,但也没有表现出剂量依赖性。二者对Caco-2细胞上P-gp功能的影响呈相反的趋势;Caco-2细胞与药物孵育72 h后,中、高浓度(10,60μmol.L-1)α-GL下调MDR1 mRNA表达,β-GL只在高浓度(60μmol.L-1)上调了MDR1 mRNA表达;高浓度(60μmol.L-1)β-GL在蛋白水平对P-gp有诱导作用,α-GL各浓度没有表现出对P-gp表达的影响。结论:转录水平上α-GL,β-GL对P-gp的影响与α-GL,β-GL对CYP3A的影响呈现一定的同向性,甘草酸18位差向异构体对CYP3A与P-gp的影响有相似的立体选择性,其机制是否与孕烷X受体(PXR)有关,有待进一步研究。     

Modulation of P-glycoprotein-mediated resistance by kaempferol derivatives isolated from Zingiber zerumbet

This study examined the effects of the kaempferol derivatives extracted from Zingiber zerumbet on the accumulation and efflux of [(3)H]-daunomycin (DNM) in P-glycoprotein (P-gp) overexpressing multidrug resistant (MDR) human breast cancer cells, MCF-7/ADR. Of six kaempferol derivatives extracted from Z. zerumbet, kaempferol-3-O-methyl ether (1) and kaempferol-3,4'-O-dimethyl ether (2) showed a potent P-gp inhibitory effect as great as verapamil, a well-known P-gp inhibitor. The P-gp inhibitory activity of these two compounds was through a 3-fold increase of the level of [(3)H]-DNM accumulation and a decrease of P-gp-mediated efflux. These results suggest that the kaempferol derivative components of Z. zerumbet can be used as a scaffold for developing agents that reverse P-gp-mediated MDR in human cancer chemotherapy.     

复方中药紫龙金克服肿瘤细胞多药耐药性的机制

目的探讨中药紫龙金(Zilongjin,ZLJ)对多药耐药肿瘤细胞的作用机制。方法采用MTT法检测ZLJ对细胞增殖的影响;流式细胞术检测细胞周期以及罗丹明123的荧光强度变化;Western blot方法检测相关蛋白的表达变化。结果 ZLJ分别处理人乳腺癌MCF-7和MCF-7/DOX耐药细胞,以及人口腔上皮癌KB和KBV200耐药细胞。MTT法测定表明:ZLJ作用耐药和敏感细胞的IC50值相近,耐药细胞对ZLJ没有交叉耐药性;无论对敏感和耐药细胞,流式细胞术分析发现ZLJ阻断细胞于S期;ZLJ单独处理MCF-7/DOX和KBV200耐药细胞,可以微弱地降低其耐药性,分别与多柔比星(doxorubicin,DOX)和长春新碱(vincris-tine,VCR)合用,可以明显地增加DOX和VCR的活性;ZLJ处理耐药细胞MCF-7/DOX后,检测到细胞内耐药蛋白P-糖蛋白(P-glyco protein,P-gp)呈时间依赖性降低。Western blot检测表明,ZLJ的抑制作用是通过使凋亡标志蛋白PARP出现切割,启动凋亡通路实现的。结论中药ZLJ抑制耐药细胞增殖,没有交叉耐药性;其抑制作用与诱导细胞凋亡以及降低P-gp表达有关。     

Paris saponin VII from trillium tschonoskii reverses multidrug resistance of adriamycin-resistant MCF-7/ADR cells via P-glycoprotein inhibition and apoptosis augmentation

Ethnopharmacological relevance

Saponins of several herbs are known to induce apoptosis in some cancer cells and are proposed to be promising modulators of drug resistance. In the present study, we extracted Paris saponin VII (PS VII), a kind of saponin, from Trillium tschonoskii Maxim. and observed its effect on adriamycin-resistant breast cancer cells.

Materials and methods

An adriamycin-resistant human breast cancer cell line, MCF-7/ADR cells were exposed to different concentrations of PS VII (0–100 μmol/L). Then, flow cytometric assays and a human apoptosis array were used to detect apoptotic cells and apoptosis related protein expression. P-glycoprotein levels and intracellular rhodamine 123 (RH-123) accumulations were measured to evaluate the expression and activity of P-glycoprotein.

Results

PS VII dose dependently suppressed cell viability as well as triggered apoptosis and modulated drug resistance of MCF-7/ADR cells. Further results showed that PS VII treatment in MCF-7/ADR cells led to increased TNFR1, TRAIL R1/DR4, TRAIL R2/DR5, and FADD expression, and activation of PARP, caspase-8, and 3. In parallel to the alterations, P-glycoprotein expression and activity were also reduced.

Conclusion

These findings showed that PS VII might be an effective tumouristatic agent for the treatment of MDR breast cancer.     

Pentasaccharide resin glycosides from Ipomoea pes-caprae

Pescapreins XXI-XXX (1-10), pentasaccharide resin glycosides, together with the known pescapreins I-IV and stoloniferin III were isolated from the aerial parts of Ipomoea pes-caprae (beach morning-glory). The pescapreins are macrolactones of simonic acid B, partially esterified with different fatty acids. The lactonization site of the aglycone, jalapinolic acid, was located at C-2 or C-3 of the second saccharide moiety. Their structures were established by a combination of spectroscopic and chemical methods. Compounds 1-10 were evaluated for their potential to modulate multidrug resistance in the human breast cancer cell line MCF-7/ADR. The combined use of these new compounds at a concentration of 5 μg/mL increased the cytotoxicity of doxorubicin by 1.5-3.7-fold.     

�����������������������������ת���ٰ���ҩ��ҩ����������

??OBJECTIVE To prepare a redox and pH dual sensitive nano-carrier based on PAMAM in order to co-loading chemotherapeutics doxorubicin and breast cancer multidrug resistance reversal agent elacridar, and study their in vitro reversal effect. METHODS The infrared spectrum FTIR was used to characterize the carrier. Confocal was used to investigate the intracellular triggered drug release. The reversal effect of breast cancer multidrug resistance and the in vitro anti-tumor activity of doxorubicin and elacridar co-loaded nanoparticles were investigated using flow cytometry and cell toxicity tests, respectively. RESULTS The doxorubicin and elacridar co-loaded nanoparticles (PSSP/DOX/ELC) were successfully prepared, and pH-redox dual sensitive of carrier was proved by cell experiments.And the carrier was uptaken into cells and delivery to lysosome, and drug release was triggered in the lysosome acid condition, then the released drug diffused to the nucleus. The trial of rhodamine 123 accumulation and efflux assay revealed that the accumulation of rhodamine 123 was notably increased after incubation of elacridar in MCF-7/ADR cells. The cytotoxicity of PSSP/DOX/ELC nanoparticles against MCF-7/ADR cell line was significantly stronger than that of either free doxorubicin or only doxorubicin loaded nanoparticles (PSSP/DOX). CONCLUSION The reversal effect of multidrug resistance and the cytotoxicity of cancer cells were significantly enhanced by PSSP/DOX/ELC nanoparticles. PSSP/DOX/ELC nanoparticles is a promising delivery system.     

香叶木素通过线粒体凋亡途径对人乳腺癌MCF-7细胞增殖及凋亡的影响

目的探讨香叶木素对人乳腺癌MCF-7细胞增殖、凋亡的影响及其作用机制。方法将MCF-7细胞分为对照组(0μmol·L^-1)和香叶木素5、10、20、30、40、50、60、70μmol·L^-1浓度组。采用CCK-8法及2,4二硝基苯肼法检测MCF-7细胞活力、乳酸脱氢酶(LDH)泄露量;采用罗丹明123(Rh123)荧光染色法、流式细胞术及荧光显微镜检测MCF-7细胞线粒体膜电位(MMP);DCFH-DA法检测细胞内活性氧(ROS)含量;Annexin V-FITC/PI双染法检测细胞凋亡;Western Blot法检测p53、Bax、Caspase 3及Bcl-2蛋白的表达。结果与对照组比较,香叶木素在5~70μmol·L^-1范围内能够浓度依赖性地抑制MCF-7的细胞活力及促进LDH的泄露(P<0.01,P<0.001),选取浓度10、30、50μmol·L^-1进行后续实验;香叶木素10、30、50μmol·L^-1浓度组能显著降低MCF-7细胞线粒体膜电位(P<0.05,P<0.01,P<0.001),促进细胞内ROS的积累(P<0.01,P<0.001),提高细胞凋亡率(P<0.001),显著上调p53(30、50μmol·L^-1)、Bax及Caspase 3蛋白的表达(P<0.001),下调Bcl-2蛋白的表达(P<0.01,P<0.001)。结论香叶木素可能通过ROS及p53介导的线粒体凋亡途径抑制MCF-7细胞的增殖及促进其凋亡。     

苦参碱与粉防己碱逆转人MCF-7细胞耐药性的作用与机制

目的:探讨中药有效成分苦参碱与粉防己碱合用能否逆转人乳腺癌MCF-7细胞的多药耐药性.方法:使用人乳腺癌MCF-7和耐多柔比星的MCF-7/DOX细胞进行研究.采用MTT法检测药物对细胞增殖的影响;流式细胞术检测罗丹明123荧光强度的变化;Western blot方法检测P-糖蛋白的表达变化.结果:苦参碱作用耐药和敏感细胞的IC50相近,耐药细胞对苦参碱没有明显的交叉耐药性;流式细胞术分析发现苦参碱单独处理MCF-7/DOX耐药细胞,可以微弱地降低其耐药性,并且与粉防己碱有协同作用;苦参碱与多柔比星合用,可以增加多柔比星对MCF-7/DOX耐药细胞的生长抑制作用,且与粉防己碱有协同作用;苦参碱和粉防己碱、多柔比星合用处理细胞,MCF-7/DOX耐药细胞株P-糖蛋白表达水平下降.结论:苦参碱与粉防己碱合用具有协同作用,能逆转人乳腺癌MCF-7细胞的多药耐药性,降低P-糖蛋白的表达.     

Norlignans, acylphloroglucinols, and a dimeric xanthone from Hypericum chinense

Two new norlignans, hyperiones A (1) and B (2), three new acylphloroglucinols, aspidinol C (3) and hyperaspidinols A (5) and B (6), the known compound aspidinol D (4), and the symmetrical dimeric xanthone hyperidixanthone (7) were isolated from Hypericum chinense. Their structures were established by spectroscopic analysis. In an antibacterial assay using a panel of multidrug-resistant (MDR) strains, compounds 3 and 4 exhibited promising activity against the NorA efflux protein overexpressing MDR Staphylococcus aureus strain SA-1199B with a minimum inhibitory concentration (MIC) of 2 μg/mL (8.4 μM) and 4 μg/mL (16.8 μM), respectively. The positive control antibiotic norfloxacin showed activity at MIC 32 μg/mL (100 μM).     

Inhibitory effect of Thai plant extracts on P-glycoprotein mediated efflux

Curcuminoids from Curcuma longa L. and extracts of Psidium guajava L., Andrographis paniculata (Burm. f.) Nees, Phyllanthus emblica L. and Solanum trilobatum L. were investigated for their inhibitory effect on P-glycoprotein (P-gp) on the efflux transport of rhodamine 123 (Rho-123 ) in Caco-2 cells and rat ileum. Of the five tested samples, curcuminoids and an extract of P. guajava showed the highest inhibitory effect on P-gp mediated efflux of Rho-123 in Caco-2 cells. Additionally, they were found to have equal potential in inhibiting Rho-123 efflux transport from serosal to mucosal surfaces of the rat ileum.     

欧白芷素对K562/A02细胞阿霉素耐药的逆转作用(英文)

目的:探讨欧白芷素对耐药细胞株K562/A02中P-糖蛋白(P-glycoprotein,P-gp)的影响,为抗白血病多药耐药(multi-drug resistance,MDR)提供新方法。方法:采用MTT法观察阿霉素对细胞活力的影响。应用流式细胞术检测欧白芷素对K562和K562/A02细胞内阿霉素累积和细胞中P-gp功能的影响。采用实时定量RT-PCR技术检测MDR1基因在mRNA表达水平的变化。结果:欧白芷素对耐药细胞株K562/A02有显著的逆转耐药活性,最大逆转倍数为7.36。在K562/A02细胞中,欧白芷素明显增加阿霉素的累积,增加了罗丹明123(rhodaminel123,Rh123)蓄积,抑制了Rh123的外排,同时欧白芷素还在mRNA水平抑制了K562/A02细胞中P-gp的表达。结论:欧白芷素能够抑制K562/A02耐药细胞株中MDR1基因表达和P-gp的功能。     

Effects of propolis extract on sensitivity to chemotherapeutic agents in HeLa and resistant sublines

The effects of a propolis extract obtained by supercritical fluid extraction on sensitivity to chemotherapeutic agents were examined in HeLa cells and resistant sublines thereof. In addition, the actions of propolis and caffeic acid phenethyl ester (CAPE), a constituent of propolis, on the multidrug efflux transporter P-glycoprotein/MDR1, were evaluated in paclitaxel-resistant HeLa/TXL cells (MDR1-overexpressing cells). In HeLa cells, the sensitivity to paclitaxel and doxorubicin, substrates of MDR1, was unchanged in the presence of propolis. In HeLa/TXL cells, propolis increased sensitivity to these MDR1 substrates. The accumulation of Rhodamine123, also a substrate for MDR1, by HeLa/TXL cells increased in the presence of 50 microg/mL, but not 10 microg/mL, of the extract. However, the growth inhibition of HeLa/TXL cells by paclitaxel was not changed by CAPE, although the accumulation of Rhodamine123 increased significantly in the presence of 100 microm, but not 1 nM or 1 microm, CAPE. Collectively, the extract was suggested to inhibit the function of MDR1 and to increase the sensitivity to MDR1 substrates in HeLa/TXL cells, effects likely to be caused by constituents other than CAPE.     

穿心莲内酯诱导白念珠菌生物膜分散细胞凋亡的研究

目的:探讨中药有效成分穿心莲内酯对白念珠菌生物膜分散细胞凋亡的影响。方法:Hoechst33258染色荧光显微镜检测白念珠菌生物膜细胞凋亡的形态;Rh123染色流式细胞仪检测白念珠菌生物膜细胞线粒体膜电位(MMP)变化;DHR染色流式细胞仪检测白念珠菌生物膜细胞内活性氧(ROS)水平。结果:1 000,100μmol.L-1的穿心莲内酯能诱导白念珠菌生物膜细胞核固缩、浓染致密,1 000,100,10μmol.L-1的穿心莲内酯能降低白念珠菌生物膜线粒体膜电位,提高细胞内ROS水平。结论:一定浓度的穿心莲内酯可诱导白念珠菌生物膜分散细胞凋亡。     

中药功劳木对乳腺癌耐药MCF7/ADM逆转MDR1的作用研究

目的：研究中药功劳木对乳腺癌耐药MCF7/ADM逆转MDR1的作用。方法：应用台盼蓝拒染试验测试应用功劳木后ADM对MCF7/ADM的活细胞率：MTT法测试功劳木的细胞毒性及对ADM的增效;Rh123荧光技术检测应用功劳木对耐药细胞内药物浓度的影响,流式细胞仪检测功劳木对MDR1的影响。结果：实验组浓度为10 mg/L以及20mg/L时对MCF7/ADM的抑制分别为（0.218±0.081）,（0.202±0.033）,均明显高于对照组（0.146±0.062）,（0.153±0.018）,差异有统计学意义（P〈0.05）。实验组对MCF-7/S以及MCF7/ADM的抑制分别为（0.204±0.076）,（51.08±0.63）,明显高于对照组的（0.075±0.032）及（36.55±0.53）,差异有统计学意义（P〈0.05）。结论：GLM对乳腺癌耐药MCF7/ADM逆转MDR1具有抑制作用。     

普流罗尼克对P-gp底物罗丹明123在Caco-2细胞蓄积的影响

 目的 研究普流罗尼克（Pluronic F127,F68,P85,P123对小肠P-糖蛋白（P-gp的调控作用。方法 采用MTT法检测不同质量浓度的Pluronic对Caco-2细胞生长抑制作用;以P-gp底物罗丹明123(R-123为荧光探针,评价各种Pluronic辅料和P-gp抑制剂维拉帕米对R-123细胞蓄积的影响,细胞内的R-123浓度采用高效液相-荧光检测。同时考察了Pluronic P123和F127对P-gp ATP酶活性的影响。结果 除了L61,经Pluronic处理后的细胞生存率都在80％以上,表明在蓄积实验中的细胞活性没有受到Pluronic的影响。在抑制剂维拉帕米和不同浓度的Pluronic(0.001~50 mg·mL-1作用下,可在不同程度上提高R-123在Caco-2细胞的蓄积作用,同时具有浓度依赖性,在接近或超过临界胶束浓度(CMC后,细胞蓄积达到最大值,然后随着Pluronic浓度的继续增大,蓄积作用又逐渐减弱。不同浓度的Pluronic P123和F127对P-gp ATP酶活性具有抑制作用。结论 蓄积实验结果表明,Pluronic F127,F68,P85和P123可以通过抑制P-gp的作用改善药物的吸收,抑制P-gp ATP酶活性可能是原因之一。因此,联合使用Pluronic,有望提高P-gp底物药物的口服生物利用度。