康莱特诱导具多药耐药表型的人乳腺癌细胞MCF7~(adr)凋亡及周期阻滞的研究

目的:观察康莱特对耐药细胞的作用及分子机制。方法:以敏感型乳腺癌细胞株 MCF7为对照,四氮唑蓝(MTT)法检测康莱特对耐药细胞株 MCF7~(adr)的抑制作用。以 Annexin V 标记法、DNA 含量测定法及电镜观察康莱特的诱导耐药细胞 MCF7~(adr)凋亡及周期阻滞的作用。免疫组化法检测康莱特对 MCF7~(adr)细胞 p53、p21~(WAF1/CIP1)蛋白表达的影响,RT-PCR 法检测康莱特对 MCF7~(adr)细胞 p21~(WAF1/CIP1)mRNA 表达水平的影响。结果:康莱特对 MCF7~(adr)细胞的半数抑制剂量(IC_(50))为26μ1/ml,与其对敏感细胞 MCF7的 IC_(50)(21μ1/ml)差异无显著性(P>0.05)。康莱特能诱导 MCF7~(adr)出细胞凋亡及细胞周期阻滞(G_0/G_1及 G_2/M 期),使 p53蛋白、p21~(WAF1/CIP1)mRNA 及蛋白表达水平升高。结论:康莱特对耐药细胞同样有效,这为其临床应用于已产生耐药性的肿瘤提供了依据。     

大蒜辣素对多药耐药HepG2细胞MDR1基因表达影响

目的 多药耐药(MDR)是肿瘤化疗的主要障碍,该研究旨在体外建立MDR人肝癌HepG2细胞株(HepG2/mdr),并探讨大蒜辣素(Allicin)对MDR1表达的影响.方法 采用高效液相色谱法(HPLC)提纯Allicin,采用间歇逐步增加阿霉素( adramycin,ADM)剂量法建立人肝癌MDR细胞株HepG2/mdr,运用细胞计数试剂盒(Cell counting kit-8,CCK -8)检测耐药细胞株耐药性,运用倒置显微镜观察不同浓度Allicin孵育下HepG2/mdr细胞形态学变化,运用逆转录聚合酶链反应( RT - PCR)及免疫印迹(Western blot)测定不同浓度Allicin作用下,HepG2/mdr细胞MDR1mRNA与蛋白表达情况.结果 Allicin经HPLC法提纯后其纯度达到99％.在2000nmol/L ADM作用下,HepG2/mdr细胞生长良好,而HepG2细胞生长停滞与死亡,且Allicin能阻止HepG2/mdr细胞生长,促进其死亡,随着Allicin浓度增加越加明显.Allicin能降低MDR1 mRNA和蛋白表达,呈现剂量依赖性.结论 成功建立人肝癌MDR细胞株HepG2/mdr,Allicin能通过抑制MRD1基因的表达,恢复肿瘤细胞对化疗药物的敏感性.     

大蒜辣素对多药耐药HepG2细胞中MDR1基因表达影响

目的多药耐药(MDR)是肿瘤化疗的主要障碍,该研究旨在体外建立MDR人肝癌HepG2细胞株(HepG2/mdr),并探讨大蒜辣素(Allicin)对MDR1表达的影响。方法采用高效液相色谱法(HPLC)提纯Allicin,采用间歇逐步增加阿霉素(adramycin,ADM)剂量法建立人肝癌MDR细胞株HepG2/mdr,运用细胞计数试剂盒(Cell counting kit-8,CCK-8)检测耐药细胞株耐药性,运用倒置显微镜观察不同浓度Allicin孵育下HepG2/mdr细胞形态学变化,运用逆转录聚合酶链反应(RT-PCR)及免疫印迹(Western blot)测定不同浓度Allicin作用下,HepG2/mdr细胞MDR1mRNA与蛋白表达情况。结果 Allicin经HPLC法提纯后其纯度达到99%。在2000nmol/L ADM作用下,HepG2/mdr细胞生长良好,而HepG2细胞生长停滞与死亡,且Allicin能阻止HepG2/mdr细胞生长,促进其死亡,随着Allicin浓度增加越加明显。Allicin能降低MDR1 mRNA和蛋白表达,呈现剂量依赖性。结论成功建立人肝癌MDR细胞株HepG2/mdr,Allicin能通过抑制MRD1基因的表达,恢复肿瘤细胞对化疗药物的敏感性。     

粉防己碱抗人乳腺癌细胞MCF-7/TAM对三苯氧胺的耐药性研究

目的 探讨粉防己碱（Tet）对耐三苯氧胺（TAM）的人乳腺癌细胞MCF 7/TAM逆转耐药效应及其机制。方法 采用四甲基偶氮唑蓝（MTT）法测定Tet对MCF 7/TAM 细胞的药物毒性及其逆转耐药效果;采用实时荧光定量PCR法检测Tet对MCF 7/TAM 细胞多药耐药相关蛋白1（MRP1）基因影响;采用Western blot法检测MCF 7/TAM 细胞MRP1蛋白变化。结果 Tet对MCF 7/TAM细胞有明显逆转耐药作用,非细胞毒性剂量（0.625 μg/ mL）的Tet逆转耐药倍数为2.0。Tet作用于MCF 7/TAM细胞后,能够下调MRP1基因（P<0. 05）和蛋白表达水平。结论 Tet可逆转MCF 7/TAM细胞的耐药性,逆转机制可能与下调细胞的MRP1表达有关。     

Caco-2细胞中8-羟基二氢小檗碱和小檗碱对多药耐药基因表达的研究

目的：研究8-羟基二氢小檗碱和小檗碱与多药耐药基因之间的作用。方法：采用MTT比色法测定药物的细胞毒浓度,适时定量PCR测定不同多药耐药基因的表达。结果：在1～100μM浓度范围内,8-羟基二氢小檗碱和小檗碱对Caco-2细胞无毒性作用;8-羟基二氢小檗碱和小檗碱对mdr1和mrp 1的表达作用一致,在25～100μM范围内,小檗碱能明显降低mrp2的mRNA表达,而8-羟基二氢小檗碱对其无明显影响。结论：8-羟基二氢小檗碱与小檗碱对mrp2的基因表达差异较大,其对MRP2蛋白表达的作用以及与基因调控之间的作用关系还有待更进一步的研究。     

红花黄色素对乳腺癌耐药细胞MCF－7／ADR 缝隙连接蛋白 Cx43的影响

目的：观察红花黄色素对乳腺癌耐药细胞MCF－7／ADR缝隙连接蛋白Cx43的影响。方法取对数生长期的MCF－7／ADR细胞,以2×105 cells／孔接种于6孔培养板,24 h后分为4组,每组3孔,其中3组分别加入红花黄色素至终浓度25、50、100 mg／L,另一孔设为空白对照组,作用48 h。 Western blot法检测各组MCF－7／ADR细胞中Cx43表达水平。结果不同浓度红花黄色素作用于MCF－7／ADR细胞48 h后, Cx43表达水平均增高,有明显的剂量依赖关系,以50、100 mg／L浓度作用显著（ P＜0．01）。结论红花黄色素能提高乳腺癌耐药细胞MCF－7／ADR缝隙连接蛋白Cx43的表达水平。     

EGCG对两株耐药肿瘤细胞的细胞毒增敏作用及抑瘤作用研究

目的:研究EGCG对人耐药肝癌细胞BEL7404/Adr和耐药口腔癌细胞KBV200的细胞毒增敏作用及对裸鼠移植瘤的抑瘤作用。方法:MTT法检测药物对体外培养细胞的毒性作用,采用BEL7404/ADR或KBV200细胞种植于裸鼠皮下,建立耐药肿瘤模型,观察用药后对裸鼠体重、抑瘤率、病理改变以及RTPCR和免疫组化法分别检测瘤组织mdr1和P糖蛋白的表达。结果:EGCG在100mg/L以下剂量对两株耐药肿瘤细胞的抑制率均小于10%,EGCG与抗肿瘤药物联合应用可明显提高抗肿瘤药物的细胞毒作用;体内与抗肿瘤药物联合应用对两种肿瘤抑瘤率明显高于单用抗肿瘤药物组,mdr1mRNA和Pgp的表达下降。结论:EGCG与抗肿瘤药物联合应用可增强化疗药物对耐药肿瘤细胞BEL7404/Adr和耐药口腔癌细胞KBV200的细胞毒作用,机制可能与降低MDR1mRNA表达、降低Pgp表达有关。     

粉防己碱逆转耐阿霉素的人乳腺癌MCF-7细胞的抗凋亡作用

肿瘤细胞的多药耐药性与抗细胞凋亡作用关系密切。本研究用抗肿瘤药物阿霉素 (5μmol·L-1 )处理人乳腺癌敏感和耐药的MCF 7细胞 2 4hr后 ,观察到在敏感细胞中 ,有较多的漂浮细胞 ,阿霉素主要分布在细胞核中 ;而在耐药细胞中 ,细胞形态未发生变化 ,阿霉素主要分布在细胞质中 ,其含量明显减少。阿霉素诱导MCF 7细胞的凋亡作用进一步用AnnexinV FITC染色法证实。此外 ,用高效逆转耐药性的药物粉防己碱 (2 0 μmol·L-1 )与阿霉素合用外理敏感和耐药的细胞 ,用线粒体荧光染料MitosensorTM 染色 ,证明合用组凋亡细胞明显增多。通过流式细胞术分析显示 :细胞凋亡的发生与细胞周期无关。本研究表明 :粉防己碱能逆转耐阿霉素的人乳腺癌MCF 7细胞的抗凋亡作用。     

白藜芦醇诱导K562／AO2凋亡与mdr1基因表达的关系

目的研究白藜芦醇（Res）体外对K562／AO2增殖及凋亡的影响，并探讨与耐药基因mdr1表达的关系。方法用噻唑蓝比色法（MTT）检测对照组、不同剂量Res组（25～200μmol／L）作用48h后K562／AO2增殖率，用流式细胞仪检测各组凋亡率，并用逆转录-聚合链反应（RT—PCR）法检测各组mdr1 mRNA的表达。结果Res体外对人K562／AO2细胞有显著增殖抑制及凋亡诱导作用，Res组（25μmol／L、50μmol／L、100μmol／L、200μmol／L）作用48h后其凋亡率均显著高于对照组（P〈0．05）；mdr1 mRNA表达相对强度均显著低于对照组（P〈0．05），且随浓度增加而表达减弱。结论Res体外能诱导K562／AO2细胞的凋亡，且呈一定的浓度依赖性，其作用机制可能与逆转mdr1 mRNA基因表达有关。     

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。     

Barbigerone reverses multidrug resistance in breast MCF‐7/ADR cells

Development of agents to overcome multidrug resistance (MDR) is one of the important strategies in cancer chemotherapy, and P‐glycoprotein (P‐gp) correlates with the degree of resistance. As a naturally occurring isoflavone, whether barbigerone (BA) could reverse MDR, is unknown. In this paper, we evaluated effects of BA on reversing P‐gp mediated MDR of adriamycin (ADR)‐resistant human breast carcinoma (MCF‐7/ADR) cells. BA (0.5 μM) treatment showed strong potency to increase ADR cytotoxicity toward MCF‐7/ADR cells. It was also demonstrated that BA time‐ and dose‐dependently increased accumulations of ADR and reduced the efflux in MCF‐7/ADR cells, pretreatment of these cells with BA might relocalized ADR to the nuclei. Furthermore, the results also revealed that BA did not affect P‐gp, but alter P‐gp ATPase activity. Intravenous administration of BA significantly increased anticancer efficacy of ADR to MCF‐7/ADR xenograft model in nude mice. These results revealed that BA might reverse P‐gp mediated MDR through inhibition of ATPase activity, which indicated a novel use of BA as a potent candidate for cancer chemotherapy.     

Quercetin reversed MDR in breast cancer cells through down‐regulating P‐gp expression and eliminating cancer stem cells mediated by YB‐1 nuclear translocation

Overexpression of P‐glycoprotein (P‐gp) plays an important role in mediating multidrug resistance (MDR), resulting in chemotherapy failure of tumor patients and enhancement of cancer stem cell characteristics. By preparing doxorubicin (Dox) resistant human breast cancer MCF‐7 cells, here, we wanted to evaluate the effects of quercetin (Que) on MDR reversal activity and investigate its possible mechanism. MCF‐7 and MCF‐7/dox cells were respectively treated by Dox, paclitaxel (Pac), or vincristine (Vcr) with or without Que intervention for 24 hr. Cell viability, cell apoptosis, cell cycle, intracellular drug accumulation, the expression of P‐gp and Y‐box binding protein 1 (YB‐1), and breast cancer stem cells (BCSCs) were then assessed. The results showed that Que significantly enhanced the antitumor activities of Dox, Pac, and Vcr in breast cancer cells. In addition, combined treatment of Dox, Pac, or Vcr with Que significantly downregulated P‐gp expression and eliminated BCSCs. Furthermore, combined treatment of Dox, Pac, or Vcr with Que significantly inhibited nuclear translocation of YB‐1. Thus, we speculated that Que reversed MDR in breast cancer cells through downregulating P‐gp expression and eliminating cancer stem cells mediated by YB‐1 nuclear translocation.     

六神丸逆转K562/DOX细胞阿霉素耐药的研究

目的:探讨六神丸对阿霉素耐药细胞株K562/DOX的耐药逆转作用。方法:MTT法检测单独应用阿霉素或阿霉素联合六神丸含药血清对K562/DOX细胞的生存率的影响;RT-PCR检测MDR1 mRNA表达;Western blot检测MDR1蛋白的表达。结果:阿霉素与六神丸含药血清联合作用:①细胞抑制率最高,最高值出现在72h(49.4%),且与对照组和单独应用阿霉素组相比有显著性差异;②48h细胞MDR1 mRNA表达明显降低,与单独应用阿霉素组相比有显著性差异;③48h细胞MDR1蛋白表达下降,且与对照组和单独应用阿霉素组相比有显著性差异。结论:六神丸可能通过下调MDR1表达水平进而逆转K562/DOX细胞对阿霉素的耐药性,增加肿瘤耐药细胞对化疗药物的敏感性。     

Chemoprevention and inhibition of P-glycoprotein in cancer cells by Chinese medicinal herbs

Many of the herbal extracts used in the Chinese clinical medical routine inhibit the growth of tumor cells. In the present work, extracts of 12 selected herbs were prepared with methanol, chloroform, ethyl acetate and water, and the effects of these on the multidrug resistance (MDR) and P-glycoprotein of mouse lymphoma cells transfected with the human mdr1 gene and on a human lung alveolar epithelial cell line were investigated. The extracts were tested for antiproliferative effects, and the reversal of MDR in mouse lymphoma cells. The possible chemopreventive effect of the chloroform extracts was studied on the expression of cytomegalovirus (CMV) immediate-early (IE) antigen in human lung cancer cells (A549). The antimicrobial effects of the extracts were tested on some representative micro-organisms. Certain of the chloroform extracts of the plant materials were the most effective compounds on the reversal of MDR. Two of the chloroform extracts enhanced the antiproliferative effect of doxorubicin on MDR mouse lymphoma cells. The selected extracts did not show any antibacterial effect with the agar diffusion method. Certain chloroform extracts decreased the intermediate IE antigen expression of CMV in A459 cells.     

肠胃清对长春新碱诱导的人结肠癌耐药细胞株Y盒结合蛋白核移位及P-糖蛋白表达的影响

目的观察肠胃清对长春新碱诱导的人结肠癌耐药细胞株HCT8/V的Y盒结合蛋白(YB-1)核移位、多药耐药基因MDR1及P-糖蛋白(P-gp)表达的影响,进一步探讨该方逆转肿瘤多药耐药的分子机制。方法制备大鼠灌胃后的肠胃清药物血清,以Western blot方法检测肠胃清药物血清作用下HCT8/V细胞胞浆、胞核YB-1表达情况,EMSA法分析YB-1与MDR1基因启动子结合活性,RT-PCR检测MDR1、YB-1、多药耐药相关蛋白(MRP)mRNA转录水平,流式细胞仪检测细胞膜表面P-gp表达。结果在1.25%、2.5%、5%药物血清作用下,HCT8/V细胞核内YB-1表达逐渐减弱,细胞质内YB-1表达则逐渐增强;YB-1与MDR1基因启动子结合活性逐渐下降(P0.01);MDR1 mRNA转录水平逐渐降低(P0.05,P0.01),YB-1和MRPmRNA的水平无明显变化(P0.05);细胞膜表面P-gp表达荧光强度值减弱(P0.05,P0.01)。结论肠胃清可通过影响耐药细胞YB-1的核移位、降低MDR1/P-gp的表达,逆转结肠癌细胞的耐药。     

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。     

Danshen (Salvia miltiorrhiza) extract inhibits proliferation of breast cancer cells via modulation of Akt activity and p27 level

Danshen is widely used in traditional Chinese medicine, often in combination with other herbs. To check the effect of Danshen on the proliferation of breast cancer cells, Danshen extract was used to treat MCF‐7 and MCF‐7 HER2 cells, the latter of which overexpresses HER2. HER2 is a receptor tyrosine kinase, and is involved in signal transduction pathways leading to tumor cell proliferation. MTT and cell proliferation assays revealed that Danshen strongly inhibited the proliferation of both MCF‐7 vec cells and MCF‐7 HER2 cells. Flow cytometry analyses indicated that Danshen induced cell cycle delay in the G1 phase. HER2 expression was shown to confer resistance to Danshen‐induced inhibition of proliferation and cell cycle delay, suggesting that HER2 is responsible for the resistance to Danshen. Danshen treatment induced the down‐regulation of Akt phosphorylation and an increase in p27 in MCF‐7 vec and MCF‐7 HER2 cells. Nevertheless, MCF‐7 HER2 cells were more resistant to the Danshen‐induced inhibition of Akt phosphorylation and p27 up‐regulation. Copyright © 2009 John Wiley & Sons, Ltd.