叶酸受体及二氢叶酸还原酶与人乳腺癌细胞MCF-7/ADR多药耐药的关系

目的以人乳腺癌多药耐药细胞系MCF-7/ADR及其敏感亲本系MCF-7为对象,探讨叶酸受体(FOLRα)及其下游基因二氢叶酸还原酶(DHFR)的表达与乳腺癌细胞多药耐药的关系。方法 MTT法测定阿霉素的细胞毒性作用及DHFR抑制剂对MCF-7/ADR细胞多药耐药的逆转作用;RT-PCR检测细胞FOLRα和DHFR mRNA的表达水平;免疫细胞化学检测FOLRα、P-gp的表达水平。结果 MCF-7细胞增殖速度快于MCF-7/ADR细胞,MCF-7细胞FOLRα mRNA转录水平较MCF-7/ADR细胞高,而MCF-7/ADR细胞DHFR mRNA较MCF-7细胞转录水平高;免疫细胞化学显示MCF-7细胞FOLRα的表达高于MCF-7/ADR细胞,而MCF-7/ADR细胞P-gp的表达较MCF-7细胞高;MCF-7/ADR对甲氨蝶啶无耐药性,甲氨蝶啶对MCF-7/ADR细胞多药耐药有逆转作用。结论 MCF-7/ADR细胞FOLRα的表达水平下调可能与细胞增殖水平有关,其下游基因DHFR表达水平与MCF-7/ADR细胞的MDR可能存在相关性。     

功劳木中异汉防己碱对P-糖蛋白介导的人乳腺癌细胞多药耐药性的逆转作用

本文探讨了异汉防己碱对P-糖蛋白(P-gp)介导的人乳腺癌细胞多药耐药性的逆转作用。首先以RT-PCR和免疫组化方法分别从RNA和蛋白水平检测MCF-7/DOX细胞P-gp表达情况，以明确MCF-7/DOX细胞的耐药特征；然后采用MTT法检测异汉防己碱的内在细胞毒性及其对阿霉素(DOX)的增敏作用，并以RF(reversal fold)值评价其逆转效果；同时应用流式细胞仪(FCM)对细胞内DOX的蓄积量进行了分析；再以免疫组化方法检测异汉防己碱对MCF-7/DOX细胞P-gp表达水平的影响；最后采用罗丹明蓄积和外排试验检测了异汉防己碱对P-gp功能的影响。整个试验以维拉帕米作为阳性对照。实验结果表明：MCF-7/DOX细胞是具有多药耐药表型且P-gp表达阳性的细胞株；无毒剂量异汉防己碱可明显增强DOX对MCF-7/DOX细胞的细胞毒性(RF=3.89)，明显高于维拉帕米(RF=2.54)的逆转活性(P<0.05)，但其几乎不影响DOX对MCF-7细胞的抑制作用；异汉防己碱对MCF-7/DOX细胞P-gp表达水平无明显影响，但其可有效抑制P-gp的药物外排功能。因此，异汉防己碱可有效逆转P-gp介导的人乳腺癌细胞的多药耐药性，它可能成为有效多药耐药逆转剂的候选药物。     

高胰岛素对 MCF-7/ADR 细胞化疗敏感性的影响

摘要:目的 分析高胰岛素对 MCF-7/ADR 细胞 P-糖蛋白（P-gp）表达和功能的影响, 并初步探讨胰岛素对 MCF-7/ADR 细胞化疗敏感性的影响。方法 用不同胰岛素浓度（0.001、 0.005、 0.01、 0.05、 0.10 μmol/L）的全细胞培 养基干预 MCF-7/ADR 细胞, 分别采用 Real-time PCR 法检测细胞中 P-gp mRNA 表达, Western Blot 法检测细胞中 P-gp 蛋白表达水平, 罗丹明 123 荧光实验测定 P-gp 的外排功能变化, MTT 法检测 MCF-7/ADR 细胞活性及化疗敏 感性。结果 0.10 μmol/L 的胰岛素可促进 MCF-7/ADR 细胞增殖, 0.05 及 0.10 μmol/L 浓度的胰岛素可增加 MCF-7/ ADR 细胞 P-gp mRNA 及蛋白水平表达, 并能增加 P-gp 的外排功能, 降低 MCF-7/ADR 细胞对表阿霉素的化疗敏感 性。结论 高浓度胰岛素可能通过促进 MCF-7/ADR 细胞 P-gp 的表达和功能, 增加乳腺癌细胞化疗耐药性, 从而降 低 MCF-7/ADR 细胞的化疗敏感性。     

钙调素拮抗剂O-(4-乙氧基)-丁基-小檗胺增强阿霉素杀伤MCF-7/ADR细胞的机制研究

目的研究钙调素拮抗剂0-4-乙氧基-丁基-小檗胺(EBB)增强阿霉素诱导乳腺癌多药耐药细胞系MCF-7/ADR细胞的杀伤作用及其相关机制。方法用MTT法测定阿霉素、EBB单独及联合用药对阿霉素杀伤乳腺癌多药耐药细胞系(MCF-7/ADR)及其亲代细胞系(MCF-7)的作用的IC50值,用不同浓度EBB处理MCF-7/ADR细胞后用FACS法分析EBB对阿霉素诱导细胞凋亡及对mdr1mRNA和P-gp蛋白水平表达的影响,通过激光共聚焦显微镜观察EBB处理前后及用EBB预处理24和48h后MCF-7/ADR和MCF-7细胞内阿霉素浓度的改变。结果MTT结果显示EBB对MCF-7和MCF-7/ADR都具有抗肿瘤活性;EBB还能协同提高阿霉素的细胞毒作用,MCF-7组两药相互作用指数(CDI)值为0.73,MCF-7/ADR组CDI值为0.49,其对耐药细胞的协同作用更为明显。随EBB剂量增加,低剂量阿霉素诱导MCF-7/ADR细胞凋亡增加而且P-gp蛋白表达水平逐渐下降,细胞内阿霉素浓度逐渐提高,而且用EBB预处理MCF-7/ADR细胞24和48h后细胞内阿霉素和罗丹明浓度也逐渐提高。结论EBB是有效的肿瘤细胞化疗药物,它不但能直接抑制P-gp功能还具有下调P-gp蛋白表达的作用,从而有效逆转MCF-7/ADR细胞的耐药现象,协同增强化疗药物对耐药细胞的杀伤作用。     

Gomisin A alters substrate interaction and reverses P-glycoprotein-mediated multidrug resistance in HepG2-DR cells

Through an extensive herbal drug screening program, we found that gomisin A, a dibenzocyclooctadiene compound isolated from Schisandra chinensis, reversed multidrug resistance (MDR) in Pgp-overexpressing HepG2-DR cells. Gomisin A was relatively non-toxic but without altering Pgp expression, it restored the cytotoxic actions of anticancer drugs such as vinblastine and doxorubicin that are Pgp substrates but may act by different mechanisms. Several lines of evidence suggest that gomisin A alters Pgp-substrate interaction but itself is neither a Pgp substrate nor competitive inhibitor. (1) First unlike Pgp substrates gomisin A inhibited the basal Pgp-associated ATPase (Pgp-ATPase) activity. (2) The cytotoxicity of gomisin A was not affected by Pgp competitive inhibitors such as verapamil. (3) Gomisin A acted as an uncompetitive inhibitor for Pgp-ATPase activity stimulated by the transport substrates verapamil and progesterone. (4) On the inhibition of rhodamine-123 efflux the effects of gomisin A and the competitive inhibitor verapamil were additive, so were the effects of gomisin A and the ATPase inhibitor vanadate. (5) Binding of transport substrates with Pgp would result in a Pgp conformational change favoring UIC-2 antibody reactivity but gomisin A impeded UIC-2 binding. (6) Photocrosslinking of Pgp with its transport substrate [125I]iodoarylazidoprazosin was inhibited by gomisin A in a concentration-dependent manner. Taken together our results suggest that gomisin A may bind to Pgp simultaneously with substrates and alters Pgp-substrate interaction.     

盐酸千金藤碱逆转K562/ADR细胞多药耐药性及其机制

研究盐酸千金藤碱(cepharanthine hydrochloride,CH)逆转K562/ADR细胞多药耐药性及其机制。采用MTT法检测多柔比星(adriamycin,ADR)单用及分别与CH、维拉帕米(verapamil,VER)合用的细胞毒作用;采用流式细胞仪,测定CH对细胞内ADR蓄积、罗丹明123(Rho123)蓄积和泵出及P糖蛋白(P-gp)表达的影响。结果表明,CH(4μmol.L1)使K562/ADR细胞对ADR的敏感性增加7.43倍,逆转活性是VER的3.19倍,但对K562敏感株基本无影响。同时CH浓度依赖性地增加K562/ADR细胞内ADR和Rho123的蓄积,减少Rho123的泵出,抑制P糖蛋白的表达,但对K562细胞均无明显影响。CH在体外逆转肿瘤细胞多药耐药性的作用可能与其抑制P糖蛋白的功能和表达有关。     

洛美利嗪逆转K562/ADM细胞多药耐药性

目的研究洛美利嗪(lomerizine,Lom)逆转K562/ADM细胞多药耐药性的作用及机制。方法MTT法检测细胞毒作用,流式细胞仪研究Lom对ADM和长春新碱(vincristine,VCR)的K562/ADM细胞凋亡诱导作用的影响及对罗丹明123(rhodamine 123,Rh123)外排和P-糖蛋白(P-glycoprotein,P-gp)表达的作用。结果Lom明显提高ADM对K562/ADM多药耐药细胞的细胞毒作用及ADM和VCR的凋亡诱导作用,3,10和30 μmol·L^-1 Lom使K562/ADM对ADM的IC₅₀值由79.03 μmol·L^-1分别降至28.14,8.16和3.16 μmol·L^-1。Lom增加胞内ADM的蓄积浓度并抑制Rh123外排;但作用72 h后对K562/ADM细胞P-gp表达无影响。结论Lom通过抑制P-gp的活性逆转K562/ADM细胞的多药耐药性。     

Reversal of P-glycoprotein-mediated multidrug resistance in human hepatoma cells by hedyotiscone A,a compound isolated from Hedyotis corymbosa

1. Multidrug resistance is a major problem in hepatocellular carcinoma. Hedyotiscone A, a compound isolated from Chinese herbal medicine Hedyotis corymbosa (HC, family Rubiaceae), was used as the chemical marker to distinguish between HC and an anticancer herb Hedyotis diffusa (HD) in our previous study.

2. The present study aimed to investigate whether HA exhibited antiproliferative activities in multidrug-resistant hepatocellular carcinoma cells R-HepG2 and the parental cells HepG2 using MTT assay and [³H]-thymidine incorporation assay.

3. Our results showed that HA could significantly inhibit cell proliferation in R-HepG2 and HepG2 (IC₅₀?=?43.7 and 56.3 µg/mL, respectively), but not in normal human liver cells WRL-68 (IC₅₀ > 100 µg/mL) cells, suggesting its selective cytotoxic effects. Besides, HA induced apoptosis in R-HepG2 cells, as confirmed by annexin-V & propidium iodide staining, and DNA fragmentation assay. The caspase cascade was activated as shown by a significant increase of cleaved caspases-3, -7 and -9 in HA-treated R-HepG2 cells. The activities and protein expression of P-glycoprotein as well as mRNA expression of MDR1 were also decreased in HA-treated R-HepG2 cells.

4. Our study demonstrated for the first time the antiproliferative activities of hedyotiscone A in multidrug-resistant R-HepG2 cells. The findings revealed the potential of this compound in treating multidrug-resistant tumor.

     

Acquired multidrug resistance in human K562/ADM cells is associated with enhanced autophagy

Abstract

Autophagy, as a necessary process for survival in mammalian cells deprived of nutrients or growth factors, will be activated in many tumor cells while treated with chemotherapeutic drugs, but the role of autophagy in acquired multidrug resistance of human acute myelogenous leukemia to adriamycin-based chemotherapy remains to be clarified. Our aim was to address that question by surveying the autophagic activity in parental acute myelogenous leukemia cell line K562 and resistant sub cell line, K562/ADM, which were obtained by treating adriamycin with increasing concentrations. K562/ADM and K562 cells were exposed to PBS culture medium for 3 hours, then the stress-induced autophagy was measured. Real-time quantitative RT-PCR revealed the expression of LC3 mRNA was higher in K562/ADM than in K562 cells. LC3-II, as an autophagosomal marker, was more abundant in K562/ADM than in K562 cells measured by Western blotting. To determine the effect of 3-MA, a known specific inhibitor of autophagy, on overcoming acquired multidrug resistance induced by adriamycin, the MTT assay and flow cytometry were performed. We also found that 3-MA can enhance the growth inhibition and apoptotic effect of adriamycin in acquired resistant cells (K562/ADM). Collectively, our results provide evidence that the upregulation of autophagy plays a major role in multidrug resistance of K562/ADM cells induced by adriamycin.     

补骨脂素逆转多药耐药细胞系K562/ADR耐药性研究

目的　研究补骨脂素对白血病细胞阿霉素耐药株(K5 6 2 /ADR)多药耐药 (multidrugresistance,MDR)性的逆转作用及其机制。方法　采用MTT法检测药物细胞毒性作用 ,高效液相色谱法检测细胞内阿霉素 (ADR)的浓度 ,流式细胞术测定细胞P 糖蛋白 (P gp)的表达。 结果　补骨脂素 (1～ 2 0 μmol·L-1)能不同程度地降低ADR对K5 6 2 /ADR细胞的IC50 。 2 0 μmol·L-1能显著提高ADR在K5 6 2 /ADR细胞内的浓度 ,降低K5 6 2 /ADR细胞P gp的表达。 结论　补骨脂素能逆转K5 6 2 /ADR细胞的MDR ,其机制与抑制P gp的功能及其表达 ,增加细胞内ADR的积累有关     

A Novel Submicron Emulsion System Loaded with Doxorubicin Overcome Multi-Drug Resistance in MCF-7/ADR Cells

The purpose of the present study was to develop the Solutol HS15-based doxorubicin submicron emulsion with good stability and overcoming multi-drug resistance. In this study, we prepared doxorubicin submicron emulsion, and examined the stability after autoclaving, the in vitro cytotoxic activity, the intracellular accumulation and apoptpsis of doxorubicin submicron emulsion in MCF-7/ADR cells. The physicochemical properties of doxorubicin submicron emulsion were not significantly affected after autoclaving. The doxorubicin submicron emulsion significantly increased the intracellular accumulation of doxorubicin submicron emulsion and enhanced cytotoxic activity and apoptotic effects of doxorubicin. These results may be correlated to doxorubicin submicron emulsion inhibitory effects on efflux pumps through the progressive release of intracellular free Solutol HS15 from doxorubicin submicron emulsion. Furthermore, these in vitro results suggest that the Solutol HS15-based submicron emulsion may be a potentially useful drug delivery system to circumvent multi-drug resistance of tumor cells.     

蛇床子素对耐阿霉素人乳腺癌细胞耐药的逆转作用

目的:研究蛇床子素(Ost)对人乳腺癌细胞阿霉素耐药株多药耐药(MDR)的逆转作用及其机制。方法:采用MTT比色法测定药物的细胞毒性,高效液相-紫外检测法检测细胞内ADR浓度。结果:Ost对MCF-7及MCF-7/ADR细胞均有增殖抑制作用,IC50值分别为(58.1±2.3)μmol.L-1和(59±5)μmol.L-1;在5~15μmol.L-1范围内,Ost可以不同程度地增强ADR对MCF-7细胞杀伤作用,与ADR联合用药降低ADR对MCF-7/ADR的IC50值,显著增加MCF-7/ADR细胞内ADR的积累。结论:Ost对人乳腺癌细胞株MCF-7及其阿霉素耐药株MCF-7/ADR细胞均有增殖抑制作用,而且Ost通过明显增加MCF-7/ADR细胞内ADR的浓度,逆转其阿霉素耐药性。     

冬凌草甲素逆转SGC7901/ADR细胞的多药耐药性及其机制

目的 观察冬凌草甲素(Ori)对胃癌细胞株SGC7901/ADR多药耐药性的逆转及其作用机制.方法 用MTT法检测Ori的非细胞毒浓度及对SGC7901/ADR细胞株阿霉素敏感性的影响;免疫荧光法检测Ori对SGC7901/ADR细胞p-糖蛋白(P-gp)活性及表达的影响;RT-PCR法检测Ori对细胞MDR1基因转录的影响.结果 用Ori无毒剂量处理细胞后,细胞对阿霉素的逆转倍数为4.53,MDR1基因的转录降低了0.78,P-gp的表达率降低了18.83％.结论 Ori能逆转SGC7901/ADR细胞的多药耐药性,其机制为降低MDR1基因的转录和下调P-gp的表达.     

低频超声联合微泡对比剂通过PI3K/AKT通路对乳腺癌耐药株MCF-7/ADR多药耐药的逆转机制研究

目的 探索低频超声联合微泡（LFUSMB）通过磷脂酰肌醇3激酶（PI3K）/蛋白激酶B（AKT）通路对乳腺癌 耐药株MCF-7/阿霉素（ADR）多药耐药的逆转效果及机制。方法 CCK-8法筛选LFUSMB作用时间并测定ADR对 MCF-7/ADR 细胞的半抑制浓度（IC50）。将 MCF-7/ADR 细胞分组为：对照（C）组,采用 MCF 细胞培养基正常培养; ADR组,采用加入ADR（终质量浓度20 mg/L）的MCF细胞培养基培养;LFUSMB+ADR组,采用加入ADR（终质量浓度 20 mg/L）的MCF细胞培养基培养并经LFUSMB处理30 s;LFUSMB+ADR+740 YP（PI3K/AKT通路活化剂）组,采用加 入 ADR（终质量浓度 20 mg/L）和 740 YP（终质量浓度 50 mg/L）的 MCF 细胞培养基培养并经 LFUSMB 处理 30 s。 Annexin V-FIFC/PI 法检测 MCF-7/ADR 细胞凋亡率,Western blot 检测 P 糖蛋白（P-gp）、抗多药耐药蛋白（MRP）1、 MRP2、乳腺癌抗性蛋白（BCRP/ABCG2）、PI3K、AKT、p-PI3K、p-AKT蛋白表达。结果 采用LFUSMB干预30 s进行 后续研究。LFUSMB 30 s组ADR对MCF/ADR细胞的IC50低于未超声处理组,相对耐药逆转倍数约为8.20倍。ADR 组细胞凋亡率较C组增高（P＜0.05）,MRP1、MRP2、P-gp、BCRP/ABCG2、p-PI3K/PI3K和p-AKT/AKT 蛋白水平无明 显变化;LFUSMB+ADR组细胞凋亡率较ADR组增高,MRP1、MRP2、P-gp、BCRP/ABCG2、p-PI3K/PI3K和p-AKT/AKT 蛋白水平显著下调（P＜0.05）。与 LFUSMB+ADR 组比较,LFUSMB+ADR+740 YP 组细胞凋亡率显著降低,MRP1、 MRP2、P-gp、BCRP/ABCG2 蛋白水平显著增高（P＜0.05）。结论 LFUSMB 可通过抑制 PI3K/AKT 通路活化,下调 MRP1等腺苷三磷酸结合盒（ABC）家族蛋白表达,逆转乳腺癌MCF-7/ADR细胞耐药性。     

Increased rate of glutathione synthesis from cystine in drug-resistant MCF-7 cells

The rate of glutathione synthesis was determined in drug-sensitive and -resistant MCF-7 cells by monitoring the rate of label uptake from [3,3'-13C(2)]-cystine using NMR spectroscopy and mass spectrometry. Compared with the wild-type human mammary adenocarcinoma cell line (MCF-7wt), the isotope incorporation rate was increased 1.6-, 2.4-, and 5.3-fold in the etoposide-resistant MCF-7 cell line (MCF-7vp), doxorubicin-resistant MCF-7 cell line (MCF-7adr), and 4-hydroperoxycyclophosphamide-resistant MCF-7 cell line (MCF-7hc), respectively. The increase in glutathione metabolism in the MCF-7hc line correlated with steady-state levels as determined by biochemical assay. In contrast, both the MCF-7vp and MCF-7adr lines showed increased metabolic synthesis of glutathione but displayed lower steady-state levels compared with the MCF-7wt line. The increased synthetic rates of all resistant lines reflected, in part, contributions from the increased activities of both gamma-glutamyltranspeptidase and gamma-glutamylcysteine synthetase. These results emphasize the importance of monitoring glutathione metabolic rates, rather than steady-state levels of enzymes or substrates, for assessing drug resistance.     

Crizotinib (PF-02341066) reverses multidrug resistance in cancer cells by inhibiting the function of P-glycoprotein

BACKGROUND AND PURPOSE

Besides targeting the well-known oncogenic c-Met, crizotinib is the first oral tyrosine kinase inhibitor inhibiting anaplastic lymphoma kinase (ALK) in clinical trials for the treatment of non-small cell lung cancer. Here, we assessed the possible reversal of multidrug resistance (MDR) by crizotinib in vitro and in vivo.

EXPERIMENTAL APPROACH

1-(4,5-Dimethylthiazol-2-yl)-3,5- diphenylformazan was used in vitro and xenografts in nude mice were used in vivo to investigate reversal of MDR by crizotinib. To understand the mechanisms for MDR reversal, the alterations of intracellular doxorubicin or rhodamine 123 accumulation, doxorubicin efflux, ABCB1 expression level, ATPase activity of ABCB1 and crizotinib-induced c-Met, Akt and ERK1/2 phosphorylation were examined.

KEY RESULTS

Crizotinib significantly enhanced the cytotoxicity of chemotherapeutic agents which are also ABCB1 substrates, in MDR cells with no effect found on sensitive cells in vitro and in vivo. Additionally, crizotinib significantly increased intracellular accumulation of rhodamine 123 and doxorubicin and inhibited the drug efflux in ABCB1-overexpressing MDR cells. Further studies showed that crizotinib enhanced the ATPase activity of ABCB1 in a concentration-dependent manner. However, expression of ABCB1 was not affected, and reversal of MDR by crizotinib was not related to the phosphorylation of c-Met, Akt or ERK1/2. Importantly, crizotinib significantly enhanced the effect of paclitaxel against KBv200 cell xenografts in nude mice.

CONCLUSIONS AND IMPLICATIONS

Crizotinib reversed ABCB1-mediated MDR by inhibiting ABCB1 transport function without affecting ABCB1 expression or blocking the Akt or ERK1/2 pathways. These findings are useful for planning combination chemotherapy of crizotinib with conventional chemotherapeutic drugs.     

Reversal effect of Dioscin on multidrug resistance in human hepatoma HepG2/adriamycin cells

Multidrug resistance is a serious obstacle encountered in cancer treatment. Since drug resistance in human cancer is mainly associated with overexpression of the multidrug resistance gene 1 (MDR1), the promoter of the human MDR1 gene may be a target for multidrug resistance reversion drug screening. In the present study, HEK293T cells were transfected with pGL3 reporter plasmids containing the 2 kb of MDR1 promoter, and the transfected cells were used as models to screen for candidate multidrug resistance inhibitors from over 300 purified naturally occurring compounds extracted from plants and animals. Dioscin was found to have an inhibiting effect on MDR1 promoter activity. The resistant HepG2 cell line (HepG2/adriamycin) was used to validate the activity of multidrug resistance reversal by Dioscin. Results showed that Dioscin could decrease the resistance degree of HepG2/adriamycin cells, and significantly inhibit P-glycoprotein expression, as well as increase the accumulation of adriamycin in HepG2/adriamycin cells as measured by Flow Cytometric analysis. These results suggest that Dioscin is a potent multidrug resistance reversal agent and may be a potential adjunctive agent for tumor chemotherapy.     

三萜皂苷Saxifragifolin D抑制人肝癌耐药细胞HepG2/ADM生长并诱导细胞凋亡作用研究

目的研究Saxifragifolin D(SD)对人肝癌耐药细胞HepG2/ADM的生长抑制及诱导凋亡作用。方法采用MTT法观察SD对HepG2/ADM细胞的增殖抑制作用,应用流式细胞仪分析SD对细胞周期的影响,AnnexinⅤ-FITC/PI双染检测凋亡细胞比率,JC-1染色观察SD对细胞内线粒体膜电位的影响,Western blot检测凋亡相关蛋白caspase-9,caspase-3和PARP的激活及c-Raf,MEK和ERK蛋白的表达和磷酸化水平。结果 SD可以明显抑制人肝癌耐药细胞HepG2/ADM的增殖。细胞周期检测发现SD诱导细胞产生亚二倍体凋亡峰,同时细胞凋亡率也由对照组的5.3%增加到34.8%和47.8%。线粒体膜电位检测结果显示SD导致细胞内线粒体膜电位的明显降低。Western blot检测结果表明caspase-9,caspase-3被激活,PARP被剪切活化,cytochrome C由线粒体释放至胞质,c-Raf、MEK和ERK蛋白的磷酸化水平降低。结论 SD可以抑制人肝癌耐药细胞HepG2/ADM增殖并诱导其凋亡,作用机制可能与线粒体功能障碍及抑制c-Raf/MEK/ERK通路的活化有关。     

薯蓣皂苷下调ABCC1表达对人肝癌细胞HepG2/ADM耐药的影响

目的:探究薯蓣皂苷对人肝癌细胞HepG2/多柔比星(ADM)耐药性的影响及其可能的作用机制。方法:采用含0,10,20,30,40,50μmol·L^-1薯蓣皂苷的培养液培养HepG2、HepG2/ADM细胞,CCK法检测细胞增殖抑制率;MTT检测薯蓣皂苷对HepG2/ADM细胞、HepG2细胞半抑制浓度(IC50)值的影响;流式细胞术检测HepG2、HepG2/ADM细胞摄取ADM的能力。薯蓣皂苷联合ABCC1抑制剂处理HepG2/ADM细胞,MTT检测细胞IC50值变化;流式细胞术检测细胞摄取ADM的能力、细胞凋亡情况,免疫印迹法检测细胞人多药耐药相关蛋白1(ABCC1)、半胱天冬酶(caspase-3)、B淋巴细胞瘤-2(bcl-2)、Bcl-2相关X蛋白(Bax)蛋白表达情况。结果:HepG2/ADM细胞中ABCC1蛋白表达显著高于HepG2细胞(P<0.05)。薯蓣皂苷0~30μmol·L^-1对HepG2/ADM细胞、HepG2细胞毒性小。随着薯蓣皂苷处理浓度的升高,HepG2/ADM细胞IC50值均降低,摄取ADM量明显升高,具有剂量依赖性(P<0.05)。薯蓣皂苷联合ABCC1抑制剂处理HepG2/ADM细胞后能够进一步降低细胞IC50,提高细胞ADM摄取量,加速细胞凋亡,上调caspase-3、Bax蛋白表达,下调ABCC1、bcl-2蛋白表达(P<0.05)。结论:薯蓣皂苷能够逆转HepG2/ADM细胞耐药性,逆转效果与作用剂量相关,其机制可能与下调ABCC1表达,诱导细胞凋亡有关。     

新的四氢异喹啉类化合物CPUE1逆转K562/A02细胞的多药耐药性

目的 :研究新的四氢异喹啉类化合物CPUE1逆转K5 6 2 A0 2细胞多药耐药性的作用及机制。方法 :在CPUE1的作用下 ,用MTT法检测长春新碱(vincristine ,VCR)对K5 6 2 A0 2多药耐药细胞的细胞毒作用的变化 ,使用DNA分析和Annexin Ⅴ PI双染法研究CPUE1对VCR诱导K5 6 2 A0 2细胞凋亡的影响 ,流式细胞仪检测CPUE1对P 糖蛋白 (P glycop rotein ,P gp)外排罗丹明 12 3(rhodamine12 3,Rh12 3)的作用。结果 :CPUE1能明显提高VCR对K5 6 2 A0 2多药耐药细胞的细胞毒作用以及凋亡诱导作用 ,10 μmol·L-1的CPUE1能使K5 6 2 A0 2对VCR的IC50值由 6 0 .5 4μmol·L-1降至 4 .17μmol·L-1。CPUE1还能抑制Rh12 3外排从而增加细胞内Rh12 3的蓄积浓度。结论 :CPUE1通过抑制P gp的活性逆转K5 6 2 A0 2细胞的多药耐药性。