Increased expression of sorcin is associated with multidrug resistance in leukemia cells via up-regulation of MDR1 expression through cAMP response element-binding protein

Sorcin, a 22 kDa Ca²⁺ binding protein, was first identified in a vincristine-resistant Chinese hamster lung cell line, and was later demonstrated to be involved in the development of multidrug-resistance (MDR) phenotypes in a variety of human cancer cell lines. However, the exact role of sorcin in MDR cells is yet to be fully elucidated. Here we explored the role of sorcin in the development of MDR in leukemia cells, and revealed that the expression level of sorcin was directly correlated to the expression of MDR1/P-glycoprotein (P-gp). In addition, it was shown that sorcin induced the expression of MDR1/P-gp through a cAMP response element (CRE) between −716 and −709 bp of the mdr1/p-gp gene. Furthermore, overexpression of sorcin increased the phosphorylation of CREB1 and the binding of CREB1 to the CRE sequence of mdr1/p-gp promoter, and induced the expression of MDR1/P-gp. These findings suggested that sorcin induces MDR1/P-gp expression markedly through activation of the CREB pathway and is associated with the MDR phenotype. The new findings may be helpful for understanding the mechanisms of MDR in human cancer cells, prompting its further investigation as a molecular target to overcome MDR.     

P-gp 和MDR1 在三种乳腺癌细胞中表达差异研究

目的:比较P-gp和MDR1在人乳腺癌敏感细胞(MCF-7/S)和耐药细胞(MCF-7/ADR、MCF-7/TAM)中的表达差异,初步探讨乳腺癌细胞对阿霉素与对三苯氧胺产生耐药机制的区别。方法:采用免疫细胞化学法、流式细胞术检测P-gp,采用实时荧光定量PCR法检测MDR1在三种乳腺癌细胞中的表达情况。结果:在MCF-7/ADR细胞中P-gp和MDR1均呈高表达,阳性表达率与MCF-7/S细胞比较,有统计学意义(P<0.01)。在MCF-7/TAM细胞中P-gp、MDR1均呈低表达,与MCF-7/S细胞比较,无统计学意义(P>0.05)。结论:P-gp和MDR1的高表达是乳腺癌细胞对阿霉素产生耐药的主要机制,而并非是乳腺癌细胞对三苯氧胺产生耐药的机制。     

MDR1基因多态性及其临床相关性研究进展

体内外研究证明,人体中P—gP在药物的吸收、分布、代谢和排泄（ADME）过程中发挥了非常重要的作用。多药耐药基因MDR1（ABCB1）是P-gP的编码基因。药物基因组学和遗传药理学研究发现在不同个体中MDR1基因多态性与P—gP表达和功能的改变密切相关,而且这些多态位点存在基因型分布和等位基因频率的种族差异性。近几年,已陆续发现在MDR1基因中有50处单核苷酸多态性（SNPs）和3处插入与缺失多态性。随后,大量文献报道某些位点的SNPs如C3435T会使个体患病的易感性增加。因此人们相信,深入研究MDR1基因多态性与P—gP的生理和生化方面的相关性将对个体医疗有着非常深远的意义。文章总结了国外最新的研究进展并结合本实验室的工作着重讨论了4个方面：1）P—gP对药代动力学性质的影响：2）MDR1基因多态性及其对遗传药理学性质的影响;3）MDR1^C3435T的单核苷酸多态性与P-gP表达和功能之间的相关性：4）MDR1基因多态性与人类某些疾病之间的相关性。     

Influence of IGF-I and cell density on MDR1 expression in the T-lymphoblastoid cell line CCRF-CEM

The debate about a direct or indirect effect of GH and IGF-I on the recurrence of malignancy, especially in the case of rhGH therapy in patients with leukemia, is still going on. Recent studies suggested that IGF-I plays a role in drug resistance during anticancer therapy. This resistance to diverse cytotoxic drugs, named multidrug-resistance (MDR), is mainly due to high levels of P-glycoprotein (P-gp). The gene encoding this membrane-associated transporter protein was named MDR1, and increased levels of P-gp are linked to enhanced MDR1 mRNA expression. Our aim was to investigate a possible effect of rhIGF-I on MDR1 gene expression in vitro. We cultured the T-lymphoblastoid cell line CCRF-CEM with different rhIGF-I concentrations (0, 5, 20 and 50 ng/ml) in serum-free medium for 3 days. CCRF-CEM cells are drug-sensitive and express MDR1 at low levels. MDR1 mRNA expression was measured by semiquantitative RT-PCR using a competitive assay with a heterologous DNA construct. In addition, GAPDH mRNA was amplified as an internal control for RNA integrity. P-gp activity was determined by a flow cytometric assay measuring rhodamine 123 accumulation. Furthermore, cell proliferation was monitored in all experiments. Our data do not support an effect of rhIGF-I on MDR1 mRNA expression, P-gp activity or cell proliferation in the CCRF-CEM cell line. MDR1 mRNA levels were inversely correlated to cell density with high significance (p < 0.0001). In conclusion, multidrug resistance linked to P-gp is not induced by IGF-I in CCRF-CEM cells. At high density, CCRF-CEM cells downregulate MDR1 gene expression. Our experimental model provides a very useful tool for monitoring the influence of growth factors on multidrug resistance in vitro.     

长春新碱诱导建立人结肠癌多药耐受性小鼠模型及MDR1和MRP1基因表达

目的建立人结肠癌多药耐受性动物模型并初步探索其耐药机制。方法结合体内外诱导方法建立人结肠癌多药耐受性动物模型,利用VCR和CTX的肿瘤抑制实验评价其MDR特性;利用real-time PCR和West-ern blotting等方法分析其P-gp/MDR1和MRP1基因和蛋白的表达。结果肿瘤抑制实验结果显示,MDR和敏感型结肠癌模型的肿瘤生长速度差异不显著,MDR结肠癌动物模型对于VCR和CTX的耐药性均有较大程度的提高;表达分析结果显示,人结肠癌MDR动物模型的P-gp/MDR1表达水平有较大提高,而MRP1表达没有显著变化。结论人结肠癌多药耐受性动物模型具有较好的多药耐受性,其多药耐受性表型主要是由于P-gp/MDR1过量表达所导致。     

P—gp和细胞容积调节

本实验用基因阻抑技术阻抑牛眼睫状体非色素上皮(NPE)细胞MDR1基因表达,在激光共聚焦显微镜下检测细胞MDRI基因产物P-gp免疫荧光,研究MDRI基因及P-gp与细胞容积调节的关系。结果表明:NPE细胞表达MDR1基因,存在P-gp蛋白。人反义MDR1特异性阻抑NPE细胞MDR1基因表达,剂量依赖性抑制P-gp免疫荧光(r=0.95,P<0.01),减少P-gp合成,导致细胞容积调节减弱,鼠反义MDR1对NPE细胞MDR1基因表达及容积调节没有影响。结果提示P-gp在细胞容积调节中起重要作用。     

Stereoselective regulation of MDR1 expression in Caco-2 cells by cetirizine enantiomers

MDR1-encoded P-glycoprotein (P-gp) is a drug efflux transporter mainly expressed in liver, kidney, intestine, brain (at the level of the blood-brain barrier), and placenta. It thus plays important roles in drug absorption, distribution, and excretion. Cetirizine is a second-generation nonsedating antihistamine used to treat allergic disease of respiratory system, skin and eyes. To evaluate P-gp expression and function in Caco-2 cells pretreated with cetirizine enantiomers, we assessed the sensitivity of Caco-2 cells to paclitaxel using the MTT assay and the polarized transport of rhodamine-123 and doxorubicin across Caco-2 monolayers. RT-PCR and flow cytometry were used to assay MDR1 mRNA and P-gp protein respectively. The sensitivity of Caco-2 cells to paclitaxel decreased significantly after cells were pretreated with 100 microM R-cetirizine but increased upon treatment with S-cetirizine. The efflux of rhodamine-123 and doxorubicin was enhanced significantly after Caco-2 monolayers were pretreated with 100 microM R-cetirizine but was reduced by S-cetirizine. The MDR1 mRNA and P-gp levels in Caco-2 cells were increased by 100 microM R-cetirizine and decreased by 100 microM S-cetirizine. These results suggest that R-cetirizine up-regulates MDR1 expression while S-cetirizine down-regulates MDR1 expression.     

Tryptanthrin inhibits MDR1 and reverses doxorubicin resistance in breast cancer cells

Development of agents to overcome multidrug resistance (MDR) is important in cancer chemotherapy. Up to date, few chemicals have been reported to down-regulate MDR1 gene expression. We evaluated the effect of tryptanthrin on P-glycoprotein (P-gp)-mediated MDR in a breast cancer cell line MCF-7. Tryptanthrin could depress overexpression of MDR1 gene. We observed reduction of P-gp protein in parallel with decreases in mRNA in MCF-7/adr cells treated with tryptanthrin. Tryptanthrin suppressed the activity of MDR1 gene promoter. Tryptanthrin also enhanced interaction of the nuclear proteins with the negatively regulatory CAAT region of MDR1 gene promoter in MCF-7/adr. It might result in suppression of MDR1 gene. In addition, tryptanthrin decreased the amount of mutant p53 protein with decreasing mutant p53 protein stability. It might contribute to negative regulation of MDR1 gene. In conclusion, tryptanthrin exhibited MDR reversing effect by down-regulation of MDR1 gene and might be a new adjuvant agent for chemotherapy.     

以重组P-gp为抗原建立检测MDR 1抗体间接ELISA方法的研究

目的:构建MDR 1基因原核表达质粒,表达P-gp重组蛋白,建立检测MDR1抗体的间接ELISA方法。方法:利用重组PCR技术扩增MDR 1基因的1kb片段,克隆至pET-28b（＋）中,构建原核表达质粒pETP-gp,转染感受态菌BL21（DE3）和BL21（DE3）plyss;以E.coli高效表达的P-gp基因主要抗原编码区重组蛋白为抗原,以HRP标记的兔抗人IgG为二抗,建立间接ELISA检测方法。结果:正确构建了pETP-gp原核表达质粒,并可在E.coli中高效表达,表达蛋白可用作检测MDR 1抗体ELISA抗原。结论:成功表达出重组蛋白P-gp,建立了检测MDR 1抗体的间接ELISA方法。     

Effect of Stemona curtisii root extract on P-glycoprotein and MRP-1 function in multidrug-resistant cancer cells

Multidrug resistance (MDR) is the result of overexpression of membrane bound proteins that efflux chemotherapeutic drugs from the cells. Two proteins, P-glycoprotein (P-gp) and multidrug-resistance associated protein-1 (MRP-1) efflux chemotherapeutic agents out of the cancer cell that decrease intracellular drug accumulation, thereby decreasing the effectiveness of many chemotherapeutic agents. In the present study, the ethanolic extract of the roots of Stemona curtisii Hook. was tested for the potential ability to modulate the MDR phenotype and function of P-gp and MRP-1. The S. curtisii extract reversed the resistance to putative chemotherapeutic agents, including vinblastine, paclitaxel and colchicine of KB-V1 cells (MDR human cervical carcinoma with high P-gp expression) in a dose-dependent manner, but not in KB-3-1 cells (drug sensitive human cervical carcinoma, which lack P-gp expression). The root extract also increased the intracellular uptake and retention of (3)[H]-vinblastine in KB-V1 cells dose dependently. The extract did not influence MDR phenotype-mediated MRP-1 in MRP1-HEK293 (human embryonic kidney cells stably transfected with pcDNA3.1-MRP1-H10 which show high MRP-1 expression) and pcDNA3.1-HEK293 (wild type). In summary, the S. curtisii root extract modulated P-gp activity but not MRP-1 activity. The result obtained from this study strongly indicated that S. curtisii extract may play an important role as a P-gp modulator as used in vitro and may be effective in the treatment of multidrug-resistant cancers. The purified form of the active components of S. curtisii extract should be investigated in more details in order to explain the molecular mechanisms involved in P-gp modulation. This is the first report of new biological activity in this plant, which could be a potential source of a new chemosensitizer.