A new multidrug resistance protein at the blood-brain barrier

Porcine brain capillary endothelial cells (PBCEC) cultured in serum-free and hydrocortisone supplemented medium are characterised by high transendothelial electrical resistances and low cell monolayer permeabilities for small solutes very similar to the blood-brain barrier (BBB) in vivo. Differential screening of a subtracted cDNA library disclosed a 2.1-kb mRNA that is overexpressed in hydrocortisone treated PBCEC relative to untreated cells. The mRNA encodes a 656-aa member of the ATP-binding cassette (ABC) superfamily of transporters that we named brain multidrug resistance protein (BMDP). Phylogenetic analysis and multiple sequence alignment showed that porcine BMDP is most related to the human and mouse breast cancer resistance protein (BCRP). Northern blot analysis revealed that BMDP is expressed in brain tissue in vivo and was predominantly localised within the endothelial cells isolated from brain capillaries. Thus, we identified a new transport protein at the BBB that might play an important role in the exclusion of xenobiotics from the brain.     

Use of ribozymes and antisense oligodeoxynucleotides to investigate mechanisms of drug resistance

Chemotherapy can cure a number of human cancers but resistance (either intrinsic or acquired) remains a significant problem in many patients and in many types of solid tumour. Combination chemotherapy (using drugs with different cellular targets/mechanisms) was introduced in order to kill cells which had developed resistance to a specific drug, and to allow delivery of a greater total dose of anti-cancer chemicals by combining drugs with different side-effects (Pratt et al., 1994). Nearly all anti-cancer drugs kill tumour cells by activating an endogenous bio-chemical pathway for cell suicide, known as programmed cell death or apoptosis. This revised version was published online in August 2006 with corrections to the Cover Date.     

Human MDR1 and MRP1 Recognize Berberine as Their Transport Substrate

To examine whether human ATP-binding cassette (ABC) transporters play a role in the detoxification of plant alkaloid berberine, we investigated berberine transport using multidrug resistance protein1 (MDR1) and multidrug resistance-associated protein1 (MRP1). Cells expressing MDR1 or MRP1 accumulated less berberine. Berberine accumulation depended on the cellular ATP level, and was reversed by typical inhibitors of MDR1, suggesting that human MDR1 and MRP1 directly efflux berberine as their substrate.     

Combination of dihydromyricetin and ondansetron strengthens antiproliferative efficiency of adriamycin in K562/ADR through downregulation of SORCIN: A new strategy of inhibiting P-glycoprotein

Though the advancement of chemotherapy drugs alleviates the progress of cancer, long-term therapy with anticancer agents gradually leads to acquired multidrug resistance (MDR), which limits the survival outcomes in patients. It was shown that dihydromyricetin (DMY) could partly reverse MDR by suppressing P-glycoprotein (P-gp) and soluble resistance-related calcium-binding protein (SORCIN) independently. To reverse MDR more effectively, a new strategy was raised, that is, circumventing MDR by the coadministration of DMY and ondansetron (OND), a common antiemetic drug, during cancer chemotherapy. Meanwhile, the interior relation between P-gp and SORCIN was also revealed. The combination of DMY and OND strongly enhanced antiproliferative efficiency of adriamycin (ADR) because of the increasing accumulation of ADR in K562/ADR-resistant cell line. DMY could downregulate the expression of SORCIN and P-gp via the ERK/Akt pathways, whereas OND could not. In addition, it was proved that SORCIN suppressed ERK and Akt to inhibit P-gp by the silence of SORCIN, however, not vice versa. Finally, the combination of DMY, OND, and ADR led to G2/M cell cycle arrest and apoptosis via resuming P53 function and restraining relevant proteins expression. These fundamental findings provided a promising approach for further treatment of MDR.     

乳腺癌耐药蛋白基因的转录调控机制

乳腺癌耐药蛋白(Breast cancer resistance protein, BCRP), 又名ABCG2, 是ATP结合盒(ATP-binding cas-sette, ABC)转运蛋白超家族成员之一, 在肿瘤多药耐药中具有十分重要的作用。BCRP基因启动子区无TATA盒, 含CAAT盒、AP1位点、AP2位点以及CpG岛下游的多个Sp-1位点。近年来的研究发现, 转录因子孕激素受体(PR)、雌激素受体(ER)、核因子-κB (NF-κB)、缺氧诱导因子(HIF)、Nrf2、芳香烃受体(AhR)、过氧化物酶体增殖活化受体(PPAR)和KLF5等可与BCRP启动子或增强子区的特定反应元件结合进而激活BCRP的转录。促炎细胞因子、生长因子、同源盒基因MSX2、Sonic hedgehog信号通路、Notch信号通路和RAR/RXR信号通路等均参与了BCRP的转录调控。此外, 启动子甲基化和组蛋白乙酰化在BCRP转录调控尤其是药物诱导BCRP表达中发挥重要作用。文章综述了这一研究领域的进展, 着重讨论了转录因子及表观遗传学在BCRP转录调控中的作用。     

A new shuttle vector for gene expression in biopolymer-producing Ralstonia eutropha

Ralstonia eutropha (formerly Alcaligenes eutrophus) is a fascinating microorganism with a great scientific importance and an immense commercial potential. A new genetic transformation system for the organism would greatly facilitate the biological study and molecular engineering of this organism. We report here a versatile gene expression method for the genetic engineering of R. eutropha. This method, based on a simplified electroporation protocol, uses a recombinant plasmid, pBS29-P2, containing a Pseudomonas syringae promoter (P2) and two antibiotic-resistance markers (i.e., genes coding for kanamycin (Km)- and tetracycline (Tc)-resistance). Using this method, we successfully achieved transformation of wild-type R. eutropha and its poly(hydroxyalkanoate)-negative mutant, R. eutropha PHB⁻4, with various pBS29-P2-based recombinants. A transformation frequency as high as 4 × 10³ Km-resistance colonies/μg DNA was obtained per electroporation experiment. We further demonstrated the successful expression of a heterologous gene coding for green-fluorescent-protein by fluorescence measurement. In addition, our results indicated the expression of a truncated but active Streptomyces coelicolor α-galactosidase in R. eutropha.     

Isolation and Characterization of Mustard (Sinapis alba L.) Seed Storage Proteins

A total storage protein fraction was prepared from mustard (Sinapis alba L.) seeds via isolated protein bodies and characterized by sedimentation, immunological, and electrophoretic techniques. Mustard seed storage protein consists of three fractions (1) a “legumin-like” 13-S complex composed of two pairs of disulfide-linked polypeptides (16.5 + 28.5 kDa and 19.5 + 34 kDa, respectively) and two single polypeptides (18 kDa and 26 kDa), (2) a “vicilin-like” 9-S complex composed of two glycoproteins (64 kDa and 77 kDa), and (3) two small polypeptides (10 kDa and 11 kDa) which probably represent the 1.7-S complex found in other Cruciferae. In contrast to related species, no glycosylated polypeptide was found in the 13-S complex. Immunological relationships were found between the paired polypeptides of the 13-S complex but not between polypeptides of the 13-S complex and polypeptides of the 9-S complex. Pulse-chase labeling and in vitro translation of polysomal RNA from young embryos demonstrated that the polypeptides of the 13-S complex originate from high molecular mass precursors, except for the 18 kDa polypeptide which appears to be synthesized in its final size. The amino-acid composition of the major polypeptides of the mustard storage protein is given.     

FOXO1 associated with sensitivity to chemotherapy drugs and glial-mesenchymal transition in glioma

Mesenchymal subtype of glioblastoma (GBM), identified as one of four clinically relevant molecular subtypes, has worst prognosis because of its close relation with the malignant biological properties induced by glial-mesenchymal transition (GMT). However, the molecular mechanism of GMT and its characterized molecule of GBM have not been studied. Forkhead box protein O1 (FOXO1) is at a convergence point of receptor tyrosine kinase signaling as one of the three core pathways implicated in GBM. Our previous study indicated that the inactivation of FOXO1 involved in the inhibition of GMT is an independent prognosis factor of GBM. In this study, we will further confirm the role of FOXO1 in GMT through cytological experiments to clarify how FOXO1 regulates GMT and its clinical significance. We established virus-infected FOXO1 overexpression and FOXO1 knockdown cells of U373 MG and U251 mediated by lentivirus, based on the effect of which FOXO1-correlated-GMT experiments were performed in vitro and in vivo. Our data suggested that FOXO1 played a crucial role in resistance to TMZ, BCNU, and CDDP; migration and invasion; and stem cell properties of glioma cells. FOXO1 may serve as a targeted biomarker for prediction of sensitivity to chemotherapy drugs, metastasis, and prognosis, which provides a new idea for mesenchymal GBM treatment.     

Ascofuranone prevents ER stress-induced insulin resistance via activation of AMP-activated protein kinase in L6 myotube cells

The current study presents that ascofuranone isolated from a phytopathogenic fungus, Ascochyta viciae, has antitumor activity against various transplantable tumors and a considerable hypolipidemic activity. AMP-activated protein kinase (AMPK) plays a critical role in cellular glucose and lipid homeostasis. We found that ascofuranone improves ER stress-induced insulin resistance by activating AMPK through the LKB1 pathway. In L6 myotube cells, ascofuranone treatment increased the phosphorylation of the Thr-172 residue of the AMPKα subunit and the Ser-79 subunit of acetyl-CoA carboxylase (ACC) and cellular glucose uptake. Ascofuranone-induced phosphorylation of AMPK and ACC was not increased in A549 cells lacking LKB1. Interestingly, ascofuranone treatment also improved insulin signaling impaired by ER stress in L6 myotube cells. These effects were all reversed by pretreatment with Compound C, an AMPK inhibitor or with adenoviral-mediated dominant-negative AMPKα2. Taken together, these results indicated that ascofuranone-mediated enhancement of glucose uptake and reduction of impaired insulin sensitivity in L6 cells is predominantly accomplished by activating AMPK, thereby mediating beneficial effects in type 2 diabetes and insulin resistance.     

PKD1 Inhibits AMPKα2 through Phosphorylation of Serine 491 and Impairs Insulin Signaling in Skeletal Muscle Cells

AMP-activated protein kinase (AMPK) is an energy-sensing enzyme whose activity is inhibited in settings of insulin resistance. Exposure to a high glucose concentration has recently been shown to increase phosphorylation of AMPK at Ser^485/491 of its α1/α2 subunit; however, the mechanism by which it does so is not known. Diacylglycerol (DAG), which is also increased in muscle exposed to high glucose, activates a number of signaling molecules including protein kinase (PK)C and PKD1. We sought to determine whether PKC or PKD1 is involved in inhibition of AMPK by causing Ser^485/491 phosphorylation in skeletal muscle cells. C2C12 myotubes were treated with the PKC/D1 activator phorbol 12-myristate 13-acetate (PMA), which acts as a DAG mimetic. This caused dose- and time-dependent increases in AMPK Ser^485/491 phosphorylation, which was associated with a ∼60% decrease in AMPKα2 activity. Expression of a phosphodefective AMPKα2 mutant (S491A) prevented the PMA-induced reduction in AMPK activity. Serine phosphorylation and inhibition of AMPK activity were partially prevented by the broad PKC inhibitor Gö6983 and fully prevented by the specific PKD1 inhibitor CRT0066101. Genetic knockdown of PKD1 also prevented Ser^485/491 phosphorylation of AMPK. Inhibition of previously identified kinases that phosphorylate AMPK at this site (Akt, S6K, and ERK) did not prevent these events. PMA treatment also caused impairments in insulin-signaling through Akt, which were prevented by PKD1 inhibition. Finally, recombinant PKD1 phosphorylated AMPKα2 at Ser⁴⁹¹ in cell-free conditions. These results identify PKD1 as a novel upstream kinase of AMPKα2 Ser⁴⁹¹ that plays a negative role in insulin signaling in muscle cells.     

Cyclo‐oxygenase 2 up‐regulates the effect of multidrug resistance

COX‐2 (cyclo‐oxygenase 2), an inducible form of the enzyme that catalyses the first step in the synthesis of prostanoids, is associated with inflammatory diseases and carcinogenesis, which is suspected to promote angiogenesis and tissue invasion of tumours and resistance to apoptosis. COX‐2 is also involved in drug resistance and poor prognosis of many neoplastic diseases or cancers. The activation of the COX‐2/PGE₂ (prostaglandin E₂)/prostaglandin E receptor signal pathway can up‐regulate the expression of all three ABC (ATP‐binding‐cassette) transporters, MDR1/P‐gp (multidrug resistance/P‐glycoprotein), MRP1 (multidrug‐resistance protein 1) and BCRP (breast‐cancer‐resistance protein), which encode efflux pumps, playing important roles in the development of multidrug resistance. In addition, COX inhibitors inhibit the expression of MDR1/P‐gp, MRP1 and BCRP and enhance the cytotoxicity of anticancer drugs. Therefore we can use the COX inhibitors to potentialize the effects of chemotherapeutic agents and reverse multidrug resistance to facilitate the patient who may benefit from addition of COX inhibitors to standard cytotoxic therapy.     

ABCG transporters: structure, substrate specificities and physiological roles

The ATP-binding cassette (ABC) transporter superfamily is one of the largest protein families with representatives in all kingdoms of life. Members of this superfamily are involved in a wide variety of transport processes with substrates ranging from small ions to relatively large polypeptides and polysaccharides. The G subfamily of ABC transporters consists of half-transporters, which oligomerise to form the functional transporter. While ABCG1, ABCG4 and ABCG5/8 are involved in the ATP-dependent translocation of steroids and, possibly, other lipids, ABCG2 (also termed the breast cancer resistance protein) has been identified as a multidrug transporter that confers resistance on tumor cells. Evidence will be summarized suggesting that ABCG2 can also mediate the binding/transport of non-drug substrates, including free and conjugated steroids. The characterization of the substrate specificities of ABCG proteins at a molecular level might provide further clues about their potential physiological role(s), and create new opportunities for the modulation of their activities in relation to human disease.     

从干扰素诱导蛋白MxA筛选抗乙肝病毒活性肽

黏病毒抗性蛋白A（myxovirus resistance protein A,MxA）是由干扰素诱导的具有重要抗乙肝病毒（hepatitis B virus,HBV）功能的蛋白质,我们前期工作发现,MxA主要依赖其中心互作结构域（central interactive domain,CID）与病毒直接相互作用发挥功能,但其具体的抗病毒功能区以及功能区是否具有独立的抗病毒活性仍不清楚。本研究拟鉴定MxA蛋白上的抗乙肝病毒活性肽。首先从全长MxA构建缺失突变体ΔCID和截短体CID,以HepG2-2-15细胞为病毒模型,分别转染空载质粒、MxA、ΔCID和CID,免疫荧光法检测转染效率,Western印迹法检测质粒表达,酶联免疫法测定细胞培养液中HBsAg、HBeAg的量及荧光定量PCR法测定乙肝病毒 DNA的量,评估CID段的抗乙肝病毒活性。根据CID段的晶体结构,缩短肽段长度,构建α1、α2、α3等9段肽段质粒,鉴定各段的抗乙肝病毒活性和细胞毒性（MTT法）。运用计算生物学手段--分子对接法预测MxA蛋白与病毒相互作用的模式和位点。结果显示,ΔCID、CID和9段肽段质粒的序列及表达正确,9段肽段的表达量未见显著性差异,无显著的细胞毒性。CID组和黏病毒抗性蛋白A组较对照组乙肝病毒的复制水平显著降低,CID组细胞上清中HBsAg、HBeAg及乙肝病毒 DNA的量分别减少了55.57%±8.48%、68.37%±6.24%、66.67%±6.40%,P＜0.01;MxA组的3个指标分别减少了61.63%±3.11%、70.77%±7.25%、73.73%±6.18%,P＜0.01;ΔCID组较对照组无明显变化。9段肽段中α1组较对照组HBsAg、HBeAg及乙肝病毒 DNA的量有显著下降,分别减少了48.33%±1.70%、70.67%±3.30%、68.95%±2.55%,P＜0.001,表明α1对乙肝病毒具有强抑制活性。分子对接的结果显示,384 ～ 408位氨基酸是MxA蛋白与病毒互作的关键位点,该区域落在α1肽段上,验证了α1是MxA蛋白抗乙肝病毒及与乙肝病毒相互作用中的关键区段。本研究筛选并鉴定出人干扰素诱导蛋白MxA上最有效的抗乙肝病毒活性肽α1,研究结果为抗乙肝病毒多肽类新药的研发奠定了基础。     

Interaction of ivermectin with multidrug resistance proteins (MRP1, 2 and 3)

Ivermectin is a potent antiparasitic drug from macrocyclic lactone (ML) family, which interacts with the ABC multidrug transporter P-glycoprotein (Pgp). We studied the interactions of ivermectin with the multidrug resistance proteins (MRPs) by combining cellular and subcellular approaches. The inhibition by ivermectin of substrate transport was measured in A549 cells (calcein or 2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein, BCECF) and in HL60-MRP1 (calcein). Ivermectin induced calcein and BCECF retention in A549 cells (IC(50) at 1 and 2.5microM, respectively) and inhibited calcein efflux in HL60-MRP1 (IC(50)=3.8microM). The action of ivermectin on the transporters ATPase activity was followed on membranes from Sf9 cells overexpressing human Pgp, MRP1, 2 or 3. Ivermectin inhibited the Pgp, MRP1, 2 and 3 ATPase activities after stimulation by their respective activators. Ivermectin showed a rather good affinity for MRPs, mainly MRP1, in the micromolar range, although it was lower than that for Pgp. The transport of BODIPY-ivermectin was followed in cells overexpressing selectively Pgp or MRP1. In both cell lines, inhibition of the transporter activity induced intracellular retention of BODIPY-ivermectin. Our data revealed the specific interaction of ivermectin with MRP proteins, and its transport by MRP1. Although Pgp has been considered until now as the sole active transporter for this drug, the MRPs should be taken into account for the transport of ivermectin across cell membrane, modulating its disposition in addition to Pgp. This could be of importance for optimizing clinical efficacy of ML-based antiparasitic treatments. This offers fair perspectives for the use of ivermectin or non-toxic derivatives as multidrug resistance-reversing agents.     

蛋白降解靶向嵌合体临床前及临床研究进展

蛋白降解靶向嵌合体(proteolysis targeting chimera,PROTAC)是一种可以同时结合E3连接酶和靶蛋白的异双功能小分子,能够借助泛素-蛋白酶体系统特异性降解靶蛋白。目前PROTAC药物大多处于临床试验阶段,配体主要为非共价化合物,具有克服耐药性、降解“不可用药”靶蛋白的优势,但非共价配体会使PROTAC产生钩效应(hook effect),影响药效发挥。而共价配体凭借自身优势,可以避免该现象的发生,对于PROTAC的发展具有极大的帮助。本文总结了临床前及临床研究阶段,PROTAC分子在核内蛋白、跨膜蛋白和胞浆蛋白3种蛋白靶点中的应用,并以此为基础进行了讨论与展望,以期为今后PROTAC的发展提供一定的研究思路和参考。     

Resistance to wheat streak mosaic virus in transgenic wheat engineered with the viral coat protein gene

Wheat (Triticum aestivum) plants were stably transformed with the coat protein (CP) gene of wheat streak mosaic virus (WSMV) by the biolistic method. Eleven independently transformed plant lines were obtained and five were analyzed for gene expression and resistance to WSMV. One line showed high resistance to inoculations of two WSMV strains. This line had milder symptoms and lower virus titer than control plants after inoculation. After infection, new growth did not show symptoms. The observed resistance was similar to the recovery type resistance described previously using WSMV NIb transgene and in other systems. This line looked morphologically normal but had an unusually high transgene copy number (approximately 90 copies per 2C homozygous genome). Northern hybridization analysis indicated a high level of degraded CP mRNA expression. However, no coat protein expression was detected.