Dibenzocyclooctadiene lignans: a class of novel inhibitors of multidrug resistance-associated protein 1

We recently reported that dibenzocyclooctadiene lignans were a novel class of P-glycoprotein (P-gp) inhibitors. In this study, we demonstrated that the lignans of this class were also effective inhibitors of multidrug resistance-associated protein 1 (MRP1). The activities of 5 dibenzocyclooctadiene lignans (schisandrin A, schisandrin B, schisantherin A, schisandrol A, and schisandrol B) to reverse MRP1-mediated drug resistance were tested using HL60/Adriamycin (ADR) and HL60/Multidrug resistance-associated protein (MRP), two human promyelocytic leukemia cell lines with overexpression of MRP1 but not P-gp. The five lignans could effectively reverse drug resistance of the two cell lines to vincristine, daunorubicin, and VP-16. This study, together with our previous reports, proves that dibenzocyclooctadiene lignans have multiple activities against cancer multidrug resistance, including inhibition of P-gp and MRP1, and enhancement of apoptosis. Considering that cancer multidrug resistance (MDR) is multifactorial, agents with broad activities are preferable to the use of combination of several specific modulators to prevent drug-drug interaction and cumulative toxicity.     

HSP60 interacts with YB-1 and affects its polysome association and subcellular localization

YB-1 is a DNA/RNA-binding protein which, in the cytoplasm, associates with polysomes and regulates translation. However, YB-1 has a novel nuclear localization signal, and its nuclear accumulation is correlated with cancer induction. Here we designated the amino-acid sequence as YB-NLS and demonstrated that YB-NLS is necessary for the nuclear translocation of overexpressed YB-1 in NG108-15 cells. In addition, we found that a heat shock protein, HSP60, binds to YB-NLS in the cytoplasm. Interestingly, when HSP60 expression was repressed, an increase of polysome-associated YB-1 was observed in heavy-sedimenting fractions on a sucrose gradient. Overexpression of HSP60 resulted in a decrease of YB-1 in the heavy-sedimenting fractions and suppression of YB-NLS activity. Furthermore, the NLS-deleted YB-1 was apparently associated with the heavy-sedimenting polysomes. These results suggest that HSP60 interacts with YB-1 at the YB-NLS region and acts as a regulator of polysome association and the subcellular distribution of YB-1.     

YB-1与肿瘤发生及治疗

Y-box结合蛋白(Y-box binding protein,YB)是一类广泛存在于从低等到高等多种生物中的蛋白质家族,在体内行使多种生物学功能.大量研究表明,YB-1作为该家族成员之一,与许多重要的生物大分子存在密切联系,并对细胞、组织和机体的生理机能产生重大影响.更为重要的是,YB-1在多种疾病,尤其是恶性肿瘤的发生和发展中也起到十分关键的作用,对癌细胞表型的维持及肿瘤多药耐药性(MDR)的产生具有全方位的影响.以YB-1为作用靶点的新型肿瘤治疗策略可望有效控制癌症患者病情恶化,改善耐药状况.现就对YB-1与肿瘤发生和发展之间关系的研究进展,以及针对YB-1治疗策略的制定作一评述和展望.     

Participation of mTOR in the regulation of multidrug resistance of tumor cells

Molecular mechanisms of the influence of PI3K/Akt/PTEN/mTOR-signaling pathway on survival of tumor cells treated with cytotoxic drugs was studied using rapamycin (Rapa), mTOR specific inhibitor, and 9 human tumor cell lines of different origin and with different Akt kinase activity. Three of these cell lines were selected for drug resistance due to P-glycoprotein (Pgp or ABCB1) overexpression. Rapa inhibited phosphorylation of mTOR downstream effectors. Rapa sensitivity of the cells was Akt-dependent but did not correlate with ABCB1 overexpression. Suppression of mTOR function increased drug resistance in 8 out of 9 cell lines studied. The influence of Rapa on the ABC-transporter gene expression was examined. It was shown that in half of the cell lines studied Rapa exerted differential effects on the amount of ABC-transporter proteins: in some cases the protein amount decreased and in others, increased. The amount of mRNA remained unchanged. These data suggest that mTOR can regulate ABC transporters at the level of translation.     

多重耐药菌在人类、动物和环境的耐药和传播机制

抗生素等抗菌药物的滥用在全球范围内造成了多重耐药菌的传播。多重耐药菌(Multidrug resistant organisms,MDRO)以及耐药基因(Antibiotic resistance genes,ARGs)可在人类、动物和环境之间进行传播,尤其是ARGs可以通过水平转移的方式在同种属或者不同种属的菌群之间进行传播,使得细菌耐药问题日益严重,耐药机制趋于复杂,疾病治疗更加困难,对人类公众健康造成严重的威胁。因此抗生素等抗菌药物的使用应加以规范。     

Regulation of MDR1 gene expression in multidrug-resistant cancer cells is independent from YB-1

The MDR1 gene encoded transmembrane ABC-transporter MDR1/P-glycoprotein can mediate the phenotype of multidrug resistance (MDR), a major obstacle in the clinical management of cancer patients. It was hypothesized that YB-1 is a fundamental regulatory factor of the MDR1 gene in tumor cells and can therewith enhance drug resistance. To analyze the potential impact of YB-1 in MDR cancer cells, two specific anti-YB-1 small interfering RNAs (siRNAs) were designed for transient triggering the gene-silencing RNA interference (RNAi) pathway in the MDR cell lines EPG85-257RDB and EPP85-181RDB as well as in their drug-sensitive counterparts EPG85-257P and EPP85-181P. Since both siRNAs showed biological activity, for stable inhibition of YB-1 corresponding tetracycline-inducible short hairpin RNA (shRNA)-encoding expression vectors were designed. By treatment of the cancer cells with these constructs, the expression of the targeted YB-1 encoding mRNA and protein was completely inhibited following tetracycline exposure. These gene-silencing effects were not accompanied by modulation of the MDR1 expression or by reversal of the drug-resistant phenotype. In conclusion, the data demonstrate the utility of the analyzed RNAs as powerful laboratory tools and indicate that YB-1 is not involved in the regulation of the MDR1 gene or the development of the drug-resistant phenotype in MDR cancer cells.     

B4GALT family mediates the multidrug resistance of human leukemia cells by regulating the hedgehog pathway and the expression of p-glycoprotein and multidrug resistance-associated protein 1

β-1, 4-Galactosyltransferase gene (B4GALT) family consists of seven members, which encode corresponding enzymes known as type II membrane-bound glycoproteins. These enzymes catalyze the biosynthesis of different glycoconjugates and saccharide structures, and have been recognized to be involved in various diseases. In this study, we sought to determine the expressional profiles of B4GALT family in four pairs of parental and chemoresistant human leukemia cell lines and in bone marrow mononuclear cells (BMMC) of leukemia patients with multidrug resistance (MDR). The results revealed that B4GALT1 and B4GALT5 were highly expressed in four MDR cells and patients, altered levels of B4GALT1 and B4GALT5 were responsible for changed drug-resistant phenotype of HL60 and HL60/adriamycin-resistant cells. Further data showed that manipulation of these two gene expression led to increased or decreased activity of hedgehog (Hh) signaling and proportionally mutative expression of p-glycoprotein (P-gp) and MDR-associated protein 1 (MRP1) that are both known to be related to MDR. Thus, we propose that B4GALT1 and B4GALT5, two members of B4GALT gene family, are involved in the development of MDR of human leukemia cells, probably by regulating the activity of Hh signaling and the expression of P-gp and MRP1.     

Identification of novel leads applying in silico studies for Mycobacterium multidrug resistant (MMR) protein

Multidrug efflux mechanism is the main cause of intrinsic drug resistance in bacteria. Mycobacterium multidrug resistant (MMR) protein belongs to small multidrug resistant family proteins (SMR), causing multidrug resistance to proton (H⁺)-linked lipophilic cationic drug efflux across the cell membrane. In the present work, MMR is treated as a novel target to identify new molecular entities as inhibitors for drug resistance in Mycobacterium tuberculosis. In silico techniques are applied to evaluate the 3D structure of MMR protein. The putative amino acid residues present in the active site of MMR protein are predicted. Protein–ligand interactions are studied by docking cationic ligands transported by MMR protein. Virtual screening is carried out with an in-house library of small molecules against the grid created at the predicted active site residues in the MMR protein. Absorption distribution metabolism and elimination (ADME) properties of the molecules with best docking scores are predicted. The studies with cationic ligands and those of virtual screening are analysed for identification of new lead molecules as inhibitors for drug resistance caused by the MMR protein.     

Nonspecific and specific interactions of Y-box-binding protein 1 (YB-1) with mRNA and posttranscriptional regulation of protein synthesis in animal cells

Y-box-binding protein 1 (YB-1) is an animal multifunctional DNA/RNA-binding protein that is involved in reproduction, storing, and expression of genetic information. YB-1 accompanies mRNA throughout its life, from synthesis to degradation, and has a high specific and nonspecific affinity for RNA. In the nucleus YB-1 regulates mRNA processing. In the cytoplasm YB-1 is responsible for global and selective regulation of protein synthesis, as well as the mRNA life. This review focuses on the role of YB-1 in regulating translation. The possible mechanisms of the positive and negative effects of YB-1 on this process are considered. The recent original data are described, supporting the role of YB-1 as a major structural component of mRNP. Data about specific interactions of YB-1 with RNA are summarized for the first time.     

YB-1与EGFR在子宫内膜异位症中表达的研究进展

YB-1,EGFR各自作为冷激蛋白和糖蛋白家族的成员表达于各种生理和环境的损伤之后,保护细胞的生存。近年来发现YB-1和EGFR在多种癌症及内异症中高表达,并且参与肿瘤和子宫内膜异位组织的发生,发展,分化及转移的各个方面。因而YB-1和EGFR的高表达能影响子宫内膜异位症的发生和发展;也说明YB-1和EGFR可以作为子宫内膜异位症患者一个潜在的诊断和治疗靶点。     

Noncoding RNAs that associate with YB-1 alter proliferation in prostate cancer cells

The highly conserved, multifunctional YB-1 is a powerful breast cancer prognostic indicator. We report on a pervasive role for YB-1 in which it associates with thousands of nonpolyadenylated short RNAs (shyRNAs) that are further processed into small RNAs (smyRNAs). Many of these RNAs have previously been identified as functional noncoding RNAs (http://www.johnlab.org/YB1). We identified a novel, abundant, 3′-modified short RNA antisense to Dicer1 (Shad1) that colocalizes with YB-1 to P-bodies and stress granules. The expression of Shad1 was shown to correlate with that of YB-1 and whose inhibition leads to an increase in cell proliferation. Additionally, Shad1 influences the expression of additional prognostic markers of cancer progression such as DLX2 and IGFBP2. We propose that the examination of these noncoding RNAs could lead to better understanding of prostate cancer progression.     

Structure prediction and docking-based molecular insights of human YB-1 and nucleic acid interaction

Y-box-binding protein 1 (YB-1), a cold shock domain protein, is one of the most conserved nucleic acid-binding proteins. The multifunctional human YB-1 is a member of a large family of proteins with an evolutionary ancient cold shock domain. The presence of a cold shock domain is a specific feature of Y-box-binding proteins and allows attributing them to a wider group of proteins containing a cold shock domain. This protein is involved in a number of cellular processes including proliferation, differentiation and stress response. The YB-1 performs its function both in the cytoplasm and in the cell nucleus. In this study, we present the structure of full-length human YB-1 protein along with investigation of their nucleic acid-binding preferential. The study also focuses on biases for particular purine and pyrimidine bases. The overall goal of this study was to model and validate full-length YB-1 protein and to compare its nucleic acid-binding studies with previous reports.     

Roles of YB-1 under arsenite-induced stress: Translational activation of HSP70 mRNA and control of the number of stress granules

Background

When cells become stressed, they form stress granules (SGs) and show an increase of the molecular chaperone HSP70. The translational regulator YB-1 is a component of SGs, but it is unclear whether it contributes to the translational induction of HSP70 mRNA. Here we examined the roles of YB-1 in SG assembly and translational regulation of HSP70 mRNA under arsenite-induced stress.

Method

Using arsenite-treated NG108-15 cells, we examined whether YB-1 was included in SGs with GluR2 mRNA, a target of YB-1, and investigated the interaction of YB-1 with HSP70 mRNA and its effect on translation of the mRNA. We also investigated the distribution of these mRNAs to SGs or polysomes, and evaluated the role of YB-1 in SG assembly.

Results

Arsenite treatment reduced the translation level of GluR2 mRNA; concomitantly, YB-1-bound HSP70 mRNA was increased and its translation was induced. Sucrose gradient analysis revealed that the distribution of GluR2 mRNA was shifted from heavy-sedimenting to much lighter fractions, and also to SG-containing non-polysomal fractions. Conversely, HSP70 mRNA was shifted from the non-polysomal to polysome fractions. YB-1 depletion abrogated the arsenite-responsive activation of HSP70 synthesis, but SGs harboring both mRNAs were still assembled. The number of SGs was increased by YB-1 depletion and decreased by its overexpression.

Conclusion

In arsenite-treated cells, YB-1 mediates the translational activation of HSP70 mRNA and also controls the number of SGs through inhibition of their assembly.

General significance

Under stress conditions, YB-1 exerts simultaneous but opposing actions on the regulation of translation via SGs and polysomes.     

Role of the PTEN protein in the multidrug resistance of prostate cancer cells

At present, there is no doubt that the signal transduction pathway P13K/Akt/PTEN/mTOR, controlled by phosphatidylinositol-3-kinase, is involved in tumor cell resistance to a number of drugs. Another well-known mechanism determining drug resistance in tumors is associated with the activity of drug transporters of the ABC superfamily (first of all, P-glycoprotein (Pgp), MRP1, BCRP, and LRP). Several mechanisms of cell defense can simultaneously operate in one cell. The interplay of different mechanisms involved in drug resistance is poorly understood. The PC3 and DU145 human prostate cell lines were used to show that the PTEN functional status determined the cell resistance to some drugs and that correlated with the levels of MRP1 and BCRP. Pgp was not involved in drug resistance of these cells. Introduction of PTEN into PTEN-deficient PC3 cells, as well as rapamycin treatment, inhibited Akt and mTOR and sensitized cells to doxorubicin and vinblastine. Exogenous PTEN altered the MRP1 and BCRP expression. The results indicate that at least two mechanisms of drug resistance operate in prostate cancer cells: the PI3K/Akt/PTEN/mTOR pathway and an elevated MRP1 expression. The mechanisms are interconnected: PTEN and mTOR signaling is involved in MRP1 and BCRP expression regulation.     

Substrate-dependent transport mechanism in AcrB of multidrug resistant bacteria

The multidrug resistance (MDR) system effectively expels antibiotics out of bacteria causing serious issues during bacterial infection. In addition to drug, indole, a common metabolic waste of bacteria, is expelled by MDR system of gram-negative bacteria for their survival. Experimental results suggest that AcrB, one of the key components of MDR system, undergoes large scale conformation changes during the pumping due to proton-motive process. However, due to extremely short time scale, it is difficult to observe (experimentally) those changes in the AcrB, which might facilitate the pumping process. Molecular simulations can shed light to understand the conformational changes for transport of indole in AcrB. Examination of conformational changes using all-atom simulation is, however, impractical. Here, we develop a hybrid coarse-grained force field to study the conformational changes of AcrB in presence of indole in the porter domain of monomer II. Using the coarse-grained force field, we investigated the conformational changes of AcrB for a number of model systems considering the effect of protonation in aspartic acid (Asp) residues Asp407 and Asp408 in the transmembrane domain of monomer II. Our results show that in the presence of indole, protonation of Asp408 or Asp407 residue causes conformational changes from binding state to extrusion state in monomer II, while remaining two monomers (I and III) approach access state in AcrB protein. We also observed that all three AcrB monomers prefer to go back to access state in the absence of indole. Steered molecular dynamics simulations were performed to demonstrate the feasibility of indole transport mechanism for protonated systems. Identification of indole transport pathway through AcrB can be very helpful in understanding the drug efflux mechanism used by the MDR bacteria.     

CIAPIN1基因与肿瘤

细胞因子诱导的凋亡抑制因子1(cytokine induced apoptosis inhibitor1,CIAPIN1)是最新发现的一个细胞因子依赖性抗凋亡分子,并已经被证实是独立于Bcl家族、胱天蛋白酶(caspase)家族等之外的Ras信号转导通路中的另一个调节分子。CIAPIN1广泛分布于胎儿和成人的正常组织中,特别在分化型组织和活性代谢组织中具有很高的表达水平,但是在某些癌症发生时表达受到抑制。通过基因转染、RNA干扰等技术手段研究CIAPIN1与肿瘤发生、发展的关联,揭示了CIAPIN1表达水平的改变与肿瘤进展具有相关性,CIAPIN1有望成为一个新的肿瘤治疗靶分子。     

The proteolytic YB-1 fragment interacts with DNA repair machinery and enhances survival during DNA damaging stress

The Y-box binding protein 1 (YB-1) is a DNA/RNA-binding nucleocytoplasmic shuttling protein whose regulatory effect on many DNA and RNA-dependent events is determined by its localization in the cell. We have shown previously that YB-1 is cleaved by 20S proteasome between E219 and G220, and the truncated N-terminal YB-1 fragment accumulates in the nuclei of cells treated with DNA damaging drugs. We proposed that appearance of truncated YB-1 in the nucleus may predict multiple drug resistance. Here, we compared functional activities of the full-length and truncated YB-1 proteins and showed that the truncated form was more efficient in protecting cells against doxorubicin treatment. Both forms of YB-1 induced changes in expression of various genes without affecting those responsible for drug resistance. Interestingly, although YB-1 cleavage did not significantly affect its DNA binding properties, truncated YB-1 was detected in complexes with Mre11 and Rad50 under genotoxic stress conditions. We conclude that both full-length and truncated YB-1 are capable of protecting cells against DNA damaging agents, and the truncated form may have an additional function in DNA repair.     

Identification of a 170 kDa membrane kinase with increased activity in KB-V1 multidrug resistant cells

Using an in situ kinase assay we have identified kinases that are elevated in some multidrug resistant cells. Kinases were detected by measurement of ³²P incorporation in proteins that were renatured after being subjected to SDS-polyacrylamide gel electrophoresis and transferred to polyvinylidene difluoride membranes [Ferrell and Martin: J Biol Chem 264:20723–20729, 1989; Mol Cell Biol 10:3020–3026, 1990]. Kinases at 79, 84, and 92 kDa showed increased activity in the multidrug resistant human KB-V1 cells as compared to the sensitive parental KB-3-1 cells. The KB-V1 multidrug resistant cell line exhibited a 170 kDa membrane associated kinase activity that was not present in the parental drug sensitive line. The 170 kDa kinase activity was not affected by Ca⁺⁺, phosphatidylserine, or cAMP, but was diminished after incubation in the presence of the kinase inhibitors staurosporine, K252a and KT5720. The 170 kDa kinase activity phosphorylated mainly threonine, with no evidence of tyrosine phosphorylation, and was not identical to either the multidrug resistance associated P-glycoprotein or the EGF receptor. Other multidrug resistant cell lines also showed elevated 170 kDa kinase activity, such as the human breast cancer MCF-7/Adr^R and murine melanoma B16/Adr^R. cells, but the activity was not present in murine leukemia P-388 sensitive or multidrug resistant cells.