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.     

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.     

Talazoparib nanoparticles for overcoming multidrug resistance in triple-negative breast cancer

Herein, we investigated efflux pumps-mediated talazoparib-resistance in the treatment of triple-negative breast cancer (TNBC). Furthermore, we produced a novel talazoparib-solid lipid nanoparticles (SLNs) and then explored in vitro therapeutic efficacy of talazoparib-SLNs to overcome talazoparib-resistance in TNBC cells. Talazoparib-SLNs formulation was produced and then characterized. Calcein and Rho-123 were used to analyze the functional activity of drug efflux pumps in these cells. Additionally, RT-PCR, western blot and immunofluorescence analysis were used to detect the messenger RNA, and protein expression level, and cellular localization of the multidrug resistance (MDR1), breast cancer resistance protein (BCRP), and MRP1. We found that talazoparib efflux was mediated by BCRP and MRP1 pumps in TNBC cells. Talazoparib-SLNs could significantly enhance therapeutic efficacy of talazoparib. Furthermore, talazoparib-SLNs were more effective in the suppression of MDR1, BCRP, and MRP1 gene and protein expression levels than talazoparib. Consequently, this study suggests that talazoparib-SLNs formulation represents a promising therapeutic carrier to reverse MDR-mediated resistance in TNBC.     

Involvement of ABC transporters in the efflux and toxicity of MPA‐COOH‐CdTe quantum dots in human breast cancer SK‐BR‐3 cells

This paper aimed to study the possible involvement of adenosine triphosphate‐binding cassette (ABC) transporters in the detoxification of quantum dots (QDs) in human breast carcinoma (SK‐BR‐3) cells. The effects of QD sizes on such interactions were also evaluated. For this purpose, we used monodispersed MPA‐COOH‐CdTe QDs with different diameters (emission length at 560 and 625 nm, named as QD‐560 and QD‐625). Such QDs tended to accumulate in cells and cause significant toxicity. Using specific inhibitors of ABC transporters, the cellular accumulation and toxicity of QDs in SK‐BR‐3 cells were significantly affected. Moreover, treatment of QDs caused concentration‐ and time‐dependent induction of ABC transporters. Furthermore, the induction effects of smaller QDs were found to be greater than larger ones at equivalent concentrations, suggesting a size‐dependent recognition of substrates by ABC transporters. Overall, these results provided important support for the modulation of QDs toxicity by ABC transporters.     

p53对乳腺癌耐药蛋白基因的转录调控

乳腺癌耐药蛋白（breast cancer resistance protein,BCRP）是ATP结合盒转运蛋白超家族成员之一,其通过主动外排化疗药物如米托蒽醌、托泊替康和甲氨蝶呤,进而介导肿瘤化疗耐受. 最近有研究发现,在野生型p53（wild type p53, wt-p53）低表达的乳腺癌细胞系MCF-7中,外源性wt-p53通过抑制核转录因子-κB （nuclear factor-κB, NF-κB）的活性进而抑制BCRP的表达,但其详细的分子机制有待进一步阐明. 本研究选用p53缺失的骨肉瘤细胞系Saos-2,通过瞬时转染技术发现,wt-p53可以激活BCRP的表达,而突变型p53的激活作用消失;报告基因试验显示,wt-p53可以上调BCRP启动子活性;通过生物信息学软件MatInspector对BCRP启动子区进行预测,未发现p53结合元件;同时,通过转染IκB抑制Saos-2细胞中NF-κB的活性后发现,Saos-2细胞中NF-κB活性越低,p53对BCRP启动子的激活作用越弱甚至完全消失. 上述结果提示,p53对Saos-2细胞中BCRP的激活作用是NF-κB依赖性的.     

NDV‐D90 inhibits 17β‐estradiol‐mediated resistance to apoptosis by differentially modulating classic and nonclassic estrogen receptors in breast cancer cells

Newcastle disease virus (NDV) is endowed with the oncolytic ability to kill tumor cells, while rarely causing side effects in normal cells. Both estrogen receptor α (ERα) and the G protein estrogen receptor (GPER) modulate multiple biological activities in response to estrogen, including apoptosis in breast cancer (BC) cells. Here, we investigated whether NDV‐D90, a novel strain isolated from natural sources in China, promoted apoptosis by modulating the expression of ERα or the GPER in BC cells exposed to 17β‐estradiol (E2). We found that NDV‐D90 significantly killed the tumor cell lines MCF‐7 and BT549 in a time‐ and dose‐dependent manner. We also found that NDV‐D90 exerted its effects on the two cell lines mainly by inducing apoptosis but not necrosis. NDV‐D90 induced apoptosis via the intrinsic and extrinsic signaling pathways in MCF‐7 cells (ER‐positive cells) during E2 exposure not only by disrupting the E2/ERα axis and enhancing GPER expression but also by modulating the expression of several apoptosis‐related proteins through ERα‐and GPER‐independent processes. NDV‐D90 promoted apoptosis via the intrinsic signaling pathway in BT549 cells (ER‐negative cells), possibly by impairing E2‐mediated GPER expression. Furthermore, NDV‐D90 exerted its antitumor effects in vivo by inducing apoptosis. Overall, these results demonstrated that NDV‐D90 promotes apoptosis by differentially modulating the expression of ERα and the GPER in ER‐positive and negative BC cells exposed to estrogen, respectively, and can be utilized as an effective approach to treating BC.     

Protein tyrosine phosphatase interacting protein 51 (PTPIP51) mRNA expression and localization and its in vitro interacting partner protein tyrosine phosphatase 1B (PTP1B) in human placenta of the first, second, and third trimester

The cellular localization of protein tyrosine phosphatase 51 (PTPIP51) and its in vitro interacting partner protein tyrosine phosphatase 1B (PTP1B) was studied in human placentae of different gestational stages. The expression of PTPIP51 protein and mRNA was observed in the syncytiotrophoblast and cytotrophoblast layer of placentae from the first, second, and third trimesters. In contrast, PTP1B expression was restricted to the syncytiotrophoblast during all gestational stages. Cells of the cytotrophoblasts and parts of the syncytiotrophoblasts expressing high amounts of PTPIP51 were found to execute apoptosis as shown by TdT-mediated dUTP-biotin nick end labeling assay, cytokeratin 18f, and caspase 3 expression. PTPIP51 could also be traced in the endothelium and smooth muscle cells of placental arterial and venous vessels, identified by double immunostainings with antibodies directed against van Willebrand factor and alpha-smooth muscle actin. Some of these cells showing a high PTPIP51 reactivity were Ki67 positive, indicating proliferation. Additionally, a small population of placental CD14-positive macrophages and mesenchymal cells within the villous stroma were detected as PTPIP51 positive. Our data suggest that both proteins, PTPIP51 and PTP1B, play a role in differentiation and apoptosis of the cytotrophoblast and syncytiotrophoblast, respectively. Moreover, PTPIP51 may also serve as a cellular signaling partner in angiogenesis and vascular remodeling.     

Involvement of p38 MAPK in haemozoin‐dependent MMP‐9 enhancement in human monocytes

The lipid moiety of natural haemozoin (nHZ, malarial pigment) was previously shown to enhance expression and release of human monocyte matrix metalloproteinase‐9 (MMP‐9), and a major role for 15‐(S,R)‐hydroxy‐6,8,11,13‐eicosatetraenoic acid (15‐HETE), a nHZ lipoperoxidation product, was proposed. Here, the underlying mechanisms were investigated, focusing on the involvement of mitogen‐activated protein kinases (MAPKs). Results showed that nHZ promoted either early or late p38 MAPK phosphorylation; however, nHZ did not modify basal phosphorylation/expression ratios of extracellular signal‐regulated kinase‐1/2 and c‐jun N‐terminal kinase‐1/2. 15‐HETE mimicked nHZ effects on p38 MAPK, whereas lipid‐free synthetic (s)HZ and delipidized (d)HZ did not. Consistently, both nHZ and 15‐HETE also promoted phosphorylation of MAPK‐activated protein kinase‐2, a known p38 MAPK substrate; such an effect was abolished by SB203580, a synthetic p38 MAPK inhibitor. SB203580 also abrogated nHZ‐dependent and 15‐HETE‐dependent enhancement of MMP‐9 mRNA and protein (latent and activated forms) levels in cell lysates and supernatants. Collectively, these data suggest that in human monocytes, nHZ and 15‐HETE upregulate MMP‐9 expression and secretion through activation of p38 MAPK pathway. The present work provides new evidence on mechanisms underlying MMP‐9 deregulation in malaria, which might be helpful to design new specific drugs for adjuvant therapy in complicated malaria. Copyright © 2013 John Wiley & Sons, Ltd.     

Nicotine and cotinine increases the brain penetration of saquinavir in rat

Endothelial tight junctions and efflux transporters of the blood-brain barrier (BBB) significantly limit brain accumulation of many drugs, including protease inhibitors such as saquinavir. The cholinergic agonist nicotine is one of the most commonly used drugs in the world and the incidence is even higher in the human immune deficiency virus population (～ 70%). We examined the ability of nicotine and its primary metabolite cotinine to modify brain uptake of saquinavir in rats. Both nicotine and cotinine at pharmacological concentrations matching those in smokers, increased brain saquinavir uptake by two fold. Co-perfusion with nicotinic receptor antagonists and passive permeability markers showed that the effect was not caused by receptor activation or BBB permeability disruption. Transport inhibition studies demonstrated that brain saquinavir uptake is limited by multiple efflux transporters, P-glycoprotein (P-gp), breast cancer resistance protein and multidrug resistance-associated protein. In situ perfusion and in vitro experiments using a classical P-gp substrate rhodamine 123 linked the effect of nicotine to inhibition of BBB P-gp transport. The effect was confirmed in vivo in chronic 14 day nicotine administration animals. These data suggest nicotine increases antiretroviral drug exposure to brain and may represent a significant in vivo drug-drug interaction at the BBB. Although this may slightly benefit CNS antiretroviral efficacy, it may also expose the brain to potential serious neurotoxicity.     

Fibronectin promotes proplatelet formation in the human megakaryocytic cell line UT-7/TPO

We investigated PPF (proplatelet formation) in the human megakaryocytic cell line UT-7/TPO in vitro and signal transduction pathways responsible for PPF. The megakaryocytic cell lines are useful for studying megakaryocyte biology, although PPF is induced only in the presence of phorbol ester. TPO (thrombopoietin) stimulates megakaryocyte proliferation and differentiation; however, no PPF occurred in the megakaryocytic cell lines, even after the addition of TPO. Therefore, factors other than TPO may play an important role in the process of PPF. As PPF occurs in the bone marrow in vivo, we noted extracellular matrix proteins and found that soluble FN (fibronectin) induced potent PPF in UT-7/TPO without phorbol ester. A Western blot analysis showed that the expression of integrins was not increased by FN treatment. Anti-β1 antibody and the RGD (arginine-glycine-aspartate) peptide inhibited FN-induced PPF. This result indicates that the signal originated from integrin β1, which is essential to inducing PPF in UT-7/TPO. Results of the experiments using several inhibitors suggest that activation of the MEK [MAPK (mitogen-activated protein kinase)/ERK (extracellular-signal-regulated kinase) kinase]-ERK and PI3K (phosphoinositide 3-kinase) pathways are necessary for PPF. The phosphorylation of ERK gradually increased for 2 h after the addition of soluble FN, which suggests that activation of ERK is essential for the initial induction of FN-induced PPF in UT-7/TPO. UT-7/TPO is a useful cell line that enables us to study the signals of PPF without effects of chemical compounds.     

Interferon‐Gamma test for the detection of Mycobacterium tuberculosis complex infection in Macaca mulatta and other non‐human primates

We have formatted an assay to detect Mycobacterium tuberculosis complex infections of non‐human primates. Commercially available reagents were used to elicit a specific immune response that was measured by interferon‐gamma release. Initial evaluation using blood samples from Rhesus macaques experimentally infected with M tuberculosis distinguished infected versus uninfected animals.     

A novel protein,sperm head and tail associated protein (SHTAP), interacts with cysteine‐rich secretory protein 2 (CRISP2) during spermatogenesis in the mouse

Background information. CRISP2 (cysteine‐rich secretory protein 2) is a sperm acrosome and tail protein with the ability to regulate Ca²⁺ flow through ryanodine receptors. Based on these properties, CRISP2 has a potential role in fertilization through the regulation of ion signalling in the acrosome reaction and sperm motility. The purpose of the present study was to determine the expression, subcellular localization and the role in spermatogenesis of a novel CRISP2‐binding partner, which we have designated SHTAP (sperm head and tail associated protein). Results. Using yeast two‐hybrid screens of an adult testis expression library, we identified SHTAP as a novel mouse CRISP2‐binding partner. Sequence analysis of all Shtap cDNA clones revealed that the mouse Shtap gene is embedded within a gene encoding the unrelated protein NSUN4 (NOL1/NOP2/Sun domain family member 4). Five orthologues of the Shtap gene have been annotated in public databases. SHTAP and its orthologues showed no significant sequence similarity to any known protein or functional motifs, including NSUN4. Using an SHTAP antiserum, multiple SHTAP isoforms (～20–87 kDa) were detected in the testis, sperm, and various somatic tissues. Interestingly, only the ～26 kDa isoform of SHTAP was able to interact with CRISP2. Furthermore, yeast two‐hybrid assays showed that both the CAP (CRISP/antigen 5/pathogenesis related‐1) and CRISP domains of CRISP2 were required for maximal binding to SHTAP. SHTAP protein was localized to the peri‐acrosomal region of round spermatids, and the head and tail of the elongated spermatids and sperm tail where it co‐localized with CRISP2. During sperm capacitation, SHTAP and the SHTAP—CRISP2 complex appeared to be redistributed within the head. Conclusions. The present study is the first report of the identification, annotation and expression analysis of the mouse Shtap gene. The redistribution observed during sperm capacitation raises the possibility that SHTAP and the SHTAP—CRISP2 complex play a role in the attainment of sperm functional competence.     

Single amino acid (482) variants of the ABCG2 multidrug transporter: major differences in transport capacity and substrate recognition

The human ABCG2 protein is an ATP binding cassette half-transporter, which protects our cells and tissues against various xenobiotics, while overexpression of ABCG2 in tumor cells confers multidrug resistance. It has been documented that single amino acid changes at position 482 resulted in altered drug resistance and transport capacity. In this study, we have generated nine Arg-482 mutants (G, I, M, S, T, D, N, K, Y) of ABCG2, and expressed them in insect cells. All ABCG2 variants showed cell surface expression and, in isolated membranes, an ABCG2-specific ATPase activity. When methotrexate accumulation was measured in inside-out membrane vesicles, this transport was supported only by the wild-type ABCG2. In intact cells, mitoxantrone was transported by all ABCG2 variants, except by R482K. Rhodamine 123 was extruded by most of the mutants, except by R482K, Y and by wild-type ABCG2. Hoechst 33342 was pumped out from cells expressing the wild-type and all Arg-482 variants, but not from those expressing R482K and Y. Our study demonstrates that the substrate specificity of the Arg (wild-type) form is unique and that amino acid replacements at position 482 induce major alterations in both the transport activity and substrate specificity of this protein.     

Ins(1,4,5)P3 receptor type 1 associates with AKAP9 (AKAP450 variant) and protein kinase A type IIβ in the Golgi apparatus in cerebellar granule cells

Background information. Interconnections between the Ca²⁺ and cAMP signalling pathways can determine the specificity and diversity of the cellular effects mediated by these second messengers. Most cAMP effects are mediated by PKA (protein kinase A), which is anchored close to its membranous substrates by AKAPs (A kinase‐anchoring proteins). In many cell types, the activation of InsP₃R (inositol 1,4,5‐trisphosphate receptor), an endoplasmic reticulum Ca²⁺ channel, is a key event of Ca²⁺ signalling. The phosphorylation of InsP₃R1 by PKA stimulates Ca²⁺ mobilization. This control is thought to be tight, involving the association of PKA with InsP₃R1. The InsP₃R1 isoform predominates in central nervous tissue and its concentration is highest in the cerebellar microsomes. We investigated the complex formed by InsP₃R1 and PKA in this fraction, vith a view to identifying its components and determining its distribution in the cerebellar cortex. Results. Immunoprecipitation experiments showed that InsP₃R1 associated with PKA type IIβ and AKAP450, the longer variant of AKAP9, in sheep cerebellar microsomes. The co‐purification of AKAP450 with InsP₃R1 on heparin‐agarose provided further evidence of the association of these proteins. Immunohistofluorescence experiments on slices of cerebellar cortex showed that AKAP450 was colocalized with InsP₃R1 and RIIβ (regulatory subunit of PKA IIβ) in granule cells, but not in Purkinje cells. AKAP450 was localized in the Golgi apparatus of these two cell types whereas InsP₃R1 was detected in this organelle only in granule cells. Conclusions. Taken together these results suggest that InsP₃R1 forms a complex with AKAP450 and PKAIIβ, localized in the Golgi apparatus of cerebellar granule cells. In contrast, the association of InsP₃R1 with PKA in Purkinje cells would require a different macromolecular complex.     

Transport and transporters in the endoplasmic reticulum

Enzyme activities localized in the luminal compartment of the endoplasmic reticulum are integrated into the cellular metabolism by transmembrane fluxes of their substrates, products and/or cofactors. Most compounds involved are bulky, polar or even charged; hence, they cannot be expected to diffuse through lipid bilayers. Accordingly, transport processes investigated so far have been found protein-mediated. The selective and often rate-limiting transport processes greatly influence the activity, kinetic features and substrate specificity of the corresponding luminal enzymes. Therefore, the phenomenological characterization of endoplasmic reticulum transport contributes largely to the understanding of the metabolic functions of this organelle. Attempts to identify the transporter proteins have only been successful in a few cases, but recent development in molecular biology promises a better progress in this field.