Immunolocalization and cell expression of lung resistance-related protein (LRP) in normal and tumoral human respiratory cells.

Lung resistance-related protein (LRP) is an integral part of the multidrug resistance (MDR) phenotype involved in cell resistance toward xenobiotics or chemotherapy. The aim of this study was to compare the intracellular localization and cell expression of LRP in normal bronchial cells and their tumoral counterparts from non-small cell lung cancer (NSCLC). LRP expression was also investigated concurrently with DNA ploidy and chromosome 16 (lrp gene locus) aberrations. Confocal microscopy showed that LRP localization was exclusively intracytoplasmic regardless of the cell type and was never observed in the nuclear pore complex. Flow cytometry demonstrated a similar level of LRP expression in normal bronchial cells and in cancer cells from NSCLC samples. FISH analysis, performed to evaluate the number of chromosome 16 and lrp loci, demonstrated a significant gain of chromosome 16 in DNA aneuploid tumors. Furthermore, we did not find any link between LRP expression and DNA ploidy status or chromosome 16 number. These results suggest that LRP expression observed in NSCLC, maintained through the carcinogenesis process of respiratory cells, is not altered by the increased number of copies of chromosome 16 and probably controlled by mechanisms different from those of MRP1 expression, whereas both proteins are associated with the MDR phenotype.     

Expression of the lung resistance-related protein in human and rat hepatocarcinogenesis

Lung resistance-related protein (LRP) plays an important role in chemoresistance of tumor cells probably by altering nuclear-cytoplasmic transport processes. We analyzed the association between LRP expression and hepatocarcinogenesis in humans and rats by RT-PCR, immunoblotting, and immunohistochemistry. LRP was found in hepatocytes and bile epithelia of normal human and rat liver showing distinct interindividual variations. In human tissues, the LRP expression levels of dysplastic liver nodules, hepatocellular adenomas, and carcinomas were highly variable, including decreased but also distinctly increased staining intensities. Mean expression levels, however, were comparable to the surrounding tissue. Considerable levels of LRP mRNA and protein were also found in human hepatoma cell lines. To study LRP expression from the beginning of hepatocarcinogenesis onward, rats were subjected to a tumor initiation/promotion protocol leading to preneoplastic hepatocytes present as single cells or multicellular clones, followed by adenoma and carcinoma. All of the (pre)neoplastic rat liver lesions expressed, comparable to the surrounding tissue, considerable amounts of LRP. We conclude that LRP might be one mechanism involved in the intrinsically high but variable chemoresistance of normal and (pre)neoplastic hepatocytes.     

Low density lipoprotein (LDL) receptor-related protein 6 (LRP6) regulates body fat and glucose homeostasis by modulating nutrient sensing pathways and mitochondrial energy expenditure

Body fat, insulin resistance, and type 2 diabetes are often linked together, but the molecular mechanisms that unify their association are poorly understood. Wnt signaling regulates adipogenesis, and its altered activity has been implicated in the pathogenesis of type 2 diabetes and metabolic syndrome. LRP6(+/-) mice on a high fat diet were protected against diet-induced obesity and hepatic and adipose tissue insulin resistance compared with their wild-type (WT) littermates. Brown adipose tissue insulin sensitivity and reduced adiposity of LRP6(+/-) mice were accounted for by diminished Wnt-dependent mTORC1 activity and enhanced expression of brown adipose tissue PGC1-α and UCP1. LRP6(+/-) mice also exhibited reduced endogenous hepatic glucose output, which was due to diminished FoxO1-dependent expression of the key gluconeogenic enzyme glucose-6-phosphatase (G6pase). In addition, in vivo and in vitro studies showed that loss of LRP6 allele is associated with increased leptin receptor expression, which is a likely cause of hepatic insulin sensitivity in LRP6(+/-) mice. Our study identifies LRP6 as a nutrient-sensitive regulator of body weight and glucose metabolism and as a potential target for pharmacological interventions in obesity and diabetes.     

Induction of cytochrome P450 (CYP)1A1, CYP1A2, and CYP3A4 but not of CYP2C9, CYP2C19, multidrug resistance (MDR-1) and multidrug resistance associated protein (MRP-1) by prototypical inducers in human hepatocytes

Human hepatocytes cultured serum-free for up to 6 weeks were used to study expression and induction of enzymes and membrane transport proteins involved in drug metabolism. Phase I drug metabolizing enzymes cytochrome P450 (CYP)1A1, CYP1A2, CYP2C9, CYP2C19, CYP2E1, and CYP3A4 were detected by Western blot analyses and, when appropriate, by enzymatic assays for ethoxyresorufin-O-deethylase(EROD)-activity and testosterone-6beta-hydroxylase(T6H)-activity. Expression of the membrane transporter multi-drug resistance protein (P-glycoprotein, MDR-1), multidrug resistance-associated protein (MRP-1), and lung-resistance protein (LRP) was maintained during the culture as detected by RT-PCR and Western blot analyses. Model inducers like rifampicin, phenobarbital, or 3-methylcholanthrene and beta-naphtoflavone were able to induce CYP1A or CYP3A4 as well as EROD or T6H activities for up to 30 days. CYP2C9, CYP2C19 and CYP2E1 expression was maintained but not inducible for 48 days. Also, rifampicin and phenobarbital were unable to increase MDR-1 and MRP-1 protein levels significantly.     

Myeloid cell function in MRP-14 (S100A9) null mice

Myeloid-related protein 14 (MRP-14) and its heterodimeric partner, MRP-8, are cytosolic calcium-binding proteins, highly expressed in neutrophils and monocytes. To understand the function of MRP-14, we performed targeted disruption of the MRP-14 gene in mice. MRP-14(-/-) mice showed no obvious phenotype and were fertile. MRP-8 mRNA but not protein is present in the myeloid cells of these mice, suggesting that the stability of MRP-8 protein is dependent on MRP-14 expression. A compensatory increase in other proteins was not detected in cells lacking MRP-8 and MRP-14. Although the morphology of MRP-14(-/-) myeloid cells was not altered, they were significantly less dense. When Ca(2+) responses were investigated, there was no change in the maximal response to the chemokine MIP-2. At lower concentrations, however, there was reduced responsiveness in MRP-14(-/-) compared with MRP-14(+/+) neutrophils. This alteration in the ability to flux Ca(2+) did not impair the ability of the MRP-14(-/-) neutrophils to respond chemotactically to MIP-2. In addition, the myeloid cell functions of phagocytosis, superoxide burst, and apoptosis were unaffected in MRP-14(-/-) cells. In an in vivo model of peritonitis, MRP-14(-/-) mice showed no difference from wild-type mice in induced inflammatory response. The data indicate that MRP-14 and MRP-8 are dispensable for many myeloid cell functions.     

Folate decorated dual drug loaded nanoparticle: role of curcumin in enhancing therapeutic potential of nutlin-3a by reversing multidrug resistance

Retinoblastoma is the most common intraocular tumor in children. Malfunctioning of many signaling pathways regulating cell survival or apoptosis, make the disease more vulnerable. Notably, resistance to chemotherapy mediated by MRP-1, lung-resistance protein (LRP) is the most challenging aspect to treat this disease. Presently, much attention has been given to the recently developed anticancer drug nutlin-3a because of its non-genotoxic nature and potency to activate tumor suppressor protein p53. However, being a substrate of multidrug resistance protein MRP1 and Pgp its application has become limited. Currently, research has step towards reversing Multi drug resistance (MDR) by using curcumin, however its clinical relevance is restricted by plasma instability and poor bioavailability. In the present investigation we tried to encapsulate nutlin-3a and curcumin in PLGA nanoparticle (NPs) surface functionalized with folate to enhance therapeutic potential of nutlin-3a by modulating MDR. We document that curcumin can inhibit the expression of MRP-1 and LRP gene/protein in a concentration dependent manner in Y79 cells. In vitro cellular cytotoxicity, cell cycle analysis and apoptosis studies were done to compare the effectiveness of native drugs (single or combined) and single or dual drug loaded nanoparticles (unconjugated/folate conjugated). The result demonstrated an augmented therapeutic efficacy of targeted dual drug loaded NPs (Fol-Nut-Cur-NPs) over other formulation. Enhanced expression or down regulation of proapoptotic/antiapoptotic proteins respectively and down-regulation of bcl2 and NFκB gene/protein by Fol-Nut-Cur-NPs substantiate the above findings. This is the first investigation exploring the role of curcumin as MDR modulator to enhance the therapeutic potentiality of nutlin-3a, which may opens new direction for targeting cancer with multidrug resistance phenotype.     

Upregulation of Autophagy-Related Gene-5 (ATG-5) Is Associated with Chemoresistance in Human Gastric Cancer

Autophagy-related gene-5 (ATG-5) is one of the key regulators of autophagic cell death. It has been widely regarded as a protective molecular mechanism for tumor cells during the course of chemotherapy. In the present study, we investigated the expression pattern of ATG-5 and multidrug resistance-associated protein-1 (MRP-1) in 135 gastric cancers (GC) patients who were treated with epirubicin, cisplatin and 5-FU adjuvant chemotherapy (ECF) following surgical resection and explored their potential clinical significance. We found that both ATG-5 (77.78%) and MRP-1 (79.26%) were highly expressed in GC patients. ATG-5 expression was significantly associated with depth of wall invasion, TNM stages and distant metastasis of GC (P<0.05), whereas MRP-1 expression was significantly linked with tumor size, depth of wall invasion, lymph node metastasis, TNM stages and differentiation status (P<0.05). ATG-5 expression was positively correlated with MRP-1 (rp = 0.616, P<0.01). Increased expression of ATG-5 and MPR-1 was significantly correlated with poor overall survival (OS; P<0.01) and disease free survival (DFS; P<0.01) of our GC cohort. Furthermore, we demonstrated that ATG-5 was involved in drug resistant of GC cells, which was mainly through regulating autophagy. Our data suggest that upregulated expression of ATG-5, an important molecular feature of protective autophagy, is associated with chemoresistance in GC. Expression of ATG-5 and MRP-1 may be independent prognostic markers for GC treatment.     

Low level of low-density lipoprotein receptor-related protein 1 predicts an unfavorable prognosis of hepatocellular carcinoma after curative resection

Background

Low-density lipoprotein receptor-related protein 1 (LRP1) is a multifunctional receptor involved in receptor-mediated endocytosis and cell signaling. The aim of this study was to elucidate the expression and mechanism of LRP1 in hepatocellular carcinoma (HCC).

Methods

LRP1 expression in 4 HCC cell lines and 40 HCC samples was detected. After interruption of LRP1 expression in a HCC cell line either with specific lentiviral-mediated shRNA LRP1 or in the presence of the LRP1-specific chaperone, receptor-associated protein (RAP), the role of LRP1 in the migration and invasion of HCC cells was assessed in vivo and in vitro, and the expression of matrix metalloproteinase (MMP) 9 in cells and the bioactivity of MMP9 in the supernatant were assayed. The expression and prognostic value of LRP1 were investigated in 327 HCC specimens.

Results

Low LRP1 expression was associated with poor HCC prognosis, with low expression independently related to shortened overall survival and increased tumor recurrence rate. Expression of LRP1 in non-recurrent HCC samples was significantly higher than that in early recurrent samples. LRP1 expression in HCC cell lines was inversely correlated with their metastatic potential. After inhibition of LRP1, low-metastatic SMCC-7721 cells showed enhanced migration and invasion and increased expression and bioactivity of MMP9. Correlation analysis showed a negative correlation between LRP1 and MMP9 expression in HCC patients. The prognostic value of LRP1 expression was validated in the independent data set.

Conclusions

LRP1 modulated the level of MMP9 and low level of LRP1 expression was associated with aggressiveness and invasiveness in HCCs. LRP1 offered a possible strategy for tumor molecular therapy.     

Striking differences of LDL receptor-related protein 1B expression in mouse and human

The low density lipoprotein (LDL) receptor-related protein 1B (LRP1B) is a member of the expanding LDL receptor family, and is closely related to LRP. It was discovered as a putative tumor suppressor, and is frequently inactivated in human malignant tissues. However, the expression pattern of LRP1B in normal human tissues was unclear. In the present study, we analyzed LRP1B expression in normal mouse and human tissues. By using RT-PCR, we found that, while mouse LRP1B expression is mostly restricted to the brain, human LRP1B expression is more widespread with highest expression levels detected in the brain, adrenal gland, salivary gland, and testis. Although mouse LRP1B expresses in the forms of both full-length receptor tail and an alternatively spliced form lacking a 33-amino acid insert, human LRP1B is expressed exclusively in the form of full-length receptor tail. Finally, we found that, unlike mouse LRP1B, human LRP1B is cleaved by furin. Taken together, these data demonstrate that there are striking differences between LRP1B expression in mouse and human tissues. The broader expression pattern of LRP1B in human tissues suggests that this putative tumor suppressor may play roles in several types of human cancer.     

Multidrug resistance-associated protein MRP-1 regulates dauer diapause by its export activity in Caenorhabditis elegans

Multidrug resistance-associated proteins (MRPs), when overexpressed, confer drug resistance to cancer cells by exporting anti-cancer agents through the cell membrane, but their role in animal development has not been elucidated. Here we show that an MRP homolog regulates larval development in the nematode Caenorhabditis elegans. C. elegans forms a special third-stage larva called a dauer larva under conditions inappropriate for growth. By contrast, we found that mutants in mrp-1, an MRP homolog gene, form dauer larvae even under conditions appropriate for growth, in the background of certain mutations that partially block the insulin signaling pathway. A functional mrp-1::GFP gene was shown to be expressed in many tissues, and the wild-type mrp-1 gene must be expressed in multiple tissues for a wild-type phenotype. Human MRP1 could substitute for C. elegans MRP-1 in dauer larva regulation, and an inhibitor of the human MRP1 transport activity impaired this function, showing that export activity is required for normal dauer larva regulation. Epistasis studies revealed that MRP-1 acts in neither the TGF-beta nor the cGMP signaling pathway. mrp-1 mutations enhanced the dauer-constitutive phenotype of mutants in the insulin signaling pathway more strongly than that in other pathways. Thus, MRP-1, through its export activity, supports the induction of the normal (non-dauer) life cycle by the insulin signaling pathway.     

LRP6 protein regulates low density lipoprotein (LDL) receptor-mediated LDL uptake

Genetic variations in LRP6 gene are associated with high serum LDL cholesterol levels. We have previously shown that LDL clearance in peripheral B-lymphocytes of the LRP6(R611C) mutation carriers is significantly impaired. In this study we have examined the role of wild type LRP6 (LRP6(WT)) and LRP6(R611C) in LDL receptor (LDLR)-mediated LDL uptake. LDL binding and uptake were increased when LRP6(WT) was overexpressed and modestly reduced when it was knocked down in LDLR-deficient CHO (ldlA7) cells. These findings implicated LRP6 in LDLR-independent cellular LDL binding and uptake. However, LRP6 knockdown in wild type CHO cells resulted in a much greater decline in LDL binding and uptake compared with CHO-ldlA7 cells, suggesting impaired function of the LDLR. LDLR internalization was severely diminished when LRP6 was knocked down and was restored after LRP6 was reintroduced. Further analysis revealed that LRP6(WT) forms a complex with LDLR, clathrin, and ARH and undergoes a clathrin-mediated internalization after stimulation with LDL. LDLR and LRP6 internalizations as well as LDL uptake were all impaired in CHO-k1 cells expressing LRP6(R611C). These studies identify LRP6 as a critical modulator of receptor-mediated LDL endocytosis and introduce a mechanism by which variation in LRP6 may contribute to high serum LDL levels.     

The drug resistance proteins, multidrug resistance-associated protein and P-glycoprotein, do not confer resistance to Fas-induced cell death

BACKGROUND: Multidrug resistance (MDR) is mediated by the drug resistance proteins, the multidrug resistance-associated protein (MRP) and P-glycoprotein, both of which confer resistance by the active efflux of chemotherapeutic drugs from the cell. Reduced Fas (CD95/APO-1) expression and resistance to Fas-mediated apoptosis have also been correlated with P-glycoprotein-mediated MDR. METHODS: We investigated cell surface Fas expression (using anti-Fas monoclonal antibody DX2.1) in a series of MRP-expressing drug-resistant leukemia sublines, and P-glycoprotein-expressing leukemia sublines, and their susceptibility to apoptosis induced by anti-Fas treatment (CH-11 monoclonal antibody). Caspase-3 activation was detected by Western blot and apoptosis was determined by flow cytometry with 7-aminoactinomycin D (7-AAD) staining of cells. RESULTS: Fas expression was not reduced in either the MRP- or P-glycoprotein-expressing drug-resistant cell lines, although expression was reduced by 15% in one low-level drug-resistant subline. Expression of MRP or P-glycoprotein did not confer resistance to caspase-3 activation or to anti-Fas-induced cell death. CONCLUSIONS: MDR mediated by the drug transport proteins MRP and P-glycoprotein does not correlate with resistance to Fas-mediated cell death or resistance to caspase-3 activation.     

Tyrosine-phosphorylated low density lipoprotein receptor-related protein 1 (Lrp1) associates with the adaptor protein SHC in SRC-transformed cells

v-Src transforms fibroblasts in vitro and causes tumor formation in the animal by tyrosine phosphorylation of critical cellular substrates. Exactly how v-Src interacts with these substrates remains unknown. One of its substrates, the adaptor protein Shc, is thought to play a crucial role during cellular transformation by v-Src by linking v-Src to Ras. We used Shc proteins with mutations in either the phosphotyrosine binding (PTB) or Src homology 2 domain to determine that phosphorylation of Shc in v-Src-expressing cells depends on the presence of a functional PTB domain. We purified a 100-kDa Shc PTB-binding protein from Src-transformed cells that was identified as the beta chain of the low density lipoprotein receptor-related protein LRP1. LRP1 acts as an import receptor for a variety of proteins and is involved in clearance of the beta-amyloid precursor protein. This study shows that LRP1 is tyrosine-phosphorylated in v-Src-transformed cells and that tyrosine-phosphorylated LRP1 binds in vivo and in vitro to Shc. The association between Shc and LRP1 may provide a mechanism for recruitment of Shc to the plasma membrane where it is phosphorylated by v-Src. It is at the membrane that Shc is thought to be involved in Ras activation. These observations further suggest that LRP1 could function as a signaling receptor and may provide new avenues to investigate its possible role during embryonal development and the onset of Alzheimer's disease.     

A comparison of human S100A12 with MRP-14 (S100A9)

MRP-8 and -14 are two S100 proteins highly expressed as a complex by neutrophils, and to a lesser extent by monocytes and certain squamous epithelia. However, less is known about the close homologue S100A12. This S100 protein is expressed by neutrophils and here we show that it is also expressed by monocytes, but not lymphocytes. An absence of coimmunoprecipitation of MRP-14 and S100A12 indicates that S100A12 is not associated with the MRP proteins in vivo. When directly compared to MRP-14, S100A12 expression by squamous epithelia is more restricted. In esophagus and psoriatic skin, S100A12 is differentially regulated, like MRP-14, but the expression pattern of the two S100 proteins is quite different.     

PABP interacting protein 2A (PAIP2A) regulates specific key proteins during spermiogenesis in the mouse

During spermiogenesis, expression of the specific proteins needed for proper differentiation of male germ cells is under translational control. We have shown that PAIP2A is a major translational regulator involved in the maturation of male germ cells and male fertility. To identify the proteins controlled by PAIP2A during spermiogenesis, we characterized the proteomic profiles of elongated spermatids from wild-type (WT) mice and mice that were Paip2a/Paip2b double-null mutants (DKO). Elongated spermatid populations were obtained and proteins were extracted and separated on gradient polyacrylamide gels. The gels were digested with trypsin and peptides were identified by mass spectrometry. We identified 632 proteins with at least two unique peptides and a confidence level of 95%. Only 209 proteins were consistently detected in WT or DKO replicates with more than five spectra. Twenty-nine proteins were differentially expressed with at least a 1.5-fold change; 10 and 19 proteins were down- and up-regulated, respectively, in DKO compared to WT mice. We confirmed the significantly different expression levels of three proteins, EIF4G1, AKAP4, and HK1, by Western blot analysis. We have characterized novel proteins that have their expression controlled by PAIP2A; of these, 50% are involved in flagellar structure and sperm motility. Although several proteins affected by abrogation of Paip2a have established roles in reproduction, the roles of many others remain to be determined.     

Detection of the Mr 110,000 lung resistance-related protein LRP/MVP with monoclonal antibodies.

The Mr 110,000 lung resistance-related protein (LRP), also termed the major vault protein (MVP), constitutes >70% of subcellular ribonucleoprotein particles called vaults. Overexpression of LRP/MVP and vaults has been linked directly to MDR in cancer cells. Clinically, LRP/MVP expression can be of value to predict response to chemotherapy and prognosis. Monoclonal antibodies (MAbs) against LRP/MVP have played a critical role in determining the relevance of this protein in clinical drug resistance. We compared the applicability of the previously described MAbs LRP-56, LMR-5, LRP, 1027, 1032, and newly isolated MAbs MVP-9, MVP-16, MVP-18, and MVP-37 for the immunodetection of LRP/MVP by immunoblotting analysis and by immunocyto- and histochemistry. The availability of a broader panel of reagents for the specific and sensitive immunodetection of LRP/MVP should greatly facilitate biological and clinical studies of vault-related MDR.     

Evaluation of Somatostatin and CXCR4 Receptor Expression in a Large Set of Prostate Cancer Samples Using Tissue Microarrays and Well-Characterized Monoclonal Antibodies

BACKGROUND: Prostate cancer (PCa) is the most common type of cancer among men in Western countries. Despite numerous therapeutic options, few treatments are available for patients with end-stage disease. In the present study, different somatostatin receptors (SSTs) and the chemokine receptor CXCR4 were evaluated for their suitability as novel therapeutic targets in PCa. MATERIALS AND METHODS: The expression of SST subtypes 1, 2A, 3, and 5 and of CXCR4 was evaluated in 276 PCa tumor samples on a tissue microarray (TMA) in 23 whole-block tumor samples and in 3 PCa cell lines by immunohistochemistry using well-characterized monoclonal antibodies. RESULTS: Overall, the frequency and intensity of expression of SSTs and CXCR4 were very low among the PCa samples investigated. Specifically, SST5, SST2A, and SST3 were expressed, albeit at low intensity, in 10.5%, 9.1%, and 0.7% of the TMA samples, respectively. None of the TMA samples showed SST1 or CXCR4 expression. Only a single small-cell-type neuroendocrine carcinoma that was coincidentally included among the whole-block samples exhibited strong SST2A, SST5, and CXCR4 and moderate SST3 expression. Independent of the tumor cells, the tumor capillaries in many of the PCa samples were strongly positive for SST2A, SST3, SST5, or CXCR4 expression. SST expression in the tumor cells was associated with advanced tumor grade and stage. CONCLUSION: Overall, SST and CXCR4 expression levels are clearly of no therapeutic relevance in PCa. SST- or CXCR4-based therapy might be feasible, however, in rare cases of small-cell-type neuroendocrine carcinoma of the prostate.