The Small Non-coding Vault RNA1-1 Acts as a Riboregulator of Autophagy.

Vault RNAs (vtRNA) are small non-coding RNAs transcribed by RNA polymerase III found in many eukaryotes. Although they have been linked to drug resistance, apoptosis, and viral replication, their molecular functions remain unclear. Here, we show that vault RNAs directly bind the autophagy receptor sequestosome-1/p62 in human and murine cells. Overexpression of human vtRNA1-1 inhibits, while its antisense LNA-mediated knockdown enhances p62-dependent autophagy. Starvation of cells reduces the steady-state and p62-bound levels of vault RNA1-1 and induces autophagy. Mechanistically, p62 mutants that fail to bind vtRNAs display increased p62 homo-oligomerization and augmented interaction with autophagic effectors. Thus, vtRNA1-1 directly regulates selective autophagy by binding p62 and interference with oligomerization, a critical step of p62 function. Our data uncover a striking example of the potential of RNA to control protein functions directly, as previously recognized for protein-protein interactions and post-translational modifications.

Structure, Dynamics and Functional Implications of the Eukaryotic Vault Complex.

Vault ribonucleoprotein particles are naturally designed nanocages, widely found in the eukaryotic kingdom. Vaults consist of 78 copies of the major vault protein (MVP) that are organized in 2 symmetrical cup-shaped halves, of an approximate size of 70x40x40 nm, leaving a huge internal cavity which accommodates the vault poly(ADP-ribose) polymerase (vPARP), the telomerase-associated protein-1 (TEP1) and some small untranslated RNAs. Diverse hypotheses have been developed on possible functions of vaults, based on their unique capsular structure, their rapid movements and the distinct subcellular localization of the particles, implicating transport of cargo, but they are all pending confirmation. Vault particles also possess many attributes that can be exploited in nanobiotechnology, particularly in the creation of vehicles for the delivery of multiple molecular cargoes. Here we review what is known about the structure and dynamics of the vault complex and discuss a possible mechanism for the vault opening process. The recent findings in the characterization of the vaults in cells and in its natural microenvironment will be also discussed.

Proteomic analyses identify major vault protein as a prognostic biomarker for fatal prostate cancer.

The demographic shift toward an older population will increase the number of prostate cancer cases. A challenge in the treatment of prostate cancer is to avoid undertreatment of patients at high risk of progression following curative treatment. These men can benefit from early salvage treatment. An explorative cohort consisting of tissue from 16 patients who underwent radical prostatectomy, and were either alive or had died from prostate cancer within 10 years postsurgery, was analyzed by mass spectrometry analysis. Following proteomic and bioinformatic analyses, major vault protein (MVP) was identified as a putative prognostic biomarker. A publicly available tissue proteomics dataset and a retrospective cohort of 368 prostate cancer patients were used for validation. The prognostic value of the MVP was verified by scoring immunohistochemical staining of a tissue microarray. High level of MVP was associated with more than 4-fold higher risk for death from prostate cancer (hazard ratio = 4.41, 95% confidence interval: 1.45-13.38; P = 0.009) in a Cox proportional hazard models, adjusted for Cancer of the Prostate Risk Assessments Post-surgical (CAPRA-S) score and perineural invasion. Decision curve analyses suggested an improved standardized net benefit, ranging from 0.06 to 0.18, of adding MVP onto CAPRA-S score. This observation was confirmed by receiver operator characteristics curve analyses for the CAPRA-S score versus CAPRA-S and MVP score (area under the curve: 0.58 versus 0.73). From these analyses, one can infer that MVP levels in combination with CAPRA-S score might add onto established risk parameters to identify patients with lethal prostate cancer.

Zebrafish MVP Recruits and Degrades TBK1 To Suppress IFN Production.

IFN production is crucial for hosts to defend against viral infection, yet it must be tightly controlled to maintain immune homeostasis. TANK-binding kinase 1 (TBK1) is a pivotal kinase in the IFN induction signaling pathway, but it is negatively regulated by multiple molecules to avoid the excessive expression of IFN in mammals. However, the identified TBK1 suppressors and the mechanisms are rare in fish. In this study, we show that zebrafish major vault protein (MVP) recruits and degrades TBK1 in a lysosome-dependent manner to inhibit IFN production. Through viral infection, polyinosinic:polycytidylic acid and RIG-I-like receptor factor stimulation upregulated IFN expression, but overexpression of MVP significantly subverted these inductions. On the protein level, MVP interacted with TBK1, and interestingly, MVP recruited TBK1 from a uniformly distributed state in the cytoplasm to an aggregated state. Finally, MVP mediated the lysosome-dependent degradation of TBK1 and decreased the IFN response and IFN-stimulated genes expression. Our findings reveal that zebrafish MVP is a negative regulator of IFN production by restricting the activation of TBK1, supplying evidence of the balanced mechanisms of IFN expression in lower vertebrates.

MVP Expression Facilitates Tumor Cell Proliferation and Migration Supporting the Metastasis of Colorectal Cancer Cells.

Cancer cells show significant dysregulation of genes expression, which may favor their survival in the tumor environment. In this study, the cellular vault's components MVP (major vault protein), TEP1 (telomerase-associated protein 1) and vPARP (vault poly(ADP-ribose) polymerase) were transiently or completely inhibited in U2OS cells (human bone osteosarcoma epithelial cells) to evaluate their impact on the cell proliferative and migratory capacity as well as on the development of their resistance to the drug vinorelbine. Comparative analysis of MVP protein expression level in normal colon tissue, primary colorectal tumor, and metastasis showed that the expression of this protein does not increase significantly in the primary tumor, but its expression increases in metastatic cells. Further comparative molecular analysis using the whole transcriptome microarrays for MVP-positive and MVP-negative cells showed that MVP is involved in regulating proliferation and migration of cancer cells. MVP may facilitate metastasis of colon cancer due to its impact on cell migration. Moreover, two vault proteins, MVP and TEP1, contribute the resistance to vinorelbine, while vPARP does not.

The vault RNA of Trypanosoma brucei plays a role in the production of trans-spliced mRNA.

The vault ribonucleoprotein (RNP), comprising vault RNA (vtRNA) and telomerase-associated protein 1 (TEP1), is found in many eukaryotes. However, previous studies of vtRNAs, for example in mammalian cells, have failed to reach a definitive conclusion about their function. vtRNAs are related to Y RNAs, which are complexed with Ro protein and influence Ro's function in noncoding RNA (ncRNA) quality control and processing. In Trypanosoma brucei, the small noncoding TBsRNA-10 was first described in a survey of the ncRNA repertoire in this organism. Here, we report that TBsRNA-10 in T. brucei is a vtRNA, based on its association with TEP1 and sequence similarity to those of other known and predicted vtRNAs. We observed that like vtRNAs in other species, TBsRNA-10 is transcribed by RNA polymerase III, which in trypanosomes also generates the spliceosomal U-rich small nuclear RNAs. In T. brucei, spliced leader (SL)-mediated trans-splicing of pre-mRNAs is an obligatory step in gene expression, and we found here that T. brucei's vtRNA is highly enriched in a non-nucleolar locus in the cell nucleus implicated in SL RNP biogenesis. Using a newly developed permeabilized cell system for the bloodstream form of T. brucei, we show that down-regulated vtRNA levels impair trans-spliced mRNA production, consistent with a role of vtRNA in trypanosome mRNA metabolism. Our results suggest a common theme for the functions of vtRNAs and Y RNAs. We conclude that by complexing with their protein-binding partners TEP1 and Ro, respectively, these two RNA species modulate the metabolism of various RNA classes.

RNAs, Phase Separation, and Membrane-Less Organelles: Are Post-Transcriptional Modifications Modulating Organelle Dynamics?

Membranous organelles allow sub-compartmentalization of biological processes. However, additional subcellular structures create dynamic reaction spaces without the need for membranes. Such membrane-less organelles (MLOs) are physiologically relevant and impact development, gene expression regulation, and cellular stress responses. The phenomenon resulting in the formation of MLOs is called liquid-liquid phase separation (LLPS), and is primarily governed by the interactions of multi-domain proteins or proteins harboring intrinsically disordered regions as well as RNA-binding domains. Although the presence of RNAs affects the formation and dissolution of MLOs, it remains unclear how the properties of RNAs exactly contribute to these effects. Here, the authors review this emerging field, and explore how particular RNA properties can affect LLPS and the behavior of MLOs. It is suggested that post-transcriptional RNA modification systems could be contributors for dynamically modulating the assembly and dissolution of MLOs.

Major Vault Protein, a Candidate Gene in 16p11.2 Microdeletion Syndrome, Is Required for the Homeostatic Regulation of Visual Cortical Plasticity.

Microdeletion of a region in chromosome 16p11.2 increases susceptibility to autism. Although this region contains exons of 29 genes, disrupting only a small segment of the region, which spans five genes, is sufficient to cause autistic traits. One candidate gene in this critical segment is MVP, which encodes for the major vault protein (MVP) that has been implicated in regulation of cellular transport mechanisms. MVP expression levels in MVP+/- mice closely phenocopy those of 16p11.2 mutant mice, suggesting that MVP+/- mice may serve as a model of MVP function in 16p11.2 microdeletion. Here we show that MVP regulates the homeostatic component of ocular dominance (OD) plasticity in primary visual cortex. MVP+/- mice of both sexes show impairment in strengthening of open-eye responses after several days of monocular deprivation (MD), whereas closed-eye responses are weakened as normal, resulting in reduced overall OD plasticity. The frequency of miniature EPSCs (mEPSCs) in pyramidal neurons is decreased in MVP+/- mice after extended MD, suggesting a reduction of functional synapses. Correspondingly, upregulation of surface GluA1 AMPA receptors is reduced in MVP+/- mice after extended MD, and is accompanied by altered expression of STAT1 and phosphorylated ERK, which have been previously implicated in OD plasticity. Normalization of STAT1 levels by introducing STAT1 shRNA rescues surface GluA1 and open-eye responses, implicating STAT1 as a downstream effector of MVP. These findings demonstrate a specific role for MVP as a key molecule influencing the homeostatic component of activity-dependent synaptic plasticity, and potentially the corresponding phenotypes of 16p11.2 microdeletion syndrome.SIGNIFICANCE STATEMENT Major vault protein (MVP), a candidate gene in 16p11.2 microdeletion syndrome, has been implicated in the regulation of several cellular processes including transport mechanisms and scaffold signaling. However, its role in brain function and plasticity remains unknown. In this study, we identified MVP as an important regulator of the homeostatic component of experience-dependent plasticity, via regulation of STAT1 and ERK signaling. This study helps reveal a new mechanism for an autism-related gene in brain function, and suggests a broader role for neuro-immune interactions in circuit level plasticity. Importantly, our findings might explain specific components of the pathophysiology of 16p11.2 microdeletion syndrome.

Adipocytes promote breast cancer resistance to chemotherapy, a process amplified by obesity: role of the major vault protein (MVP).

INTRODUCTION: Clinical studies suggest that obesity, in addition to promoting breast cancer aggressiveness, is associated with a decrease in chemotherapy efficacy, although the mechanisms involved remain elusive. As chemotherapy is one of the main treatments for aggressive or metastatic breast cancer, we investigated whether adipocytes can mediate resistance to doxorubicin (DOX), one of the main drugs used to treat breast cancer, and the mechanisms associated. METHODS: We used a coculture system to grow breast cancer cells with in vitro differentiated adipocytes as well as primary mammary adipocytes isolated from lean and obese patients. Drug cellular accumulation, distribution, and efflux were studied by immunofluorescence, flow cytometry, and analysis of extracellular vesicles. Results were validated by immunohistochemistry in a series of lean and obese patients with cancer. RESULTS: Adipocytes differentiated in vitro promote DOX resistance (with cross-resistance to paclitaxel and 5-fluorouracil) in a large panel of human and murine breast cancer cell lines independently of their subtype. Subcellular distribution of DOX was altered in cocultivated cells with decreased nuclear accumulation of the drug associated with a localized accumulation in cytoplasmic vesicles, which then are expelled into the extracellular medium. The transport-associated major vault protein (MVP), whose expression was upregulated by adipocytes, mediated both processes. Coculture with human mammary adipocytes also induced chemoresistance in breast cancer cells (as well as the related MVP-induced DOX efflux) and their effect was amplified by obesity. Finally, in a series of human breast tumors, we observed a gradient of MVP expression, which was higher at the invasive front, where tumor cells are at close proximity to adipocytes, than in the tumor center, highlighting the clinical relevance of our results. High expression of MVP in these tumor cells is of particular interest since they are more likely to disseminate to give rise to chemoresistant metastases. CONCLUSIONS: Collectively, our study shows that adipocytes induce an MVP-related multidrug-resistant phenotype in breast cancer cells, which could contribute to obesity-related chemoresistance.

Major vault protein suppresses lung cancer cell proliferation by inhibiting STAT3 signaling pathway.

BACKGROUND: Major vault protein (MVP) is the major component of vault, a eukaryotic organelle involved in multiple cellular processes, and is important in multiple cellular processes and diseases including the drug resistance in cancer chemotherapies. However, the role of MVP in lung cancer remains unclear. METHODS: We examined MVP expression in 120 non-small cell lung cancer (NSCLC) tumors and matched normal tissues by immunohistochemistry. Its relationship with NSCLC prognosis was determined by investigating the patient cohort and analyzing the data from a published dataset consisting with more than 1900 lung cancer patients. We further performed shRNA-introduced knockdown of MVP in Lewis lung carcinoma (LLC) cells and examined its effects on the tumor formation in a xenograft mouse model and the tumor cell proliferation, apoptosis, and signal transduction in vitro. RESULTS: We found that MVP was up-regulated significantly in tumor tissues compared with the matched tumor-adjacent normal tissues. The increased expression of MVP in lung adenocarcinoma was associated with a better prognosis. Knockdown of MVP in LLC cells promoted xenografted lung cancer formation in mice, which was accompanied with accelerated tumor cell proliferation and suppressed cell apoptosis in vitro. Knockdown of MVP stimulated STAT3 phosphorylation, nuclear localization, and activation of JAK2 and RAF/MEK/ERK pathways in LLC cells. Administration of STAT3 inhibitor WP1066 could prevent MVP knockdown induced tumorigenesis. CONCLUSIONS: Our findings demonstrate that MVP may act as a lung tumor suppressor via inhibiting STAT3 pathway. MVP would be a potential target for novel therapies of lung adenocarcinoma.

Synthesis and assembly of human vault particles in yeast.

Vault particles are the largest naturally occurring ribonucleoprotein complexes found in the cytoplasm. In all 78 copies of major vault protein (MVP) assemble on polyribosome templates, forming recombinant vault particles, which are of great interest as encapsulation carriers for therapeutics delivery and enzyme stabilization. Baculovirus-insect cell expression is the only system that has been developed for recombinant vault synthesis, but it has low scalability and slow production rate. In this study, we demonstrated the first use of yeast cells for the production of vault particles with full integrity and functionality solely by expressing the complementary DNA (cDNA) encoding MVP. Vaults synthesized in Pichia pastoris yeast cells are morphologically indistinguishable from recombinant vault particles produced in insect cells, and are able to package and stabilize enzymes resulting in improved longevity and catalytic efficiency. Thus, our results imply that the yeast system is an economical alternative to insect cells for the production of recombinant vaults. The consistency of vault morphology between yeast and insect cell systems also underlines that polyribosome templating may be conserved among eukaryotes, which promises the synthesis and assembly of recombinant human vault particles in other eukaryotic organisms.

Inducible Major Vault Protein Plays a Pivotal Role in Double-Stranded RNA- or Virus-Induced Proinflammatory Response.

Pathogen invasion triggers robust antiviral cytokine production via different transcription factor signaling pathways. We have previously demonstrated that major vault protein (MVP) induces type I IFN production during viral infection; however, little is known about the role of MVP in proinflammatory responses. In this study, we found in vitro that expression of MVP, IL-6, and IL-8 was inducible upon dsRNA stimulation or viral infection. Moreover, MVP was essential for the induction of IL-6 and IL-8, as impaired expression of IL-6 and IL-8 in MVP-deficient human PBMCs, human lung epithelial cells (A549), and THP-1 monocytes, as well as in murine splenocytes, peritoneal macrophages, and PBMCs from MVP-knockout (MVP(-/-)) mice, was observed. Upon investigation of the underlying mechanisms, we demonstrated that MVP acted in synergy with AP-1 (c-Fos) and CCAAT/enhancer binding protein (C/EBP)ß-liver-enriched transcriptional activating protein to activate the IL6 and IL8 promoters. Introduction of mutations into the AP-1 and C/EBPß binding sites on the IL6 and IL8 promoters resulted in the loss of synergistic activation with MVP. Furthermore, we found that MVP interacted with both c-Fos and C/EBPß. The interactions promoted nuclear translocation and recruitment of these transcription factors to IL6 and IL8 promoter regions. In the MVP(-/-) mouse model, significantly decreased expression of early antiviral cytokines resulted in higher viral titer in the lung, higher mortality, and heavier lung damage after infection with lethal influenza A virus. Taken together, our findings help to delineate a novel role of MVP in host proinflammatory response.

Synergistic Effect of Baicalin and Adriamycin in Resistant HL-60/ADM Leukaemia Cells.

BACKGROUND/AIMS: The present study was designed to investigate the expression of multidrug resistance (MDR)-related genes, verify the synergistic effects of baicalin and Adriamycin (ADM) and investigate the related mechanisms in ADM-resistant leukaemic HL-60/ADM cells. METHODS: We used a HL-60/ADM cell line. Cytotoxicity and flow cytometry assays were employed to verify the cytotoxic effects of baicalin. Real-time polymerase chain reaction and Western blotting assays were used to assess the expression of MDR-related genes and the changes in gene expression (both MDR-related and PI3K/Akt pathway-related) induced by administration of baicalin. RESULTS: We found that only multidrug resistance protein 1 (MRP1), lung resistance-related protein (LRP) and Bcl-2 genes were expressed in both HL-60 and HL-60/ADM cells. HL-60/ADM cells exhibited significantly higher expression (p < 0.05). We also observed that low-dose baicalin (5 and 10 µmol/L) can induce growth inhibition and apoptotic effects on HL-60/ADM cells by increasing the intracellular accumulation of ADM. The synergistic effect of baicalin and ADM was verified. Concerning the potential mechanisms involved in this process, we showed that baicalin down-regulated the expression of several MDR-related and PI3K/Akt pathway-related genes. CONCLUSIONS: We confirmed the increased expression of MRP1, LRP and Bcl-2 genes in HL-60/ADM cells compared to regular HL-60 cells, which are recommended for future investigation on MDR. The present study provided evidence of the synergistic effect of baicalin and ADM in HL-60/ADM cells. Therefore, baicalin may be considered as a potential therapeutic agent against resistant leukaemia. Suppression of the PI3K/Akt signalling pathway, followed by inhibition of the expression of MDR-related genes may be a common mechanism in combination treatments with ADM for the reduction of resistance to ADM.

Altered mRNP granule dynamics in FTLD pathogenesis.

In neurons, RNA-binding proteins (RBPs) play a key role in post-transcriptional gene regulation, for example alternative splicing, mRNA localization in neurites and local translation upon synaptic stimulation. There is increasing evidence that defective or mislocalized RBPs - and consequently altered mRNA processing - lead to neuronal dysfunction and cause neurodegeneration, including frontotemporal lobar degeneration and amyotrophic lateral sclerosis. Cytosolic RBP aggregates containing TAR DNA-binding protein of 43 kDa (TDP-43) or fused in sarcoma (FUS) are a common hallmark of both disorders. There is mounting evidence that translationally silent mRNP granules, such as stress granules or transport granules, play an important role in the formation of these RBP aggregates. These granules are thought to be 'catalytic convertors' of RBP aggregation by providing a high local concentration of RBPs. As recently shown in vitro, RBPs that contain a so-called low-complexity domain start to 'solidify' and eventually aggregate at high protein concentrations. The same may happen in mRNP granules in vivo, leading to 'solidified' granules that lose their dynamic properties and ability to fulfill their physiological functions. This may result in a disturbed stress response, altered mRNA transport and local translation, and formation of pathological TDP-43 or FUS aggregates, all of which may contribute to neuronal dysfunction and neurodegeneration. Here, we discuss the general functional properties of these mRNP granules, how their dynamics may be disrupted in frontotemporal lobar degeneration/amyotrophic lateral sclerosis, for example by loss or gain of function of TDP-43 and FUS, and how this may contribute to the development of RBP aggregates and neurotoxicity. In this review, we discuss how dynamic mRNP granules, such as stress granules or neuronal transport granules, may be converted into pathological aggregates containing misfolded RNA-binding proteins (RBPs), such as TDP-43 and FUS. Abnormal interactions between low-complexity domains in RBPs may cause dynamic mRNP granules to solidify and become dysfunctional. This may result in a disturbed stress response, altered mRNA transport and local translation, as well as RBP aggregation, all of which may contribute to neuronal dysfunction and neurodegeneration.

Connection between Proliferation Rate and Temozolomide Sensitivity of Primary Glioblastoma Cell Culture and Expression of YB-1 and LRP/MVP.

Glioblastomas (GBL) are the most common and aggressive brain tumors. They are distinguished by high resistance to radiation and chemotherapy. To find novel approaches for GBL classification, we obtained 16 primary GBL cell cultures and tested them with real-time PCR for mRNA expression of several genes (YB-1, MGMT, MELK, MVP, MDR1, BCRP) involved in controlling cell proliferation and drug resistance. The primary GBL cultures differed in terms of proliferation rate, wherein a group of GBL cell cultures with low proliferation rate demonstrated higher resistance to temozolomide. We found that GBL primary cell cultures characterized by high proliferation rate and lower resistance to temozolomide expressed higher mRNA level of the YB-1 and MDR1 genes, whereas upregulated expression of MVP/LRP mRNA was a marker in the group of GBL with low proliferation rate and high resistance. A moderate correlation between expression of YB-1 and MELK as well as YB-1 and MDR1 was found. In the case of YB-1 and MGMT expression, no correlation was found. A significant negative correlation was revealed between mRNA expression of MVP/LRP and MELK, MDR1, and BCRP. No correlation in expression of YB-1 and MVP/LRP genes was observed. It seems that mRNA expression of YB-1 and MVP/LRP may serve as a marker for GBL cell cultures belonging to distinct groups, each of which is characterized by a unique pattern of gene activity.

Reversal of cisplatin resistance in non-small cell lung cancer stem cells by Taxus chinensis var.

Drug resistance in cells is a major impedance to successful treatment of lung cancer. Taxus chinensis var. inhibits the growth of tumor cells and promotes the synthesis of interleukins 1 and 2 and tumor necrosis factor, enhancing immune function. In this study, T. chinensis var.-induced cell death was analyzed in lung cancer cells (H460) enriched for stem cell growth in a defined serum-free medium. Taxus-treated stem cells were also analyzed for Rhodamine 123 (Rh-123) expression by flow cytometry, and used as a standard functional indicator of MDR. The molecular basis of T. chinensis var.-mediated drug resistance was established by real-time PCR analysis of ABCC1, ABCB1, and lung resistance-related protein (LRP) mRNA, and western blot analysis of MRP1, MDR1, and LRP. Our results revealed that stem cells treated with higher doses of T. chinensis var. showed significantly lower growth inhibition rates than did H460 cells (P < 0.05). The growth of stem and H460 cells treated with a combination of T. chinensis var. and cisplatin was also significantly inhibited (P < 0.05). Rh-123 was significantly accumulated in the intracellular region and showed delayed efflux in stem cells treated with T. chinensis var. (P < 0.05), compared to those treated with verapamil. T. chinensis var.-treated stem cells showed significant downregulation of the ABCC1, ABCB1, and LRP mRNA and MRP1, MDR1, and LRP (P < 0.05) compared to H460 cells. Thus, T. chinensis var.-mediated downregulation of MRP1, MDR1, and LRP might contribute to the reversal of drug resistance in non-small cell lung cancer stem cells.

The Effects and Mechanisms of Periplaneta americana Extract Reversal of Multi-Drug Resistance in BEL-7402/5-FU Cells.

The present study reports the reversing effects of extracts from P. americana on multidrug resistance of BEL-7402/5-FU cells, as well as a preliminary investigation on their mechanism of action. A methylthiazolyl tetrazolium (MTT) method was applied to determine the multidrug resistance of BEL-7402/5-FU, while an intracellular drug accumulation assay was used to evaluate the effects of a column chromatography extract (PACC) and defatted extract (PADF) from P. americana on reversing multi-drug resistance. BEL-7402/5-FU reflected high resistance to 5-FU; PACC and PADF could promote drug accumulation in BEL-7402/5-FU cells, among which PADF was more effective than PACC. Moreover, results from the immunocytochemical method showed that PACC and PADF could downregulate the expression of drug resistance-associated proteins (P-gp, MRP, LRP); PACC and PADF had no effects on the expression of multidrug resistance-associated enzymes (GST-π), but PACC could increase the expression of multidrug resistance-associated enzymes (PKC). Results of real-time fluorescence quantitative PCR revealed that PACC and PADF were able to markedly inhibit the expression of multidrug resistance-associated genes (MDR1, LRP and MRP1); PACC presented a significant impact on the gene expression of multidrug resistance-associated enzymes, which increased the gene expression of GST-π and PKC. However, PADF had little impact on the expression of multidrug resistance-associated enzymes. These results demonstrated that PACC and PADF extracted from P. americana could effectively reverse MDR in BEL-7402/5-FU cells, whose mechanism was to inhibit the expression of P-gp, MRP, and LRP, and that PADF was more effective in the reversal of MDR than did PACC. In addition, some of extracts from P. americana altered (sometimes increasing) the expression of multidrug resistance-associated enzymes.

ABCB1, ABCC1, and LRP gene expressions are altered by LDL, HDL, and serum deprivation in a human doxorubicin-resistant uterine sarcoma cell line.

Multidrug resistance (MDR) is the major cause of cancer treatment failure. The ATP-binding cassette-B1 (ABCB1) transporter, also known as MDR1 or P-glycoprotein, is thought to promote the efflux of drugs from cells. MDR is also associated with the multidrug resistance-associated protein 1 (ABCC1) and the lung resistance-related protein (LRP), a human major vault protein. Moreover, MDR has a complex relationship with lipids. The ABCB1 has been reported to modulate cellular cholesterol homeostasis. Conversely, cholesterol has been reported to modulate multidrug transporters. However, results reported to date are contradictory and confusing. The aim of this study was to investigate whether LDL, HDL, and serum deprivation could influence ABCB1, ABCC1, and LRP expression in a human doxorubicin-resistant uterine sarcoma cell line. ABCB1 and ABCC1 expression increased after 24 h of serum deprivation, and expression returned to basal levels after 72 h. LDL, depending on concentration, increased ABCB1, ABCC1, and LRP expression. ABCB1 expression increased at low HDL, and decreased at high HDL concentrations. We demonstrated that serum deprivation and lipoproteins, particularly LDL, modulated ABCB1 expression and, to a lesser extent, ABCC1 expression. This finding may link the phenomena of drug transport, cholesterol metabolism and cancer.

Allelic methylation levels of the noncoding VTRNA2-1 located on chromosome 5q31.1 predict outcome in AML.

Deletions of chromosome 5q are associated with poor outcomes in acute myeloid leukemia (AML) suggesting the presence of tumor suppressor(s) at the locus. However, definitive identification of putative tumor suppressor genes remains controversial. Here we show that a 106-nucleotide noncoding RNA vault RNA2-1 (vtRNA2-1), previously misannotated as miR886, could potentially play a role in the biology and prognosis of AML. vtRNA2-1 is transcribed by polymerase III and is monoallelically methylated in 75% of healthy individuals whereas the remaining 25% of the population have biallelic hypomethylation. AML patients without methylation of VTRNA2-1 have a considerably better outcome than those with monoallelic or biallelic methylation (n = 101, P = .001). We show that methylation is inversely correlated with vtRNA2-1 expression, and that 5-azanucleosides induce vtRNA2-1 and down-regulate the phosphorylated RNA-dependent protein kinase (pPKR), whose activity has been shown to be modulated by vtRNA2-1. Because pPKR promotes cell survival in AML, the data are consistent with vtRNA2-1 being a tumor suppressor in AML. This is the first study to show that vtRNA2-1 might play a significant role in AML, that it is either mono- or biallelically expressed in the blood cells of healthy individuals, and that its methylation state predicts outcome in AML.