Expression and function of SLC38A5, an amino acid-coupled Na+/H+ exchanger, in triple-negative breast cancer and its relevance to macropinocytosis.

Metabolic reprogramming in cancer necessitates increased amino acid uptake, which is accomplished by up-regulation of specific amino acid transporters. However, not all tumors rely on any single amino acid transporter for this purpose. Here, we report on the differential up-regulation of the amino acid transporter SLC38A5 in triple-negative breast cancer (TNBC). The up-regulation is evident in TNBC tumors, conventional and patient-derived xenograft TNBC cell lines, and a mouse model of spontaneous TNBC mammary tumor. The up-regulation is confirmed by functional assays. SLC38A5 is an amino acid-dependent Na+/H+ exchanger which transports Na+ and amino acids into cells coupled with H+ efflux. Since cell-surface Na+/H+ exchanger is an established inducer of macropinocytosis, an endocytic process for cellular uptake of bulk fluid and its components, we examined the impact of SLC38A5 on macropinocytosis in TNBC cells. We found that the transport function of SLC38A5 is coupled to the induction of macropinocytosis. Surprisingly, the transport function of SLC38A5 is inhibited by amilorides, the well-known inhibitors of Na+/H+ exchanger. Down-regulation of SLC38A5 in TNBC cells attenuates serine-induced macropinocytosis and reduces cell proliferation significantly as assessed by multiple methods, but does not induce cell death. The Cancer Genome Atlas database corroborates SLC38A5 up-regulation in TNBC. This represents the first report on the selective expression of SLC38A5 in TNBC and its role as an inducer of macropinocytosis, thus revealing a novel, hitherto unsuspected, function for an amino acid transporter that goes beyond amino acid delivery but is still relevant to cancer cell nutrition and proliferation.

Clinical effects of Lactobacillus strains as probiotics in the treatment of irritable bowel syndrome. Results from the LAPIBSS trial: Future objectives.

The objective of this communication is to present and analyze the recent results from the LAPIBSS study in order to improve future clinical trials on the effects of Lactobacillus strains in the treatment of irritable bowel syndrome (IBS). Using a tightly-controlled clinical trial protocol with the highest Jadad score of 5/5, the current trial aimed to demonstrate the efficacy of a 2-strain mixture of Lactobacillus acidophilus (L. acidophilus) to improve IBS symptoms. Eighty patients diagnosed with IBS according to Rome III criteria were recruited to a multicentric, double-blind, in parallel groups, placebo-controlled, randomized clinical trial. Patients were provided with a daily dose of two capsules containing either two probiotic strains (5 x 109 cfu/capsule) or placebo for 8 weeks. The primary endpoint was abdominal pain score assessed with a 100-mm visual analogue scale (VAS). Secondary endpoints included scores of bloating, flatus and rumbling assessed with a 100-mm VAS, a composite score that consisted of the sum of the 4 VAS scores, and the stool frequency and consistency assessed with the Bristol Stool Form Scale. Our study has failed to demonstrate a significant improvement of the primary endpoint of abdominal pain. Significant differences between groups were observed for flatus score at week 4 (P=0.04) and week 8 (P=0.03) and for composite score at week 8 (P=0.04). The consumption of the 2-strain mixture of L. acidophilus over 8 weeks is safe, significantly decreases flatus and composite scores. The significant effect on flatus could result from the species-specific homofermentative properties of L. acidophilus strains. The negative results on abdominal pain and the gained experience are discussed for the future clinical trials in IBS.

Cabazitaxel regimens inhibit the growth of prostate cancer cells and enhances the anti-tumor properties of PEDF with various efficacy and toxicity.

BACKGROUND: Taxanes chemotherapies represent the major therapeutic alternative for symptomatic mCRPC. While docetaxel is the most commonly prescribed Taxane for mCRPC; cabazitaxel has been approved for patients unresponsive to docetaxel. Still mCRPC remains incurable and patients often experience severe side effects. Recently, the FIRSTANA trial first demonstrated the absence of superiority in overall survival between cabazitaxel and docetaxel in mCRPC patients. Inversely, different toxicity were reported suggesting that cabazitaxel may provide a first line treatment option for some patients urging for a deeper characterization of cabazitaxel mechanisms of action as well as a re-evaluation of cabazitaxel conventional dose and schedule. In this study, our goal was therefore to evaluate the anti-tumor efficacy of various cabazitaxel regimens delivered as monotherapy or in combination with PEDF, a known anti-angiogenic and anti-neoplastic agent. METHODS: CRPC cells undergoing Taxane treatment were evaluated for cell proliferation, migration and death, and apoptosis using crystal violet staining, chemotaxis, cell cycle, and TUNEL assays. In vitro data were corroborated in CL1 CRPC xenografts where mice received intermittent or metronomic low-doses cabazitaxel ± PEDF. RESULTS: We found that cabazitaxel inhibits the proliferation of CRPC cells with a higher efficacy than docetaxel in vitro. As expected, high-doses of Taxanes blocked the cells in mitosis. Surprisingly, low-doses of cabazitaxel induced more cell death than docetaxel mainly through apoptosis. In vivo, intermittent cabazitaxel lead to disease stabilization when combined with PEDF. Unexpectedly, low-doses of cabazitaxel delayed tumor growth with severe toxicity for some of the doses tested. Other results showed that PEDF and low-doses of cabazitaxel combination inhibited the migration of tumor cell and increased the tumoricidal activity of macrophages toward prostate tumor cells. CONCLUSIONS: Our findings highlight the great promise of cabazitaxel drug and predict a possible move of cabazitaxel forward within the therapeutic sequence of prostate cancer.

Cross-species prophylactic efficacy of Sm-p80-based vaccine and intracellular localization of Sm-p80/Sm-p80 ortholog proteins during development in Schistosoma mansoni, Schistosoma japonicum, and Schistosoma haematobium.

Schistosomiasis remains a major global health problem. Despite large-scale schistosomiasis control efforts, clear limitations such as possible emergence of drug resistance and reinfection rates highlight the need for an effective schistosomiasis vaccine. Schistosoma mansoni large subunit of calpain (Sm-p80)-based vaccine formulations have shown remarkable efficacy in protecting against S. mansoni challenge infections in mice and baboons. In this study, we evaluated the cross-species protective efficacy of Sm-p80 vaccine against S. japonicum and S. haematobium challenge infections in rodent models. We also elucidated the expression of Sm-p80 and Sm-p80 ortholog proteins in different developmental stages of S. mansoni, S. haematobium, and S. japonicum. Immunization with Sm-p80 vaccine reduced worm burden by 46.75% against S. japonicum challenge infection in mice. DNA prime/protein boost (1 + 1 dose administered on a single day) resulted in 26.95% reduction in worm burden in S. haematobium-hamster infection/challenge model. A balanced Th1 (IFN-γ, TNF-α, IL-2, and IL-12) and Th2 (IL-4, IgG1) type of responses were observed following vaccination in both S. japonicum and S. haematobium challenge trials and these are associated with the prophylactic efficacy of Sm-p80 vaccine. Immunohistochemistry demonstrated that Sm-p80/Sm-p80 ortholog proteins are expressed in different life cycle stages of the three major human species of schistosomes studied. The data presented in this study reinforce the potential of Sm-p80-based vaccine for both hepatic/intestinal and urogenital schistosomiasis occurring in different geographical areas of the world. Differential expression of Sm-p80/Sm-p80 protein orthologs in different life cycle makes this vaccine potentially useful in targeting different levels of infection, disease, and transmission.

Vacuolar H+-ATPase in the nuclear membranes regulates nucleo-cytosolic proton gradients.

The regulation of the luminal pH of each organelle is crucial for its function and must be controlled tightly. Nevertheless, it has been assumed that the nuclear pH is regulated by the cytoplasmic proton transporters via the diffusion of H+ across the nuclear pores because of their large diameter. However, it has been demonstrated that ion gradients exist between cytosol and nucleus, suggesting that the permeability of ions across the nuclear pores is restricted. Vacuolar H+-ATPase (V-H+-ATPase) is responsible for the creation and maintenance of trans-membrane electrochemical gradient. We hypothesize that V-H+-ATPase located in the nuclear membranes functions as the primary mechanism to regulate nuclear pH and generate H+ gradients across the nuclear envelope. We studied the subcellular heterogeneity of H+ concentration in the nucleus and cytosol using ratio imaging microscopy and SNARF-1, a pH indicator, in prostate cells. Our results indicate that there are proton gradients across the nuclear membranes that are generated by V-H+-ATPase located in the outer and inner nuclear membranes. We demonstrated that these gradients are mostly dissipated by inhibiting V-H+-ATPase. Immunoblots and V-H+-ATPase activity corroborated the existence of V-H+-ATPase in the nuclear membranes. This study demonstrates that V-H+-ATPase is functionally expressed in nuclear membranes and is responsible for nuclear H+ gradients that may promote not only the coupled transport of substrates, but also most electrochemically driven events across the nuclear membranes. This study represents a paradigm shift that the nucleus can regulate its own pH microenvironment, providing new insights into nuclear ion homeostasis and signaling.

Activity of plasma membrane V-ATPases is critical for the invasion of MDA-MB231 breast cancer cells.

The vacuolar (H(+))-ATPases (V-ATPases) are a family of ATP-driven proton pumps that couple ATP hydrolysis with translocation of protons across membranes. Previous studies have implicated V-ATPases in cancer cell invasion. It has been proposed that V-ATPases participate in invasion by localizing to the plasma membrane and causing acidification of the extracellular space. To test this hypothesis, we utilized two separate approaches to specifically inhibit plasma membrane V-ATPases. First, we stably transfected highly invasive MDA-MB231 cells with a V5-tagged construct of the membrane-embedded c subunit of the V-ATPase, allowing for extracellular expression of the V5 epitope. We evaluated the effect of addition of a monoclonal antibody directed against the V5 epitope on both V-ATPase-mediated proton translocation across the plasma membrane and invasion using an in vitro Matrigel assay. The addition of anti-V5 antibody resulted in acidification of the cytosol and a decrease in V-ATPase-dependent proton flux across the plasma membrane in transfected but not control (untransfected) cells. These results demonstrate that the anti-V5 antibody inhibits activity of plasma membrane V-ATPases in transfected cells. Addition of the anti-V5 antibody also inhibited in vitro invasion of transfected (but not untransfected) cells. Second, we utilized a biotin-conjugated form of the specific V-ATPase inhibitor bafilomycin. When bound to streptavidin, this compound cannot cross the plasma membrane. Addition of this compound to MDA-MB231 cells also inhibited in vitro invasion. These studies suggest that plasma membrane V-ATPases play an important role in invasion of breast cancer cells.

Regulation of retinoid mediated cholesterol efflux involves liver X receptor activation in mouse macrophages.

Removal of cholesterol from macrophage-derived foam cells is a critical step to the prevention of atherosclerotic lesions. We have recently demonstrated the functional importance of retinoids in the regulation of the steroidogenic acute regulatory (StAR) protein that predominantly mediates the intramitochondrial transport of cholesterol in target tissues. In the present study, treatment of mouse macrophages with retinoids, particularly all-trans retinoic acid (atRA) and 9-cis RA, resulted in increases in cholesterol efflux to apolipoprotein AI (Apo-A1). Activation of the PKA pathway by a cAMP analog, (Bu)2cAMP, markedly augmented retinoid mediated cholesterol efflux. Macrophages overexpressing hormone-sensitive lipase increased the hydrolysis of cholesteryl esters and concomitantly enhanced the efficacy of retinoic acid receptor and liver X receptor (LXR) ligands on StAR and ATP-binding cassette transporter A1 (ABCA1) protein levels. RAs elevated StAR promoter activity in macrophages, and an increase in StAR levels augmented cholesterol efflux to Apo-A1, suggesting retinoid-mediated efflux of cholesterol involves enhanced oxysterol production. Further studies revealed that retinoids activate the LXR regulated genes, sterol receptor-element binding protein-1c and ABCA1. These findings provide insights into the regulatory events in which retinoid signaling effectively enhances macrophage cholesterol efflux and indicate that retinoid therapy may have important implications in limiting and/or regressing atherosclerotic cardiovascular disease.

Molecular phenotypes segregate missense mutations in SLC13A5 Epilepsy.

The sodium-coupled citrate transporter (NaCT, SLC13A5) mediates citrate uptake across the plasma membrane via an inward Na + gradient. Mutations in SLC13A5 cause early infantile epileptic encephalopathy type-25 (EIEE25, SLC13A5 Epilepsy) due to impaired citrate uptake in neurons. Despite clinical identification of disease-causing mutations, underlying mechanisms and cures remain elusive. We mechanistically classify the molecular phenotypes of six mutations. C50R, T142M, and T227M exhibit impaired citrate transport despite normal expression at the cell surface. G219R, S427L, and L488P are hampered by low protein expression, ER retention, and reduced transport. Mutants' mRNA levels resemble wildtype, suggesting post-translational defects. Class II mutations display immature core-glycosylation and shortened half-lives, indicating protein folding defects. These experiments provide a comprehensive understanding of the mutation's defects in SLC13A5 Epilepsy at the biochemical and molecular level and shed light into the trafficking pathway(s) of NaCT. The two classes of mutations will require fundamentally different treatment approaches to either restore transport function, or enable correction of protein folding defects. Summary: Loss-of-function mutations in the SLC13A5 causes SLC13A5-Epilepsy, a devastating disease characterized by neonatal epilepsy. Currently no cure is available. We clarify the molecular-level defects to guide future developments for phenotype-specific treatment of disease-causing mutations.

Impact of Oncogenic Changes in p53 and KRAS on Macropinocytosis and Ferroptosis in Colon Cancer Cells and Anticancer Efficacy of Niclosamide with Differential Effects on These Two Processes.

Mutations in p53 and KRAS are seen in most cases of colon cancer. The impact of these mutations on signaling pathways related to cancer growth has been studied in depth, but relatively less is known on their effects on amino acid transporters in cancer cells. This represents a significant knowledge gap because amino acid nutrition in cancer cells profoundly influences macropinocytosis and ferroptosis, two processes with opposing effects on tumor growth. Here, we used isogenic colon cancer cell lines to investigate the effects of p53 deletion and KRAS activation on two amino acid transporters relevant to macropinocytosis (SLC38A5) and ferroptosis (SLC7A11). Our studies show that the predominant effect of p53 deletion is to induce SLC7A11 with the resultant potentiation of antioxidant machinery and protection of cancer cells from ferroptosis, whereas KRAS activation induces not only SLC7A11 but also SLC38A5, thus offering protection from ferroptosis as well as improving amino acid nutrition in cancer cells via accelerated macropinocytosis. Niclosamide, an FDA-approved anti-helminthic, blocks the functions of SLC7A11 and SLC38A5, thus inducing ferroptosis and suppressing macropinocytosis, with the resultant effective reversal of tumor-promoting actions of oncogenic changes in p53 and KRAS. These findings underscore the potential of this drug in colon cancer treatment.

Induction of Oxidative Stress and Ferroptosis in Triple-Negative Breast Cancer Cells by Niclosamide via Blockade of the Function and Expression of SLC38A5 and SLC7A11.

The amino acid transporters SLC38A5 and SLC7A11 are upregulated in triple-negative breast cancer (TNBC). SLC38A5 transports glutamine, methionine, glycine and serine, and therefore activates mTOR signaling and induces epigenetic modifications. SLC7A11 transports cystine and increases the cellular levels of glutathione, which protects against oxidative stress and lipid peroxidation via glutathione peroxidase, a seleno (Se)-enzyme. The primary source of Se is dietary Se-methionine (Se-Met). Since SLC38A5 transports methionine, we examined its role in Se-Met uptake in TNBC cells. We found that SLC38A5 interacts with methionine and Se-Met with comparable affinity. We also examined the influence of Se-Met on Nrf2 in TNBC cells. Se-Met activated Nrf2 and induced the expression of Nrf2-target genes, including SLC7A11. Our previous work discovered niclosamide, an antiparasitic drug, as a potent inhibitor of SLC38A5. Here, we found SLC7A11 to be inhibited by niclosamide with an IC50 value in the range of 0.1-0.2 µM. In addition to the direct inhibition of SLC38A5 and SLC7A11, the pretreatment of TNBC cells with niclosamide reduced the expression of both transporters. Niclosamide decreased the glutathione levels, inhibited proliferation, suppressed GPX4 expression, increased lipid peroxidation, and induced ferroptosis in TNBC cells. It also significantly reduced the growth of the TNBC cell line MB231 in mouse xenografts.

AFM nano-mechanics and calcium dynamics of prostate cancer cells with distinct metastatic potential.

BACKGROUND: Despite recent advances, it is not clear to correlate the mechanical compliances and the metastatic potential of cancer cells. In this study, we investigated combined signatures of mechanical compliances, adhesions, and calcium dynamics correlated with the metastatic potential of cancer cells. SCOPE OF REVIEW: We used the lowly (LNCaP) and highly (CL-1, CL-2) metastatic human prostate cancer cells. The AFM-based nanomechanics was performed to determine the elastic moduli and the cell-to-substrate adhesion. The intracellular calcium dynamics was evaluated by fluorescence spectroscopy. Cell migration and the distribution of cytoskeleton were evaluated using the wounded monolayer model and immunofluorescence, respectively. The elastic moduli, the calcium dynamics, and the migratory ability are greater in CL-1 and CL-2 than LNCaP. CL-1 and CL-2 also display a significantly larger area of cell-to-substrate adhesions while the LNCaP displays a limited adhesion. These properties were slightly reduced in CL-2 compared with CL-1 cells. The enhanced elastic moduli and calcium dynamics found in CL-1 and CL-2 can be consistently explained by the intensified tensile stress generated by actin cytoskeletons anchored at more focal adhesion sites. MAJOR CONCLUSIONS: Although the suppressed mechanical compliance of highly metastatic cells may not support the enhanced cancer metastasis, the enhanced adhesion and calcium dynamics are favorable for invasion and extra-vasation required for malignant progression. GENERAL SIGNIFICANCE: Our results suggest that the mechanical compliance alone may fail to indicate the metastatic progression, but the combined biomechanical signatures of mechanical compliance, adhesion, and calcium dynamics can provide critical clues to determine the metastatic potential of cells.

Potent Inhibition of Macropinocytosis by Niclosamide in Cancer Cells: A Novel Mechanism for the Anticancer Efficacy for the Antihelminthic.

Niclosamide, a drug used to treat tapeworm infection, possesses anticancer effects by interfering with multiple signaling pathways. Niclosamide also causes intracellular acidification. We have recently discovered that the amino acid transporter SLC38A5, an amino acid-dependent Na+/H+ exchanger, activates macropinocytosis in cancer cells via amino acid-induced intracellular alkalinization. Therefore, we asked whether niclosamide will block basal and SLC38A5-mediated macropinocytosis via intracellular acidification. We monitored macropinocytosis in pancreatic and breast cancer cells using TMR-dextran and the function of SLC38A5 by measuring Li+-stimulated serine uptake. The peptide transporter activity was measured by the uptake of glycylsarcosine. Treatment of the cancer cells with niclosamide caused intracellular acidification. The drug blocked basal and serine-induced macropinocytosis with differential potency, with an EC50 of ~5 µM for the former and ~0.4 µM for the latter. The increased potency for amino acid-mediated macropinocytosis is due to direct inhibition of SLC38A5 by niclosamide in addition to the ability of the drug to cause intracellular acidification. The drug also inhibited the activity of the H+-coupled peptide transporter. We conclude that niclosamide induces nutrient starvation in cancer cells by blocking macropinocytosis, SLC38A5 and the peptide transporter. These studies uncover novel, hitherto unknown, mechanisms for the anticancer efficacy of this antihelminthic.

Helicase-like transcription factor (Hltf)-deletion activates Hmgb1-Rage axis and granzyme A-mediated killing of pancreatic ß cells resulting in neonatal lethality.

Epigenetic mechanisms are integral to pancreatic ß cell function. Promoter hypermethylation of the helicase like-transcription factor (HLTF) gene-a component of the cellular DNA damage response that contributes to genome stability-has been implicated in age-associated changes in ß cells. To study HLTF, we generated global and ß cell-specific (ß) Hltf knockout (KO) immune competent (IC) and immune deficient (ID) Rag2-/IL2- mice. IC global and ß Hltf KO mice were neonatal lethal whereas ID global and ß Hltf KO newborn mice had normal survival. This focused our investigation on the effects of Rag2 interruption with common gamma chain interruption on ß cell function/survival. Three-way transcriptomic (RNAseq) analyses of whole pancreata from IC and ID newborn ß Hltf KO and wild type (Hltf +/+) controls combined with spatially resolved transcriptomic analysis of formalin fixed paraffin embedded tissue, immunohistochemistry and laser scanning confocal microscopy showed DNA damage caused by ß Hltf KO in IC mice upregulated the Hmgb1-Rage axis and a gene signature for innate immune cells. Perforin-delivered granzyme A (GzmA) activation of DNase, Nme1, showed damaged nuclear single-stranded DNA (γH2AX immunostaining). This caspase-independent method of cell death was supported by transcriptional downregulation of Serpinc1 gene that encodes a serine protease inhibitor of GzmA. Increased transcriptional availability of complement receptors C3ar1 and C5ar1 likely invited crosstalk with Hmgb1 to amplify inflammation. This study explores the complex dialog between ß cells and immune cells during development. It has implications for the initiation of type I diabetes in utero when altered gene expression that compromises genome stability invokes a localized inflammatory response.

Amino acid transporter SLC38A5 is a tumor promoter and a novel therapeutic target for pancreatic cancer.

Pancreatic ductal adenocarcinoma (PDAC) cells have a great demand for nutrients in the form of sugars, amino acids, and lipids. Particularly, amino acids are critical for cancer growth and, as intermediates, connect glucose, lipid and nucleotide metabolism. PDAC cells meet these requirements by upregulating selective amino acid transporters. Here we show that SLC38A5 (SN2/SNAT5), a neutral amino acid transporter is highly upregulated and functional in PDAC cells. Using CRISPR/Cas9-mediated knockout of SLC38A5, we show its tumor promoting role in an in vitro cell line model as well as in a subcutaneous xenograft mouse model. Using metabolomics and RNA sequencing, we show significant reduction in many amino acid substrates of SLC38A5 as well as OXPHOS inactivation in response to SLC38A5 deletion. Experimental validation demonstrates inhibition of mTORC1, glycolysis and mitochondrial respiration in KO cells, suggesting a serious metabolic crisis associated with SLC38A5 deletion. Since many SLC38A5 substrates are activators of mTORC1 as well as TCA cycle intermediates/precursors, we speculate amino acid insufficiency as a possible link between SLC38A5 deletion and inactivation of mTORC1, glycolysis and mitochondrial respiration, and the underlying mechanism for PDAC attenuation. Overall, we show that SLC38A5 promotes PDAC, thereby identifying a novel, hitherto unknown, therapeutic target for PDAC.

A Proton-Coupled Transport System for ß-Hydroxy-ß-Methylbutyrate (HMB) in Blood-Brain Barrier Endothelial Cell Line hCMEC/D3.

ß-Hydroxy-ß-methylbutyrate (HMB), a leucine metabolite, is used as a nutritional ingredient to improve skeletal muscle health. Preclinical studies indicate that this supplement also elicits significant benefits in the brain; it promotes neurite outgrowth and prevents age-related reductions in neuronal dendrites and cognitive performance. As orally administered HMB elicits these effects in the brain, we infer that HMB crosses the blood-brain barrier (BBB). However, there have been no reports detailing the transport mechanism for HMB in BBB. Here we show that HMB is taken up in the human BBB endothelial cell line hCMEC/D3 via H+-coupled monocarboxylate transporters that also transport lactate and ß-hydroxybutyrate. MCT1 (monocarboxylate transporter 1) and MCT4 (monocarboxylate transporter 4) belonging to the solute carrier gene family SLC16 (solute carrier, gene family 16) are involved, but additional transporters also contribute to the process. HMB uptake in BBB endothelial cells results in intracellular acidification, demonstrating cotransport with H+. Since HMB is known to activate mTOR with potential to elicit transcriptomic changes, we examined the influence of HMB on the expression of selective transporters. We found no change in MCT1 and MCT4 expression. Interestingly, the expression of LAT1 (system L amino acid transporter 1), a high-affinity transporter for branched-chain amino acids relevant to neurological disorders such as autism, is induced. This effect is dependent on mTOR (mechanistic target of rapamycine) activation by HMB with no involvement of histone deacetylases. These studies show that HMB in systemic circulation can cross the BBB via carrier-mediated processes, and that it also has a positive influence on the expression of LAT1, an important amino acid transporter in the BBB.

Dishevelled-1 DIX and PDZ domain lysine residues regulate oncogenic Wnt signaling.

DVL proteins are central mediators of the Wnt pathway and relay complex input signals into different branches of the Wnt signaling network. However, molecular mechanism(s) that regulate DVL-mediated relay of Wnt signals still remains unclear. Here, for the first time, we elucidate the functional significance of three DVL-1 lysines (K/Lys) which are subject to post-translational acetylation. We demonstrate that K34 Lys residue in the DIX domain regulates subcellular localization of ß-catenin, thereby influencing downstream Wnt target gene expression. Additionally, we show that K69 (DIX domain) and K285 (PDZ domain) regulate binding of DVL-1 to Wnt target gene promoters and modulate expression of Wnt target genes including CMYC, OCT4, NANOG, and CCND1, in cell line models and xenograft tumors. Finally, we report that conserved DVL-1 lysines modulate various oncogenic functions such as cell migration, proliferation, cell-cycle progression, 3D-spheroid formation and in-vivo tumor growth in breast cancer models. Collectively, these findings highlight the importance of DVL-1 domain-specific lysines which were recently shown to be acetylated and characterize their influence on Wnt signaling. These site-specific modifications may be subject to regulation by therapeutics already in clinical use (lysine deacetylase inhibitors such as Panobinostat and Vorinostat) or may possibly have prognostic utility in translational efforts that seek to modulate dysfunctional Wnt signaling.

Studying Proton Gradients Across the Nuclear Envelope.

The existence of nuclear pore complexes in the nuclear envelope has led to the assumption that ions move freely from the cytosol into the nucleus, and that the molecular mechanisms at the plasma membrane that regulate cytosolic pH also regulate nuclear pH. Furthermore, studies to measure pH in the nucleus have produced contradictory results, since it has been found that the nuclear pH is either similar to the cytosol or more alkaline than the cytosol. However, most studies of nuclear pH have lacked the rigor needed to understand pH regulation in the nucleus. A major problem has been the lack of in situ titrations in the nucleus and cytosol, since the intracellular environment is different in the cytosol and nucleus and the behavior of fluorescent pH probes is different in these environments. Here we present a method that uses the fluorescence of SNARF-1 that labels both cytosol and nucleus. Using ratio imaging microscopy, regions of interest corresponding to the nucleus and cytosol to perform steady-state pH measurements followed by in situ titrations, to correctly assign pH in those cellular domains.

The Wnt non-canonical signaling modulates cabazitaxel sensitivity in prostate cancer cells.

BACKGROUND: Despite new drugs, metastatic prostate cancer remains fatal. Growing interest in the latest approved cabazitaxel taxane drug has markedly increased due to the survival benefits conferred when used at an earlier stage of the disease, its promising new therapeutic combination and formulation, and its differential toxicity. Still cabazitaxel's mechanisms of resistance are poorly characterized. The goal of this study was thus to generate a new model of acquired resistance against cabazitaxel in order to unravel cabazitaxel's resistance mechanisms. METHODS: Du145 cells were cultured with increasing concentrations of cabazitaxel, docetaxel/ taxane control or placebo/age-matched control. Once resistance was reached, Epithelial-to-Mesenchymal Translation (EMT) was tested by cell morphology, cell migration, and E/M markers expression profile. Cell transcriptomics were determined by RNA sequencing; related pathways were identified using IPA, PANTHER or KEGG software. The Wnt pathway was analyzed by western blotting, pharmacological and knock-down studies. RESULTS: While age-matched Du145 cells were sensitive to both taxane drugs, docetaxel-resistant cells were only resistant to docetaxel and cabazitaxel-resistant cells showed a partial cross-resistance to both drugs concomitant to EMT. Using RNA-sequencing, the Wnt non-canonical pathway was identified as exclusively activated in cabazitaxel resistant cells while the Wnt canonical pathway was restricted to docetaxel-resistant cells. Cabazitaxel-resistant cells showed a minimal crossover in the Wnt-pathway-related genes linked to docetaxel resistance validating our unique model of acquired resistance to cabazitaxel. Pharmacological and western blot studies confirmed these findings and suggest the implication of the Tyrosine kinase Ror2 receptor in cabazitaxel resistant cells. Variation in Ror2 expression level altered the sensitivity of prostate cancer cells to both drugs identifying a possible new target for taxane resistance. CONCLUSION: Our study represents the first demonstration that while Wnt pathway seems to play an important role in taxanes resistance, Wnt effectors responsible for taxane specificity remain un-identified prompting the need for more studies.

In vitro study of transportation of porphyrin immobilized graphene oxide through blood brain barrier.

Blood brain barrier (BBB), a barrier formed by endothelial cells, separates the brain from the circulatory system and protects the stability of central neural system normally, however, it also results in low permeability of vast majority of drugs for brain disease therapy. In this work, the cytotoxicity, uptake and transportation of 2D graphene nanosheet through BBB were investigated in in vitro models of BBB constructed by human brain microvascular endothelia cells (hBMECs). Permeability of two types of graphene nanosheet, including graphene oxide (GO) and porphyrin conjugated graphene oxide (PGO) through BBB were studied. With hydrophobic chemicals conjugation on its surface, permeability of PGO was greatly improved compared to GO. Furthermore, transportation behavior of assorted sizes of PGO obtained by differential velocity centrifugation through BBB was also explored, revealing that PGO with larger size has higher permeability than smaller-size PGO. The significant improved permeability of 2D graphene nanosheet through BBB compared to traditional drugs provides promising applications in drug delivery and disease therapy for brain disease in the near future.

A 2-strain mixture of Lactobacillus acidophilus in the treatment of irritable bowel syndrome: A placebo-controlled randomized clinical trial.

BACKGROUND: In the absence of a well-established therapeutic approach, patients with irritable bowel syndrome seek alternative strategies such as probiotics. AIMS: The current trial named LAPIBSS aimed to demonstrate the efficacy of a 2-strain mixture of Lactobacillus acidophilus to improve irritable bowel syndrome symptoms. METHODS: Eighty patients diagnosed for irritable bowel syndrome were recruited to a multicentre, double-blinded, in parallel groups, placebo-controlled, randomized clinical trial. Patients were provided with a daily dose of two capsules containing either probiotics (5 × 109 cfu/capsule) or placebo for 8 weeks. The primary outcome was abdominal pain score assessed with a 100-mm visual analogue scale. Secondary outcomes included scores of bloating, flatus and rumbling assessed with a 100-mm visual analogue scale, a composite score and bowel habits. RESULTS: Abdominal pain score was significantly improved in both groups at weeks 4 and 8 (P < 0.0001), but no significant differences were found between groups at week 8 (19.0 ±â€¯2.5 vs 25.1 ±â€¯2.6, respectively; LS Means differences = 6.0 ±â€¯3.2; P = 0.06). Significant differences between groups were observed for flatus score at week 4 (P = 0.04) and week 8 (P = 0.03) and composite score (P = 0.04) at week 8. CONCLUSIONS: The consumption of the 2-strain mixture of L. acidophilus over 8 weeks is safe and decreases significantly flatus and composite scores. TRIAL REGISTRATION NUMBER: EudraCT No 2008 A00844-51.