The hepatic phosphatidylcholine (PC) transporter ATP-binding cassette (ABC) B4 flops PC from hepatocytes into bile, and its dysfunction causes chronic cholestasis and fibrosis. Because a nuclear receptor-dependent PC pathway has been determined to exert antidiabetic effects, we now analyzed the role of ABCB4 in glucose metabolism. We bred congenic Abcb4-knockout (Abcb4(-/-)) mice on the fibrosis-susceptible BALB/cJ background. Knockout mice and wild-type controls were phenotyped by measuring plasma glucose concentrations, intraperitoneal glucose tolerance, hepatic RNA expression profiles, and liver histology. In addition, 4 procholestatic ABCB4 gene variants were correlated with blood glucose levels in 682 individuals from 2 independent European cohorts. Systemic glucose levels differ significantly between Abcb4(-/-) mice and wild-type controls, and knockout mice display improved glucose tolerance with significantly lower area under the curve values on intraperitoneal glucose challenge. Of note, hepatic expression of the antidiabetic nuclear receptor 5A2 (LRH-1) is induced consistently in Abcb4(-/-) mice, and its specific rare PC ligands are detected in liver by mass spectrometry imaging. In humans, serum glucose levels are associated significantly with the common ABCB4 variant c.711A>T. In summary, ABCB4 might play a critical role in glucose homeostasis in mice and humans. We speculate that the effects could be mediated via LRH-1-dependent PC pathways.

On the canalicular membranes of hepatocytes, several ABC transporters are responsible for the secretion of bile lipids. Among them, ABCB4, also called MDR3, is essential for the secretion of phospholipids from hepatocytes into bile. The biliary phospholipids are associated with bile salts and cholesterol in mixed micelles, thereby reducing the detergent activity and cytotoxicity of bile salts and preventing cholesterol crystallization. Mutations in the ABCB4 gene result in progressive familial intrahepatic cholestasis type 3, intrahepatic cholestasis of pregnancy, low-phospholipid-associated cholelithiasis, primary biliary cirrhosis, and cholangiocarcinoma. In vivo and cell culture studies have demonstrated that the secretion of biliary phospholipids depends on both ABCB4 expression and bile salts. In the presence of bile salts, ABCB4 located in nonraft membranes mediates the efflux of phospholipids, preferentially phosphatidylcholine. Despite high homology with ABCB1, ABCB4 expression cannot confer multidrug resistance. This review summarizes our current understanding of ABCB4 functions and physiological relevance, and discusses the molecular mechanism for the ABCB4-mediated efflux of phospholipids.

BACKGROUND

Pyrethroid resistance is a threat to effective vector control of Aedes aegypti, the vector of dengue, Zika and other arboviruses, but there are many major knowledge gaps on the mechanisms of resistance. In Jeddah and Makkah, the principal dengue-endemic areas of Saudi Arabia, pyrethroids are used widely for Ae. aegypti control but information about resistance remains sparse, and the underlying genetic basis is unknown. Findings from an ongoing study in this internationally significant area are reported here.

METHODS

Aedes aegypti collected from each city were raised to adults and assayed for resistance to permethrin, deltamethrin (with and without the synergist piperonyl butoxide, PBO), fenitrothion, and bendiocarb. Two fragments of the voltage-gated sodium channel (Vgsc), encompassing four previously identified mutation sites, were sequenced and subsequently genotyped to determine associations with resistance. Expression of five candidate genes (CYP9J10, CYP9J28, CYP9J32, CYP9M6, ABCB4) previously associated with pyrethroid resistance was compared between assay survivors and controls.

RESULTS

Jeddah and Makkah populations exhibited resistance to multiple insecticides and a similarly high prevalence of resistance to deltamethrin compared to a resistant Cayman strain, with a significant influence of age and exposure duration on survival. PBO pre-exposure increased pyrethroid mortality significantly in the Jeddah, but not the Makkah strain. Three potentially interacting Vgsc mutations were detected: V1016G and S989P were in perfect linkage disequilibrium in each strain and strongly predicted survival, especially in the Makkah strain, but were in negative linkage disequilibrium with 1534C, though some females with the Vgsc triple mutation were detected. The candidate gene CYP9J28 was significantly over-expressed in Jeddah compared to two susceptible reference strains, but none of the candidate genes was consistently up-regulated to a significant level in the Makkah strain.

CONCLUSIONS

Despite their proximity, Makkah and Jeddah exhibit significant differences in pyrethroid resistance phenotypes, with some evidence to suggest a different balance of mechanisms, for example with more impact associated with CYP450s in the Jeddah strain, and the dual kdr mutations 989P and 1016G in the more resistant Makkah strain. The results overall demonstrate a major role for paired target site mutations in pyrethroid resistance and highlight their utility for diagnostic monitoring.

NorUDCA (24-norursodeoxycholic acid), the C₂₃-homolog of ursodeoxycholic acid (UDCA), showed remarkable therapeutic effects in cholestatic Mdr2 (Abcb4) (multidrug resistance protein 2/ATP-binding cassette b4) knockout mice with sclerosing/fibrosing cholangitis. In contrast to UDCA, norUDCA is inefficiently conjugated in human and rodent liver, and conjugation has been discussed as a key step for the anticholestatic action of UDCA in cholestasis. We compared the choleretic, anticholestatic, and antiapoptotic properties of unconjugated and taurine-conjugated UDCA (C₂₄) and norUDCA (C₂₃) in isolated perfused rat liver (IPRL) and in natrium/taurocholate cotransporting polypeptide (Ntcp)-transfected human hepatoma (HepG2) cells. Taurolithocholic acid (TLCA) was used to induce a predominantly hepatocellular cholestasis in IPRL. Bile flow was determined gravimetrically; bile acids determined by gas chromatography and liquid chromatography/tandem mass spectrometry; the Mrp2 model substrate, 2,4-dinitrophenyl-S-glutathione (GS-DNP) was determined spectrophotometrically; and apoptosis was determined immunocytochemically. The choleretic effect of C₂₃-bile acids was comparable to their C₂₄-homologs in IPRL. In contrast, TnorUDCA, but not norUDCA antagonized the cholestatic effect of TLCA. Bile flow (percent of controls) was 8% with TLCA-induced cholestasis, and unchanged by coinfusion of norUDCA (14%). However, it was increased by TnorUDCA (83%), UDCA (73%) and TUDCA (136%). Secretion of GS-DNP was markedly reduced by TLCA (5%), unimproved by norUDCA (4%) or UDCA (17%), but was improved modestly by TnorUDCA (26%) or TUDCA (58%). No apoptosis was observed in IPRL exposed to low micromolar TLCA, but equivalent antiapoptotic effects of TUDCA and TnorUDCA were observed in Ntcp-HepG2 cells exposed to TLCA.

CONCLUSIONS

Conjugation is essential for the anticholestatic effect of norUDCA in a model of hepatocellular cholestasis. Combined therapy with UDCA and norUDCA may be superior to UDCA or norUDCA monotherapy in biliary disorders in which hepatocyte as well as cholangiocyte dysfunction contribute to disease progression.

Cholestasis develops as a consequence of impaired bile formation and/or bile flow and can be classified as intra- or extrahepatic. Chronic cholestatic diseases are mostly intrahepatic with the exception of primary and secondary sclerosing cholangitis affecting intra- and extrahepatic bile ducts. Recent genome-wide association studies have confirmed major histocompatibility complex associations and discovered multiple susceptibility loci in primary biliary cirrhosis and primary sclerosing cholangitis, providing new insights into disease pathogenesis, which may translate into more precise therapeutic prevention and intervention in the future. Diagnostic steps in cholestatic conditions comprise a thorough patient history, abdominal imaging and distinct serological studies including antimitochondrial antibodies and IgG4 levels; if the diagnosis remains unclear, liver biopsy is warranted. Genetic testing should also be considered, as mutations in the hepatobiliary transporters ATP8B1, ABCB11 and ABCB4 are causative for three different forms of familial intrahepatic cholestasis. Disease severity is dependent on the genotypic variants of these transporters, ranging from mildly elevated liver enzymes in adults to cirrhosis in early childhood. Ligands of nuclear receptors, which represent important regulators of hepatobiliary transporters, and modified bile salts are new promising therapeutic options in cholestatic liver disease and are currently being investigated in clinical trials.

Doxorubicin is one of the most widely used chemotherapy agents for the treatment of breast cancer. However, the development of doxorubicin resistance limits the long‑term treatment benefits in patients with breast cancer. Curcumin, a well‑known dietary polyphenol derived from the rhizomes of turmeric (Curcuma longa), enhances the sensitivity of breast cancer cells to chemotherapeutic agents; however, the mechanisms underlying this phenomenon remain unclear. The aim of the present study was to evaluate the effect of curcumin on chemoresistance in doxorubicin‑resistant breast cancerMCF‑7/DOX and MDA‑MB‑231/DOX cell lines. Cell Counting Kit‑8, monolayer transport, western blot and ATPase activity assays were performed during the study. The results revealed that curcumin significantly enhanced the effect of doxorubicin in doxorubicin‑resistant breast cancer cells. The intracellular accumulation of doxorubicin was substantially increased following curcumin treatment in doxorubicin‑resistant breast cancer cells, in a manner that was inversely dependent on the activity of ATP binding cassette subfamily B member 4 (ABCB4). Treatment with a combination of curcumin and doxorubicin decreases the efflux of doxorubicin in ABCB4‑overexpressing cells. Furthermore, curcumin inhibited the ATPase activity of ABCB4 without altering its protein expression. In conclusion, curcumin reversed doxorubicin resistance in human breast cancer MCF‑7/DOX and MDA‑MB‑231/DOX cells by inhibiting the ATPase activity of ABCB4. The study highlights the promising use of curcumin as a chemosensitizer in the treatment of breast cancer.

Primary biliary cirrhosis (PBC) is a multifactorial disease in which genetic factors rather than environmental factors may predominantly contribute to the pathogenesis. In order to identify the genetic determinants of the disease severity and progression of PBC, we examined an association of seven tag single-nucleotide polymorphisms (SNPs) in the multidrug resistance protein 3 (MDR3/ABCB4) gene in 148 Japanese PBC patients and 150 age- and sex-matched healthy control subjects. SNPs were detected via polymerase chain reaction (PCR) restriction fragment length polymorphism and PCR direct DNA sequencing methods. Subsequently, haplotypes were constructed from three tag SNPs (rs31658, rs31672, and rs1149222) that were significantly associated with progression of PBC. Logistic regression analyses revealed that a Hap 2 haplotype and its homozygous diplotype, Hap 2/Hap 2, in MDR3 were closely associated with the susceptibility to jaundice-type progression of PBC [P = 0.004, odds ratio (OR) 3.93, 95% confidence interval (CI) 1.56-9.90 and P = 0.0003, OR 17.73, 95% CI 3.77-83.42, respectively]. Conversely, another haplotype, Hap 1, and its homozygous diplotype, Hap 1/Hap 1, were associated with the insusceptibility to the progression to late-stage PBC (P = 0.021, OR 0.55, 95% CI 0.33-0.91 and P = 0.011, OR 0.24, 95% CI 0.08-0.71, respectively).

CONCLUSIONS

The present study is the first report of an association of MDR3 haplotypes and diplotypes with progression of PBC. The Hap 2/Hap 2 diplotype in MDR3 could therefore be potentially applied to DNA-based diagnosis in Japanese patients with PBC as a strong genetic biomarker for predicting the progression and prognosis of PBC.

The phosphatidylethanolamine N-methyltransferase (PEMT) pathway of phosphatidylcholine (PC) biosynthesis is not essential for the highly specific acyl chain composition of biliary PC. We evaluated whether the PEMT pathway is quantitatively important for biliary PC secretion in mice under various experimental conditions. Biliary bile salt and PC secretion were determined in mice in which the gene encoding PEMT was inactivated (Pemt(-/-)) and in wild-type mice under basal conditions, during acute metabolic stress (intravenous infusion of the bile salt tauroursodeoxycholate), and during chronic metabolic stress (feeding a taurocholate-containing diet for 1 week). The activity of CTP:phosphocholine cytidylyltransferase, the rate-limiting enzyme of PC biosynthesis via the CDP-choline pathway, and the abundance of multi-drug-resistant protein 2 (Mdr2; encoded by the Abcb4 gene), the canalicular membrane flippase essential for biliary PC secretion, were determined. Under basal conditions, Pemt(-/-) and wild-type mice exhibited similar biliary secretion rates of bile salt and PC ( approximately 145 and approximately 28 nmol/min/100 g body weight, respectively). During acute or chronic bile salt administration, the biliary PC secretion rates increased similarly in Pemt(-/-) and control mice. Mdr2 mRNA and protein abundance did not differ between Pemt(-/-) and wild-type mice. The cytidylyltransferase activity in hepatic lysates was increased by 20% in Pemt(-/-) mice fed the basal (bile salt-free) diet (P < 0.05). We conclude that the biosynthesis of PC via the PEMT pathway is not quantitatively essential for biliary PC secretion under acute or chronic bile salt administration.

Most ovarian cancer patients are chemosensitive initially, but finally relapse with acquired chemoresistance. Multidrug-resistance is the extremely terrible situation. The mechanism for the acquired chemoresistance of ovarian cancer patients is still not clear. LncRNAs have been recognized as the important regulator of a variety of biological processes, including the multidrug-resistant process. Here, we carried out the lncRNA sequencing of the ovarian cancer cell line A2780 and the paxitaxel resistant cell line A2780/PTX which is also cross resistant to the cisplatin and epirubicin. Through integrating the published data with the cisplatin resistant lncRNAs in ovarian cancer cell line or ovarian cancer patients, 5 up-regulated and 21 down-regulated lncRNAs are considered as the multidrug-resistant lncRNAs. By real-time PCR analysis, we confirmed the 5 up-regulated and 4 down-regulated multidrug resistant lncRNAs were similarly changed in both the multidrug resistant ovarian cancer cell lines and the multidrug resistant colon cancer cell lines. Furthermore, we conducted the lncRNA-mRNA co-expression network to predict the potential multidrug resistant lncRNAs' targets. Interestingly, the multidrug resistant genes ABCB1, ABCB4, ABCC3, and ABCG2 are all co-expressed with lncRNA CTD-2589M5.4. Our results provide the valuable information for the understanding of the lncRNA function in the multidrug resistant process.

BACKGROUND

The discovery of genetic mutations in children with inherited syndromes of intrahepatic cholestasis allows for diagnostic specificity despite similar clinical phenotypes. Here, we aimed to determine whether mutation screening of target genes could assign a molecular diagnosis in children with idiopathic cholestasis.

METHODS

DNA samples were obtained from 51 subjects with cholestasis of undefined etiology and surveyed for mutations in the genes SERPINA1, JAG1, ATP8B1, ABCB11, and ABCB4 by a high-throughput gene chip. Then, the sequence readouts for all 5 genes were analyzed for mutations and correlated with clinical phenotypes. Healthy subjects served as controls.

RESULTS

Sequence analysis of the genes identified 14 (or 27%) subjects with missense, nonsense, deletion, and splice site variants associated with disease phenotypes based on the type of mutation and/or biallelic involvement in the JAG1, ATP8B1, ABCB11, or ABCB4 genes. These patients had no syndromic features and could not be differentiated by biochemical markers or histopathology. Among the remaining subjects, 10 (or ∼20%) had sequence variants in ATP8B1 or ABCB11 that involved only 1 allele, 8 had variants not likely to be associated with disease phenotypes, and 19 had no variants that changed amino acid composition.

CONCLUSIONS

Gene sequence analysis assigned a molecular diagnosis in 27% of subjects with idiopathic cholestasis based on the presence of variants likely to cause disease phenotypes.

Deficiency of ABCB4 is associated with several forms of cholestasis in humans. Abcb4(-/-) mice also develop cholestasis, but it remains uncertain what role other canalicular transporters play in the development of this disease. We examined the expression of these transporters in Abcb4(-/-) mice compared with their wild-type littermate controls at ages of 10 days and 3, 6, and 12 wk. Elevated plasma bile acid levels were already detected at 10 days and at all ages thereafter in Abcb4(-/-) mice. The expression of Bsep, Mrp2, Atp8b1, Abcg5, and Abcg8 liver proteins did not change at 10 days, but Bsep, Mrp2, and Atp8b1 were reduced, whereas Abcg5 and Abcg8 expression were increased in Abcb4(-/-) mice at all later ages. Lower bile acid concentrations were also detected in the bile of 6-wk-old Abcb4(-/-) mice. Immunofluorescence labeling revealed distorted canalicular architecture in the liver tissue by 12 wk in Abcb4(-/-) mice. Whereas Bsep and Mrp2 remained associated with the apical membrane, Atp8b1 was now localized in discrete punctuate structures adjacent to the canalicular membrane in these mice. Expression of Bsep mRNA was increased in the livers of 10-day-old Abcb4(-/-) mice, whereas Ost-α was decreased. By 12 wk, Bsep, Mrp2, and Abcg5 mRNA were all increased, whereas Ost-α and Ntcp were reduced. These findings indicate that canalicular transporters that determine the formation of bile are altered early in the development of cholestasis in Abcb4(-/-) mice and may contribute to the pathogenesis of cholestasis in this disorder.

Gallstone disease is one of the most prevalent gastrointestinal diseases with a substantial burden to health care systems that is expected to increase in ageing populations at risk. This review summarizes recent data on the genetic background of cholesterol gallstones and the role of biliary lipid composition. Three previously unknown non-synonymous mutations in the ABCB4 gene encoding the hepatobiliary phospholipid-flippase MDR3 are presented.

We tested the hypothesis that differential expression of liver plasma membrane transporters might account for variations in biliary lipid secretion rates between gallstone-susceptible C57L/J and gallstone-resistant AKR/J mice. Plasma membrane fractions and total RNA isolated from livers of mice fed with a control or lithogenic (15% fat/1.25% cholesterol/0.5% cholic acid) diet were used for measurements of steady-state gene expression of hepatobiliary transport systems for bile salts (Ntcp1/Slc10a1, Oatp1/Slc21a1 and Bsep/Abcb11), phospholipids (Mdr2/Abcb4), organic anions (Mrp2/Abcc2) and organic cations (Oct1/Slc22a1). Irrespective of the diet, the steady-state gene expression of hepatobiliary transporters did not differ significantly between the two strains. Despite a higher basal bile flow and bile-salt secretion in C57L mice, Mrp2 (Abcc2) and Bsep (Abcb11) expression did not differ between the two strains. Elevated biliary phospholipid secretion in response to the lithogenic diet was linked to increased Mdr2 (Abcb4) protein expression, whereas the induction of Oct1 (Slc22a1) might reflect an enhanced uptake of choline for augmented phospholipid synthesis. In response to the lithogenic diet, Bsep (Abcb11) protein expression was up-regulated only marginally and bile salt secretion did not increase. The down-regulation of Ntcp1 (Slc10a1) protein expression might protect hepatocytes from high intracellular bile-salt loads. We conclude that variations in protein function rather than in the gene expression of liver plasma membrane transporters might account for variations in biliary lipid secretion rates. Our findings support the concept that the formation of lithogenic bile is caused by the hypersecretion of bile salts as a result of augmented availability of canalicular membrane cholesterol, possibly amplified by bile-salt-phospholipid uncoupling due to the increased bile flow.

BACKGROUND

Cystic fibrosis is the most common lethal recessive disorder among Caucasians. Over 1500 mutations have been identified in cystic fibrosis transmembrane conductance regulator disease-gene so far. A large variability of the clinical phenotype has been observed both in cystic fibrosis patients bearing the same genotype, and in affected sibpairs. Thus, genes inherited independently from cystic fibrosis transmembrane conductance regulator could modulate the clinical expression of cystic fibrosis.

METHODS

We analysed some putative modifier genes of liver cystic fibrosis phenotype (serpin 1, hemochromatosis, transferrin receptor 2, ferroportin 1, mannose binding lectin and adenosine triphospate-binding cassette subfamily B member 4) in 108 unrelated cystic fibrosis patients with and without liver involvement.

RESULTS

HYPD mannose binding lectin haplotype was significantly (p<0.05) more frequent in cystic fibrosis patients with liver disease versus those without liver disease. This haplotype already related to a more severe pulmonary cystic fibrosis phenotype, is associated to a reduced MBL immunological activity. The c.834-66G>T variant of adenosine triphospate-binding cassette subfamily B member 4 gene was significantly (p<0.05) less frequent in cystic fibrosis patients with liver disease as compared to those with no liver disease.

CONCLUSIONS

The HYPD mannose binding lectin haplotype may predispose a subgroup of cystic fibrosis patients to a more severe liver involvement impairing the local defence mechanisms whereas the c.834-66G>T adenosine triphospate-binding cassette subfamily B member 4 variant may enhance the activity of the protein and thus exert a protective effect toward liver disease.

Resistance to taxane-based therapy in breast cancer patients is a major clinical problem that may be addressed through insight of the genomic alterations leading to taxane resistance in breast cancer cells. In the current study we used whole exome sequencing to discover somatic genomic alterations, evolving across evolutionary stages during the acquisition of docetaxel resistance in breast cancer cell lines.

Two human breast cancer in vitro models (MCF-7 and MDA-MB-231) of the step-wise acquisition of docetaxel resistance were developed by exposing cells to 18 gradually increasing concentrations of docetaxel. Whole exome sequencing performed at five successive stages during this process was used to identify single point mutational events, insertions/deletions and copy number alterations associated with the acquisition of docetaxel resistance. Acquired coding variation undergoing positive selection and harboring characteristics likely to be functional were further prioritized using network-based approaches. A number of genomic changes were found to be undergoing evolutionary selection, some of which were likely to be functional. Of the five stages of progression toward resistance, most resistance relevant genomic variation appeared to arise midway towards fully resistant cells corresponding to passage 31 (5 nM docetaxel) for MDA-MB-231 and passage 16 (1.2 nM docetaxel) for MCF-7, and where the cells also exhibited a period of reduced growth rate or arrest, respectively. MCF-7 cell acquired several copy number gains on chromosome 7, including ABC transporter genes, including ABCB1 and ABCB4, as well as DMTF1, CLDN12, CROT, and SRI. For MDA-MB-231 numerous copy number losses on chromosome X involving more than 30 genes was observed. Of these genes, CASK, POLA1, PRDX4, MED14 and PIGA were highly prioritized by the applied network-based gene ranking approach. At higher docetaxel concentration MCF-7 subclones exhibited a copy number loss in E2F4, and the gene encoding this important transcription factor was down-regulated in MCF-7 resistant cells.

Our study of the evolution of acquired docetaxel resistance identified several genomic changes that might explain development of docetaxel resistance. Interestingly, the most relevant resistance-associated changes appeared to originate midway through the evolution towards fully resistant cell lines. Our data suggest that no single genomic event sufficiently predicts resistance to docetaxel, but require genomic alterations affecting multiple pathways that in concert establish the final resistance stage.

In this chapter, we describe techniques used to determine the efficiency of hepatocyte transplantation in animal models of liver disease. We have included the Gunn rat as a model of an inherited liver disease without hepatocyte damage and Abcb4 knockout mice as a model for an inherited liver disease with hepatocyte damage. Immunodeficient mice are included as an animal model for human hepatocyte transplantation.We describe problems that can be encountered in the maintenance and breeding of Gunn rats and immunodeficient Rag2/gamma common knockout mice. Protocols for the collection of bile in rats and mice are described, and we have also detailed the detection of green fluorescent protein (GFP)-labelled human hepatocytes in immunodeficient mice in this chapter.

The purpose of the present study was to determine the absolute protein expression levels of various transporters in renal brush-border membrane (BBM) and basolateral membrane (BLM) fractions, in order to understand the quantitative differences in average transport activities among different transporters at each cellular membrane. BBM and BLM fractions of rat kidney were prepared and digested with trypsin, and simultaneous absolute quantification of 28 transporters and a BLM marker, Na(+)/K(+)-ATPase, was performed using our established quantitative-targeted absolute proteomics (QTAP) technique. In BBM fraction, the protein expression levels of bcrp, urat1, mate1, octl1, mrp4, mdr1a, and abca3 were 40.3, 22.2, 8.90, 4.85, 4.69, 3.22, and 0.976 fmol/μg protein, respectively. In BLM fraction, the protein expression levels of oat1, oat3, oct1, mrp6, and mrp1 were 10.6, 10.2, 4.59, 0.724, and 0.271 fmol/μg protein, respectively. The expression levels of abca2, abca4, abca5, abca12, abcb4, mrp5, abcc9, abcg1, abcg5, lat1, ntcp, pgt, oatp2b1, oatp1b2, oatp3a1, and oct3 were under the limit of quantification in both fractions. The quantitative transporter protein expression profiles at these membranes, as determined by QTAP analysis, should be helpful to understand the contributions of individual transporters to renal excretion of xenobiotics and endogenous compounds.

Cellular senescence is a potent tumor-suppressive program that prevents neoplastic events. Paradoxically, senescent cells develop an inflammatory secretome, termed the senescence-associated secretory phenotype (SASP), which is implicated in age-related pathologies including cancer. Here we report that senescent cells actively synthesize and release small extracellular vesicles (sEVs) with a distinctive size distribution. Mechanistically, SIRT1 loss supported accelerated sEV production despite enhanced proteome-wide ubiquitination, a process correlated with ATP6V1A downregulation and defective lysosomal acidification. Once released, senescent stromal sEVs significantly altered the expression profile of recipient cancer cells and enhanced their aggressiveness, specifically drug resistance mediated by expression of ATP binding cassette subfamily B member 4 (ABCB4). Targeting SIRT1 with agonist SRT2104 prevented development of cancer resistance by restraining sEV production by senescent stromal cells. In clinical oncology, sEVs in peripheral blood of posttreatment cancer patients were readily detectable by routine biotechniques, presenting an exploitable biomarker to monitor therapeutic efficacy and predict long-term outcome. Together, this study identifies a distinct mechanism supporting pathological activities of senescent cells and provides a potent avenue to circumvent advanced human malignancies by co-targeting cancer cells and their surrounding microenvironment, which contributes to drug resistance via secretion of sEVs from senescent stromal cells.

Colorectal cancer (CRC) is a leading cause of cancer death. Chemoresistance is a pivotal feature of cancer cells leading to treatment failure and ATP-binding cassette (ABC) transporters are responsible for the efflux of several molecules, including anticancer drugs. The Hedgehog-GLI (HH-GLI) pathway is a major signalling in CRC, however its role in chemoresistance has not been fully elucidated. Here we show that the HH-GLI pathway favours resistance to 5-fluorouracil and Oxaliplatin in CRC cells. We identified potential GLI1 binding sites in the promoter region of six ABC transporters, namely ABCA2, ABCB1, ABCB4, ABCB7, ABCC2 and ABCG1. Next, we investigated the binding of GLI1 using chromatin immunoprecipitation experiments and we demonstrate that GLI1 transcriptionally regulates the identified ABC transporters. We show that chemoresistant cells express high levels of GLI1 and of the ABC transporters and that GLI1 inhibition disrupts the transporters up-regulation. Moreover, we report that human CRC tumours express high levels of the ABCG1 transporter and that its expression correlates with worse patients' prognosis. This study identifies a new mechanism where HH-GLI signalling regulates CRC chemoresistance features. Our results indicate that the inhibition of Gli1 regulates the ABC transporters expression and therefore should be considered as a therapeutic option in chemoresistant patients.

OBJECTIVE

It is to characterize the underlying molecular mechanisms of the anti-atherosclerotic effects of hydrogen (dihydrogen; H(2)), a novel antioxidant. In particular, to examine the effects of hydrogen on athero-susceptibility in lipoproteins and aorta of apolipoprotein E knockout (apoE-/-) mice.

RESULTS

Plasma analysis by enzymatic method and spectrophotometric measurement showed that eight weeks intraperitoneally injection of hydrogen-saturated saline remarkably decreased plasma total and non-high-density lipoprotein (non-HDL) cholesterol, and malondialdehyde in apoE-/- mice fed either chow or high fat diet. Western blot analysis showed hydrogen treatment reduced the contents of apolipoprotein B (apoB), a major protein constituent of non-HDL in either plasma or hepatic tissues. Moreover, ELISA assay revealed that the production of tumor necrosis factor-α and interleukin-6 were significantly suppressed by hydrogen in RAW264.7 macrophages, after stimulation with the isolated non-HDL from treated or untreated mice. Immunohistochemistry of aortic valve sections revealed that hydrogen suppressed the expression of several proinflammatory factors and decreased vessel wall infiltration of macrophages. Besides, real-time PCR and Western blot analysis disclosed that hepatic scavenger receptor class B type I (SR-BI), ATP-binding cassette (ABC) transporters ABCG8, ABCB4, ABCB11, and macrophage SR-BI, were all induced by hydrogen treatment. Finally arterial wall lipid disposition displayed by oil red O staining was reduced significantly in aortic root and whole aorta en face in hydrogen administrated mice. In addition, hydrogen significantly improved HDL functionality in C57BL/6J mice assessed in two independent ways, namely (i) stimulation of cholesterol efflux from macrophage foam cells by measuring HDL-induced [(3)H]cholesterol efflux, and (ii) protection against LDL oxidation as a measure of Cu(2+)-induced TBARS formation.

CONCLUSIONS

These results reveal that administration of hydrogen-saturated saline decreases athero-susceptibility in apoB-containing lipoprotein and aortic atherosclerosis in apoE-/- mice and improves HDL functionality in C57BL/6J mice.

OBJECTIVE

Mutations in the gene encoding the ABCB4 [adenosine triphosphate (ATP)-binding cassette, sub-family B (MDR/TAP), member 4] transporter lower phosphatidylcholine output into bile and contribute to cholesterol gallstone formation by decreasing the solubility of cholesterol in bile. Mutations in ABCB4 have been identified in patients with low phospholipid-associated cholelithiasis. The aim of the present study was to determine the types and frequencies of ABCB4 mutations in cholecystectomized patients aged <40 years.

METHODS

Hundred and four patients (mean age 30.6 years, range 12-39) were included in the study and the ABCB4 gene was sequenced. The frequency of missense mutations found in the patient material was measured in 95 healthy controls. The potential functional implications of the ABCB4 missense variations were assessed by computerized analysis (BLOSUM62 and Grantham substitution matrices, polymorphism phenotyping and sorting intolerant from tolerant).

RESULTS

One patient was heterozygous for a frameshift mutation (c.1399_1400ins10/p.Y467F fsX25). Another patient was heterozygous for a nonsense mutation (c.3136C>T/p.R1046X). These two mutations are considered detrimental to ABCB4 protein function. In addition, six missense mutations were found in the ABCB4 gene, and three of these were only present in patients.

CONCLUSIONS

In our study, <2% of young gallstone patients were found to be heterozygous for detrimental ABCB4 mutations. The functional implication of several missense mutations remains to be clarified. Thus, mutations in the ABCB4 gene are a rare cause of gallstone disease.

Bile is synthesized in the liver and is essential for the emulsification of dietary lipids and lipid-soluble vitamins. It is a complex mixture of amphiphilic bile acids (BAs; which act as detergent molecules), the membrane phospholipid phosphatidylcholine (PC), cholesterol and a variety of endogenous metabolites and waste products. Over the last 20 years, the combined effort of clinicians, geneticists, physiologists and biochemists has shown that each of these bile components is transported across the canalicular membrane of the hepatocyte by its own specific ATP-binding cassette (ABC) transporter. The bile salt export pump (BSEP) ABCB11 transports the BAs and drives bile flow from the liver, but it is now clear that two lipid transporters, ABCB4 (which flops PC into the bile) and the P-type ATPase ATP8B1/CDC50 (which flips a different phospholipid in the opposite direction) play equally critical roles that protect the biliary tree from the detergent activity of the bile acids. Understanding the interdependency of these lipid floppases and flippases has allowed the development of an assay to measure ABCB4 function. ABCB4 harbours numerous mis-sense mutations which probably reflects the spectrum of liver disease rooted in ABCB4 aetiology. Characterization of the effect of these mutations at the protein level opens the possibility for the development of personalized prognosis and treatment.

The organic anion (99m)Tc-N-[2-[(3-bromo-2,4,6-trimethylphenyl)-amino]-2-oxoethyl]-N-(carboxymethyl)-glycine ((99m)Tc-mebrofenin) and its analogs are widely used for hepatobiliary imaging. Identification of the mechanisms directing bile canalicular transport of these agents will provide insights into the basis of their hepatic handling for assessing perturbations.

METHODS

We performed studies in animals, including healthy Fischer 344 rats or rats treated with carbon tetrachloride or intrasplenic cell transplantation and healthy Wistar rats or HsdAMC:TR-Abcc2 mutant rats in Wistar background. Onset of hepatic inflammation was verified by analysis of carbon uptake in Kupffer cells. Hepatic clearance of (99m)Tc-mebrofenin was studied with dynamic imaging, and fractional retention of peak hepatic mebrofenin activity after 60 min was determined. Changes in the expression of bile canalicular transporters were analyzed by real-time polymerase chain reaction and Western blots.

RESULTS

Carbon tetrachloride and cell transplantation produced hepatic inflammation with activation of Kupffer cells, resulting in a rapid decline in the expression of the bile canalicular transporters Abcb4, Abcb11, and Abcc2. Among these transporters, decreased expression of Abcc2 was most prominent, and this decline persisted for 4 wk. Next, we examined (99m)Tc-mebrofenin excretion in HsdAMC:TR-Abcc2 mutant rats (in which Abcc2 expression is naturally inactivated), compared with their healthy counterparts. In healthy HsdRccHan:WIST rats, only 23% +/- 3% of the peak (99m)Tc-mebrofenin activity was retained after 60 min. By contrast, in HsdAMC:TR-Abcc2 mutant rats, 73% +/- 5% of the peak (99m)Tc-mebrofenin activity was retained (P < 0.001). Moreover, the administration of cyclosporin A markedly inhibited (99m)Tc-mebrofenin excretion in healthy rats, with no further effect on already impaired (99m)Tc-mebrofenin excretion in HsdAMC:TR-Abcc2 mutant rats. Hepatic excretion of (99m)Tc-mebrofenin was largely dependent on Abcc2. This molecular basis of (99m)Tc-mebrofenin excretion will advance studies of pathophysiologic mechanisms in hepatic Abcc2 pathways.

In this study we investigated 36 single nucleotide polymorphisms within 10 genes previously associated with major depression and bipolar disorder, as well as with the response to their treatment (ABCB1, ABCB4, TAP2, CLOCK, CPLX1, CPLX2, SYN2, NRG1, 5HTR1A and GPRIN2). No association with mood disorders and clinical outcomes was observed.