AKAP350 Is involved in the development of apical "canalicular" structures in hepatic cells HepG2

Hepatocytes are epithelial cells whose apical poles constitute the bile canaliculi. The establishment and maintenance of canalicular poles is a finely regulated process that dictates the efficiency of primary bile secretion. Protein kinase A (PKA) modulates this process at different levels. AKAP350 is an A-kinase anchoring protein that scaffolds protein complexes involved in modulating the dynamic structures of the Golgi apparatus and microtubule cytoskeleton, facilitating microtubule nucleation at this organelle. In this study, we evaluated whether AKAP350 is involved in the development of bile canaliculi-like structures in hepatocyte derived HepG2 cells. We found that AKAP350 recruits PKA to the centrosomes and Golgi apparatus in HepG2 cells. De-localization of AKAP350 from these organelles led to reduced apical cell polarization. A decrease in AKAP350 expression inhibited the formation of canalicular structures and impaired F-actin organization at canalicular poles. Furthermore, loss of AKAP350 expression led to diminished polarized expression of the p-glycoprotein (MDR1/ABCB1) at the apical "canalicular" membrane. AKAP350 knock down effects on canalicular structures formation and actin organization could be mimicked by inhibition of Golgi microtubule nucleation by depletion of CLIP associated proteins (CLASPs). Our data reveal that AKAP350 participates in mechanisms which determine the development of canalicular structures as well as accurate canalicular expression of distinct proteins and actin organization, and provide evidence on the involvement of Golgi microtubule nucleation in hepatocyte apical polarization.     

ABCB4 exports phosphatidylcholine in a sphingomyelin-dependent manner

ABCB4, which is specifically expressed on the canalicular membrane of hepatocytes, exports phosphatidylcholine (PC) into bile. Because SM depletion increases cellular PC content and stimulates PC and cholesterol efflux by ABCA1, a key transporter involved in generation of HDL, we predicted that SM depletion also stimulates PC efflux through ABCB4. To test this prediction, we compared the lipid efflux activity of ABCB4 and ABCA1 under SM depletion induced by two different types of inhibitors for SM synthesis, myriocin and (1R,3S)-N-(3-hydroxy-1-hydroxymethyl-3-phenylpropyl)dodecanamide, in human embryonic kidney 293 and baby hamster kidney cells. Unexpectedly, SM depletion exerted opposite effects on ABCB4 and ABCA1, suppressing PC efflux through ABCB4 while stimulating efflux through ABCA1. Both ABCB4 and ABCA1 were recovered from Triton-X-100-soluble membranes, but ABCB4 was mainly recovered from CHAPS-insoluble SM-rich membranes, whereas ABCA1 was recovered from CHAPS-soluble membranes. These results suggest that a SM-rich membrane environment is required for ABCB4 to function. ABCB4 must have evolved to exert its maximum activity in the SM-rich membrane environment of the canalicular membrane, where it transports PC as the physiological substrate.     

Stimulation of ABCB4/MDR3 ATPase activity requires an intact phosphatidylcholine lipid

ABCB4/MDR3 is located in the canalicular membrane of hepatocytes and translocates PC-lipids from the cytoplasmic to the extracellular leaflet. ABCB4 is an ATP-dependent transporter that reduces the harsh detergent effect of the bile salts by counteracting self-digestion. To do so, ABCB4 provides PC lipids for extraction into bile. PC lipids account for 40% of the entire pool of lipids in the canalicular membrane with an unknown distribution over both leaflets. Extracted PC lipids end up in so-called mixed micelles. Mixed micelles are composed of phospholipids, bile salts, and cholesterol. Ninety to ninety-five percent of the phospholipids are members of the PC family, but only a subset of mainly 16.0-18:1 PC and 16:0-18:2 PC variants are present. To elucidate whether ABCB4 is the key discriminator in this enrichment of specific PC lipids, we used in vitro studies to identify crucial determinants in substrate selection. We demonstrate that PC-lipid moieties alone are insufficient for stimulating ABCB4 ATPase activity, and that at least two acyl chains and the backbone itself are required for a productive interaction. The nature of the fatty acids, like length or saturation has a quantitative impact on the ATPase activity. Our data demonstrate a two-step enrichment and protective function of ABCB4 to mitigate the harsh detergent effect of the bile salts, because ABCB4 can translocate more than just the PC-lipid variants found in bile.     

Effects of cellular, chemical, and pharmacological chaperones on the rescue of a trafficking-defective mutant of the ATP-binding cassette transporter proteins ABCB1/ABCB4

The ATP-binding cassette transporter ABCB4 is a phosphatidylcholine translocator specifically expressed at the bile canalicular membrane in hepatocytes, highly homologous to the multidrug transporter ABCB1. Variations in the ABCB4 gene sequence cause progressive familial intrahepatic cholestasis type 3. We have shown previously that the I541F mutation, when reproduced either in ABCB1 or in ABCB4, led to retention in the endoplasmic reticulum (ER)/Golgi. Here, Madin-Darby canine kidney cells expressing ABCB1-GFP were used as a model to investigate this mutant. We show that ABCB1-I541F is not properly folded and is more susceptible to in situ protease degradation. It colocalizes and coprecipitates with the ER chaperone calnexin and coprecipitates with the cytosolic chaperone Hsc/Hsp70. Silencing of calnexin or overexpression of Hsp70 have no effect on maturation of the mutant. We also tested potential rescue by chemical and pharmacological chaperones. Thapsigargin and sodium 4-phenyl butyrate were inefficient. Glycerol improved maturation and exit of the mutant from the ER. Cyclosporin A, a competitive substrate for ABCB1, restored maturation, plasma membrane expression, and activity of ABCB1-I541F. Cyclosporin A also improved maturation of ABCB4-I541F in Madin-Darby canine kidney cells. In HepG(2) cells transfected with ABCB4-I541F cDNA, cyclosporin A allowed a significant amount of the mutant protein to reach the membrane of bile canaliculi. These results show that the best strategy to rescue conformation-defective ABCB4 mutants is provided by pharmacological chaperones that specifically target the protein. They identify cyclosporin A as a potential novel therapeutic tool for progressive familial intrahepatic cholestasis type 3 patients.     

Down-regulation of ABCB1 transporter by atorvastatin in a human hepatoma cell line and in human peripheral blood mononuclear cells

PURPOSE: The effect of atorvastatin, an HMG-CoA reductase inhibitor, on expression and activity of the drug transporter ABCB1 in HepG2 cells and peripheral blood mononuclear cells (PBMCs) was examined. METHODS: Localization and expression of ABCB1 in hepatocytes was examined by indirect immunofluorescence. Expression of ABCB1 mRNA and ABCB1 activity were examined in atorvastatin-treated and control cells and PBMCs using real-time PCR and Rhodamine 123 efflux assay. RESULTS: Immunohistochemical analysis revealed that ABCB1 is located at the apical membrane of the bile canaliculi. Atorvastatin at 10 and 20 microM up-regulated ABCB1 expression resulting in a significant 1.4-fold increase of the protein levels. Treatment of HepG2 cells with 20 microM atorvastatin caused a 60% reduction on mRNA expression (p<0.05) and a 41% decrease in ABCB1-mediated efflux of Rhodamine123 (p<0.01) by flow cytometry. Correlation was found between ABCB1 mRNA levels and creatine kinase (r=0.30; p=0.014) and total cholesterol (r=-0.31; p=0.010). CONCLUSIONS. Atorvastatin leads to decreased ABCB1 function and modulates ABCB1 synthesis in HepG2 cells and in PBMCs. ABCB1 plays a role in cellular protection as well as in secretion and/or disposition, therefore, inhibition of ABCB1 synthesis may increase the atorvastatin efficacy, leading to a more pronounced reduction of plasma cholesterol.     

Intracellular trafficking of bile salt export pump (ABCB11) in polarized hepatic cells: constitutive cycling between the canalicular membrane and rab11-positive endosomes

The bile salt export pump (BSEP, ABCB11) couples ATP hydrolysis with transport of bile acids into the bile canaliculus of hepatocytes. Its localization in the apical canalicular membrane is physiologically regulated by the demand to secrete biliary components. To gain insight into how such localization is regulated, we studied the intracellular trafficking of BSEP tagged with yellow fluorescent protein (YFP) in polarized WIF-B9 cells. Confocal imaging revealed that BSEP-YFP was localized at the canalicular membrane and in tubulo-vesicular structures either adjacent to the microtubule-organizing center or widely distributed in the cytoplasm. In the latter two locations, BSEP-YFP colocalized with rab11, an endosomal marker. Selective photobleaching experiments revealed that single BSEP-YFP molecules resided in canalicular membranes only transiently before exchanging with intracellular BSEP-YFP pools. Such exchange was inhibited by microtubule and actin inhibitors and was unaffected by brefeldin A, dibutyryl cyclic AMP, taurocholate, or PI 3-kinase inhibitors. Intracellular carriers enriched in BSEP-YFP elongated and dissociated as tubular elements from a globular structure adjacent to the microtubule-organizing center. They displayed oscillatory movement toward either canalicular or basolateral membranes, but only fused with the canalicular membrane. The pathway between canalicular and intracellular membranes that BSEP constitutively cycles within could serve to regulate apical pools of BSEP as well as other apical membrane transporters.     

A PDZ-Like Motif in the Biliary Transporter ABCB4 Interacts with the Scaffold Protein EBP50 and Regulates ABCB4 Cell Surface Expression

ABCB4/MDR3, a member of the ABC superfamily, is an ATP-dependent phosphatidylcholine translocator expressed at the canalicular membrane of hepatocytes. Defects in the ABCB4 gene are associated with rare biliary diseases. It is essential to understand the mechanisms of its canalicular membrane expression in particular for the development of new therapies. The stability of several ABC transporters is regulated through their binding to PDZ (PSD95/DglA/ZO-1) domain-containing proteins. ABCB4 protein ends by the sequence glutamine-asparagine-leucine (QNL), which shows some similarity to PDZ-binding motifs. The aim of our study was to assess the potential role of the QNL motif on the surface expression of ABCB4 and to determine if PDZ domain-containing proteins are involved. We found that truncation of the QNL motif decreased the stability of ABCB4 in HepG2-transfected cells. The deleted mutant ABCB4-ΔQNL also displayed accelerated endocytosis. EBP50, a PDZ protein highly expressed in the liver, strongly colocalized and coimmunoprecipitated with ABCB4, and this interaction required the QNL motif. Down-regulation of EBP50 by siRNA or by expression of an EBP50 dominant-negative mutant caused a significant decrease in the level of ABCB4 protein expression, and in the amount of ABCB4 localized at the canalicular membrane. Interaction of ABCB4 with EBP50 through its PDZ-like motif plays a critical role in the regulation of ABCB4 expression and stability at the canalicular plasma membrane.     

Effects of phosphatidylethanolamine N-methyltransferase on phospholipid composition, microvillus formation and bile salt resistance in LLC-PK1 cells

Bile salts are potent detergents and can disrupt cellular membranes, which causes cholestasis and hepatocellular injury. However, the mechanism for the resistance of the canalicular membrane against bile salts is not clear. Phosphatidylethanolamine (PE) is converted to phosphatidylcholine (PC) in the liver by phosphatidylethanolamine N-methyltransferase (PEMT). In this study, to investigate the effect of PEMT expression on the resistance to bile salts, we established an LLC-PK1 cell line stably expressing PEMT. By using enzymatic assays, we showed that the expression of PEMT increased the cellular PC content, lowered the PE content, but had no effect on the sphingomyelin content. Consequently, PEMT expression led to reductions in PE/PC and sphingomyelin/PC ratios. Mass spectrometry demonstrated that PEMT expression increased the levels of PC species containing longer acyl chains and almost all ether-linked PC species. PEMT expression enhanced the resistance to duramycin and lysenin, suggesting decreased ratios of PE and sphingomyelin in the apical membrane, respectively. In addition, SEM revealed that PEMT expression increased the diameter of microvilli. The expression of PEMT resulted in reduced resistance to unconjugated bile salts, but surprisingly in increased resistance to conjugated bile salts, which might be attributable to modifications of the phospholipid composition and/or structure in the apical membrane. Because most bile salts exist as conjugated forms in the bile canaliculi, PEMT may be important in the protection of hepatocytes from bile salts and in cholestatic liver injury.     

Sphingolipid Transport to the Apical Plasma Membrane Domain in Human Hepatoma Cells Is Controlled by PKC and PKA Activity: A Correlation with Cell Polarity in HepG2 Cells

The regulation of sphingolipid transport to the bile canalicular apical membrane in the well differentiated HepG2 hepatoma cells was studied. By employing fluorescent lipid analogs, trafficking in a transcytosis-dependent pathway and a transcytosis-independent (‘direct') route between the trans-Golgi network and the apical membrane were examined. The two lipid transport routes were shown to operate independently, and both were regulated by kinase activity. The kinase inhibitor staurosporine inhibited the direct lipid transport route but slightly stimulated the transcytosis-dependent route. The protein kinase C (PKC) activator phorbol-12 myristate-13 acetate (PMA) inhibited apical lipid transport via both transport routes, while a specific inhibitor of this kinase stimulated apical lipid transport. Activation of protein kinase A (PKA) had opposing effects, in that a stimulation of apical lipid transport via both transport routes was seen. Interestingly, the regulatory effects of either kinase activity in sphingolipid transport correlated with changes in cell polarity. Stimulation of PKC activity resulted in a disappearance of the bile canalicular structures, as evidenced by the redistribution of several apical markers upon PMA treatment, which was accompanied by an inhibition of apical sphingolipid transport. By contrast, activation of PKA resulted in an increase in the number and size of bile canaliculi and a concomitant enhancement of apical sphingolipid transport. Taken together, our data indicate that apical membrane-directed sphingolipid transport in HepG2 cells is regulated by kinases, which could play a role in the biogenesis of the apical plasma membrane domain.     

Enhancing effect of taurohyodeoxycholate on ABCB4-mediated phospholipid efflux

Hydrophilic bile salts, ursodeoxycholate and hyodeoxycholate, have choleretic effects. ABCB4, a member of the ABC transporter family, is essential for the secretion of phospholipids from hepatocytes into bile. In this study, we assessed the effects of taurine- or glycine-conjugated cholate, ursodeoxycholate and hyodeoxycholate on the ABCB4-mediated phosphatidylcholine (PC) efflux using Abcb4 knockout mice and HEK293 cells stably expressing ABCB4. To evaluate the effects of bile salts on bile formation in Abcb4^+/+ or Abcb4^−/− mice, the bile was collected during intravenous infusion of saline or bile salts. The biliary PC secretion in Abcb4^+/+ mice was significantly increased by the infusions of all tested bile salts, especially taurohyodeoxycholate. On the other hand, Abcb4^−/− mice exhibited extremely low secretion of PC into bile, which was not altered by bile salt infusions. We also showed that the PC efflux from ABCB4-expressing HEK293 cells was stimulated by taurohyodeoxycholate much more strongly than the other tested bile salts. However, taurohyodeoxycholate did not restore the activities of ABCB4 mutants. Furthermore, light scattering measurements demonstrated a remarkable ability of taurohyodeoxycholate to form mixed micelles with PC. Therefore, the enhancing effect of taurohyodeoxycholate on the ABCB4-mediated PC efflux may be due to the strong mixed micelle formation ability.     

Liver cell plasma membrane lipids and the origin of biliary phospholipid.

The liver cell plasma membranes of fed male Wistar rats were separated into a fraction rich in bile canaliculi and the remainder of the plasma membrane. Electron-microscopically, the bile canalicular fraction consisted almost exclusively of intact bile canaliculi with thier contiguous membranes. The remaining plasma membrane fraction consisted primarily of vesicles and sheets of membranes essentially free from the bile canaliculi. The bile canalicular membrane fraction contained relatively more total lipid, cholesterol, and phospholipid, and relatively less protein. Although the phospholipid composition of the two fractions was the same, the specific activity of the bile canalicular membrane phosholipids, up to 12 h following in vivo administration of [2-3H]glycerol, was always significantly greater than that of the remaining plasma membranes, and showed a biphasic response not found in the latter. The specific activity of the phosphatidylcholine, phosphatidylethanolamine and lysophosphatidylcholine of the bile canalicular membranes rose to a peak within 40 min after administration of the label, fell sharply and then rose to a second peak after 120 min. The specific activity of the sphingomyelin and phosphatidylserine plus phosphatidylinositol of the bile canalicular membranes and of all the phospholipids of the remaining plasma membranes diphasic pattern but increased steadily to reach a maximum at 120 min. The specific activity of biliary phosphatidylcholine followed a pattern identical to that of the phosphatidylcholine, phosphatidylethanolamine and lysophosphatidylcholine of the bile canalicular membrane fraction. These results show that the average rate of turnover of phospholipid in the bile canalicular membranes is considerably greater than that in the remaining plasma membrane and other cell membrane fractions; they indicate that the phospholipid of the bile canalicular membranes exists in two or more pools, turning over a different rates; and they support the concept that biliary phospholipid is derived from the bile canalicular membrane. The results also suggest that bile canalicular phospholipid may be derived from two different sources, in contrast to the remainong plasma membrane.     

Knockdown of hepatocyte aquaporin-8 by RNA interference induces defective bile canalicular water transport

Aquaporin-8 (AQP8) water channels, which are expressed in rat hepatocyte bile canalicular membranes, are involved in water transport during bile formation. Nevertheless, there is no conclusive evidence that AQP8 mediates water secretion into the bile canaliculus. In this study, we directly evaluated whether AQP8 gene silencing by RNA interference inhibits canalicular water secretion in the human hepatocyte-derived cell line, HepG2. By RT-PCR and immunoblotting we found that HepG2 cells express AQP8 and by confocal immunofluorescence microscopy that it is localized intracellularly and on the canalicular membrane, as described in rat hepatocytes. We also verified the expression of AQP8 in normal human liver. Forty-eight hours after transfection of HepG2 cells with RNA duplexes targeting two different regions of human AQP8 molecule, the levels of AQP8 protein specifically decreased by 60-70%. We found that AQP8 knockdown cells showed a significant decline in the canalicular volume of approximately 70% (P < 0.01), suggesting an impairment in the basal (nonstimulated) canalicular water movement. We also found that the decreased AQP8 expression inhibited the canalicular water transport in response either to an inward osmotic gradient (-65%, P < 0.05) or to the bile secretory agonist dibutyryl cAMP (-80%, P < 0.05). Our data suggest that AQP8 plays a major role in water transport across canalicular membrane of HepG2 cells and support the notion that defective expression of AQP8 causes bile secretory dysfunction in human hepatocytes.     

Structural polarity and functional bile canaliculi in rat hepatocyte spheroids

Primary hepatocytes self-assemble into spheroids that possess tight junctions and microvilli-lined channels. We hypothesized that polarity develops gradually and that the channels structurally and functionally resemble bile canaliculi. Immunofluorescence labeling of apical and basolateral proteins demonstrated reorganization of the membrane proteins into a polarized distribution during spheroid culture. By means of fluorescent dextran diffusion and confocal microscopy, an extensive network of channels was revealed in the interior of the spheroids. These channels connected over several planes and opened to pores on the surface. To examine the content of apical proteins in the channel membranes, the bile canalicular enzyme dipeptidyl peptidase IV (DPPIV) was localized using a fluorogenic substrate, Ala-Pro-cresyl violet. The results show that DPPIV activity is heterogeneously distributed in spheroids and localized in part to channels. Bile acid excretion was then investigated to demonstrate functional polarity. A fluorescent bile acid analogue, fluorescein isothiocyanate-labeled glycocholate, was taken up into the spheroids and excreted into bile canalicular channels. Due to the structural polarity of spheroids and their ability to excrete bile into channels, they are a unique three-dimensional model of in vitro liver tissue self-assembly. (Videoanimations of some results are available at http://hugroup.cems.umn.edu/research_movies).     

Vesicular and nonvesicular transport of phosphatidylcholine in polarized HepG2 cells

We have investigated the transport and canalicular enrichment of fluorescent phosphatidylcholine (PC) in HepG2 cells using the fluorescent analogs of PC C6-NBD-PC and β-BODIPY-PC. Fluorescent PC was efficiently transported to the biliary canaliculus (BC) and became enriched on the lumenal side of the canalicular membrane as shown for C6-NBD-PC. Some fluorescent PC was transported in vesicles to a subapical compartment (SAC) or apical recycling compartment (ARC) in polarized HepG2 cells as shown by colocalization with fluorescent sphingomyelin (C6-NBD-SM) and fluorescent transferrin, respectively. Extensive trafficking of vesicles containing fluorescent PC between the basolateral domain, the SAC/ARC and the BC as well as endocytosis of PC analogs from the canalicular membrane were found. Evidence for nonvesicular transport included enrichment of the PC-analog β-BODIPY-PC in the BC (t_1/2 = 3.54 min) prior to its accumulation in the SAC/ARC (t_1/2 = 18.5 min) at 37 °C. Transport of fluorescent PC to the canalicular membrane also continued after disruption of the actin or microtubule cytoskeleton and at 2 °C. These results indicate that: (i) a nonvesicular transport pathway significantly contributes to the canalicular enrichment of PC in hepatocytic cells, and (ii) vesicular transport of fluorescent PC occurs from both membrane domains via the SAC/ARC.     

Motility of bile canaliculi in the living animal: implications for bile flow

Modern fluorescence microscopic techniques were used to image the bile canalicular system in the intact rat liver, in vivo. By combining the use of sodium fluorescein secretion into bile, with digitally enhanced fluorescence microscopy and time-lapse video, it was possible to capture and record the canalicular motility events that accompany the secretion of bile in life. Active bile canalicular contractions were found predominantly in zone 1 (periportal) hepatocytes of the liver. The contractile movements were repetitive, forceful, and appeared unidirectional moving bile in a direction towards the portal bile ducts. Contractions were not seen in the network of canaliculi on the surface of the liver. Cytochalasin B administration resulted in reduced canalicular motility, progressive dilation of zone 1 canaliculi, and impairment of bile flow. Canalicular dilations invariably involved the branch points of the canalicular network. The findings add substantively to previous in vitro studies using couplets, and suggest that canalicular contractions contribute physiologically to bile flow in the liver.     

Development of bile canaliculi between chicken embryo liver cells in vivo and in vitro.

The development of an organized network of bile canaliculi is essential for the normal functioning of the liver. We have characterized bile canaliculus development in situ from Days 3-19 and in vitro in cultured hepatocyte monolayers using electron microscopical and immunofluorescent staining with antibodies that specifically recognize antigens of the bile canaliculus. Although the liver first forms as a discrete epithelial bud of endodermal tissue at stage 12-14 (45-53 h after laying), canaliculi were first detected by our antibodies at low levels in 4-day embryos and at high levels in stage 27 (5 days after laying) and later embryos. During Days 4, 5, and 6 the canaliculi near the periphery of the rudiment do not stain while canaliculi in central areas, closer to the gut, are strongly stained. During this transition period the ultrastructure of the canaliculi in the peripheral regions is also less developed than the central canaliculi where the antigens appear. By 7 days post laying, canaliculi throughout the entire liver rudiment express the marker antigens equally and have the ultrastructural characteristics of mature, functional canaliculi. Cells prepared from liver of embryos of 11 days incubation and grown in monolayer culture reformed discernible canalicular specializations, as determined by immunofluorescent staining and electron microscopy, but only transiently (for 1 to 3 days after plating). Not all of the antigens were expressed or polarized in these cultures. The capacity of the embryonic parenchymal cells to develop and maintain polarity appears to depend on factors possibly including age-dependent changes in the cells themselves, interactions with other cell types or extracellular matrix, or the shape of the cells.     

Delivery of short hairpin RNAs by transkingdom RNA interference modulates the classical ABCB1-mediated multidrug-resistant phenotype of cancer cells

Delivery of RNA interference (RNAi)-mediating agents to target cells is one of the major obstacles for the development of RNAi-based therapies. One strategy to overcome this barrier is transkingdom RNAi (tkRNAi). This technology uses non-pathogenic bacteria to produce and deliver therapeutic short hairpin RNA (shRNA) into target cells to induce RNAi. In this study, the tkRNAi approach was used for modulation of the “classical” ABCB1-mediated multidrug resistance (MDR) in human cancer cells. Subsequent to treatment with anti-ABCB1 shRNA expression vector bearing E. coli, MDR cancer cells (EPG85 257RDB) showed 45% less ABCB1 mRNA expression. ABCB1 protein expression levels were reduced to a point at which merely a weak band could be detected. Drug accumulation was enhanced 11-fold, to an extent that it reached 45% of the levels in non-resistant cells and resistance to daunorubicin was decreased by 40%. The data provide the proof-of-concept that tkRNAi is suitable for modulation of “classical” MDR in human cancer cells. Overall, the prototype tkRNAi system tested here did not yet attain the levels of gene silencing seen with conventional siRNAs nor virally delivered shRNAs; but the tkRNAi system for gene-silencing of ABCB1 is still being optimized, and may become a powerful tool for delivery of RNAi effectors for the reversal of cancer MDR in future.     

Inhibition of Transient Receptor Potential Channel 5 Reverses 5-Fluorouracil Resistance in Human Colorectal Cancer Cells

5-Fluorouracil (5-Fu) is commonly used in the chemotherapy of colorectal cancer (CRC), but resistance to 5-Fu occurs in most cases, allowing cancer progression. Suppressing ABCB1 (ATP-binding cassette, subfamily B, member 1), which is a pump overproduced in cancer cells to export cytotoxic drugs, is an attractive strategy to overcome drug resistance. In the present study, transient receptor potential channel TrpC5 was found to be overproduced at the mRNA and protein levels together with ABCB1 in 5-Fu-resistant human CRC HCT-8 (HCT-8/5-Fu) and LoVo (LoVo/5-Fu) cells. More nuclear-stabilized β-catenin accumulation was found in HCT-8/5-Fu and LoVo/5-Fu cells than in HCT-8 and LoVo cells. Suppressing TrpC5 expression with TrpC5-specific siRNA inhibited the canonical Wnt/β-catenin signal pathway, reduced the induction of ABCB1, weakened the ABCB1 efflux pump, and caused a remarkable reversal of 5-Fu resistance in HCT-8/5-Fu and LoVo/5-Fu cells. On the contrary, enforcing TrpC5 expression resulted in an activated Wnt/β-catenin signal pathway and up-regulation of ABCB1. Taken together, we demonstrated an essential role of TrpC5 in ABCB1 induction and drug resistance in human CRC cells via promoting nuclear β-catenin accumulation.     

Fluorescence anisotropy from diphenylhexatriene in rat liver plasma membranes

Rat livers were fractionated to obtain intracellular membrane preparations and a highly purified preparation of bile canaliculi. The fraction containing bile canaliculi was homogenized and subfractionated to give fractions representing fragments of contiguous membrane and of canalicular microvilli. The relative purity and extent of contamination of each preparation was determined. When the fluorescent probe 1,6-diphenyl-1,3,5-hexatriene was incorporated into aliquots of each fraction at the same probe: lipid ratio and the steady-state anisotropy of its fluorescence measured, it was found that the plasma membrane preparations were much more ordered than the intracellular membrane preparations. Of the plasma membrane preparations, that containing the canalicular microvilli was the most ordered, even allowing for any contribution of contaminants. Thus the microvillus membrane of the bile canaliculus appears to be the most ordered domain of the plasma membrane of the hepatocyte. The high order in this domain may be a factor in reducing the susceptibility to bile salt damage during bile secretion, since it is this region which is exposed to high concentrations of bile salts in vivo.