Effect Of Bafilomycin A1 Research Articles

The role of bafilomycin as a therapeutic agent in the modulation of endo-lysosomal store of rat hepatocytes

Endo-lysosomal system through the process of autophagy is involved in the pathogenesis of many diseases. Acidification of these organelles is carried out by V-type H+-ATPases, which is inhibited by bafilomycin A1. Endosomes and lysosomes are also important Ca2+-storage in a cell. Nіcotіnіc acіd adenіne dіnucleotіde phosphate (NAADP) releases Cа2+ from endo-lysosomes. The main purpose of the study was to found out the effect of bafilomycin A1 and NAADP on stored Ca2+ and on the ATPase activity of rat hepatocytes. The stored Ca2+ was estimated using chlorotetracycline in permeabilized hepatocytes of rats. ATPase activity was determined by level of orthophosphate spectrophotometrically. It was found that bafilomycin A1 reduces stored Ca2+ in permeabilized hepatocytes of rats in the micromolar range of concentration (20 and 0.04 mkM) and averted the effect of NAADP on calcium content. Lower concentrations of bafilomycin A1 (0.001 mkM) did not alter the content of stored calcium, but prevented the influence of NAADP in permeabilized hepatocytes of rats. In the subcellular fraction of rat liver bafi­lomycin A1 (0.001 mkM) increased Ca2+-ATPase and basal Mg2+-ATPase activities and reduced Na+/K+-ATPase acti­vity. Preincubation of the subcellular fraction with bafilomycin A1 completely averts any changes in the activity of estimated ATPases by means of NAADP. It was concluded that the bafilomycin-sensitive store in hepatocytes of rats is NAADP-sensitive endo-lysosomal Ca2+-store. Using of bafilomycin A1 may be useful in treating autophagy-depended diseases.

Детекция окислительного стресса в артериальных эндотелиальных клетках человека при воздействии кальций-фосфатных бионов

Введение. Кальций-фосфатные бионы (КФБ) формируются в организме человека при перенасыщении сыворотки ионами кальция и фосфора и вызывают дисфункцию эндотелия, однако молекулярные механизмы нарушения функционирования эндотелия при воздействии КФБ не ясны. Цель исследования - выяснение роли кальций-фосфатных бионов различной формы в развитии окислительного стресса в артериальных эндотелиальных клетках (ЭК) человека. Методика. Для детекции окислительного стресса к конфлюэнтным культурам первичных ЭК коронарной и внутренней грудной артерии человека добавляли равные концентрации КФБ сферической или игольчатой формы (СКФБ и ИКФБ соответственно) с последующим культивированием в течение 1 и 4 ч, добавлением флюоресцентных индикаторов окислительного стресса MitoSOX Red и CellROX Green и конфокальной микроскопией. Измеряли концентрацию продуктов перекисного окисления липидов в культуральной жидкости через 24 ч экспозиции эндотелиальных клеток КФБ. Анализ нейтрализации цитотоксических эффектов перекисного окисления липидов проводили путем добавления к ЭК супероксиддисмутазы и каталазы на 4 или 24 ч (одновременно с КФБ). Для сравнения механизмов клеточной гибели при воздействии СКФБ и ИКФБ анализировали цитотоксичность обоих типов бионов при одновременном воздействии лизосомального ингибитора бафиломицина А1. Результаты. Значимого увеличения генерации активных форм кислорода (АФК) в результате экспозиции СКФБ (независимо от линии ЭК и продолжительности экспозиции) не было выявлено. В то же время наблюдалось повышение генерации супероксида через 4 ч, а иных свободных радикалов через 1 ч после добавления ИКФБ к ЭК. Предварительная нейтрализация АФК супероксиддисмутазой и каталазой частично защищала ЭК от индуцируемой ИКФБ гибели. При этом добавление бафиломицина А1 к ЭК частично защищало их от гибели только при воздействии СКФБ, но не ИКФБ. Заключение. Гибель ЭК при воздействии СКФБ происходит в результате первичного повреждения лизосом, а при воздействии ИКФБ - в первую очередь вследствие окислительного стресса. Background. Calcium phosphate bions (CPB) form in the human blood upon its supersaturation with calcium and phosphate and provoke endothelial dysfunction; however, the molecular mechanisms of these pathological processes remain unclear. Aim. To elucidate the role of differently shaped CPBs in induction of oxidative stress in human arterial endothelial cells (Ecs). Methods. For detection of oxidative stress, equal concentrations of spherical CPB (CPB-S) or needle-shaped CPB (CPB-N) were added to confluent cultures of primary human coronary artery and internal thoracic artery ECs for 1 and 4 h; this was followed by MitoSOX Red and CellROX Green staining and subsequent confocal microscopy. Concentration of thiobarbituric acid-reactive substances was measured in the EC culture supernatant at 24 h of the CPB exposure. The lipid peroxidation cytotoxicity was neutralized by adding superoxide dismutase and catalase to ECs for 4 or 24 h. To compare cell death subroutines induced by CPB-S and CPB-N, the effect of bafilomycin A1, a lysosomal inhibitor, on CRB cytotoxicity was studied. Results. No increase in reactive oxygen species generation was observed in the CPB-S exposure, regardless of the EC line and exposure duration. However, addition of CPB-N to ECs increased the production of superoxide and other free radicals after four- and one-hour exposure, respectively. Prior neutralization of reactive oxygen species with superoxide dismutase and catalase partially protected ECs from CPB-N- but not CPB-S-induced death while bafilomycin A1, vice versa, protected ECs from CPB-S- but not CPB-N-induced death. Conclusion. CPB-S cause cell death due to primary damage of lysosomes whereas CPB-N induce apoptosis due to oxidative stress.

C-jun N-terminal kinase dependent autophagic cell death in cancer cells induced by Zanthoxylum fruit extract from Japanese pepper.

653 Background: Natural products constitute a promising resource for drug development including an anticancer drug. Zanthoxylum fruit, obtained from the Japanese pepper plant (Zanthoxylum piperitum De Candolle), and its extract (Zanthoxylum fruit extract, ZFE) is an important component of Daikenchuto, which is a form of Japanese traditional medicine. Recently, we have reported that Daikenchuto has an anticancer activity in vivo, however precise mechanism is still unclear. Therefore, we investigated the potential anticancer activity of ZFE as an inducer of autophagic cell death (ACD). Methods: ZFE powder was provided by Tsumura (Japan). We investigated the effect of ZFE on the morphology of six types of human cancer cells and normal cells by using phase contrast microscopy and electron microscopy. Knockdown of autophagy-related gene 5 (ATG5), which is an essential gene for autophagy, by transfecting small interfering RNA was performed and confirmed by quantitative RT-qPCR and Western blot analysis. Effect of bafilomycin A1 (Baf A1), an inhibitor of vacuolar type H+-ATPases, on the anticancer activity of ZFE was investigated. Western blot analysis revealed LC3-II levels, a marker of autophagy. Results: ZFE caused remarkable autophagy-like cytoplasmic vacuolization with the inhibition of cell proliferation and subsequent induction of cell death in human cancer cell lines, DLD-1, HepG2 and Caco-2 cells but not in A549, MCF-7 or WiDr cells. ZFE increased LC3-II protein levels. Suppression of an ATG5 using siRNA inhibited ZFE-induced cytoplasmic vacuolization and cell death. Moreover, ZFE increased the phosphorylation of c-jun N-terminal kinase (JNK) in cancer cells which can be induced cell death by ZFE and JNK inhibitor SP600125 attenuated both vacuolization and cell death induced by ZFE. Instead, ZFE-induced cell death was neither apoptosis nor necrosis according to the morphological perspective and the marker of apoptosis or necrosis. And normal intestinal cell was not affected by ZFE. Conclusions: ZFE induces JNK-dependent ACD, which appears to be the main mechanism underlying its anticancer activity, suggesting a promising starting point for anticancer drug development.

Endosomal escape and siRNA delivery with cationic shell crosslinked knedel-like nanoparticles with tunable buffering capacities

Cationic shell crosslinked knedel-like nanoparticles (cSCKs) have emerged as a highly efficient transfection agent for nucleic acids delivery. In this study, a new class of cSCKs with tunable buffering capacities has been developed by altering the amounts of histamines and primary amines incorporated into their crosslinked shell regions. The effect of histamine content of these nanoparticles with a hydrodynamic diameter of ca. 20 nm, on the siRNA-binding affinity, cytotoxicity, immunogenicity, and transfection efficiency was investigated. The modification of cSCKs with histamine was found to reduce the siRNA-binding affinity and cellular binding. On the other hand, it significantly reduced the toxicity and immunogenicity of the nanoparticles with subsequent increase in the transfection efficiency. In addition, escape from endosomes was facilitated by having two species of low and high pKas (i.e. histamine and primary amine groups, respectively), as demonstrated by the potentiometric titration experiments and the effect of bafilomycin A1, an inhibitor of the endosomal acidification, on the transfection efficiency of cSCKs. Histamine modification of 15 mol% was a threshold, above which cSCKs with higher histamine content completely lost the ability to bind siRNA and to transfect cells. This study highlights the potential of histamine incorporation to augment the gene silencing activity of cationic nanoparticles, reduce their toxicity, and increase their biocompatibility, which is of particular importance in the design of nucleic acids delivery vectors.

Synaptic vesicles control the time course of neurotransmitter secretion via a Ca2+/H+ antiport

We investigated the physiological role of the vesicular Ca2+/H+ antiport in rapid synaptic transmission using the Torpedo electric organ (a modified neuromuscular system). By inhibiting V-type H+-transporting ATPase (V-ATPase), bafilomycin A1 dissipates the H+ gradient of synaptic vesicles, thereby abolishing the Ca2+/H+ antiport driving force. In electrophysiology experiments, bafilomycin A1 significantly prolonged the duration of the evoked electroplaque potential. A biochemical assay for acetylcholine (ACh) release showed that the effect of bafilomycin A1 was presynaptic. Indeed, bafilomycin A1 increased the amount of radio-labelled ACh released in response to paired-pulse stimulation. Bafilomycin A1 also enhanced Ca2+-dependent ACh release from isolated nerve terminals (synaptosomes). The bafilomycin-induced electroplaque potential lengthening did not arise from cholinesterase inhibition, since eserine (which also prolonged the electroplaque potential) strongly decreased evoked ACh release. Bafilomycin A1 augmented the amount of calcium accumulating in nerve terminals following a short tetanic stimulation and delayed subsequent calcium extrusion. By reducing stimulation-dependent calcium accumulation in synaptic vesicles, bafilomycin A1 diminished the corresponding depletion of vesicular ACh, as tested using both intact tissue and isolated synaptic vesicles. Strontium ions inhibit the vesicular Ca2+/H+ antiport, while activating transmitter release at concentrations one order of magnitude higher than Ca2+ does. In the presence of Sr2+ the time course of the electroplaque potential was also prolonged but, unlike bafilomycin A1, Sr2+ enhanced facilitation in paired-pulse experiments. It is therefore proposed that the vesicular Ca2+/H+ antiport function is to shorten 'phasic' transmitter release, allowing the synapse to transmit briefer impulses and so to work at higher frequencies.

Efficient Gene Delivery with Osmotically Active and Hyperbranched Poly(ester amine)s

Degradable and hyperbranched poly (ester amine)s (PEAs) were successfully synthesized by Michael addition reaction between hydrophilic glycerol triacrylate (GTA) and low-molecular-weight polyethylenimine (LMW-PEI) and evaluated as nonviral gene carriers. PEAs effectively condensed DNA with particle sizes below 200 nm and suitable surface charges (15-45 mV), suitable for intracellular delivery. PEAs degraded in a controlled fashion showing half-lives of more than 12 days and were essentially nontoxic in three different cell lines. Elevated transfection levels by luciferase assay revealed the superiority of PEAs over PEI 25K and Lipofectamine. PEAs synthesized using 1:4 mol ratio of GTA to PEI [GTA/PEI-1.2(1:4)] showed highest transfection efficiency in HepG2 cells. PEAs showed significant gene expression in vitro as well as in vivo through aerosol administration. Reduction in packed cell volume (PCV) of cells when treated with polyplexes supported the hyperosmotic effect of PEAs. Effect of bafilomycin A1 on transfection efficiency of PEAs on 293T cells indicated its endosomal buffering capacity. High transfection efficiency was attributed to the synergism from hyperosmotic glycerol backbone in the PEAs and endosomal buffering capacity of PEI amine groups. Therefore, this convergence of osmotically active biodegradable PEAs suggests their potential as a safe and efficient gene delivery vector.

The effect of bafilomycin A1 and protease inhibitors on the degradation and recycling of a Class 5-mutant LDLR

The low-density lipoprotein receptor (LDLR) mediates cholesterol homeostasis through endocytosis of lipoprotein particles, particularly low-density lipoproteins (LDLs). Normally, the lipoprotein particles are released in the endosomes and the receptors recycle to the cell surface. Familial hypercholesterolemia (FH) is an autosomal dominant disease caused by mutations in the gene encoding the LDLR. These mutations are divided into five functional classes where Class 5 mutations encode receptors that suffer from ligand-induced degradation and recycling deficiency. The aim of this study was to investigate whether it is possible to prevent the fast ligand-induced degradation of Class 5-mutant LDLR and to restore its ability to recycle to the cell surface. E387K is a naturally occurring Class 5 mutation found in FH patients, and in the present study, we used Chinese hamster ovary cells transfected with an E387K-mutant LDLR. Abrogation of endosomal acidification by adding bafilomycin A1 or addition of the irreversible serine protease inhibitors, 4-(2-aminoethyl)-benzenesulfonyl fluoride (AEBSF) and 3,4-dichloroisocoumarin (DCI), prevented the degradation of the E387K-mutant LDLR. However, the undegraded receptor did not recycle to the cell surface in the presence of LDL. Unexpectedly, AEBSF caused aggregation of early endosome antigen-1- positive endosomes and the intracellular trapped LDLR co-localized with these aggregated early endosomes.

Acquisition of the Vacuolar ATPase Proton Pump and Phagosome Acidification Are Essential for Escape of Francisella tularensis into the Macrophage Cytosol

The Francisella tularensis-containing phagosome (FCP) matures to a late-endosome-like phagosome prior to bacterial escape into the cytosols of macrophages, where bacterial proliferation occurs. Our data show that within the first 15 min after infection of primary human monocyte-derived macrophages (hMDMs), approximately 90% of the FCPs acquire the proton vacuolar ATPase (vATPase) pump and the lysomotropic dye LysoTracker, which concentrates in acidic compartments, similar to phagosomes harboring the Listeria monocytogenes control. The acquired proton vATPase pump and lysomotropic dye are gradually lost by 30 to 60 min postinfection, which coincides with bacterial escape into the cytosols of hMDMs. Colocalization of phagosomes harboring the iglD mutant with the vATPase pump and the LysoTracker dye was also transient, and the loss of colocalization was faster than that observed for the wild-type strain, which is consistent with the faster escape of the iglD mutant into the macrophage cytosol. In contrast, colocalization of both makers with phagosomes harboring the iglC mutant was persistent, which is consistent with fusion to the lysosomes and failure of the iglC mutant to escape into the macrophage cytosol. We have utilized a fluorescence microscopy-based phagosome integrity assay for differential labeling of vacuolar versus cytosolic bacteria, using antibacterial antibodies loaded into the cytosols of live hMDMs. We show that specific inhibition of the proton vATPase pump by bafilomycin A1 (BFA) blocks rapid bacterial escape into the cytosols of hMDMs, but 30% to 50% of the bacteria escape into the cytosol by 6 to 12 h after BFA treatment. The effect of BFA on the blocking of bacterial escape into the cytosol is completely reversible, as the bacteria escape after removal of BFA. We also show that the limited fusion of the FCP to lysosomes is not due to failure to recruit the late-endosomal fusion regulator Rab7. Therefore, within few minutes of its biogenesis, the FCP transiently acquires the proton vATPase pump to acidify the phagosome, and this transient acidification is essential for subsequent bacterial escape into the macrophage cytosol.

Role of H+-ATPase-mediated Acidification in Sorting and Release of the Regulated Secretory Protein Chromogranin A: EVIDENCE FOR A VESICULOGENIC FUNCTION

The constitutive and regulated secretory pathways represent the classical routes for secretion of proteins from neuroendocrine cells. Selective aggregation of secretory granule constituents in an acidic, bivalent cation-rich environment is considered to be a prerequisite for sorting to the regulated secretory pathway. The effect of selective vacuolar H+-ATPase (V-ATPase) inhibitor bafilomycin A1 on the pH gradient along the secretory pathway was used here to study the role of acidification on the trafficking of the regulated secretory protein chromogranin A (CgA) in PC12 cells. Sorting of CgA was assessed by three-dimensional deconvolution microscopy, subcellular fractionation, and secretagogue-stimulated release, examining a series of full-length or truncated domains of human CgA (CgA-(1-115), CgA-(233-439)) fused to either green fluorescent protein or to a novel form of secreted embryonic alkaline phosphatase (EAP). We show that a full-length CgA/EAP chimera is sorted to chromaffin granules for exocytosis. Inhibition of V-ATPase by bafilomycin A1 markedly reduced the secretagogue-stimulated release of CgA-EAP by perturbing sorting of the chimera (at the trans-Golgi network or immature secretory granule) rather than the late steps of exocytosis. The effect of bafilomycin A1 on CgA secretion depends on a sorting determinant located within the amino terminus (CgA-(1-115)) but not the C-terminal region of the granin. Moreover, examination of chromaffin granule abundance in PC12 cells exposed to bafilomycin A1 reveals a substantial decrease in the number of dense-core vesicles. We propose that a V-ATPase-mediated pH gradient in the secretory pathway is an important factor for the formation of dense-core granules by regulating the ability of CgA to form aggregates, a crucial step that may underlie the granulogenic function of the protein.

Specific biological functions of vacuolar-type H(+)-ATPase and lysosomal cysteine proteinase, cathepsin K, in osteoclasts.

We report the effects of specific and potent inhibitors of vacular-type H(+)-ATPase and lysosomal cysteine proteinases, cathepsins, on the ultrastructure, expression of these enzymes, and resorptive functions of cultured osteoclasts. Osteoclasts were formed by co-culture of marrow cells and calvarial primary osteoblasts of ddY mice. Formed osteoclasts were cultured on dentine slices for 6-48 hr with either an H(+)-ATPase inhibitor, bafilomycin A1, or a cysteine proteinase inhibitor, E-64. In control cultures with no additive, osteoclasts were structurally characterized by the development of ruffled borders and clear zones, and formed many resorption lacunae on dentine slices. Both H(+)-ATPase and cathepsin K were strongly expressed in the ruffled borders of these osteoclasts. In bafilomycin A1-treated cultures, osteoclasts lacked ruffled borders, and resorption lacuna formation was markedly diminished. This effect of bafilomycin A1 on osteoclast structure was reversible by removal of the compound. Bafilomycin A1 treatment altered the subcellular localization and decreased the expression of H(+)-ATPase molecules. H(+)-ATPase expression was observed throughout the cytoplasm, but not along the plasma membranes facing dentine slices. On the other hand, E-64 treatment did not affect the ultrastructure of osteoclasts and the expression of enzyme molecules. Although E-64 showed no effect on demineralization of dentine slices, it dose-dependently reduced resorption lacuna formation. Our results suggest that 1) bafilomycin A1 dose-dependently inhibits resorption lacuna formation via inhibition of ruffled border formation, 2) H(+)-ATPase expression is closely associated with the cytoskeleton of osteoclasts, and 3) E-64 treatment decreases the depth of resorption lacunae, by inhibition of secreted cathepsin K activity, but does not impair ruffled border formation and the associated expression of H(+)-ATPase and cathepsin K in osteoclasts.

Effect of bafilomycin A1, a specific inhibitor of vacuolar (V-type) proton ATPases, on the capacitation of rabbit spermatozoa

Mammalian spermatozoa maintain precisely regulated ionic gradients that must be modified during capacitation and the acrosome reaction. In other cell types, ionic gradients are mainly regulated by the presence in plasma membranes of three metabolically different types of ATPases. The modifications induced during in vitro capacitation of rabbit spermatozoa by the specific inhibition of V-type H+-ATPases with bafilomycin A were studied. We used chlortetracycline binding to rabbit spermatozoa to monitor capacitation, and the coomassie brilliant blue method to identify acrosome-reacted sperm cells. There was a significant difference between the percentage of epididymal (66 +/- 7%) and ejaculated (43 +/- 11%) spermatozoa capacitated in vitro, after a 6-h incubation period in the presence of Ca2+ without ATPase inhibitor. The presence of bafilomycin significantly reduced these numbers (25 +/- 11 and 16+/- 8%, epididymal and ejaculated spermatozoa, respectively) and eliminated the difference. Ejaculated spermatozoa capacitated in the absence of bafilomycin showed a linear increase in the percentage of acrosome reactions induced by the addition of A23187 (12 +/- 5, 23+/- 6 and 31 +/- 5 after 15, 30 and 45 min). The presence of 0.2 micromol l-1 bafilomycin during the capacitation incubation induced a significant decrease in the acrosome reaction percentages (4 +/- 2, 8 +/- 3 and 14 +/- 4 after 15, 30 and 45 min). The addition of bafilomycin after the capacitating period had no effect upon the induction of the acrosome reaction by A23187. These results indicate that vacuolar ATPases play an important role during rabbit sperm capacitation. However, once the spermatozoa have been capacitated, V-type ATPases do not have a significant participation during the acrosome reaction.

Effect of bafilomycin A1 and nocodazole on endocytic transport in HeLa cells: implications for viral uncoating and infection.

Bafilomycin A1 (baf), a specific inhibitor of vacuolar proton ATPases, is commonly employed to demonstrate the requirement of low endosomal pH for viral uncoating. However, in certain cell types baf also affects the transport of endocytosed material from early to late endocytic compartments. To characterize the endocytic route in HeLa cells that are frequently used to study early events in viral infection, we used 35S-labeled human rhinovirus serotype 2 (HRV2) together with various fluid-phase markers. These virions are taken up via receptor-mediated endocytosis and undergo a conformational change to C-antigenic particles at a pH of <5.6, resulting in release of the genomic RNA and ultimately in infection (E. Prchla, E. Kuechler, D. Blaas, and R. Fuchs, J. Virol. 68:3713-3723, 1994). As revealed by fluorescence microscopy and subcellular fractionation of microsomes by free-flow electrophoresis (FFE), baf arrests the transport of all markers in early endosomes. In contrast, the microtubule-disrupting agent nocodazole was found to inhibit transport by accumulating marker in endosomal carrier vesicles (ECV), a compartment intermediate between early and late endosomes. Accordingly, lysosomal degradation of HRV2 was suppressed, whereas its conformational change and infectivity remained unaffected by this drug. Analysis of the subcellular distribution of HRV2 and fluid-phase markers in the presence of nocodazole by FFE revealed no difference from the control incubation in the absence of nocodazole. ECV and late endosomes thus have identical electrophoretic mobilities, and intraluminal pHs of <5.6 and allow uncoating of HRV2. As bafilomycin not only dissipates the low endosomal pH but also blocks transport from early to late endosomes in HeLa cells, its inhibitory effect on viral infection could in part also be attributed to trapping of virus in early endosomes which might lack components essential for uncoating. Consequently, inhibition of viral uncoating by bafilomycin cannot be taken to indicate a low pH requirement only.

Intracellular trafficking of tropoelastin.

Elastin is secreted as soluble tropoelastin monomers which are then cross-linked in the presence of extracellular microfibrils to form insoluble elastic fibers. Although the secretion of tropoelastin is thought to be mediated and targeted by an intracellular chaperone complex, the intracellular route taken by this protein and the role of such a chaperone complex remain undefined. In the present study, the specific pathway of tropoelastin secretion was investigated in fetal bovine chondrocytes and ligamentum nuchae fibroblasts by immunofluorescence staining and immunoprecipitation of tropoelastin following treatment with secretion-disrupting agents. In untreated cells, tropoelastin is secreted in approximately 30 min. In both cell types, brefeldin A and monensin inhibited secretion of tropoelastin and caused an intracellular accumulation of the protein in the fused ER/Golgi compartment or in the Golgi stacks, respectively. Incubations of longer than 1 h in the presence of brefeldin A result in eventual degradation of tropoelastin in the ER/Golgi compartment (Davis and Mecham, 1996). In contrast, the tropoelastin trapped in the Golgi as a result of monensin treatment steadily accumulated. Agents that elevate intracellular pH, such as ammonium chloride and chloroquine, also caused an intracellular accumulation of tropoelastin which appeared by immunofluorescence staining to be localized in secretory vesicles and/or endosomes. Since weak bases and ionophores alter the morphology of vacuolar compartments, the effect of bafilomycin A1 on tropoelastin secretion was also investigated. This vacuolar H+-ATPase inhibitor prevents acidification of the trans-Golgi network and endosomal compartments without disrupting intracellular organelle formation. When the elastogenic cells were treated with bafilomycin A1, tropoelastin secretion was diminished and an intracellular accumulation of tropoelastin was detected in the trans-Golgi network and small secretory vesicles. These results suggest that tropoelastin may be diverted from the constitutive pathway after exiting the Golgi and instead targeted to an acidic compartment prior to transport to the cell surface. The identity and role of such a compartment in the sorting and/or trafficking of tropoelastin has yet to be determined.

Bafilomycin A1 induces apoptosis in the human pancreatic cancer cell line Capan-1.

Bafilomycin A1, a specific inhibitor of vacuolar type H(+)-ATPase, can inhibit the growth of a variety of cultured cells in a dose-dependent manner, but its mechanism is unclear. The aim of this study was to examine whether bafilomycin A1 inhibits the growth of Capan-1 human pancreatic cancer cells through apoptosis. The effect of bafilomycin A1 on tumour growth in vitro and in vivo was examined using an MTT assay and an in vivo tumour model. The presence or absence of apoptosis was determined by morphology and DNA analysis of tumour cells. The concentration of bafilomycin A1 for 50 per cent inhibition of cell viability during 72 h by the MTT assay was 5 nm. In DNA analysis, a ladder of fragmented DNA was detected in Capan-1 cells treated with bafilomycin A1 at concentrations greater than 10 nm for 24 h. Nude mice bearing a xenografted Capan-1 cell line tumour received 4 weeks of bafilomycin A1 (1.0 mg/kg per day). This treatment significantly inhibited tumour growth compared with controls after 21 days (P < 0.05). Histopathological examination of tumour cells in the treated group demonstrated signs of apoptosis with chromatin condensation and cell shrinkage. These observations suggest that bafilomycin A1 inhibits the growth of Capan-1 human pancreatic cancer cells through apoptosis.