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PubMed1979

Hemocytes and hemocytopoiesis in Silkworms.

A brief review is presented of the current state of ultrastructure, cytochemistry, and physiology of the hemocytes and meso- and metathoracic peri-imaginal-wing organs in silkworms. According to the accepted morphological classification, five circulating types of hemocytes are recognized in Bombyx mori as well as in Antheraea pernyi. They are prophemocytes or stem cells, plasmatocytes or pre-differentiated cells and three specialized cells, granulocytes, spherule cells and oenocytoids. During post-embryonic development the last four types are the most common in the circulating hemolymph. Plasmatocytes are considered to be pluripotent cells from which granulocytes, spherule cells and oenocytoids are derived. Contrary to the situation in most insects the plasmatocytes are not phagocytic in Antheraea. The granulocytes are efficient phagocytes. Both plasmatocytes and granulocytes are involved in pinocytosis. Another possible function of the granulocytes is hemolymph coagulation. The function of the spherule cells which contain a paracrystalline material (muco- or glycoproteins) is by no means clear. The phenoloxidase activity found within the cytosol of oenocytoids appears effective against the natural monophenol and diphenol substrates. The involvement of oenocytoids in the complex metabolism of phenols and particularly in the production of plasma phenolases has been reported. The mitotic division of five circulating hemocyte types is well known and was long regarded as the only mechanism of postembryonic hemocyte production. We present for silkworms, experimental evidence of the hemocytopoietic function of the meso- and metathoracic organs surrounding the imaginal wing discs. Ablation experiments demonstrate that the mitotic activity of free hemocytes is unable to maintain the normal hemocytogram in the absence of the two paris of organs. These organs are typically divided into cell islets ensheathed by a connective tissue membrane. Two types of islets may be classified by the disposition of the cells : the compact islets or aggregations of stem cells and the reticulate islets which are mainly composed of hemocytes at different steps of differentiation. The relative number of prohemocytes in the total hemocyte population ranges from 84 to 97 p. cent in organs of Antheraea pernyi. This well-defined cell type appears to be the major hemocyte type in hemocytopoietic organs. In Antheraea, the mitotic index (the relative number of mitotic hemocytes in the total cell population) varies from 0.5 to about 3 p. cent. Finally, our data direct attention to cyclic functional changes such as mitotic divisions and hemocyte differentiation which run parallel to the molting cycle.

Animals↗
PubMed2002

Clinical implications of antigen transfer mechanisms from malignant to dendritic cells. exploiting cross-priming.

Expansion and activation of cytolytic T lymphocytes bearing high-affinity T-cell receptors specific for tumor antigens is a major goal of active cancer immunotherapy. Physiologically, T cells receive promitotic and activating signals from endogenous professional antigen-presenting cells (APC) rather than directly from malignant cells. This phenomenon fits with the broader concept of cross-presentation that earlier was demonstrated for minor histocompatibility and viral antigens. Many mechanisms have been found to be capable of transferring antigenic material from malignant cells to APC so that it can be processed and subsequently presented by MHC class I molecules expressed on APC. Dendritic cells (DC) are believed to be the most relevant APC mediating cross-presentation because they can take up antigens from apoptotic, necrotic, and even intact tumor cells. There exist specific molecular mechanisms that ensure this transfer of antigenic material: 1) opsonization of apoptotic bodies; 2) receptors for released heat shock proteins carrying peptides processed intracellularly; 3) Fc receptors that uptake immunocomplexes and immunoglobulins; and 4) pinocytosis. DC have the peculiar capability of reentering the exogenously captured material into the MHC class I pathway. Exploitation of these pieces of knowledge is achieved by providing DC with complex mixtures of tumor antigens ex vivo and by agents and procedures that promote infiltration of malignant tissue by DC. The final outcome of DC cross-presentation could be T-cell activation (cross-priming) but also, and importantly, T-cell tolerance contingent upon the activation/maturation status of DC. Artificial enhancement of tumor antigen cross-presentation and control of the immune-promoting status of the antigen-presenting DC will have important therapeutic implications in the near future.

Animals↗
PubMed1997

The molecular mechanisms of the metabolism and transport of iron in normal and neoplastic cells.

Iron uptake by mammalian cells is mediated by the binding of serum Tf to the TfR. Transferrin is then internalized within an endocytotic vesicle by receptor-mediated endocytosis and the Fe released from the protein by a decrease in endosomal pH. Apart from this process, several cell types also have other efficient mechanisms of Fe uptake from Tf that includes a process consistent with non-specific adsorptive pinocytosis and a mechanism that is stimulated by small-Mr Fe complexes. This latter mechanism appears to be initiated by hydroxyl radicals generated by the Fe complexes, and may play a role in Fe overload disease where a significant amount of serum non-Tf-bound Fe exists. Apart from Tf-bound Fe uptake, mammalian cells also possess a number of mechanisms that can transport Fe from small-Mr Fe complexes into the cell. In fact, recent studies have demonstrated that the membrane-bound Tf homologue, MTf, can bind and internalize Fe from 59Fe-citrate. However, the significance of this Fe uptake process and its pathophysiological relevance remain uncertain. Iron derived from Tf or small-Mr complexes is probably transported into mammalian cells in the Fe(II) state. Once Fe passes through the membrane, it then becomes part of the poorly characterized intracellular labile Fe pool. Iron in the labile Fe pool that is not used for immediate requirements is stored within the Fe-storage protein, ferritin. Cellular Fe uptake and storage are coordinately regulated through a feedback control mechanism mediated at the post-transcriptional level by cytoplasmic factors known as IRP1 and IRP2. These proteins bind to stem-loop structures known as IREs on the 3 UTR of the TfR mRNA and 5 UTR of ferritin and erythroid delta-aminolevulinic acid synthase mRNAs. Interestingly, recent work has suggested that the short-lived messenger molecule, NO (or its by-product, peroxynitrite), can affect cellular Fe metabolism via its interaction with IRP1. Moreover, NO can decrease Fe uptake from Tf by a mechanism separate to its effects on IRP1, and NO may also be responsible for activated macrophage-mediated Fe release from target cells. On the other hand, the expression of inducible NOS which produces NO, can be stimulated by Fe chelators and decreased by the addition of Fe salts, suggesting that Fe is involved in the control of NOS expression.

Antigens, Neoplasm↗
PubMed1990

Functional aspects of the vestibular dark cells in the guinea pig: morphological investigation using ruthenium red staining technique.

The functional significance of the vestibular dark cells in the guinea pig was investigated using ruthenium red staining technique. The apical cell surface of the dark cell was covered by the fuzzy layer of the glycocalyx, which could be responsible for water and ion transport across the apical plasma membrane. It has been suggested that the otoconia have been at first demineralized by an unknown mechanism when they are attached to the dark cell surface, and the mucopolysaccharide and mucoproteins in the otoconial matrix are then absorbed by active pinocytosis in the dark cells. These functions may be closely related to the possible regulation of the calcium ion in the vestibular endolymph.

Animals↗
PubMedDate not supplied

Mechanisms of amino acid supply to the rat conceptus in normal and abnormal development.

Using in vitro and in vivo models, we have shown that protein is an important source of amino acids from 8.5 to 17.5 d postconception, a significant portion of postimplantation development in the rat. This satisfactorily explains why inhibition of protein pinocytosis and digestion by the visceral yolk sac may lead to congenital malformations, growth retardation, and intrauterine death during organogenesis and growth retardation and death during fetal stages. In humans, this process may be just as important and possibly just as vulnerable to inhibition, but other placental or paraplacental tissues may be involved in addition to the secondary yolk sac.

Amino Acids↗
PubMed2002

CD44-mediated uptake and degradation of hyaluronan.

Hyaluronan turnover occurs systemically from the lymph and serum as well as locally by the same cells responsible for its synthesis. Local turnover involves receptor-mediated uptake and delivery to lysosomes. Of the many hyaluronan binding proteins/receptors known, the participation of CD44 in the internalization of hyaluronan has been best characterized. Some fraction of the hyaluronan bound to CD44 becomes internalized and delivered to lysosomes by a mechanism that is not dependent on clatherin, caveolae or pinocytosis. In cells such as chondrocytes, anabolic and catabolic cytokines can alter the activity of CD44 toward hyaluronan internalization. However, the mechanism of cellular regulation remains unclear. Regulation may involve the participation of alternatively spliced isoforms of CD44, changes in CD44 phosphorylation, changes in cytoskeletal binding proteins or, the activity or extracellular proteolytic activity.

Animals↗
PubMed2001

Three-dimensional high voltage electron microscopy of thick biological specimens.

The procedures recently developed in our laboratory to observe three-dimensional structures of cell organelles in thick biological specimens by means of high voltage electron microscopy are reviewed. Thick biological specimens such as whole mount cultured cells seeded and grown on grid meshes in culture vessels or thick sections cut from embedded tissues and stained by histochemical reactions can be readily observed three-dimensionally by high voltage transmission electron microscopy at 400-1000kV. Cultured cells used were both primary cultures from animal tissues and established cell lines maintained in our laboratory. The livers of adult Wistar rats were isolated by collagenase perfusion, and hepatocytes were suspended in a Leibovitz medium and seeded on formval coated gold grid meshes in Petri dishes, incubated in a CO(2) incubator in a humidified atmosphere containing 5% CO(2) in air at 37 degrees C for a few days. Established cell lines, CHO-K1 cells, were cultured in Ham's F12 medium, while HeLa cells were cultured in Eagle's MEM under the same condition. Some of the cells were cultured under experimental conditions such as hepatocyte culture in the medium containing peroxisome proliferating agents such as clofibrate or bezafibrate and some of them were labeled with (3)H-thymidine, (3)H-uridine, (3)H-labeled precursors and (14)C-bezafibrate. Also some cells were incubated in medium containing HRP to induce pinocytosis. All the whole mount cultured cells on grid meshes were prefixed in buffered 2.5% glutaraldehyde, stained with various histochemical reactions and postfixed in 1% osmium tetroxide. The histochemical reactions used were glucose-6-phosphatase (G-6-Pase), thiamine pyrophosphatase (TPPase), cytochrome oxidase, acid phosphatase (AcPase), DAB, ZIO, PA-TCH-SP reactions and radioautography was performed after labeling with radiolabeled compounds. The whole mount cultured cells were dried in a critical point dryer and were observed with JEOL JEM-4000EX or Hitachi H-1250M high voltage electron microscopes at 400-1000kV. By tilting the specimens' stereo-pair micrographs were recorded and they were observed with stereoscopes. Rat liver, mouse intestine and pancreas tissues, fixed and stained as above, were embedded in Epoxy resin, thick sectioned at 1-2 microm and were observed as for the whole mount cultured cells at 1000kV. Stereo-pairs were further analyzed with an image analyzer JEOL JIM-5000 (JEOL, Tokyo, Japan), producing two contour lines plotted from the micrographs at a thickness of 0.2 microm and were observed with anaglyph type glasses, demonstrating the depth or heights of respective cell organelles. The results show that whole mount cultured cells and thick sections stained with histochemical reactions reveal cell organelles corresponding to marker enzymes, such as G-6-Pase in endoplasmic reticulum, TPPase and ZIO in Golgi apparatus, cytochrome oxidase in mitochondria, AcPase in lysosomes, DAB in peroxisomes and pinocytotic vesicles, PA-TCH-SP in secretory granules, (3)H-thymidine and (3)H-uridine in nuclei, (3)H-animo acids in endoplasmic reticulum and secretory granules, (14)C-bezafibrate around ER and peroxisomes. The ultrastructure of these cell organelles as well as the structural relationship between them can be demonstrated three-dimensionally with stereo-pair images. Overall, these procedures are useful for analyzing stereologically the ultrastructure of cell organelles in cells and tissues.

Animals↗
PubMed1997

The GTP-binding protein Rho.

RhoA, RhoB and RhoC are three closely related proteins, and are members of the Ras super-family of small GTP-binding proteins. They bind and hydrolyse GTP, and are active in the GTP-bound form. Their activity in cells is regulated by exchange factors, GTPase activating proteins and guanine nucleotide dissociation inhibitors. Several potential downstream target proteins for Rho proteins have been identified, including protein kinases and adaptor-type proteins. Rho proteins regulate actin cytoskeletal organization; for example in fibroblasts RhoA induces the formation of actin stress fibres. Rho proteins are also involved in regulating secretion, pinocytosis and clathrin coat-mediated endocytosis, transcriptional activation and stimulation of DNA synthesis. In addition, there is evidence that Rho proteins can play a role in cell transformation, and thus Rho proteins or components of their signalling pathways may be potential targets for the development of anti-cancer therapies.

Amino Acid Sequence↗
PubMed1976

The cell coat of Crithidia fasciculata.

A prominent filamentous and granular cell coat is associated with the plasma membrane of haptomonads of Crithidia fasciculata attached to the cuticular lining of the hindgut of Anopheles gambiae. This surface material is completely absent from morphologically identical haptomonads from the mosquito foregut and from rosettes in culture. On the basis of these observations, it is suggested that cell coat formation is dependent on suitable environmental conditions. A cytochemical staining technique indicates the presence of carbohydrates in (a) the cell coat, (b) the outer leaflet of the plasma membrane of haptomonads from all locations and (c) small cytoplasmic vesicles and multivesicular bodies in all haptomonads. It is suggested that these cytoplasmic organelles represent stages in the intracellular pathway for material ingested by pinocytosis rather than elements involved in surface coat formation.

Animals↗
PubMedDate not supplied

N-(2-hydroxypropyl)methacrylamide copolymers of a glutathione (GSH)-activated glyoxalase i inhibitor and DNA alkylating agent: synthesis, reaction kinetics with GSH, and in vitro antitumor activities.

The incorporation of anticancer prodrugs into polyacrylamide conjugates has been shown to improve tumor targeting via the so-called "enhanced permeability and retention" effect. This strategy has now been expanded to include two different classes of glutathione (GSH)-activated antitumor agents prepared by radical polymerization of N-(2-hydroxypropyl)methacrylamide (HPMA) with 2-methacryloyloxy-methyl-2-cyclohexenone (7) and/or with S-(N-4-chlorophenyl-N-hydroxycarbamoyl-thioethyl)methacrylamide (8), followed by treatment with 3-chloroperoxybenzoic acid, to give the HPMA copolymers of 7 and the 8-sulfoxide, respectively. In aqueous-buffered solution at pH 6.5, GSH reacts rapidly with poly-HPMA-8-sulfoxide (k approximately 2.3 mM(-1) min(-1)) to give S-(N-4-chlorophenyl-N-hydroxycarbamoyl)glutathione (1), a tight-binding transition state analogue inhibitor of the antitumor target enzyme glyoxalase I (K(i) = 46 nM), or with poly-HPMA-7 (k approximately 0.02 mM(-1) min(-1)) to give the electrophilic antitumor agent 3-glutathio-2-methylenecyclohexenone (4). Indeed, B16 melanotic melanoma in culture is inhibited by poly-HPMA-8-sulfoxide and by poly-HPMA-7 with IC(50) values of 168 +/- 8 and 284 +/- 5 microM, respectively. These values are significantly greater than those of the unpolymerized prodrugs suggesting that the cytotoxicity of the polymer prodrugs might be limited by slow cellular uptake via pinocytosis. This prodrug strategy should be applicable to a range of different GSH-based antitumor agents.

Acrylamides↗
PubMed2006

Semitelechelic HPMA copolymers functionalized with triphenylphosphonium as drug carriers for membrane transduction and mitochondrial localization.

Semitelechelic HPMA (N-(2-hydroxypropyl)methacrylamide) copolymers possessing a single terminal lipophilic triphenylphosphonium (TPP) cation and fluorescent labels were synthesized to determine how the attached cation affected cellular uptake and intracellular trafficking. In vitro mitochondrial uptake fluorescence quenching assays using isolated mouse liver mitochondria indicated that only lower molecular weight (<5 kDa) BODIPY FL-labeled TPP-semitelechelic HPMA copolymers exhibited significant organelle localization or uptake. In vitro cellular uptake and intracellular trafficking was evaluated using cultured human ovarian carcinoma cells. Cells incubated with all types of TPP copolymers used in the study appeared to internalize the polymer by endocytosis only, and all of the internalized copolymer was confined to the lysosomal compartment after 24 h. Endocytotic uptake of the TPP-HPMA copolymer conjugates was rapid, suggesting that they were internalized by adsorptive endocytosis, rather than fluid-phase pinocytosis. Low-molecular weight (<5 kDa) and high-molecular weight (>5 kDa) semitelechelic copolymers, microinjected into cultured cells indicated that the TPP moiety did not significantly localize the polymers to mitochondria.

Biological Transport↗
PubMed2003

Remarkably size-regulated cell invasion by artificial viruses. Saccharide-dependent self-aggregation of glycoviruses and its consequences in glycoviral gene delivery.

We here report a novel example of artificial glycoviral vectors constructed via number- and size-controlled gene (pCMVluc, 7040 bp) coating with micellar glycocluster nanoparticles (GNPs) of calix[4]resorcarene-based macrocyclic glycocluster amphiphiles having eight or five saccharide moieties with terminal alpha-glucose (alpha-Glc), beta-glucose (beta-Glc), or beta-galactose (beta-Gal) residues. The resulting glycoviruses are compactly packed (approximately 50 nm) and well charge-shielded (zeta approximately equal 0 mV), undergo saccharide-dependent (alpha-Glc > beta-Gal >> beta-Glc) self-aggregation, and transfect cell (Hela and HepG2) cultures as triggered by the pinocytic form of endocytosis. The semilogarithmic linear size-activity correlation suggests that size-restricted pinocytosis (<100 nm) is effective only for monomeric viruses. The activities of oligomeric and otherwise poorly active beta-Gal-functionalized viruses toward hepatic HepG2 cells are approximately 10(2)-times higher than expected on the size basis, owing to the receptor-mediated specific pathway involving the asialoglycoprotein receptors on the hepatic cell surfaces. The scope and prospect of artificial glycoviruses are discussed.

Calixarenes↗
PubMed1997

Size-dependent dextran transport across rat alveolar epithelial cell monolayers.

The transport of dextrans (approximately 4 to approximately 150 kDa) across an in vitro model of the alveolar epithelial barrier was studied to determine the effects of molecular size on pulmonary absorption of macromolecular drugs. Fluorescein isothiocyanate (FITC)-labeled dextrans (FDs) with average molecular weights (all in kDa) of 3.86 (FD4), 9 (FD10), 19.8 (FD20), 40.5 (FD40), 71.6 (FD70), and 156.9 (FD150) were utilized as model macromolecular drugs. Unidirectional fluxes of FDs at 37 and 4 degrees C were measured from the appearance rates of FD in the receiver fluid of open-circuited monolayers (>2000 omega-cm2) of rat alveolar epithelial cells. Apparent permeability coefficients (P(app)) were estimated from the observed flux and the corresponding concentration gradient of FD. Results showed that FD fluxes were the same in both apical-to-basolateral (AB) and opposite (BA) directions at each molecular weight studied. The P(app) was not significantly different at 0.5 and 1.0 mg/mL FD40 donor concentrations. The FD P(app) (x 10(-8)cm/s) decreased gradually from 1.35 for FD4 to 0.32 for FD40, indicating an apparent inverse relationship between P(app) and molecular weight of FD. By contrast, P(app) was about the same at 0.13 for both FD70 and FD150. When experimental temperature was lowered to 4 degrees C, P(app) decreased by approximately 40% for FDs of 4 through 40 kDa, whereas the decrease in P(app) was by approximately 80% for larger FDs of both 70 and 150 kDa. Moreover, these FDs were found to be relatively intact (approximately 90%) in either receiver fluid after 5-h flux experiments without detectable levels of metabolites in the respective donor fluid, suggesting that alveolar epithelial cells allow translocation of FDs intact across the barrier. Equivalent pore analysis, assuming restricted diffusion of FDs of 4-40 kDa via cylindrical, water-filled pores across the cell monolayer revealed a population of large equivalent pores with approximately 5.6 nm radius. These data suggest that smaller macromolecules (radius <5 nm) traverse the alveolar epithelial barrier via paracellular pathways, and that larger (i.e., radius > or = 6 nm) macromolecules likely cross the barrier via other pathways (e.g., pinocytosis).

Absorption↗
PubMed2005

Biocompatibility of gold nanoparticles and their endocytotic fate inside the cellular compartment: a microscopic overview.

Macrophages are one of the principal immune effector cells that play essential roles as secretory, phagocytic, and antigen-presenting cells in the immune system. In this study, we address the issue of cytotoxicity and immunogenic effects of gold nanoparticles on RAW264.7 macrophage cells. The cytotoxicity of gold nanoparticles has been correlated with a detailed study of their endocytotic uptake using various microscopy tools such as atomic force microscopy (AFM), confocal-laser-scanning microscopy (CFLSM), and transmission electron microscopy (TEM). Our findings suggest that Au(0) nanoparticles are not cytotoxic, reduce the production of reactive oxygen and nitrite species, and do not elicit secretion of proinflammatory cytokines TNF-alpha and IL1-beta, making them suitable candidates for nanomedicine. AFM measurements suggest that gold nanoparticles are internalized inside the cell via a mechanism involving pinocytosis, while CFLSM and TEM studies indicate their internalization in lysosomal bodies arranged in perinuclear fashion. Our studies thus underline the noncytotoxic, nonimmunogenic, and biocompatible properties of gold nanoparticles with the potential for application in nanoimmunology, nanomedicine, and nanobiotechnology.

Animals↗
PubMed2000

A voltage-dependent channel involved in nutrient uptake by red blood cells infected with the malaria parasite.

Growth of the malaria parasite in human red blood cells (RBCs) is accompanied by an increased uptake of many solutes including anions, sugars, purines, amino acids and organic cations. Although the pharmacological properties and selectivity of this uptake suggest that a chloride channel is involved, the precise mechanism has not been identified. Moreover, the location of this uptake in the infected RBC is unknown because tracer studies are complicated by possible uptake through fluid-phase pinocytosis or membranous ducts. Here we have studied the permeability of infected RBCs using the whole-cell voltage-clamp method. With this method, uninfected RBCs had ohmic whole-cell conductances of less than 100 pS, consistent with their low tracer permeabilities. In contrast, trophozoite-infected RBCs exhibited voltage-dependent, non-saturating currents that were 150-fold larger, predominantly carried by anions and abruptly abolished by channel blockers. Patch-clamp measurements and spectral analysis confirmed that a small (< 10 pS) ion channel on the infected RBC surface, present at about 1,000 copies per cell, is responsible for these currents. Because its pharmacological properties and substrate selectivities match those seen with tracer studies, this channel accounts for the increased uptake of small solutes in infected RBCs. The surface location of this new channel and its permeability to organic solutes needed for parasite growth indicate that it may have a primary role in a sequential diffusive pathway for parasite nutrient acquisition.

Animals↗
PubMed1987

Anticancer agents coupled to N-(2-hydroxypropyl)methacrylamide copolymers. I. Evaluation of daunomycin and puromycin conjugates in vitro.

During recent years N-(2-hydroxypropyl)methacrylamide (HPMA) copolymers have been developed as targetable drug carriers. These soluble synthetic polymers are internalized by cells by pinocytosis and they can be tailor-made to include peptidyl side-chains degradable intracellularly by specific lysosomal enzymes. Thus they provide the opportunity fo achieve controlled intracellular delivery of anticancer agents. The anthracycline antibiotic daunomycin, and protein synthesis inhibitor puromycin, were bound to HPMA copolymers via several different peptide side-chains, including Gly-Gly, Gly-Phe-Leu-Gly and Gly-Phe-Phe-Leu. Incubation of polymer-drug conjugates with isolated lysosomal enzymes (either a mixture of rat liver lysosomal enzymes or purified thiol-dependent lysosomal proteinases, cathepsins L and B) showed that significant release of drug occurred over 20 h, more than 20% of daunomycin and more than 80% of puromycin being liberated. To test their pharmacological activity conjugates were incubated with either the mouse leukaemia L1210, or the human lymphoblastoid leukaemia CCRF in vitro. The conjugates tested were all less effective than free daunomycin, but they showed differential toxicity against L1210 depending on the aminoacid sequence of their drug-polymer linkage. Inclusion of fucosylamine-terminating side-chains into the HPMA copolymer structure increased the affinity of conjugates for the L1210 cell membrane and resulted in increased toxicity. In contrast HPMA-daunomycin conjugates with or without fucosylamine affected CCRF cells equally, but this cell line was more sensitive than the mouse leukaemia to both free and polymer-bound daunomycin. Incubation of L1210 cells in polymer-bound daunomycin for 72 h, followed by plating cells out in low density in drug-free medium, showed that a concentration of polymer-bound drug (184 micrograms ml-1) could be selected to achieve a cytotoxic effect.

Acrylates↗
PubMed1996

Human liver cancer cells and endothelial cells incorporate iodised oil.

Iodised oil (lipiodol) administered via the hepatic artery localises selectively in primary liver cell cancers (hepatocellular carcinomas or HCCs) for prolonged periods and has been used as a vehicle for cytotoxic agents. Despite clinical use, the mechanism of lipiodol retention by tumours has remained unclear, embolisation of oil droplets in the tumour vasculature being the prevailing hypothesis. We have investigated the role of tumour and endothelial cells in lipiodol retention. Human liver tumour (Hep G2) cells and human umbilical vein endothelial cells in culture were exposed to lipiodol. Light microscopy using selective silver impregnation stains and transmission electron microscopy revealed lipiodol incorporation by both cell types, probably by pinocytosis. This was not associated with cellular injury in terms of cell lysis, cell replication or radio-labelled leucine uptake. Histological analysis of 24 HCCs either surgically resected or discovered incidentally at liver transplantation (with prior arterial injection of lipiodol) revealed vesicles of lipiodol in the cytoplasm of tumour cells and endothelial cells lining tumour vessels. Thus, lipiodol is likely to deliver cytotoxic agents directly into tumour cells and endothelial cells, both in vitro and in vivo. This may also apply to other lipids and to other human tumours. These findings have significant therapeutic implications.

Carcinoma, Hepatocellular↗
PubMed2002

The correlation between hydrophilicity of hypericins and helianthrone: internalization mechanisms, subcellular distribution and photodynamic action in colon carcinoma cells.

The internalization mechanism and subcellular distribution of hypericin (Hyp), hypericin tetrasulfonic acid (HypS4) and 1,3,4,6-tetrahydroxyhelianthrone (Hel) were studied in murine colon carcinoma CT26 cells, in protein-free medium or in the presence of serum proteins. The correlation between the extent of uptake of the sensitizers by cells that were incubated in the presence of different serum components, and the internalization mechanisms, was studied. The results indicate that sensitizer internalization may be a result of three mechanisms: partitioning, pinocytosis and endocytosis, and as a direct consequence is targeted to specific subcellular sites. While Hyp and Hel, the two lipophilic sensitizers, were localized in the endoplasmic reticulum after protein-free internalization, the hydrophilic HypS4 was localized in the cytoplasmic membrane and in lysosomes. An endolysosomal internalization route was revealed for Hyp and Hel under serum-enriched conditions showing lysosomal localization, as for HypS4. The lysosomal accumulation of Hyp-serum and specifically Hyp-LDL points to an endocytotic mechanism which is supported by its higher uptake parameter in an LDL-enriched medium, compared to the medium with 10% serum. The different uptake parameters of Hyp to cells, with or without serum, reflect the different mechanisms. Smaller differences in the uptake parameter for HypS4 reflect the distinction between partitioning and endocytosis, which, in this case, are both targeted to the lysosomes. The same uptake parameter of Hel to cells incubated in media with or without serum indicates the absence of the endocytotic mechanism. The interrelationship between subcellular targeting and photodynamic treatment was shown for the three sensitizers Hyp was found to be the most efficient sensitizer for PDT under our illumination protocol and it was dependent on internalization and localization sites.

Animals↗