Tumor inhibition by metallocenes: ultrastructural localization of titanium and vanadium in treated tumor cells by electron energy loss spectroscopy

The intracellular distribution patterns of the metal atoms titanium and vanadium after in vivo as well as in vitro treatment of Ehrlich ascites tumor with the antitumor agents titanocene dichloride (TDC) or vanadocene dichloride (VDC) have been investigated by use of electron energy loss spectroscopy (EELS). The metals were found mainly accumulated in the nuclear heterochromatin and, to a minor extent, in the nucleolus and in the cytoplasmic ribosomes. In connection with other experimental results it is argued that this accumulation is indicative of the molecular interaction of the metal-containing species with the nucleic acids, especially with the DNA.     

Subcellular distribution of titanium in the liver after treatment with the antitumor agent titanocene dichloride. A study using electron spectroscopic imaging

In the present study, the subcellular distribution of titanium in the liver of mice was determined 24 and 48 h after application of a therapeutic (ED100; ED = effective dose) and a toxic (LD25; LD = lethal dose) dose (60 and 80 mg/kg, respectively) of the antitumor agent titanocene dichloride by electron spectroscopic imaging at the ultrastructural level. At 24 h, titanium was mainly accumulated in the cytoplasm of endothelial and Kupffer cells, lining the hepatic sinusoids. Titanium was detected in the nucleoli and the euchromatin of liver cells, packaged as granules together with phosphorus and oxygen. One day later titanium was still present in cytoplasmic inclusions within endothelial and Kupffer cells, whereas in hepatocyte nucleoli only a few deposits of titanium were observed at 48 h. At this time titanium was mainly accumulated in the form of highly condensed granules in the euchromatin and the perinucleolar heterochromatin. It was found in the cytoplasm of liver cells, incorporated into cytoplasmic inclusion bodies which probably represent lysosomes. Sometimes these inclusions were situated near bile canaliculi and occasionally extruded their content into the lumen of bile capillaries. This observation suggests a mainly biliary elimination of titanium-containing metabolites. These results confirm electron spectroscopic imaging to be an appropriate method for determining the subcellular distribution of light and medium-weight elements within biological tissues. Insights into the cellular mode of action of titanocene complexes or titanocene metabolites can be deduced from the findings of the present study.     

Subcellular distribution of titanium in the liver after treatment with the antitumor agent titanocene dichloride

In the present study, the subcellular distribution of titanium in the liver of mice was determined 24 and 48 h after application of a therapeutic (ED₁₀₀; ED = effective dose) and a toxic (LD₂₅; LD = lethal dose) dose (60 and 80 mg/kg, respectively) of the antitumor agent titanocene dichloride by electron spectroscopic imaging at the ultrastructural level. At 24 h, titanium was mainly accumulated in the cytoplasm of endothelial and Kupffer cells, lining the hepatic sinusoids. Titanium was detected in the nucleoli and the euchromatin of liver cells, packaged as granules together with phosphorus and oxygen. One day later titanium was still present in cytoplasmic inclusions within endothelial and Kupffer cells, whereas in hepatocyte nucleoli only a few deposits of titanium were observed at 48 h. At this time titanium was mainly accumulated in the form of highly condensed granules in the euchromatin and the perinucleolar heterochromatin. It was found in the cytoplasm of liver cells, incorporated into cytoplasmic inclusion bodies which probably represent lysosomes. Sometimes these inclusions were situated near bile canaliculi and occasionally extruded their content into the lumen of bile capillaries. This observation suggests a mainly biliary elimination of titanium-containing metabolites. These results confirm electron spectroscopic imaging to be an appropriate method for determining the subcellular distribution of light and medium-weight elements within biological tissues. Insights into the cellular mode of action of titanocene complexes or titanocene metabolites can be deduced from the findings of the present study.     

A proposed mechanism for the inhibitory effect of the anticancer agent titanocene dichloride on tumour gelatinases and other proteolytic enzymes

Titanocene dichloride, the most studied metallocene, exhibits antiproliferative activity in a wide spectrum of murine and human tumours. In this article it is demonstrated that titanocene dichloride inhibits tumour gelatinases in a dose-dependent manner. Substrate saturation experiments and the fact that the IC₅₀ values were increased in correlation with collagen substrate concentrations indicate that the titanocene dichloride induced inhibition is of a competitive type. Titanocene dichloride also specifically inhibits clostridium collagenase and trypsin, particularly when collagens are used as substrates. Binding experiments demonstrate that cyclopentadiene–Ti(IV) moieties, resulting from titanocene dichloride at physiological pH, are bound mainly to different types of collagens and to a lesser extent to casein or bovine serum albumin, forming soluble and stable adducts. These results indicate that titanocene dichloride behaves as a competitive inhibitor against various proteolytic enzymes by binding to the substrate rather than to the enzyme active site. This property may be responsible for the antiangiogenic effect of titanocene dichloride and additionally contributes to its anticancer action.     

Cytologic observations on the effects of metallocene dichlorides on human fibroblasts cultivated in vitro

The effects of the organometallic cytostatic agents titanocene dichloride (TDC) and vanadocene dichloride (VDC) and of the inorganic cytostatic drug cis-diamminedichloroplatinum(II) (DDP) on the morphologic appearance of human embryonal fibroblasts cultivated as monolayers in vitro were analyzed by light and electron microscopy. All three substances induced similar structural changes which consisted of nuclear as well as cytoplasmic alterations. Many fibroblasts enlarged but the nuclear volume increased more extensively than that of the cytoplasm. The nuclear envelopes underwent invagination so that segmented nuclei were formed and the nucleoli increased in size and density. Within the cytoplasm there was evidence of conspicuous protein synthesis, characterized morphologically by an increase in the size and number of mitochondria, Golgi apparatuses and of cisternae of the rER. The phenomena observed are interpreted as indicating unbalanced cell growth characterized by a selective inhibition of DNA synthesis, coupled with progressive RNA and protein syntheses.     

Effects of lead on hepatocyte ultrastructure in Carassius carassius (L.) var. auratus.

Subcellular modifications in hepatocytes of Carassius carassius var. auratus subjected to 24 hr and 48 hr sublethal acute lead (5mg.1-1) exposure were studied by electron microscopy. Cytological alterations were observed after 24 hr of treatment and became more evident after 48 hr. Lead induced an increase in nuclear heterochromatin and alterations in mitochondria, endoplasmic reticulum and Golgi complex ultrastructure. Glycogen granula decreased, and secondary lysosomes and lipid droplets increased. Furthermore, intracytoplasmic lumina with microvilli-bearing surfaces and numerous autophagic vacuola were observed after 48 hr of exposure.     

Pseudo-halide derivatives of titanocene: synthesis and cytotoxicity studies

The well-known anticancer drug candidate bis-[(p-methoxybenzyl)cyclopentadienyl] titanium(IV) dichloride (Titanocene ) was reacted with sodium azide or potassium cyanate, thiocyanate or selenocyanate in order to give pseudo-halide analogues of Titanocene . and were characterised by single crystal X-ray diffraction, which confirmed the expected nitrogen binding of the cyanate and thiocyanate to the titanium centre. All four titanocenes had their cytotoxicity investigated through preliminary in vitro testing on the LLC-PK (pig kidney epithelial) cell line in an MTT based assay in order to determine their IC50 values. Titanocenes were found to have IC50 values of 24 (± 8) μM, 101 (± 14) μM, 54 (± 21) μM and 27 (± 4) μM respectively. All four titanocene derivatives show significant cytotoxicity improvement when compared to unsubstituted titanocene dichloride and and showed similiar cytotoxic behaviour to Titanocene in vitro.     

Electrochemical evaluation of the interaction between antitumoral titanocene dichloride and biomolecules

Electrochemical investigation of the interaction between titanocene dichloride and several biomolecules, i.e., human and bovine serum albumin (HSA and BSA), and single-strand and double-strand deoxyribonucleic acid (ss- and ds-DNA) is reported. The addition of biomolecules to a hydroalcoholic solution of TiCp₂Cl₂ was followed by a decrease in the Ti(IV)/Ti(III) reduction peak current, due to the formation of adducts with lower diffusion coefficients. Qualitative analysis of the data reveals that titanocene dichloride has a higher affinity for proteins than for nucleic acids.     

Heteroaryl substituted titanocenes as potential anti-cancer drugs

From the reaction of Super Hydride (LiBEt(3)H) with 6-(furyl)fulvene (1a), 6-(thiophenyl)fulvene (1b) or 6-(N-methyl-pyrrole)fulvene (1c) the corresponding lithium cyclopentadienide intermediates (2a-c) were obtained. These intermediates were reacted with titanium tetrachloride and bis-[(furyl-2-cyclopentadienylmethane)] titanium(IV) dichloride (3a) and bis-[(thiophenyl-2-cyclopentadienylmethane)] titanium(IV) dichloride (3b) and bis-[(N-methylpyrrole-2-cyclopentadienylmethane)] titanium(IV) dichloride (3c) were obtained and subsequently characterised by X-ray crystallography. When titanocenes 3a-c were tested against pig kidney (LLC-PK) cells inhibitory concentrations (IC(50)) of 1.6x10(-4)M, 1.5x10(-4)M and 9.1x10(-4)M, respectively, were observed. These values represent improved cytotoxicity against LLC-PK, when compared to their corresponding ansa substituted analogues and also in comparison to unsubstituted titanocene dichloride.     

Effects of lead on hepatocyte ultrastructure in Carassius carassius (L.) var. auratus

Subcellular modifications in hepatocytes of Carassius carassius var. auratus subjected to 24 hr and 48 hr sublethal acute lead (5mg·1⁻¹) exposure were studied by electron microscopy. Cytological alterations were observed after 24 hr of treatment and became more evident after 48 hr. Lead induced an increase in nuclear heterochromatin and alterations in mitochondria, endoplasmic reticulum and Golgi complex ultrastructure. Glycogen granula decreased, and secondary lysosomes and lipid droplets increased. Furthermore, intracytoplasmic lumina with microvilli-bearing surfaces and numerous autophagic vacuola were observed after 48 hr of exposure.     

Novel titanocene anti-cancer drugs and their effect on apoptosis and the apoptotic pathway in prostate cancer cells

Advanced prostate cancer is not curable by current treatment strategies indicating a significant need for new chemotherapeutic options. Highly substituted ansa-titanocene compounds have shown promising cytotoxic activity in a range of cancers. The objectives of this study are to examine the effects of these titanocene compounds on prostate cancer cells. Prostate cell lines were treated with three novel titanocene compounds and compared to titanocene dichloride and cisplatin. Percent apoptosis, viability and cell cycle were assessed using propidium iodide DNA incorporation with flow cytometry. Cytochrome C was assessed by western blotting of mitochondrial and cytoplasmic fractions. Apoptosis Inducing Factor was assessed by confocal microscopy. These novel compounds induced more apoptosis compared to cisplatin in a dose dependent manner. Compound Y had the most significant effect on cell cycle and apoptosis. Despite the release of cytochrome C from the mitochondrial fraction there was no inhibition of apoptosis with the pan caspase inhibitor, ZVAD-FMK. AIF was shown to translocate from the cytosol to the nucleus mediating a caspase independent cell death. Bcl-2 over expressing PC-3 cells, which were resistant to cisplatin induced apoptosis, underwent apoptosis following treatment with all the titanocene compounds. This study demonstrates possible mechanisms by which these novel titanocene compounds can mediate their apoptotic effect in vitro. The fact that they can induce more apoptosis than cisplatin in advanced cancer cell lines would confer an advantage over cisplatin. They represent exciting new agents with future potential for the treatment of advanced prostate cancer.     

Compartmentalization of TNF-related apoptosis-inducing ligand (TRAIL) death receptor functions: emerging role of nuclear TRAIL-R2

Localized in the plasma membrane, death domain-containing TNF-related apoptosis-inducing ligand (TRAIL) receptors, TRAIL-R1 and TRAIL-R2, induce apoptosis and non-apoptotic signaling when crosslinked by the ligand TRAIL or by agonistic receptor-specific antibodies. Recently, an increasing body of evidence has accumulated that TRAIL receptors are additionally found in noncanonical intracellular locations in a wide range of cell types, preferentially cancer cells. Thus, besides their canonical locations in the plasma membrane and in intracellular membranes of the secretory pathway as well as endosomes and lysosomes, TRAIL receptors may also exist in autophagosomes, in nonmembraneous cytosolic compartment as well as in the nucleus. Such intracellular locations have been mainly regarded as hide-outs for these receptors representing a strategy for cancer cells to resist TRAIL-mediated apoptosis. Recently, a novel function of intracellular TRAIL-R2 has been revealed. When present in the nuclei of tumor cells, TRAIL-R2 inhibits the processing of the primary let-7 miRNA (pri-let-7) via interaction with accessory proteins of the Microprocessor complex. The nuclear TRAIL-R2-driven decrease in mature let-7 enhances the malignancy of cancer cells. This finding represents a new example of nuclear activity of typically plasma membrane-located cytokine and growth factor receptors. Furthermore, this extends the list of nucleic acid targets of the cell surface receptors by pri-miRNA in addition to DNA and mRNA. Here we review the diverse functions of TRAIL-R2 depending on its intracellular localization and we particularly discuss the nuclear TRAIL-R2 (nTRAIL-R2) function in the context of known nuclear activities of other normally plasma membrane-localized receptors.     

Loss of Rna and Protein, And Changes in Dna During A 30-Hour Cold Perchloric Acid Extraction of Cultured Cells

KB cells derived from human carcinoma were fixed in acetic-alcohol (1:3) and extracted with 10% perchloric acid (PCA) at 4 C for 1, 3, 6, 9, 12, 24 and 30 hr. Cells were then washed in water and stained for nucleic acids, proteins, polysaccharides, and lipids. Control cells were kept in water for 30 hr prior to staining. Acridine orange (AO) fluorochroming revealed color changes in residual cytoplasmic and nucleolar RNA as well as DNA during extraction--interpreted as indicative of molecular alterations. All nucleic acid stains (AO, gallocyanin chromalum, and azure B bromide) demonstrated a differential extraction of RNA, with cytoplasmic RNA being removed in about 6 hr and nucleolar RNA requiring 6 more hours for complete extraction. Large granules appeared early in nuclei. These were positive for DNA by azure B, gallocyanin chromalum, Feulgen, and fluorescent-Feulgen. These same granules stained for protein by mercuric bromphenol blue and alkaline Biebrich scarlet. At 24 hr, there was visual and Feulgen-cytophotometric evidence for a slight loss of DNA, which may amount to 10-20%. There was a progressive loss of cytoplasmic and nuclear but not nucleolar protein during PCA treatment. Concurrently, large protein-positive granules appeared in the cytoplasm. Apparently, PCA treatment in combination with an aqueous wash was responsible for some protein loss. Glycogen was gradually lost (fluorescent PAS) and redistributed in cells. Lipids were unaffected (Sudan black B).     

Detection of intracellular phosphatidylserine in living cells

To demonstrate the intracellular phosphatidylserine (PS) distribution in neuronal cells, neuroblastoma cells and hippocampal neurons expressing green fluorescence protein (GFP)-AnnexinV were stimulated with a calcium ionophore and localization of GFP-AnnexinV was monitored by fluorescence microscopy. Initially, GFP-AnnexinV distributed evenly in the cytosol and nucleus. Raising the intracellular calcium level with ionomycin-induced translocation of cytoplasmic GFP-AnnexinV to the plasma membrane but not to the nuclear membrane, indicating that PS distributes in the cytoplasmic side of the plasma membrane. Nuclear GFP-AnnexinV subsequently translocated to the nuclear membrane, indicating PS localization in the nuclear envelope. GFP-AnnexinV also localized in a juxtanuclear organelle that was identified as the recycling endosome. However, minimal fluorescence was detected in any other subcellular organelles including mitochondria, endoplasmic reticulum, Golgi complex, and lysosomes, strongly suggesting that PS distribution in the cytoplasmic face in these organelles is negligible. Similarly, in hippocampal primary neurons PS distributed in the inner leaflet of plasma membranes of cell body and dendrites, and in the nuclear envelope. To our knowledge, this is the first demonstration of intracellular PS localization in living cells, providing an insight for specific sites of PS interaction with soluble proteins involved in signaling processes.     

Ultrastructural studies on the intracellular fate of 125I-nerve growth factor in cultured rat sympathetic neurons

Primary cell cultures of sympathetic neurons from rat were exposed to 125I-nerve growth factor (NGF) and the fate of the NGF in the cell was followed using electron microscopic autoradiography. The intracellular localization of NGF was determined in the cell bodies and in the proximal neurites of neurons that had been grown in three-chamber dishes, following 5 or 24 hr of retrograde transport of NGF from the distal portions of the neurites. Label in the proximal neurites was predominantly associated with lysosomes and multivesicular bodies (MVBs), and at 5 hr elongated tubular elements were especially heavily labeled. Most of the label in the cell bodies was concentrated in lysosomes and MVBs. Lysosomes accounted for the largest fraction (45-60%) of the grains in the cell body, with a labeling density (LD = % grains/% area) of 3-5, while MVBs accounted for 5-10% of the grains with an LD of 5-20. We observed no evidence of nuclear labeling after 5 or 24 hr of retrograde transport. Mass cultures of neurons were incubated for 22 hr with NGF in the presence of the lysosomal inhibitors chloroquine (CQ, 0.05 mM) or methylamine (MA, 10 mM). In both agents the lysosomes were swollen with membranous material but still sequestered NGF, especially in CQ where the lysosomes were associated with almost 65% of the grains and had an LD of 6. CQ and MA had different effects on the morphology of the MVBs: in CQ they were few in number and compact while in MA they were numerous and appeared swollen and vacuolated. We observed no evidence for the nuclear accumulation of NGF even in the presence of the lysosomotropic agents.     

Application of sheep kidney nuclease to nucleic acid research: Enzymatic preparation of deoxy dinucleotides

Sheep kidney nuclease, which digests only single-stranded nucleic acid and has no specificity for the base and sugar moieties, was purified approximately 400-fold from sheep kidney acetone powder. By digesting denatured calf thymus DNA with this enzyme at 37°C for 72 hr and separating the fraction on DEAE-Sephadex A25, a deoxy dinucleotide fraction was obtained that after further fractionation on DEAE-Sephadex A25 equilibrated with 0.01 m formic acid and elution with a linear gradient of sodium formate (0 to 0.1 m), gave 16 peaks corresponding to all 16 deoxy dinucleotides.     

Evidence that aspartic proteinase is involved in the proteolytic processing event of procathepsin L in lysosomes

Our recent studies have shown that cathepsin L is first synthesized as an enzymatically inactive proform in endoplasmic reticulum and is successively converted into an active form during intracellular transport and we postulated that aspartic proteinases would be responsible for the intracellular propeptide-processing step of procathepsin L accompanied by the activation of enzyme (Y. Nishimura, T. Kawabata, and K. Kato (1988) Arch. Biochem. Biophys. 261, 64-71). To better understand this proposed mechanism, we investigated the effect of pepstatin, a potent inhibitor of aspartic proteinases, on the intracellular processing kinetics of cathepsin L analyzed by pulse-chase experiments in vivo with [35S]methionine in the primary cultures of rat hepatocytes. In the pepstatin-treated cells, the proteolytic conversion of cellular procathepsin L of 39 kDa to the mature enzyme was significantly inhibited and considerable amounts of proenzyme were found in the cell after 5-h chase periods. Further, the subcellular fractionation experiments demonstrated that the intracellular processing of procathepsin L in the high density lysosomal fraction was significantly inhibited and that considerable amounts of the procathepsin L form were still observed in the light density microsomal fraction after 2 h of chase. These results suggest that pepstatin treatment caused a significant inhibitory effect on the intracellular processing and also on the intracellular movement of procathepsin L from the endoplasmic reticulum to the lysosomes. These findings provide the first evidence showing that aspartic proteinase may play an important role in the intracellular proteolytic processing and activation of lysosomal cathepsin L in vivo. Therefore, we suggest that cathepsin D, a major lysosomal aspartic proteinase, is more likely to be involved in this proposed model in the lysosomes.     

Herpes Simplex Virus-Host Cell Relationships in Organized Cultures of Mammalian Nerve Tissues

Studies on the replication of herpes simplex virus in organized cultures of rat central nervous system (CNS) and peripheral nervous system (PNS) tissue demonstrated synthesis of intra- and extracellular virus, as determined by plaque assay on HEp-2 cells. Newly synthesized intracellular virus appeared 12 to 14 hr after inoculation of CNS, followed 10 hr later by the appearance of extracellular virus. In PNS cultures, where higher inputs of virus were introduced, intracellular virus appeared 6 to 8 hr after inoculation, followed by extracellular virus 12 hr later. Polykaryocyte formation was observed in CNS and PNS tissue involving neuroglial, meningeal, or Schwann cells. Neuron somas did not participate in polykaryocyte formation, but they underwent progressive morphological changes starting with increased cytoplasmic granularity followed by nucleolar distortions and disintegration, margination of nuclear chromatin, and the appearance of intranuclear inclusions. Finally, all recognizable cellular detail was lost. Immune serum globulin failed to inhibit both the progressive nature of the cytopathic effect and the synthesis of intracellular virus. These findings are discussed in relation to other in vitro systems, as well as to disease processes in man and animals.     

Gross and cellular analysis of 6-mercaptopurine-induced cleft palate in hamster

The present study analyzes the morphological, histochemical, and ultrastructural aspects of the pathogenesis of 6-mercaptopurine (6MP)-induced cleft palate in hamster fetuses. Gross and light microscopic observations indicated that 6MP stunts the growth of vertical palatal shelves and thus induces cleft palate. Ultrastructural analysis showed that, in contrast to controls, 6MP-induced alterations were first seen in the mesenchymal cells 24 hr after drug administration. The initial alterations were characterized by swelling of the nuclear membrane. During the next 12 hr, lysosomes were seen first in the mesenchymal cells and then in the cells of the medial edge epithelium (MEE) of the developing palatal primordia. The appearance of lysosomes was temporally abnormal and was interpreted as a sublethal response to 6MP treatment. Subsequently, the nuclear alterations and the lysosomes diminished; and 48 hr after 6MP administration, they were absent from the palatal tissues. Ninety hours after 6MP administration, unlike the controls (in which the palatal shelves were already fused), changes were seen at the epithelial-mesenchymal interface in the developing cleft palatal shelves. These changes were characterized by breakdown of the basal lamina and epithelial-mesenchymal contacts. Eventually, at term, the MEE of the vertical shelf stratified. It was suggested that 6MP affected cytodifferentiation in the palatal tissues during the critical phase of early vertical shelf development and thereby induced cleft palate.     

Effects of prolonged antitubulin culture on annulate lamellae in mouse alpha L929 fibroblasts

The fine structure of alpha L929 fibroblasts cultured in colchicine or vinblastine sulfate for periods as long as 48 hr was compared to control cells not exposed to antitubulins . In response to prolonged antitubulin culture, several changes in cell ultrastructure were noted: Control fibroblasts contain cytoplasmic annulate lamellae (AL), but prolonged exposure to either vinblastine sulfate or colchicine results in enhanced development of AL. Single pore complexes are present in the rough-surfaced endoplasmic reticulum (rER) in both control and antitubulin-treated cells, but stacked porous cytomembranes also occur under both conditions. Polyribosomes often are closely associated or continuous with the pore complexes. Many antitubulin-treated cells become multinucleate. Some nuclei in both control and antitubulin-treated cells contain large and multiple nucleoli. The large and multiple nucleoli are either attached directly to the inner membrane of the nuclear envelope or to infoldings of the nuclear envelope. Antitubulin-treated cells, after 48-hr exposure, appear also to contain enhanced quantities of smooth-surfaced endoplasmic reticulum (sER) and cytoplasmic filaments (and in some cells, lysosomes and rER as well) when compared to untreated cells. In both control and colchicine-treated cells, AL can exhibit continuity with either rER or sER. Further, all three membrane systems may at times be continuous, but the quantity of these membranes appears to be greater in colchicine-treated cells than in control cells. The results are discussed with respect to possible functional significance.