Ultrastructural changes in tumor cells treated with liposomal forms of anticancer drugs.

AIM: To determine the main ultrastructural changes in MCF-7 sublines sensitive and resistant to cytotoxic action of anticancer drugs, resulting from the treatment with conventional and liposomal forms of cisplatin and doxorubicin. METHODS: Electron microscopy, light microscopy, MTT-test. RESULTS: It has been shown that the phenomenon of drug resistance is associated with complication of ultrastructural organization of cells and more high differentiation by the main cytomorphologic characteristics which promote their resistance to cytotoxic action of anticancer preparations. Cytoarchitectonics of all resistant cells possesses common patterns and doesn't depend on the particular drugs toward which the resistance has been developed. It has been shown that the cells of the parental form MCF-7 line are more sensitive to cytotoxic action of doxorubicin than to cisplatin. Liposomal forms of anticancer drugs used at the same concentrations that the conventional ones, especially that of doxorubicin, caused more expressed alterations in ultrastructural organization of cells of all studied sublines with dominance of apoptotic processes. CONCLUSION: Evaluating an effect of equal concentrations of cisplatin and doxorubicin in conventional and liposomal forms, one may conclude on higher cytotoxic action of doxorubicin vs. cisplatin that is expressed in a wider spectrum of ultrastructural changes of cell architectonics in different sublines of MCF-7 cells and higher rate of apoptosis.

Variable pressure and environmental scanning electron microscopy: imaging of biological samples.

The use of elevated gas pressures in the sample chamber of a scanning electron microscope (i.e., variable pressure SEM, or VPSEM) together with specialized electron detectors create imaging conditions that allow biological samples to be examined without any preparation. Specific operating conditions of elevated pressures combined with sample cooling (usually restricted to the environmental SEM range) can allow hydrated samples to be maintained in a pristine state for long periods of time. Dynamic processes also can be easily observed. A wider range of detector options and imaging parameters introduce greater complexity to the VPSEM operation than is present in routine SEM. The current instrumentation with field emission electron sources has nanometer-scale beam resolution (approx 1 nm) and low-voltage beam capability (0.1 kV). However, under the more extreme variable pressure conditions, useful biological sample information can be achieved by skilled operators at image resolutions to 2 to 4 nm and with primary electron beam voltages down to 1.0 kV. Imaging relating to electron charge behavior in some biological samples, generally referred to as charge contrast imaging, provides information unique to this VPSEM and environmental SEM that closely relates to luminescence imaged by confocal microscopy.

Identification of 6-methylsulfinylhexyl isothiocyanate as an apoptosis-inducing component in wasabi.

The ethanol extract from Japanese horseradish wasabi was found to inhibit cell proliferation in human monoblastic leukemia U937 cells by inducing apoptotic cell death. Separation by methods including silica gel chromatography and preparative HPLC gave an active compound, which was identified as 6-methylsulfinylhexyl isothiocyanate (6-HITC). Several lines of evidence indicated that 6-HITC induced apoptosis in U937 cells and human stomach cancer MKN45 cells. Thus, 6-HITC is potentially useful as a natural anti-cancer agent.

Neuropeptides bombesin and calcitonin inhibit apoptosis-related elemental changes in prostate carcinoma cell lines.

BACKGROUND: Etoposide-induced apoptosis in prostate carcinoma cells is associated with changes in the elemental content of the cells. The authors previously reported that calcitonin and bombesin inhibited etoposide-induced apoptosis in these cells. In the current study, the authors investigated whether these neuropeptides block the etoposide-induced changes in elemental content. METHODS: Cells from the PC-3 and Du 145 prostate carcinoma cell lines were grown either on solid substrates or on thin plastic films on titanium electron microscopy grids, and they were exposed to etoposide for 48 hours in the absence or presence of calcitonin and bombesin. After the exposure, the cells were frozen and freeze dried, and their elemental content was analyzed by energy-dispersive X-ray microanalysis in both in the scanning electron microscope and the scanning transmission electron microscope. RESULTS: Etoposide treatment consistently induced an increase in the cellular Na concentration and a decrease in the cellular K concentration, resulting in a marked increase of the Na/K ratio and also an increase in the phosphorus:sulphur (P/S) ratio. Both bombesin and calcitonin inhibited the etoposide-induced changes in the cellular Na/K ratio, and calcitonin, but not bombesin, inhibited the changes in the P/S ratio. No significant elemental changes were found with bombesin or calcitonin alone. CONCLUSIONS: The neuropeptides bombesin and calcitonin, which inhibited etoposide-induced apoptosis, also inhibited the etoposide-induced elemental changes in prostate carcinoma cells. This important fact strengthens the link between apoptosis and changes in the intracellular elemental content. This correlation provides an objective basis for the study of neuropeptide target points and may be helpful for alternative therapeutic protocols using neuropeptide inhibitors in the treatment of patients with advanced prostatic carcinoma.

Blocking HSF1 by dominant-negative mutant to sensitize tumor cells to hyperthermia.

Heat shock protein 70 (HSP70), an antiapoptotic chaperon protein, is highly expressed in human breast tumors and renders them resistant to such therapy as hyperthermia. In the present study, we inhibited the expression of HSP70 by blocking the heat shock transcription factor 1 (HSF1) function with its dominant-negative mutant (mHSF1) in Bcap37 cells, a thermotolerant breast cancer cell line. Here we report that retrovirus-mediated transfer of mHSF1 led to massive cell death of Bcap37 after hyperthermia. mHSF1 sensitized Bcap37 cells to hyperthermia by promoting apoptosis induced by heat shock. We also examined the efficacy of mHSF1 gene therapy in the nude mouse. mHSF1 transfection led to diminution of tumor growth with hyperthermia therapy. Thus, disrupting HSF1 in combination with hyperthermia may open new possibilities for treatment of cancers that have acquired resistance to heat treatment.

Manipulating biological samples for environmental scanning electron microscopy observation.

Biological samples having different characteristics were observed by environmental scanning electron microscopy (ESEM). The environmental conditions for untreated biological samples was determined by optimizing sample temperature and chamber pressure. When the temperature was at 4 degrees - 6 degrees C and chamber pressure was 5.2-5.9 Torr, the relative humidity in the specimen chamber was about 85%. Under these conditions, the surface features of the sample were completely exposed and did not exhibit charging. The images obtained from the untreated samples at different ESEM conditions were also compared with fixed and coated samples observed under high vacuum.

Caulerpenyne from Caulerpa taxifolia has an antiproliferative activity on tumor cell line SK-N-SH and modifies the microtubule network.

Caulerpenyne, the major secondary metabolite synthesized by the green marine alga Caulerpa taxifolia, is cytotoxic against several cell lines. To identify possible targets of this toxin, we investigated the effect of caulerpenyne on the neuroblastoma SK-N-SH cell line. Caulerpenyne induced an inhibition of SK-N-SH cell proliferation with an IC50 of 10 +/- 2 microM after 2 hr of incubation. We observed no blockage in G2/M phase and an increase in cell death. On immunofluorescence microscopy, caulerpenyne affected the microtubule network in SK-N-SH cell line; we observed a loss of neurites and a compaction of the microtubule network at the cell periphery. In vitro, after 35 min of incubation, caulerpenyne inhibited the polymerization of pig brain purified tubulin or microtubule proteins, with an IC50 of 21 +/- 2 microM and 51 +/- 6 microM respectively. Analysis by electron microscopy indicated that caulerpenyne induced aggregation of tubulin, which may be responsible for inhibition of microtubule polymerization and bundling of residual microtubules.

Damage induction by direct electric current in tumoural target cells.

Damage induction to tumour target cells (P815) by direct electric current (DC) was investigated. A 6 min treatment of P815 cells with DC generated decreased levels of cell viability and proliferation. The ultrastructural analysis of DC-treated cells revealed the presence of blebs, loss of cell surface filopodia, and ruptures in cell membrane. Mitochondrial alterations, swelling of cells, cytoplasmic matrix rarefaction, and cellular debri formation were also observed. The study shows that tumoural target cells can be damaged by direct electric current and this approach may provide means to understand the mechanism of tumour regression induced by electrochemical therapy.

Morphological changes related to reconstituted acetylcholine release in a release-deficient cell line.

The membrane changes accompanying Ca(2+)-dependent acetylcholine release were investigated by comparing release-competent and release-incompetent clones of mouse neuroblastoma N18TG-2 cells. No release could be elicited in native N18 cells or in a N18-choline acetyltransferase clone in which acetylcholine synthesis was induced by transfection with the gene for rat choline acetyltransferase. However, acetylcholine release was operative in a To/9 clone which was co-transfected with complementary DNAs from rat choline acetyltransferase and Torpedo mediatophore 16,000 mol. wt subunit. In thin sections, the aspect of resting N18 and To/9 cells was identical: a very dense cytoplasm with practically no vesicle-like organelles. Cells were chemically fixed at different times during a stimulation using A-23187 and Ca2+, and examined following both freeze-fracture and thin section. Stimulation of To/9 cells induced a marked change affecting the intramembrane particles. The number of medium-sized particles (9.9-12.38 nm) increased, while that of the small particles decreased. This change was not observed in control, release-incompetent cell lines. In the To/9 clone (but not in control clones), this was followed by occurrence of a large new population of pits which initially had a large diameter, but subsequently became smaller as their number decreased. Coated depressions and invaginations became abundant after stimulation, suggesting an endocytosis process. By considering the succession of events and by comparison with data from experiments performed on synapses in situ, it is proposed that a particle alteration was the counterpart of acetylcholine release in co-transfected To/9 cells; this was followed by a massive endocytosis.

Protein kinase C activation upregulates intercellular adhesion of alpha-catenin-negative human colon cancer cell variants via induction of desmosomes.

The alpha-catenin molecule links E-cadherin/ beta-catenin or E-cadherin/plakoglobin complexes to the actin cytoskeleton. We studied several invasive human colon carcinoma cell lines lacking alpha-catenin. They showed a solitary and rounded morphotype that correlated with increased invasiveness. These round cell variants acquired a more normal epithelial phenotype upon transfection with an alpha-catenin expression plasmid, but also upon treatment with the protein kinase C (PKC) activator 12-O-tetradecanoyl-phorbol-13-acetate (TPA). Video registrations showed that the cells started to establish elaborated intercellular junctions within 30 min after addition of TPA. Interestingly, this normalizing TPA effect was not associated with alpha-catenin induction. Classical and confocal immunofluorescence showed only minor TPA-induced changes in E-cadherin staining. In contrast, desmosomal and tight junctional proteins were dramatically rearranged, with a conversion from cytoplasmic clusters to obvious concentration at cell-cell contacts and exposition at the exterior cell surface. Electron microscopical observations revealed the TPA-induced appearance of typical desmosomal plaques. TPA-restored cell-cell adhesion was E-cadherin dependent as demonstrated by a blocking antibody in a cell aggregation assay. Addition of an antibody against the extracellular part of desmoglein-2 blocked the TPA effect, too. Remarkably, the combination of anti-E-cadherin and anti-desmoglein antibodies synergistically inhibited the TPA effect. Our studies show that it is possible to bypass the need for normal alpha-catenin expression to establish tight intercellular adhesion by epithelial cells. Apparently, the underlying mechanism comprises upregulation of desmosomes and tight junctions by activation of the PKC signaling pathway, whereas E-cadherin remains essential for basic cell-cell adhesion, even in the absence of alpha-catenin.

Oxidizability and subsequent cytotoxicity of chylomicrons to monocytic U937 and endothelial cells are dependent on dietary fatty acid composition.

Oxidized chylomicrons may be a metabolic factor involved in the injury of the arterial wall and may constitute a potential link between postprandial lipemia and atherogenesis. It was of interest to study the influence of dietary fatty acid composition on the oxidizability and subsequent cytotoxicity of chylomicrons on cultured cells. Human chylomicrons were obtained from healthy volunteers 3 h after ingestion of a triglyceride-rich meal containing mainly either polyunsaturated fatty acids (soya oil) or monounsaturated fatty acids (olive oil) or saturated fatty acids (partly hydrogenated palm oil). Polyunsaturated fatty acid (PUFA)-rich chylomicrons exhibited a high oxidizability, whereas chylomicrons enriched with monounsaturated or saturated fatty acids were relatively resistant to oxidation. The cytotoxicity of various types of chylomicrons submitted to oxidation has been tested comparatively on cultured human monocytic U937 cells and endothelial cells. Chylomicrons enriched with saturated and monounsaturated fatty acids were not or only slightly cytotoxic to cultured cells, whereas PUFA-rich chylomicrons (highly susceptible to oxidation) were highly cytotoxic. The influence of cholesterol on the oxidizability and subsequent cytotoxicity of PUFA-rich chylomicrons has been investigated by using comparatively a soya diet supplemented or not with cholesterol. PUFA-rich cholesterol-rich chylomicrons were slightly more oxidizable and more cytotoxic than PUFA-rich (cholesterol-poor) chylomicrons, thus suggesting that the cytotoxicity of PUFA-rich chylomicrons may be due to oxidation derivatives of PUFA (for the major part) and to oxysterols (for a minor part). Furthermore, the cytotoxic effects of oxidized PUFA-rich chylomicrons and of mildly oxidized LDL were in similar range (even higher for PUFA-rich chylomicrons when expressed per lipoprotein particle), thus suggesting that oxidized PUFA-rich chylomicrons may play a nonnegligible role in cytotoxic events occurring during atherogenesis.(ABSTRACT TRUNCATED AT 250 WORDS)

Functional serotonin-2B receptors are expressed by a teratocarcinoma-derived cell line during serotoninergic differentiation.

Among immortalized teratocarcinoma-derived cells, the clone 1C11 is a committed precursor of the neuronal lineage. On day 2 of its serotoninergic differentiation, this clone expresses only one subtype of serotonin [5-hydroxytryptamine (5-HT)] receptor, which is functionally coupled to phosphatidylinositol hydrolysis. The identity of these receptors was established by comparing their properties with those of 5-HT2B receptors expressed by LMTK- fibroblasts stably transfected with the recently cloned murine cDNA NP75 (LM5 cells). In both cell types, the analysis of (+/-)-1-(2,5-dimethoxy-4-[125I]iodophenyl)- 2-aminopropane HCl ([125I]DOI) binding revealed the presence of a single class of sites, the affinity of which was 1 order of magnitude lower than that reported for 5-HT2A receptors. In 1C11 cells differentiated for 2 days, as well as in LM5 cells, DOI binding was decreased by nonhydrolyzable analogs of GTP, indicating that the 5-HT2B receptor is functionally coupled to a G protein. The DOI-induced increase of phosphoinositide hydrolysis, which was correlated with both GTPase activity and binding data, is mediated by a Gq protein. This work demonstrates that the 5-HT2B receptor is functionally expressed before complete serotoninergic differentiation of 1C11 cells. The inducible 1C11 clone thus provides an in vitro model to investigate the possible role of the 5-HT2B receptor in the expression of the serotoninergic phenotype.

Lysosomal exocytosis induced by hyperthermia: a new model of cancer cell death. II. Effect on peritoneal macrophages.

Recently we described the "macrophagic lysosomal exocytosis" (MLE) as a possible new mode of cancer cell death induced by hyperthermia (HT) [1]. In order to confirm this first report, HT was applied at the peritoneum level with perfusional procedure, after catheter insertion under local anesthesia. We evaluated the subcellular changes of peritoneal macrophages in human gastric tumor, before and after hyperthermic treatment at 42 degrees C for 90 minutes. Transmission electron microscopy showed that treatment produced the disappearance of the cytoplasmic granules, with a consequent extracellular scavenger action of the phagocytic cells, the proliferation of some organelles such as mitochondria, endoplasmic reticulum and Golgi complex that may be addressed to a subsequent restoration of the granular pool in the degranulated macrophages. This phenomenon can enhance the already documented effect of hyperthermic perfusional chemotherapy in peritoneal tumors.

Effects of K(+)-induced depolarization and purinergic receptor activation on elemental content in insulin-producing RINm5F-cells.

X-ray microanalysis was used to detect elemental changes in the insulin-producing tumor cell-line RINm5F. To improve discrimination between mobile ions and ions bound to macromolecules a new approach was employed, consisting of multivariate statistical analysis of correlations between the concentrations of Na, Mg, P, S, Cl, K, and Ca. RINm5F cells, cultured on Formvar-coated titanium grids, were stimulated with high K+ or ATP, that are both known to stimulate insulin release. The buffers used contained Ca2+ or one of the Ca(2+)-analogues Sr2+ and Ba2+, to represent Ca2+ uptake in response to stimulation. After stimulation the cells were shock-frozen and freeze-dried overnight. Incubation for 10-20 seconds in a Ca(2+)-containing buffer did not significantly affect elemental composition, whereas cellular Mg, P and K decreased in a Sr(2+)-containing buffer. Depolarization with high K+ concentration caused an increase in the cellular Na content, both in Ca(2+)- and Sr(2+)-containing buffers, but not in the buffer where Ca2+ had been replaced by Ba2+. X-ray microanalysis is useful for detection of elemental changes subsequent to stimulation of cultured cells. Moreover, multivariate statistical analysis strengthens the idea that stimulation of RINm5F cells causes redistribution of ions possibly due to changes in the state of binding of some elements to cellular proteins.

Effects of incubation with liposomes at different temperatures on cultured melanoma cells (M14).

A melanoma cell line (M14) was used in order to investigate the effect of hyperthermia on the mechanisms of interaction between liposomes and cultured cells. The treatment was performed by adding different concentrations of multilamellar liposomes (L-alpha-dipalmitoylphosphatidylcholine, stearylamine and cholesterol in the ratio 7:2:1) to cell cultures which were then incubated at 37.0 or 41.5 degrees C for 2 h. The damage induced by liposome treatment in normothermia or hyperthermia was evaluated by determining cell survival and by electron microscopy. When different concentrations of liposomes were used, a dose-dependent impairment of cell survival was observed. An enhancement of the cytotoxic effect was observed when the treatment was performed at 41.5 degrees C. This effect went on even after 24 h from the end of the treatment, but the difference between cells treated in normothermia and hyperthermia was remarkably reduced. The mechanism of the liposome-plasma membrane interaction has been investigated by electron microscopy. Our observations demonstrated that the outer bilayer of the multilamellar liposomes was capable of fusing with the plasma membrane, inducing changes in its fluidity and molecular organization. Following this process the inner liposomal bilayers entered the cell. These effects seemed to be favoured when the treatment was performed under mild hyperthermic conditions, accounting for the synergic cytotoxic action displayed by the liposome-hyperthermia association.

[An antimutagenicity study of bioginseng]. / Izuchenie antimutagennosti biozhen'shenia.

We studied antimutagenic properties of bioginseng, a biotechnological product obtained from the callus culture of the ginseng root cells by alcohol extraction (bioginseng-1) and lyophilization (bioginseng-2). We have estimated the frequency of sister-chromatid exchange and chromosome aberrations in the culture of Chinese hamster cells, of chromosome aberrations in cells of the Ehrlich-ICF ascite strain, and of micronuclei in the mouse bone marrow cells. The bioginseng exerted an antimutagenic effect with respect to nitrosomethylurea and cyclophosphamide. The sister-chromatid change in the culture of Chinese hamster cells decreased under the influence of bioginseng due, apparently, to enhanced DNA repair. The most distinct protective effect was observed when ginseng was introduced 2 h prior to the cell treatment with mutagens.

Protoporphyrin biosynthesis in melanoma B16 cells stimulated by 5-aminolevulinic acid and chemical inducers: characterization of photodynamic inactivation.

The stimulation of protoporphyrin (PP) biosynthesis in B16 melanoma cells in order to facilitate photodynamic cell killing was studied. Biosynthesis and accumulation of PP in the melanoma cells was increased from 8 to 15 pmol/mg protein by the use of dimethyl-sulfoxide (DMSO), a differentiation-inducer. Treatment of the cells with the porphyrogenic agent allylisopropyl-acetamide (AIA) stimulated an additional PP increase. The most remarkable enhancement of intracellular PP was achieved by the supplementation of 5-aminolevulinic acid (5-ALA) to the growth medium following the addition of DMSO and AIA during the induction phase. The intracellular concentration of PP exceeded 21,950 pmol/mg protein following combined stimulation by DMSO/AIA and 5-ALA. The porphyrins produced in the incubated cells, in serum-depleted medium, consisted of 95% PP; 88% of it was recovered from the cells and only 7% was excreted into the medium. Photosensitization of the B16 melanoma cells containing high PP concentrations was effective even at low light doses. Potassium (K) efflux was the first measurable sign of cell damage determined by X-ray microanalysis (XRMA) following fast liquid-nitrogen fixation. During a 1 min interval, 70% of cellular K was lost. After 5 min illumination, complete cell destruction was detected by scanning electron microscopy (SEM) and XRMA. The photodamaged cells showed influx of Na, Cl and Ca ions accompanying the immediate K losses. Ultrastructural cell damage was manifested by disintegration of the outer membrane. Total cell death of B16 melanoma cells was achieved by chemical induction of endogenous PP and photosensitization.

The cytoskeleton and proliferation of melanoma cells under hyperthermal conditions. A correlative double immunolabelling study.

Hyperthermia provides a potent therapeutical tool of cancer treatment, the cell biological effects of which are fairly understood. In the present study we applied hyperthermal shocks of 42 degrees C and 44 degrees C to human melanoma cells under tissue culture conditions. The integrity of the microtubular (mT) system and rate of DNA replication was assessed by indirect immunofluorescence using antibodies to tubulin as an mT marker and to BrdU as an indicator for DNA replication. Through this approach we obtained evidence that heat (44 degrees C for 60 min) exerts a profound damaging effect on the mT system accompanied by a change in the phenotypical appearance of melanoma cells. DNA replication, however, was still in progress in a significant number of heavily afflicted cells. From these data we conclude that a therapeutic regiment combining hyperthermia and mT inhibitors might prove useful in the treatment of human melanomas.

Ultrastructural changes of bladder cancer cells following methylene blue-sensitized photodynamic treatment.

The cytotoxic efficacy of methylene blue-sensitized photoinactivation of bladder cancer cells has been proved in vitro and in vivo in our previous work. The ultrastructural changes of MBT-2 murine bladder cancer cells following methylene blue-sensitized phototherapy have been evaluated by scanning and transmission electron microscopy. MBT-2 cells were pretreated with 0.1% methylene blue, and then exposed to ordinary light for different durations from 1 to 60 min. Both electron microscopic examinations showed that changes in tumor cells occurred at 5 min of illumination with fenestration and minipore formation on the cell membrane and mitochondrial disintegration. At 30 min, cleavage and cracking of the cell membrane with ribosomal lysis were noted. Finally, fragmentation with decomposition of the membrane and chromosomal condensation and lysis resulted at 60 min. This study showed that the cytotoxic effect induced by methylene blue phototherapy is initiated in the cell membrane and intracellular mitochondria in the early phase.

Enhancement of hyperthermic damage on M14 melanoma cells by liposome pretreatment.

Exponentially growing human melanoma cells (M14 cell line) were pretreated with various amounts of dipalmitoylphosphatidylcholine-containing multilamellar liposomes and then exposed to heat treatment (42.5 degrees C). Cell damage produced by the treatments, given separately or in combination, was evaluated in terms of cell survival. Our results demonstrate that the cell survival at 37 degrees C was not affected by liposome concentrations up to 1000 nmol of phospholipid/2.5 x 10(6) cells, while liposome treatment of cells before heat exposure determined a marked damaging effect even at 100 nmol of phospholipid/2.5 x 10(6) cells. The mechanisms of liposome-cell interaction have been investigated by electron microscopy or by electron spin resonance measurements of spin-labeled membranes of intact cells. Evidence has been obtained that liposomal lipids are either taken up by M14 cells or become incorporated in the cell membrane. The present data suggest the possibility that liposome treatments per se could be of potential value as a therapeutic approach, by increasing the effect of heat therapy.