Enhanced optical breakdown in KB cells labeled with folate-targeted silver-dendrimer composite nanodevices.

Enhanced optical breakdown of KB tumor cells folate-targeted with silver-dendrimer composite nanodevices (CNDs) is described. CNDs [(Ag(0))(25)-PAMAM_E5.(NH(2))(42)(NGly)(74)(NFA)(2.7)] were fabricated by reactive encapsulation, using a biocompatible template of dendrimer-folic acid (FA) conjugates. Preferential uptake of the folate-targeted CNDs (of various treatment concentrations and surface functionality) by KB cells was visualized with confocal microscopy and transmission electron microscopy. Intracellular laser-induced optical breakdown threshold and dynamics were detected and characterized by high-frequency ultrasonic monitoring of resulting transient bubble events. When irradiated with a near-infrared, femtosecond laser, the CND-targeted KB cells acted as well-confined activators of laser energy, enhancing nonlinear energy absorption, exhibiting a significant reduction in breakdown threshold and thus selectively promoting intracellular laser-induced optical breakdown. FROM THE CLINICAL EDITOR: This study presents a novel method to selectively destroy cancer cells by combining biochemical targeting with topical laser irradiation. A human epidermoid cancer cell line was targeted with folated silver-dendrimer composite nanodevices and the labeled cancer cells were subsequently destroyed by the microbubbles generated due the enhanced energy uptake of the silver nanoparticles from the laser irradiation, as compared to unlabeled cells.

ZnO nanocrystals shuttled by extracellular vesicles as effective Trojan nano-horses against cancer cells.

Aim: The effective application of nanoparticles in cancer theranostics is jeopardized by their aggregation in biological media, rapid degradation and clearance. The design of biomimetic nanoconstructs with enhanced colloidal stability and non-immunogenicity is therefore essential. We propose naturally stable cell-derived extracellular vesicles to encapsulate zinc oxide (ZnO) nanocrystals as efficacious nanodrugs, to obtain highly biomimetic and stable Trojan nano-horses (TNHs). Materials & methods: Coupling efficiency, biostability, cellular cytotoxicity and internalization were tested. Results:In vitro studies showed a high internalization of TNHs into cancer cells and efficient cytotoxic activity thanks to ZnO intracellular release. Conclusion: TNHs represent an efficient biomimetic platform for future nanotheranostic applications, with biomimetic extracellular vesicle-lipid envelope, facilitated ZnO cellular uptake and potential therapeutic implications.

Evidence that the penton base of adenovirus is involved in potentiation of toxicity of Pseudomonas exotoxin conjugated to epidermal growth factor.

When KB cells are incubated for 1 h with human adenovirus type 2 or type 5 (1 microgram/ml) and a conjugate of epidermal growth factor and Pseudomonas exotoxin (EGF-PE), protein synthesis is inhibited by 80 to 90%. Under these conditions, neither adenovirus nor EGF-PE alone has any effect on host protein synthesis. Thus, adenovirus enhances the toxicity of EGF-PE. A number of antibodies to intact virus and capsid components were tested for their ability to block the enhancing activity and virus uptake. At appropriate dilutions, antibodies prepared against intact virus and penton base blocked the enhancing activity without affecting virus uptake. Antibodies against hexon and fiber blocked virus uptake and enhancing activity in parallel. These studies suggest that the penton base is important in lysis of the vesicles which contain adenovirus and EGF-PE, and this base allows virus and toxin to enter the cytoplasm.

A new and rapid bioassay for the detection of gliotoxin and related epipolythiodioxopiperazines produced by fungi.

Gliotoxin is an immunosuppressive cytotoxin produced by numerous environmental or pathogenic fungal species. For this reason, it is one of the mycotoxins which must be systematically searched for in samples for biological control. In this study, a new, rapid and sensitive method for detecting gliotoxin has been developed. This bioassay is based on the induction of morphological changes in cultured cells (human KB cell line) by gliotoxin. Interpretation of the assay can be carried out after 1 h of incubation, either by direct microscopic observation, or with an automated microplate-reader at 630 nm. The limit of detection is 18-20 ng of gliotoxin in the well, depending on the used observation method. A high degree of specificity of the detection is brought about by the ability of the reducing reactant dithiothreitol to inhibit the biological activities of epipolythiodioxopiperazines (ETPs), such as gliotoxin, by reducing their polysulfide bridge. The bioassay allows a rapid primary screening of samples and a semi-quantitative evaluation of the gliotoxin concentration in extracts. The method has been used to study the gliotoxin production by different fungal strains, allowing to highlight 3 strains of Aspergillus fumigatus producing gliotoxin in various extracts.

Camptothecin resistance involving steps subsequent to the formation of protein-linked DNA breaks in human camptothecin-resistant KB cell lines.

To identify mechanisms of camptothecin (CPT) resistance/toxicity, sublines from a human KB cell line were made resistant to CPT by continuous selection in increasing concentrations of CPT. Two CPT-resistant lines, 100 and 300, were 32- and 54-fold resistant to the growth-inhibitory properties of CPT compared to the KB line. After CPT-free culturing, partial revertant lines were established from each resistant line. These partial revertant lines, 100rev and 300rev, were 2.5- and 3.2-fold resistant to CPT compared to KB. When growth inhibition and toxicity were compared, the resistant lines alone displayed an enhanced cytostatic response to CPT. The resistant and partial revertant lines displayed no cross-resistance to etoposide or cisplatin. Comparisons of topoisomerase I (TOPI) activity, content, and protein-linked DNA break production by CPT revealed that resistant and partial revertant lines had one-half the levels as KB, with TOP1 activity that was equally sensitive to CPT in all cell lines tested. However, double-stranded DNA break induction by CPT was significantly reduced only in the resistant lines. Coincubation with 3-aminobenzamide, an inhibitor of poly(ADP-ribosyl) polymerase, potentiated CPT toxicity in the resistant lines alone, without affecting CPT: TOP1 interactions. Therefore, CPT resistance in the 100 and 300 lines was characterized by factors independent of TOP1, specific for CPT, and attenuated by poly(ADP-ribosyl) polymerase inhibition. This resistant phenotype produced fewer double-stranded DNA breaks and enhanced a cytostatic response to CPT.

The farnesyl transferase inhibitor R115777 (Zarnestra) synergistically enhances growth inhibition and apoptosis induced on epidermoid cancer cells by Zoledronic acid (Zometa) and Pamidronate.

Pamidronate (PAM) and zoledronic acid (ZOL) are aminobisphosphonates (BPs) able to affect the isoprenylation of intracellular small G proteins. We have investigated the antitumor activity of BPs and R115777 farnesyl transferase inhibitor (FTI) against epidermoid cancer cells. In human epidermoid head and neck KB and lung H1355 cancer cells, 48 h exposure to PAM and ZOL induced growth inhibition (IC(50) 25 and 10 microM, respectively) and apoptosis and abolished the proliferative and antiapoptotic stimuli induced by epidermal growth factor (EGF). In these experimental conditions, ZOL induced apoptosis through the activation of caspase 3 and a clear fragmentation of PARP was also demonstrated. A strong decrease of basal ras activity and an antagonism on its stimulation by EGF was recorded in the tumor cells exposed to BPs. These effects were paralleled by impaired activation of the survival enzymes extracellular signal regulated kinase 1 and 2 (Erk-1/2) and Akt that were not restored by EGF. Conversely, farnesol induced a recovery of ras activity and antagonized the proapoptotic effects induced by BPs. The combined treatment with BPs and R115777 resulted in a strong synergism both in growth inhibition and apoptosis in KB and H1355 cells. The synergistic activity between the drugs allowed BPs to produce tumor cell growth inhibition and apoptosis at in vivo achievable concentrations (0.1 micromolar for both drugs). Moreover, the combination was highly effective in the inhibition of ras, Erk and Akt activity, while farnesol again antagonized these effects. In conclusion, the combination of BPs and FTI leads to enhanced antitumor activity at clinically achievable drug concentrations that resides in the inhibition of farnesylation-dependent survival pathways and warrants further studies for clinical translation.

Overexpression of transfected human ribonucleotide reductase M2 subunit in human cancer cells enhances their invasive potential.

The ribonucleotide reductase (RR) gene has been associated with malignant transformation and metastatic potential. In this report, the significance of the expression of RR mRNA and enzymatic activity to the invasive potential was examined by Boyden chamber invasion assay. Our results suggest that overexpression of RR M2 mRNA and RR enzymatic activity correlates to an increase in cell invasive potential. The drug-induced HURs clone expressed a higher level RR M2 mRNA and enzyme activity which contributes significantly to the 3-fold increase in invasive potential of the cells observed relative to the KB wild-type control. On the contrary, the HUr revertant clone decreased the RR M2 mRNA level and enzymatic activity, concomitantly decreasing their invasive potential. This phenomenon is most likely due to the return of RR to levels comparable to that of the KB wild-type cells. To confirm that this observation was not of a drug-resistance phenotype associated with multiple gene alterations, the panel of RR transfectants (M1-D transfected M1 subunit cDNA, M2-D transfected M2 subunit cDNA, X-D transfected M1/M2 cDNA) characterized in a previous study were also tested in the invasion assay. The M2-D clone expressed 6-fold higher RR M2 mRNA and RR activity and also demonstrated 6-fold higher invasive potential in vitro than either the parental or vector only transfected cell line (KB-V). The X-D clone demonstrated 3-fold higher M2 mRNA expression and revealed 4-fold higher invasive potential than control cells. The M1-D clone, in contrast, expressed a baseline level of RR M2 mRNA and higher M1 mRNA. In contrast to the X-D and M2-D cells, the invasive potential of M1-D reached an even lower level in the invasive assay than the control. These results, therefore, suggest that RR M2 overexpression plays an important role in a tumor's invasiveness.

Inducing the cell cycle arrest and apoptosis of oral KB carcinoma cells by hydroxychavicol: roles of glutathione and reactive oxygen species.

Hydroxychavicol (HC; 10 - 50 microM), a betel leaf component, was found to suppress the 2% H(2)O(2)-induced lucigenin chemiluminescence for 53 - 75%. HC (0.02 - 2 microM) was also able to trap superoxide radicals generated by a xanthine/xanthine oxidase system with 38 - 94% of inhibition. Hydroxyl radicals-induced PUC18 plasmid DNA breaks was prevented by HC (1.6 - 16 microM). A 24-h exposure of KB cells to HC (0.5, 1 mM) resulted in 54 - 74% cell death as analysed by a 3-(4,5-dimethyl-thiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT) assay. HC (10, 50 microM) further suppressed the growth of KB cells (15 and 76%, respectively). Long-term colony formation of KB cells was inhibited by 51% with 10 microM HC. Pretreatment of KB cells with 100 microM HC inhibited the attachment of KB cells to type I collagen and fibronectin by 59 and 29%, respectively. Exposure of KB cells to 0.1 mM HC for 24 h resulted in cell cycle arrest at late S and G2/M phase. Increasing the HC concentration to 0.25 and 0.5 mM led to apoptosis as revealed by detection of sub-G(0)/G(1) peaks with a concomitant decrease in the number of cells residing in late S and G(2)/M phase. Inducing the apoptosis of KB cells by HC was accompanied by marked depletion in reduced form of GSH (>0.2 mM) and the increasing of reactive oxygen species production (>0.1 mM) as analysed by CMF- and DCF-single cell fluorescence flow cytometry. These results indicate that HC exerts antioxidant property at low concentration. HC also inhibits the growth, adhesion and cell cycle progression of KB cells, whereas its induction of KB cell apoptosis (HC>0.1 mM) was accompanied by cellular redox changes.

Effects of coordinated gold compounds on in vitro and in situ DNA replication.

Auranofin, a coordinated gold compound, inhibits in vitro DNA synthesis and displays in vivo antitumor activity. To understand the mechanisms of inhibition of DNA replication, we have examined the effects of auranofin and other gold complexes on the activities of purified cellular and herpesvirus-induced DNA polymerases, and on in situ DNA replication in permeabilized S phase KB cells. Evaluation of the data suggests the following conclusions. (1) The gold compounds varied in their abilities to inhibit DNA polymerase activities. DNA polymerase alpha was more sensitive to inhibition by gold compounds than DNA polymerase beta; (2) Inhibition of purified DNA polymerases by gold (I) compounds was noncompetitive with both DNA template and triphosphate substrates. Inhibition by SKF 101675, a gold (III) complex was competitive with DNA. (3) None of the gold compounds tested preferentially inhibited herpesvirus-induced DNA polymerases. (4) The gold complexes that inhibited in vitro DNA replication also inhibited in situ DNA synthesis. However, the potency and order of potency of the compounds varied between the in vitro and in situ systems. (5) Auranofin and other gold compounds inhibited the clonogenic capacity of KB cells in a concentration-dependent manner. The IC50 values measured in the clonogenic assay were significantly lower than those obtained from the in vitro and in situ DNA replication assays.

Suitability of the MTT-based cytotoxicity assay to detect okadaic acid contamination of mussels.

The suitability of a cytotoxicity assay based on the MTT colorimetric method has been evaluated for the detection of okadaic acid in mussels. On KB cells, okadaic acid exhibited a dose-dependent cytotoxic effect, the IC50 being inversely related to the exposure time (IC50 = 6.3 ng/ml, 4.0 ng/ml and 1.1 ng/ml after 24, 48 and 72 hr of contact, respectively). Using a contact time of 24 hr, the MTT cytotoxicity assay is suitable for revealing okadaic acid concentrations in mussel samples as low as 50 ng/g of digestive glands, with a sensitivity higher than that of the commercially available kits for enzyme-linked immunosorbent assay (ELISA). In the okadaic acid concentration range from 50 to 1500 ng/g of digestive glands the MTT cytotoxicity assay showed satisfactory accuracy and reproducibility. A high degree of correlation was found between the okadaic acid content of 16 naturally contaminated samples measured by the MTT cytotoxicity assay and by an ELISA.

Cytostatic activity in vitro of phosphonic acid derivatives.

Cytostatic activity of 48 new amino acid analogs and derivatives of phosphonic acid type was tested against KB cell line in vitro. The tests were performed according to the recommended international protocol for screening of chemical agents against tissue culture system. Three of the compounds tested: No. No, 29, 33 and 32 revealed cytostatic activity at the concentrations: 63, 150, and 180 micrograms ml-1 respectively (ID50).

The effects of N-methyl-N'-nitro-N-nitrosoguanidine on KB cells. II. Cell sensitivity to the lethal effect of the drug as a function of the cell age and of the nature of the culture medium.

We have tested the sensitivity of KB cells to the lethal effect of N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), measured as cell loss within the 20-h period following a 1-h drug treatment, as a function of culture age and of the medium in which treated cells were incubated after elimination of MNNG. We showed that KB cell sensitivity to the lethal effect of the drug decreased with time after seeding when the treated cells were post-incubated in drug-free medium conditioned by untreated cells of the same age as treated ones but not when they were post-incubated in fresh drug-free medium. This difference was due in part to the fact that the conditioned medium had acquired with time a protective activity for treated cells and in part to an increased competence of aging cells to be protected by this medium. By post-incubating treated stationary cells sequentially in both media, we showed that a brief (15 min) post-incubation of the cells in fresh medium was sufficient to trigger cell death even if the cells were afterwards transferred to conditioned medium. In contrast, long post-incubation in fresh medium did not cause cell death if the cells were first post-incubated in conditioned medium for about 3 h. We conclude that: the medium acted on cell sensitivity to the lethal effect of MNNG through its growth regulatory ability; quiescent cells were less sensitive to the drug than growing cells; the sensitive phase of the cell was located before S; cell hypersensitivity might be due to deficient repair of cellular lesions rather than to increased lesion formation.

[Effects of cultural conditions on hexavalent chromium uptake and the cytotoxicity thereof in KB cell culture].

To reveal the effects of cultural conditions on the cytotoxicity of hexavalent chromium, the uptake of sodium chromate (Na2CrO4) by KB cells and the colony-forming efficiency of the cells were examined under various cultural conditions. The results were summarized as follows: 1) The chromium uptake by the cells after a certain period of incubation with hexavalent chromium was inhibited with the decrease of the temperature (3 degrees, 20 degrees, 37 degrees C), increase of the serum concentration (0, 10, 20, 30%) and increase of pH (6.8-8.2) of the medium. In particular, low temperatures inhibited the chromium uptake by the cells remarkably. However, in relation to the serum addition, no marked effect was found. 2) The chromium uptake by the cells increased with the volume of the medium containing an identical concentration of chromium (2 ppm) and then reached saturation when it was about 0.23 microgram per 10(6) cells. On the other hand, the chromium uptake positively correlated with the concentration of chromium and the total chromium in the medium. 3) The difference of chromium uptake by the cells in different culture media was more marked at acidic pH than that at alkaline pH. However, there was no effect of calcium chloride and glucose concentrations on the uptake of chromium. The chromium uptake by the cells in Ca-Mg-free phosphate-buffered solution (PBS(-] was higher than that in other culture media. Consequently, the above results suggested that the chromium uptake by the KB cells might be affected by the various cultural conditions, especially by temperature, pH and medium volume.

Ultrasensitive label-free photothermal imaging, spectral identification, and quantification of cytochrome c in mitochondria, live cells, and solutions.

Light-absorbing endogenous cellular proteins, in particular cytochrome c, are used as intrinsic biomarkers for studies of cell biology and environment impacts. To sense cytochrome c against real biological backgrounds, we combined photothermal (PT) thermal-lens single-channel schematic in a back-synchronized measurement mode and a multiplex thermal-lens schematic in a transient high resolution (ca. 350 nm) imaging mode. These multifunctional PT techniques using continuous-wave (cw) Ar+ laser and a nanosecond pulsed optical parametric oscillator in the visible range demonstrated the capability for label-free spectral identification and quantification of trace amounts of cytochrome c in a single mitochondrion alone or within a single live cell. PT imaging data were verified in parallel by molecular targeting and fluorescent imaging of cellular cytochrome c. The detection limit of cytochrome c in a cw mode was 5 x 10(-9) mol/L (80 attomols in the signal-generation zone); that is ca. 10³ lower than conventional absorption spectroscopy. Pulsed fast PT microscopy provided the detection limit for cytochrome c at the level of 13 zmol (13 x 10(-21) mol) in the ultrasmall irradiated volumes limited by optical diffraction effects. For the first time, we demonstrate a combination of high resolution PT imaging with PT spectral identification and ultrasensitive quantitative PT characterization of cytochrome c within individual mitochondria in single live cells. A potential of far-field PT microscopy to sub-zeptomol detection thresholds, resolution beyond diffraction limit, PT Raman spectroscopy, and 3D imaging are further highlighted.

Biphenylquinuclidines as inhibitors of squalene synthase and growth of parasitic protozoa.

In this paper we describe the preparation of some biphenylquinuclidine derivatives and their evaluation as inhibitors of squalene synthase in order to explore their potential in the treatment of the parasitic diseases leishmaniasis and Chagas disease. The compounds were screened against recombinant Leishmania major squalene synthase and against Leishmania mexicana promastigotes, Leishmania donovani intracellular amastigotes and Trypanosoma cruzi intracellular amastigotes. Compounds that inhibited the enzyme, also reduced the levels of steroids and caused growth inhibition of L. mexicana promastigotes. However there was a lower correlation between inhibition of the enzyme and growth inhibition of the intracellular parasites, possibly due to delivery problems. Some compounds also showed growth inhibition of T. brucei rhodesiense trypomastigotes, although in this case alternative modes of action other than inhibition of SQS are probably involved.

Immunocytochemical localization of cytoskeletal antigens in KB and HEp-2 cells.

KB and HEp-2 are cell lines that are commonly used for autoantibody detection. We have used conventionally produced and monoclonal antibodies as probes for cytoskeletal antigens in these cells. The presence and distribution of microfilament, microtubule, and intermediate filament antigens (vimentin and keratin) have been established. We have also confirmed that anti-vimentin antibodies of one clone do crossreact with DNA on an immunoadsorbent column, although this antibody did not appreciably stain the nucleus of either cell type.

In vitro replication of human mitochondrial DNA: accurate initiation at the origin of light-strand synthesis.

Synthesis of human light-strand mitochondrial DNA was accomplished in vitro using DNA primase, DNA polymerase, and other accessory proteins isolated from human mitochondria. Replication begins with the synthesis of primer RNA on a T-rich sequence in the origin stem-loop structure of the template DNA and absolutely requires ATP. A transition from RNA synthesis to DNA synthesis occurs near the base of the stem-loop structure and a potential recognition site for signaling that transition has been identified. The start sites of the in vitro products were mapped at the nucleotide level and were found to be in excellent agreement with those of in vivo nascent light-strand DNA. Isolated human mitochondrial enzymes recognize and utilize the bovine, but not the mouse, origin of light-strand replication.

Activation and inhibition of expression of the 72,000-Da early protein of adenovirus type 5 in mouse cells constitutively expressing an immediate early protein of herpes simplex virus type 1.

It has been previously reported that immediate early proteins of pseudorabies and cytomegalo viruses can substitute for the products of the human adenovirus type 5 (Ad5) E1A gene in the activation of early Ad5 transcription. In the present report the effect of one of the herpes simplex virus type 1 (HSV-1) immediate early genes, ICP4, on Ad5 early gene expression has been examined using mouse cell lines that constitutively express ICP4. These lines as well as nonproducers were infected with wild-type (wt) Ad5 or with various Ad5 E1A mutants and the levels of expression of the Ad5 E2A 72K DNA binding protein were measured by immunoprecipitation with a monoclonal antibody specific for 72K. With dl 312, which lacks E1A, some 72K expression was seen in nonproducer lines but levels were considerably higher in the producer lines. A similar result was also obtained using dl 312-infected nonproducer cells that were superinfected with HSV-1 virions. These data suggest that HSV-1 ICP4 can substitute for E1A in the activation of expression of early Ad5 proteins. With wt Ad5, 72K was also expressed at high levels in nonproducer mouse cells, however, in the ICP4 producer cell lines, a marked inhibition of 72K expression was observed and this inhibition correlated with the amount of ICP4 present. Using the E1A mutants pm 975 and hr 1, this inhibition was found to be specific for the products of the 1.1-kb E1A mRNA. These data suggest that ICP4 and E1A proteins either directly inhibit each other, or more likely, operate independently and competitively on factors required for viral gene activation.

Increased vinblastine binding to membrane vesicles from multidrug-resistant KB cells.

Human KB carcinoma cells resistant to high levels of colchicine, vinblastine, vincristine, adriamycin, and actinomycin D exhibit reduced accumulation of these structurally unrelated chemotherapeutic agents (Akiyama, S.-I., Fojo, A., Hanover, J. A., Pastan, I., and Gottesman, M. M. (1985) Somatic Cell Mol. Genet. 11, 117-126; Fojo, A., Akiyama, S.-I., Gottesman, M. M., and Pastan, I. (1985) Cancer Res. 45, 3002-3007). To examine the mechanism of reduced drug accumulation in these cells, we measured [3H]vinblastine ([3H]VBL) binding to membrane vesicles made from drug-sensitive (KB-3-1), drug-resistant (KB-C4), and revertant (KB-R1) cells. Membrane vesicles from KB-C4 cells bound up to 8-fold more [3H]VBL than vesicles from the parental KB-3-1 or revertant KB-R1 cell lines. No difference in binding of [3H]dexamethasone, to which the cells are equally sensitive, was observed. The difference in [3H]VBL binding by vesicles from resistant and sensitive cells was eliminated by the addition of 10 micrograms/ml verapamil, which is known to reverse the multidrug-resistance phenotype. Drug binding by KB-C4 vesicles was osmotically insensitive, temperature-dependent, and trypsin-sensitive. Binding of [3H]VBL by KB-C4 vesicles was inhibited by vinblastine, vincristine, and daunomycin (in decreasing order). Dexamethasone at 100 microM, colchicine at 100 microM, and actinomycin D at 100 microM did not significantly inhibit [3H]VBL accumulation. No significant differences in tubulin content were detected among vesicles from sensitive and resistant cells. These data demonstrate that membrane vesicles from multiply drug-resistant cells bind increased amounts of vinblastine.