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.

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.

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.

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.

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.

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.

Differential regulation of telomerase activity by six telomerase subunits.

Telomerase is a specialized reverse transcriptase responsible for synthesizing telomeric DNA at the ends of chromosomes. Six subunits composing the telomerase complex have been cloned: hTR (human telomerase RNA), TEP1 (telomerase-associated protein 1), hTERT (human telomerase reverse transcriptase), hsp90 (heat shock protein 90), p23, and dyskerin. In this study, we investigated the role of each the telomerase subunit on the activity of telomerase. Through down- or upregulation of telomerase, we found that only hTERT expression changed proportionally with the level of telomerase activity. The other components, TEP1, hTR, hsp90, p23, and dyskerin remained at high and unchanged levels throughout modulation. In vivo and in vitro experiments with antisense oligonucleotides against each telomerase component were also performed. Telomerase activity was decreased or abolished by antisense treatment. To correlate clinical sample status, four pairs of normal and malignant tissues from patients with oral cancer were examined. Except for the hTERT subunit, which showed differential expression in normal and cancer tissues, all other components were expressed in both normal and malignant tissues. We conclude that hTERT is a regulatable subunit, whereas the other components are expressed more constantly in cells. Although hTERT has a rate-limiting effect on enzyme activity, the other telomerase subunits (hTR, TEP1, hsp90, p23, dyskerin) participated in full enzyme activity. We hypothesize that once hTERT is expressed, all other telomerase subunits can be assembled to form a highly active holoenzyme.

Preparation and tumor cell uptake of poly(N-isopropylacrylamide) folate conjugates.

Folate conjugates (PNIPAM-NH-FA) of a copolymer of N-isopropylacrylamide (NIPAM) and amino-N'-ethylenedioxy-bis(ethylacrylamide) were prepared by an efficient synthesis leading to random grafting, via a short dioxyethylene spacer, of approximately 7 folic acid residues per macromolecule. The chemical composition of the copolymer was characterized by (1)H NMR and UV/vis spectroscopy. A fluorophore-labeled folate PNIPAM conjugate was tested by in vitro assays performed with cultured KB-31 cells overexpressing the folate receptor. The cellular uptake of the copolymer was found to be temperature dependent and was competitively decreased by free folic acid, indicating that the polymer uptake is mediated specifically by the folate receptor. Hydrophobically modified folate conjugates of NIPAM, amino-N'-ethylenedioxy-bis(ethylacrylamide) copolymers, bearing a small number of n-octadecyl groups were prepared following a modified synthetic procedure for use in future studies of FA-targeted liposomes.

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.

Studies leading to the identification of ZD1839 (IRESSA): an orally active, selective epidermal growth factor receptor tyrosine kinase inhibitor targeted to the treatment of cancer.

This paper describes the development of the epidermal growth factor receptor tyrosine kinase inhibitor ZD1839 from a lead series of 4-anilinoquinazoline compounds. ZD1839 has suitable properties for use as a clinically effective drug and shows activity against human tumours. In particular, the use of pharmacokinetic data in the development of ZD1839 is discussed.

R-dl-verapamil downmodulates multidrug resistance of KBv200 cells to vincristine and doxorubicin.

AIM: To study the attenuation of multidrug resistance (MDR) by R-dl-verapamil (R-Ver) and the acute animal toxicity of R-Ver, and to compare these results of R-Ver with the results of dl-verapamil (Ver). METHODS: Cytotoxicity was determined by tetrazolium (MTT) assay. Cellular accumulation of doxorubicin (Dox) was measured by fluorescence spectrophotometry. Acute animal toxicity was tested by i.p. drug administration in BALB/c mice. RESULTS: R-Ver attenuated MDR of KBv200 cells to vincristine (VCR) and Dox. This attenuation ability was dose-related, and was also dependent on drug exposure time. R-Ver 1.25 mumol.L-1 increased the sensitivity of KBv200 cells to VCR (P < 0.01) with a 24-h period of drug exposure. R-Ver downmodulated MDR and increased cellular Dox accumulation of KBv200 cells as effectively as Ver, but possessed lower acute toxicity in BALB/c mice. While LD50 of Ver was 60 (49-73) mg.kg-1, LD50 of R-Ver was 166 (137-202) mg.kg-1. CONCLUSION: R-Ver downmodulated the MDR to VCR and Dox at 1.25 mumol.L-1, and this effect on VCR can be realized with drug exposure duration of 24 h.

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.

Enhanced in vitro cytotoxicity of idarubicin compared to epirubicin and doxorubicin in rat prostate carcinoma cells.

OBJECTIVE: We evaluated the cytotoxic activity of the three anthracyclines, doxorubicin, epirubicin and idarubicin, in different sublines of the Dunning rat prostate carcinoma as well as in multidrug-resistant KB cells, expressing a high amount of the human multidrug resistance gene product, P glycoprotein. METHODS: The effectiveness of the three anthracyclines was tested in vitro in the Dunning rat prostate carcinoma sublines G, AT.1, AT.3.1, MatLu, and MatLyLu, as well as in multidrug-resistant KB cells, using an MTT assay. RESULTS: All drugs were clearly more effective in the androgen-sensitive Dunning rat prostate carcinoma subline G than in the androgen-independent growing sublines AT.1, AT.3.1, MatLu, and MatLyLu. Idarubicin was much more effective than doxorubicin or epirubicin. To further elucidate the mechanism of action of idarubicin as compared with doxorubicin and epirubicin, we tested the cytotoxicity of these anthracyclines in highly multidrug-resistant KB-V1 cells, which express high amounts of P glycoprotein, as well as in the drug-sensitive parental KB-3-1 cells. KB-V1 cells proved to be highly resistant to doxorubicin and epirubicin with IC50 values of 2,300 and 1,000 ng/ml, respectively. Idarubicin, however, was about 57.5-fold and 25-fold more active, respectively, suggesting, that it is able to overcome P-glycoprotein-mediated multidrug resistance. CONCLUSION: The strong in vitro effectiveness of idarubicin in androgen-insensitive prostate carcinoma cells suggests that this drug might be useful in the treatment of hormone-refractory prostate carcinoma.

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.

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.

9-Octadecynoic acid: a novel DNA binding agent.

A novel bioassay was developed to permit the identification of cytotoxic natural principles that bind to DNA. A hexane extract of Schoepfia californica cytotoxic to cultured KB cells displayed much less cytotoxic potential when the culture medium contained exogenously added calf thymus DNA. Fractionation of the extract afforded a purified principle shown to be 9-octadecynoic acid, an 18-carbon, unbranched acetylenic fatty acid. 9-Octadecynoic acid had an apparent DNA dissociation constant of 1.8 mM; it inhibited topoisomerase I mediated DNA filter binding but did not inhibit the DNA topoisomerase I mediated relaxation of a supercoiled plasmid DNA. The fatty acid was weakly inhibitory to DNA polymerase alpha. 9-Octadecynoic acid possesses none of the structural characteristics of known DNA binding molecules and may bind to DNA by some novel mechanism.

[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.

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.

Multiple drug-resistant human KB carcinoma cells independently selected for high-level resistance to colchicine, adriamycin, or vinblastine show changes in expression of specific proteins.

We have established four cell lines derived from the human KB carcinoma cell line which express high-level multiple drug resistance. One of these lines was selected for resistance to colchicine, one was selected for resistance to colchicine in the presence of the tumor promoter, mezerein, one for resistance to vinblastine, and one for resistance to adriamycin. All of these cell lines are cross-resistant to the other selective agents. The development of multidrug resistance in these cultured human carcinoma cells is associated with a limited number of specific protein alterations revealed by high resolution two-dimensional gel electrophoresis and Western blot analysis. These protein alterations in multidrug-resistant lines include the decreased prevalence of members of a family of proteins of molecular mass 70,000 to 80,000 daltons, pI 4.8-5.0, the increased synthesis of a protein of molecular mass 21,000 daltons, pI 5.0, in the colchicine-resistant cell lines only, and the increased expression of a 170,000-dalton protein in membrane preparations from all of the resistant cells. The loss of the 70,000- to 80,000-dalton proteins in the multidrug-resistant lines, which can also be demonstrated by immunoprecipitation of these proteins with specific antisera, is associated with a loss of translatable mRNA for these proteins. These studies suggest that only a limited number of protein changes occur in multidrug-resistant cell lines.