Genotoxicity of adriamycin in human cell lines differing in antioxidant enzymes activity

Genotoxicity of adriamycin in human cell lines was investigated by using a micronucleus assay. The result obtained was negative: the cells treated showed no increase in micronuclei. The chromosome aberration study with adriamycin indicated that the number of aberrant cells, high immediately after treatment, decreased to nearly the control level in 24 h of postincubation, probably as a result of the DNA repair process. Experiments with caffeine--a DNA repair inhibitor--indicated an increase micronuclei in the cells treated with adriamycin and caffeine together. The results obtained suggest that, in a human cells, adriamycin--induced DNA damages are quickly repaired to prevent micronuclei formation.     

Genotoxicity of nano-silica in mammalian cell lines

Nanomaterials are defined by the U.S. National Nanotechnology Initiative as materials that have at least one dimension in the 1- to 100-nm range. Due to their unique physical and chemical characteristics, nanotechnology has become one of the leading technologies over the past 10 years. This study represents data on genotoxic effects of nanoparticles and their application for assessing human health risks. Silica (SiO₂) is a multi-functional ceramic material that is being used in various industries to improve surfaces and mechanical properties of diverse materials, such as paints and coatings, plastics, synthetic rubber, adhesives, sealants, or insulation materials. However, recent studies have shown that nano-sized silica (nano-silica) (10 nm in diameter) can generate adverse effects, like liver injury and inflammation. The cytotoxicity and genotoxicity of nano-silica were investigated using the dye exclusion assay, comet assay, and mouse lymphoma thymidine kinase (tk ^+/?) mouse lymphoma assay (MLA). IC₂₀ of nano-silica in L5178Y cells was determined to be of 2,441.41 μg/mL and 2,363.28 μg/mL without and with S-9, respectively. Also IC₂₀ of nano-silica in BEAS-2B cells was determined to be of 2,324.23 μg/mL and 537.11 μg/mL without and with S-9, respectively. In the comet assay, treating L5178Y cells and BEAS-2B cells with nanosilica treatment induced approximately 2-fold increases in tail moment (P<0.05) without and with S-9. Also, the mutant frequencies in the nano-silica treated L5178Y cells were not significantly increased compared to the solvent controls. The results of this study indicate that nano-silica can cause primary DNA damage and cytotoxicity but not mutagenicity in cultured mammalian cells.     

人胰腺癌细胞中端粒酶活性的检测

目的 观察银染的端粒重复序列扩增法（TRAP）和TRAP－ELISA两种方法检测人胰腺癌细胞株中端粒酶活性的特异性和敏感性。方法 用TRAP－银染法和TRAP－ELISA两种方法检测人胰腺癌细胞株及人皮肤成纤维细胞中端粒酶活性,并检测经RNase和加热处理的阴性对照标本。结果 10个以上的P3细胞均为端粒酶阳性,而RNase和加热处理的标本均为阴性,胰腺癌细胞株Patu-8801亦为阳性,人皮肤成纤维细胞为阴性。结论 两种方法均为特异、敏感的端粒酶活性检测法,TRAP－ELISA方法更简单、方便,并证实两株人胰腺癌细胞株均为端粒酶阳性。     

Ultra-deformable liposomes containing bleomycin: in vitro stability and toxicity on human cutaneous keratinocyte cell lines

Formulations of ultra-deformable liposomes containing bleomycin (Bleosome™) have previously been described and proposed for topical treatment of skin cancer [Lau, K.G., Chopra, S., Maitani, Y., 2003. Entrapment of bleomycin in ultra-deformable liposomes. S. T. P. Pharm. Sci. 13, 237–239]. In this study, the stability of various Bleosome™ formulations was characterised and a purification process was established to isolate Bleosome™ for testing on cultures of either human cutaneous keratinocytes (NEB-1) immortalised by human papilloma virus (HPV)-type 16, or a spontaneously immortalised human squamous cell carcinoma (SCC) from a primary tumour. Bleosome™ facilitated entrapment of high concentrations of active bleomycin and samples purified by gel-filtration chromatography remained stable during 7 days of storage at 4 °C or at room temperature. Serially-diluted samples of this purified, high-strength product, ‘high dose’ were applied onto keratinocyte cell cultures to elucidate Bleosome™ LD₅₀ profiles.

In vitro data revealed that the LD₅₀ of bleomycin encapsulated in Bleosome™ was approximately three-fold higher than free bleomycin solution for SCC cells, and nearly 30 times higher for NEB-1 cells. However, Bleosome™ containing 30 μg/ml of active bleomycin killed more than twice as many SCC cells than NEB-1 cells. At that concentration, the potency of liposomal bleomycin on causing cell death of SCC cells was found to be similar to that of free bleomycin solution. This effect was not seen on NEB-1 cells. It seems that SCC cells were particularly susceptible to Bleosome™ containing high levels of bleomycin. Results from these experiments promote the development of a novel product for the topical treatment of skin cancer.     

Cytotoxic activity of styrylchromones against human tumor cell lines

A total of 6 newly-synthesized styrylchromones (SC-1 approximately SC-6) were compared for their cytotoxic activity against three normal oral human cells (gingival fibroblast HGF, pulp cell HPC, periodontal ligament fibroblast HPLF) and four human tumor cell lines (squamous cell carcinoma HSC-2, HSC-3, submandibular gland carcinoma HSG, promyelocytic leukemia HL-60). All compounds showed higher cytotoxic activity against tumor cell lines than against normal cells. Among the 6 compounds, SC-3, SC-4 and SC-5, which have one to three methoxy groups, showed higher tumor specificity and water solubility. The cytotoxic activity of SC-3 and SC-5 was slightly reduced by a lower concentration of NADH, a quinone reductase, but that of SC-3 was enhanced by higher concentrations of NADH, possibly due to demethylation of the methoxy groups. Agarose gel electrophoresis demonstrated that SC-3 and SC-5 induced intemucleosomal DNA fragmentation in HL-60 cells and production of large DNA fragment in HSC-2 cells. Both SC-3 and SC-5 enhanced the enzymatic activity to cleave the substrates for caspases 3, 8 and 9, suggesting the activation of both extrinsic and intrinsic apoptosis pathways. ESR spectroscopy showed that these compounds produced no detectable amount of radical and did not scavenge superoxide anion generated by the hypoxanthine-xanthine oxidase reaction. The highly tumor-specific cytotoxic action and apoptosis-inducing capability of SC-3 and SC-5 suggest their applicability for cancer chemotherapy.     

Is ribosomal cistron activity increased in human lymphoblastoid cell lines?

The genomic activity of nucleolar organizer regions (NORs) in eight human B-cell lymphoblastoid cell lines was studied following the routinely used Ag-NOR technique. The results demonstrate that (a) Ag-NORs are located in the short arms of D- and G-group chromosomes, (b) two out of eight cell lines have 66.7% and 49.0% of metaphases, respectively, with 9 to 10 active Ag-NORs, and (c) as a whole, Ag-NOR activity is much higher in B-cell lines as compared with conventional 72-hr peripheral blood lymphocyte (T-cell) harvests.     

Methanol teratogenicity in mutant mice with deficient catalase activity and transgenic mice expressing human catalase

The role of catalase in methanol (MeOH) teratogenesis is unclear. In rodents it both detoxifies reactive oxygen species (ROS) and metabolizes MeOH and its formic acid (FA) metabolite. We treated pregnant mice expressing either high (hCat) or low catalase activity (aCat), or their wild-type (WT) controls, with either MeOH (4 g/kg ip) or saline. hCat mice and WTs were similarly susceptible to MeOH-initiated ophthalmic abnormalities and cleft palates. aCat and WT mice appeared resistant, precluding assessment of the developmental impact of catalase deficiency. Catalase activity was respectively increased at least 1.5-fold, and decreased by at least 35%, in hCat and aCat embryos and maternal livers. MeOH and FA pharmacokinetic profiles were similar among hCat, aCat and WT strains. Although the hCat results imply no ROS involvement, embryo culture studies suggest this may be confounded by maternal factors and/or a requirement for higher catalase activity in the hCat mice.     

Cytotoxic activity of kaempferol glycosides against human leukaemic cell lines in vitro.

Two kaempferol coumaroyl glycosides (i.e. platanoside and tiliroside) isolated from the methanolic extract of Platanus orientalis L. buds, were examined for their in vitro cytotoxic activity against a panel of human leukaemic cell lines. Platanoside (1) exhibited cytotoxic activity against most of the cell lines tested, while tiliroside (2) was active against two of the nine tested cell lines. Compound 1, was examined for its effect on the uptake of [(3)H]thymidine as a marker of DNA synthesis. Kaempferol was used as a control.     

Antioxidant activity of Areca catechu extracts in human hepatocarcinoma HepG2 cell lines

The effect of aqueous and various organic extracts from the different parts of Areca catechu L. (Arecaceae) on oxidative DNA damage in human hepatocarcinoma HepG2 cells was investigated using single cell gel electrophoresis (comet) assay. Comparison of a series of organic extracts from the nut husk and different parts of this plant was performed. Incubation of methanol extract of Areca nut husk showed a dose-dependent inhibition of comet formation while other solvent extracts did not. Significant protection of H₂O₂-induced DNA damage was observed at 0.1% w/v concentration which was at the same level of protection by 1 μM butylated hydroxytoluene (BHT). The age of Areca catechu nut husk also played important role in chemical constituents exhibiting antioxidation since only two out of five different ages of husks displayed anti-oxidative effects in the comet assay. Among the different parts of this plant that were tested, the nut husk was the only consistent part that demonstrated antioxidant activity.     

Genotoxicity of wood dust in a human embryonic lung cell line

 Wood dust exposure has been found to be an occupational hazard, being linked to an enhanced incidence of various neoplasias. Here we performed an experiment to evaluate the ability of solvent extracts of natural woods to induce chromosome aberrations in respiratory cells in culture. Human embryonic lung cells, MRC-5, grown in Dolbecco’s medium were exposed to various concentrations of the dust extracts of pesticide-free (untreated) beech, oak and pine woods. Three concentrations per extract with and without metabolic activation (S9) and 100 metaphase cells per dose were examined for possible structural aberrations. Although no dose-dependent activity could be found with any extract in the presence of S9, most aberrations observed were of the chromatid type caused by oak wood. Dose-dependent chromosomal breaks caused by oak and chromatid breaks caused by both beech and oak were observed in the absence of S9. These data might support the early hypothesis that hard wood dust per se contains some in vivo genotoxic and thus possibly carcinogenic components. Received: 10 March 1995/Accepted: 19 June 1995     

Synergistic antitumor activity of troxacitabine and camptothecin in selected human cancer cell lines

Troxacitabine (L-OddC) is an L-configuration deoxycytidine analog currently in phase II trials for the treatment of cancer. The cytotoxicity of L-OddC in combination with other anticancer agents has not been studied systematically. In the present study, we assessed the cytotoxic effects produced by the combinations of L-OddC and several commonly used chemotherapy drugs in a panel of cultured human cancer cell lines. Growth inhibition resulting from simultaneous exposure to two-drug combinations was determined using the methylene blue staining method. Camptothecin (CPT) and analogs exhibited additives to synergistic interactions with L-OddC by isobologram analysis. These effects were cell type-specific, with the most pronounced synergism being observed in KB oropharyngeal carcinoma and CPT-resistant KB100 cell lines. In KB cells, the total cellular uptake and DNA incorporation of L-OddC were increased by the addition of CPT. One explanation that emerged from enzyme assays of deoxycytidine kinase (dCK) and deoxycytidine monophosphate kinase (dCMPK), key enzymes involved in L-OddC phosphorylation, was that CPT protected against L-OddC-induced reduction in dCK and dCMPK activity. The resulting increase in l-OddC metabolites and incorporation into DNA was associated with enhanced L-OddC cytotoxicity. These findings will be useful in designing future clinical trials of combination chemotherapy with l-OddC and CPT analogs with the potential for a broad use against both hematological and solid tumors.     

3-substituted 7-phenyl-pyrroloquinolinones show potent cytotoxic activity in human cancer cell lines

A novel series of 3-alkyl-substituted 7-phenyl-3H-pyrrolo[3,2-f]quinolin-9-ones (7-PPyQs) was synthesized with the aim to optimize the cytotoxic activity of recently identified PPyQs, promising inhibitors of tubulin polymerization. All compounds inhibited the growth of 11 human tumor cell lines at submicromolar concentrations as well as two human resistant cancer sublines, A549-T12 and A549-T24. FACS analysis indicated that all compounds caused significant arrest of the A549 cell cycle in G2/M phase at 0.1 and 1 muM and a good correlation between the cytotoxicity IC50 and their ability to block the cell cycle was observed.     

Oxaliplatin activity in selected and unselected human ovarian and colorectal cancer cell lines

Oxaliplatin is used for treatment of colon cancer in combination with 5-fluorouracil or irinotecan. Oxaliplatin has similar, but also different resistant mechanisms as cisplatin. We studied the activity of oxaliplatin in ovarian and colon cancer cells with different resistance patterns to cisplatin. The 40-fold cisplatin-resistant cell line ADDP was only 7.5-fold resistant to oxaliplatin. The gemcitabine-resistant AG6000 cell line, 9-fold resistant to cisplatin, was not cross-resistant. LoVo-175X2, with mutant p53 showed no resistance compared to the empty vector control. However, LoVo-Li, with inactive p53, was 3.6-fold resistant corresponding to decreased accumulation and Pt adducts. Accumulation and DNA adducts formation showed no significant correlation with oxaliplatin sensitivity. Cell cycle distribution after exposure to oxaliplatin showed arrest in G2/M (A2780) or in S-phase (LoVo-92) for wt-p53 cells. ADDP and LoVo-Li showed G1 arrest followed by S-phase arrest and no changes in distribution, respectively. The cell cycle related proteins Cyclins A and B1 and (p)CDC25C were marginally affected by oxaliplatin. Expression of hCTR1 was decreased in ADDP, LoVo-Li and AG6000, OCT1 decreased in ADDP and AG6000 and OCT3 in LoVo-175X2, compared to the parental cell lines. In ADDP and LoVo-175X2 ATP7A and B were decreased but were increased in AG6000. From this study it can be concluded that changes in cell cycle distribution were cell line dependent and not related to changes in expression of Cyclin A or B1. Oxaliplatin accumulation was related to hCTR1 and, at low concentration, ATP7A to DNA adducts formation while the retention was related to hCTR1, OCT2 and ATP7B.     

Antitumor activity of some ruthenium derivatives in human colon cancer cell lines in vitro.

Six ruthenium derivatives were evaluated in vitro in two human colon cancer cell lines (SW707 and SW948) utilizing the microculture tetrazolium test (MTT) and cell counting with a Coulter Counter. The ruthenium compound sodium-(tetrachloroimidazoledimethylsulfoxideruthenate)- bisdimethylsulfoxide (Na(RuDMSOimCl4)) showed the best efficacy in inhibiting cell proliferation of both colon cancer cell lines followed by the other DMSO ruthenium compound sodium-(tetrachloroindazoledimethylsulfoxideruthenate) - bisdimethylsulfoxide (Na(RuDMSOIndCl4)), as demonstrated by IC50 values (80 and 90 micrograms/ml in SW707 and SW948 cell lines for Na(RuDMSOImCl4); 155 and 165 micrograms/ml in SW707 and SW948 cell lines for Na(RuDMSOIndCl4), respectively). Out of the ruthenium derivatives without DMSO, transindazolium - [tetrachlorobis(1H - indazole)ruthenate (III,N2)] (HInd[RuInd2Cl4(N2)]), was as active as its DMSO-containing congener whereas trans-imidazolium- [tetrachlorobisimidazoleruthenate)(III)], (HIm(RuIm2Cl4)) was less active, as shown by the IC50 values: (HIm (RuIm2Cl4) = 250 and 260 micrograms/ml in cell lines SW707 and SW948; HInd[RuInd2Cl4(N2)] = 110 and greater than 200 micrograms/ml in cell lines SW707 and SW948, respectively). The other ruthenium derivatives containing pyrazole and triazole as ligands (trans - pyrazolium (tetrachlorobispyrazoleruthenate) (III), PzH(RuPz2Cl4) and triazolium(tetrachlorobistriazoleruthenate) (III), TrH(RuTr2Cl4)) were active only at high concentrations that cannot be regarded as realistic in vivo, as shown by the respective IC50 values: (PzH(RuPz2Cl4) = 1056 and 750 micrograms/ml in cell lines SW707 and SW948; TrH(RuTr2Cl4) = 350 and 300 micrograms/ml in cell lines SW707 and SW948). The promising activity of ruthenium compounds with DMSO, indazole and imidazole as ligands should be evaluated in vivo for elucidating their possible role in the treatment of colorectal cancer.     

Biological activity of the tryprostatins and their diastereomers on human carcinoma cell lines

Tryprostatin A 1 and B 2 are indole alkaloid-based fungal products that act in the G2/M phase of the cell cycle. Tryprostatin A and B as well as their two enantiomers and four diastereomers have been synthesized via a common strategy. As a measure of cytotoxicity, these eight stereoisomers were assayed for their growth inhibitory properties in human breast, prostate, and lung cancer cell lines. The ability of the tryprostatins and the tryprostatin stereoisomers to induce topoisomerase II-mediated DNA relaxation or to inhibit tubulin polymerization was also examined. Although none of the stereoisomers were significantly active in topoisomerase II- or tubulin-based assays, ds2-try B 11 was found to exhibit a cytotoxicity profile more potent than etoposide 3 in the human cancer cell lines examined. In addition, ds2-try B 11 is comprised of an L-tryptophan derivative coupled to a D-proline moiety, the latter stereochemistry of which may enhance the activity of 11 and potential analogues in vivo.     

Decreased GSSG reductase activity enhances cellular zinc toxicity in three human lung cell lines

Cellular reduced glutathione (GSH) levels have been identified as an essential determinant in zinc-induced cytotoxicity. However, cytotoxic effects of zinc have also been observed without depletion of GSH stores. In a previous study, the intracellular activity of GSSG reductase (GR) has come into focus (Walther et al. 2000, Biol Trace Elem Res 78:163-177). In the present paper we have tried to address this issue more deeply by inhibiting the activity of cellular GR without any appreciable decreases of cellular glutathione. In three pulmonary cell lines, GR activity was inhibited in a dose-dependent manner by the alkylating agent carmustine (BCNU), a known inhibitor of GR. Cells were pretreated with BCNU for 14 h, followed by exposure to various concentrations of zinc chloride. Then we determined the incorporation of radiolabelled methionine (to assess protein synthesis), and measured the GSH and oxidized glutathione (GSSG) levels. Additionally, GR activity of controls was measured. IC(50) values for zinc-induced inhibition of methionine incorporation, as well as GSH contents, was strongly correlated to the decreased GR activity. These results firmly suggest that GR is an important factor in the event chain of zinc cytotoxicity. Together with the results from our previously cited study where impaired regeneration of GSH levels were accompanied by a decrease in total cellular glutathione (GSH + GSSG) we conclude that GSSG itself is an important effector in zinc cytotoxicity.     

Characterization of carbonyl reducing activity in continuous cell lines of human and rodent origin.

Carbonyl reduction was investigated in the continuous cell lines V79, NCI-H322 and C2REV7 by using the ketone compound metyrapone as a substrate. Metyrapone reducing enzymes were characterized by evaluating the cosubstrate requirement and by testing the sensitivity of this reaction to specific inhibitors. All cell lines were found to produce metyrapol at a linear rate over a time course of at least 48 hr, when tested in cultured monolayers. In general, cytosolic metyrapone reduction exceeds microsomal activity several-fold in all three cell lines. Quercitrin turned out to be the strongest inhibitor in all fractions, except in NCI-H322 microsomes where it had no effect. Consequently, carbonyl reductase is suspected to be responsible for metyrapone reduction in the cytosol and microsomes of V79 and C2REV7 cells as well as in the cytosol of NCI-H322 cells. Simultaneous sensitivity towards quercitrin, dicoumarol, indomethacin and 5 alpha-dihydrotestosterone in some cases points to the existence of different isozymes of carbonyl reductase. In NCI-H322 microsomes only dicoumarol and indomethacin decrease metyrapol formation, thus pointing to an isozyme of NAD(P)H:quinone-oxidoreductase. Concerning cosubstrate requirements metyrapone reducing enzymes show a strong preference for NADPH, thus confirming the involvement of carbonyl reductase in this reaction. In conclusion, carbonyl reduction of metyrapone in continuous cell lines is mediated by carbonyl reductases due to the common sensitivity towards the diagnostic inhibitor quercitrin and due to the strong preference for NADPH as cosubstrate. According to its maintenance in permanent cell lines carbonyl reductase seems to be an essential and constitutive enzyme, which probably fills an important role in normal cell physiology.     

Antiproliferative activity of parthenolide against three human cancer cell lines and human umbilical vein endothelial cells

Parthenolide is a major sesquiterpene lactone derived from feverfew (Tanacetum parthenium) with known anti-inflammatory activity. Moreover, the anticancer potential of this compound was suggested. In this study, we determined the effect of parthenolide on proliferation of three human cancer cell lines: human lung carcinoma (A549), human medulloblastoma (TE671), human colon adenocarcinoma (HT-29) and human umbilical vein endothelial cells (HUVEC) in vitro. Cell proliferation was assessed by means of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. The IC(50) value (the concentration of drug necessary to induce 50% inhibition) together with confidence limits was calculated. Parthenolide inhibited proliferation of all three types of cancer cells (A549, TE671, HT-29) and HUVEC with the following IC(50) values (in muM): 4.3, 6.5, 7.0 and 2.8, respectively. Thus, the antiproliferative potential of parthenolide was confirmed.