醋酸曲普瑞林对人子宫内膜腺癌HEC-1B的作用及对C-myc表达的调控

目的探讨醋酸曲普瑞林对人子宫内膜腺癌HEC-1B细胞的抑制作用及其分子机制。方法体外培养人子宫内膜腺癌HEC-1B细胞,用不同浓度醋酸曲普瑞林(10-9,10-8,10-7,10-6,10-5mol/L)为实验组,0mol/L组(不含醋酸曲普瑞林的培养液)为对照组,分别和HEC-1B细胞作用24、48、72h,四甲基偶氮唑蓝(MTT)法检测细胞抑制率,绘制肿瘤细胞生长抑制率曲线。免疫组织化学染色法检测72h细胞中C-myc蛋白表达,反转录-聚合酶链反应(RT-PCR)检测72hC-mycmRNA的表达。结果①当醋酸曲普瑞林浓度为10-9mol/L时,HEC-1B细胞生长即受到抑制,抑制率为8.97%;随着浓度的增大,抑制率渐高,浓度为10-5mol/L时抑制率上升至34.12%,与对照组比较抑制率差异有统计学意义(P<0.05)。②浓度从10-9～10-5mol/L的醋酸曲普瑞林作用72h后,C-myc蛋白和C-mycmRNA表达均有不同程度的下降,与对照组相比差异有统计学意义(P<0.05)。结论醋酸曲普瑞林可以抑制HEC-1B细胞的生长,其机制可能是通过下调HEC-1B细胞C-myc蛋白的表达而实现。     

Genotoxicity analysis of the phenoxy herbicide dicamba in mammalian cells in vitro

The cytogenetic effects exerted by the phenoxy herbicide dicamba and one of its commercial formulations banvel^® (57.71% dicamba) were studied in in vitro whole blood human lymphocyte cultures. The genotoxicity of herbicides was measured by analysis of the frequency of sister chromatid exchanges (SCEs) and cell-cycle progression assays. Both dicamba and banvel^® activities were tested within 10.0–500.0 μg/ml doses range. Only concentrations of 200.0 μg/ml of dicamba and 500.0 μg/ml of banvel^® induced a significant increase in SCE frequency over control values. The highest dose of dicamba tested (500.0 μg/ml) resulted in cell culture cytotoxicity. The cell-cycle kinetics was affected by both test compounds since a significant delay in cell-cycle progression and a significant reduction of the proliferative rate index were observed after the treatment with 100.0 and 200.0 μg/ml of dicamba and 200.0 and 500.0 μg/ml of banvel^®. For both chemicals, a progressive dose-related inhibition of the mitotic activity of cultures was observed. Moreover, only the mitotic activity statistically differed from control values when doses of both chemicals higher than 100.0 μg/ml were employed. On the basis of our results, the herbicide dicamba is a DNA damage agent and should be considered as a potentially hazardous compound to humans.     

Effect of Pb2+ on Friend leukemia cells during erythroid maturation

The effect of different concentrations of Pb²⁺ on the growth rate and on the percentage of hemoglobin-synthesizing cells of Friend leukemia cells was determined. Pb²⁺ had a higher inhibitory effect on the growth and on the final percentage of hemoglobin-synthesizing cells when cultures were induced to erythroid differentiation by either dimethyl sulfoxide or hexamethylenebisacetamyde (HMBA) as compared with uninduced cultures. The increased sensitivity to Pb²⁺ of cultures containing either dimethyl sulfoxide or (HMBA) appeared to be correlated with the differentiation process triggered by these two substances, since the increased sensitivity was not observed with a Friend leukemia cells variant which did not differentiate in the presence of any inducer. The effect of Pb²⁺ on the activities of δ-aminolevulinic acid (ALA) dehydratase and URO synthetase, two enzymes involved in heme synthesis, was also determined. Induced cells continuously grown in the presence of Pb²⁺ up to 72 hr postinduction were not affected in URO synthetase activity, whereas the activity of ALA dehydratase was decreased as compared with induced cultures grown without lead. By 96 and 120 hr postinduction an increase in URO synthetase activity and a decreased inhibition of ALA dehydratase activity were observed. The inhibitory effect of Pb²⁺ on the growth rate of induced cells was much higher when cultures were treated during the first 24 hr of growth than it was when Pb²⁺ was added during the second 24 hr. In uninduced cultures the same low level of inhibition was observed following treatment during either 24-hr period. Cell agglutinability by plant lectins was also inhibited in induced cultures by the presence of Pb²⁺ during cellular growth.     

Effects of juvenile hormone and related insect growth regulators on mammalian cell proliferation: A comparative study

The effects of the synthetic C₁₈ juvenile hormone, methoprene (ZR-0515), and of the compound ZR-0619 on morphology, mitotic activity, and pattern of growth of a subline of L-929 cells were compared. The cells grew as monolayers for 24 hr in a standard medium, then for another 24 or 48 hr under one of the following conditions: (1) in the standard medium, (2) in standard medium with the addition of one of the compounds tested (20, 50, 100 μg/ml), or (3) in the presence of solvent at the appropriate concentrations (0.1, 0.25, 0.5%). The susceptibility of the cells to the compounds under examination was compared by analysis of variance of the data for mitotic indices and for the frequency of occurrence of monokaryocytes and polykaryocyts in the cultures. We found that the solvent itself did not influence either cell morphology or mitotic activity of the cells until it was at 0.5% concentration whereas juvenile hormone and either of its analogs suppressed this activity significantly at all three concentrations. At both lower concentrations applied, their officacy was similar, since the fall of the mitotic indices for the cells of the media with the hormone or its analogs did not differ significantly until their concentration was 100 μg/ml, when most of the cells died out in the media with the hormone, but grow in the media with either of the analogs, although their morphology and growth patterns were more or less affected. The number of monokaryocytes and polykaryocytes did not change in cultures of the cells under our experimental conditions.     

Inhibitory effects of mevastatin and a geranylgeranyl transferase I inhibitor (GGTI-2166) on mononuclear osteoclast formation induced by receptor activator of NF kappa B ligand (RANKL) or tumor necrosis factor-alpha (TNF-alpha)

We have previously reported that the statin mevastatin (compactin) reversibly inhibits the fusion of TRAP-positive mononuclear preosteoclasts (pOCs) into multinucleated osteoclasts and disrupts the actin ring in mature osteoclasts through the inhibition of protein prenylation. Protein geranylgeranylation, specifically, is known to be required for pOC fusion and for the function and survival of mature osteoclasts. However, it has not been determined whether protein geranylgeranylation is involved in early differentiation of osteoclasts (pOC formation). The current study shows that statins and the geranylgeranyl transferase I inhibitor GGTI-2166 inhibit the pOC formation induced by RANKL or TNF-alpha in cultures of both mouse marrow-derived macrophage-colony-stimulating factor (M-CSF) dependent monocytes (MD cells) and the mouse monocyte cell line RAW 264.7 (RAW cells). Mevastatin, 0.1-0.6 microM, inhibited the formation of pOCs induced by receptor activator of nuclear factor-kappaB ligand (RANKL) or tumor necrosis factor (TNF-alpha) in both cell cultures. The inhibitory effects of mevastatin were overcome by the addition of mevalonate, farnesyl pyrophosphate or geranylgeranyl pyrophosphate. GGTI-2166 inhibited TRAP activity induced by RANKL or TNF-alpha in both cell cultures and prevented the incorporation of [3H]all-trans geranylgeraniol into prenylated proteins in RAW cells. However, the farnesyl transferase inhibitor FTI-2153 did not inhibit TRAP activity although FTI prevented the incorporation of [14C]mevalonate into farnesylated proteins in RAW cells. Clostridium difficile cytotoxin B (toxin B) inhibited pOC formation induced by RANKL or TNF-alpha in both cell cultures. The inhibitory effects of statins and GGTI-2166 on pOC formation may result from the inhibition of the geranylgeranylation of G-proteins, such as Rho or Rac, suggesting that the geranylgeranylation of these proteins is involved in the early differentiation of progenitor cells into pOCs.     

Antitumor effect of the cinnamaldehyde derivative CB403 through the arrest of cell cycle progression in the G2/M phase

Cinnamaldehydes have been shown to have inhibitory effects on farnesyl protein transferase (FPTase; EC 2.5.1.29) in vitro, angiogenesis, cell-cell adhesion, and tumor cell growth and to be immunomodulators. However, the mechanisms responsible for these effects remain unknown. To elucidate the molecular mechanism of the cinnamaldehyde derivative CB403 for growth inhibition, CB403 was synthesized from 2'-hydroxycinnamaldehyde. CB403-treated cells were weakly adherent to the culture dishes. In addition, CB403 inhibited tumor growth in these cells in a concentration-dependent manner. FACS analysis using human cancer cells treated with this compound showed cell cycle arrest in mitosis, which was correlated with a marked increase in the amount of cyclin B1. Furthermore, CB403 blocked in vivo growth of human colon and breast tumor xenografts without loss of body weight in nude mice. These results support the hypothesis that the cinnamaldehyde derivative CB403 exerts cytostatic properties by inducing mitotic arrest in cancer cells.     

Antibacterial Potential Assessment of Jasmine Essential Oil Against E. Coli

The antibacterial activity of Jasmine (Jasminum sambac L.) flower hydro steam distilled essential oil, synthetic blends and six major individual components was assessed against Escherichia coli (MTCC-443) strain. The activity was bactericidal. Minimum inhibitory concentration was determined by tube dilution technique, and the Minimum inhibitory concentration ranged between 1.9-31.25 μl/ml. Phenolcoefficient of the oil, synthetic blends and components varied between 0.6-1.7. The activity of the chemicals was possibly due to the inhibition of cell membrane synthesis.     

异紫堇二酮体内外抗肿瘤活性研究

目的异紫堇二酮的体内外抗肿瘤活性及其作用机制的初步研究。方法通过异紫堇二酮对体外培养肿瘤细胞的生长抑制,体内荷S180瘤和荷H22瘤昆明种小鼠的肿瘤生长抑制、体重以及对小鼠免疫器官的影响(脾脏指数、胸腺指数),对其抗肿瘤活性进行综合评价,并通过流式细胞术检测异紫堇二酮作用下肺腺癌细胞(A549)的细胞凋亡及周期分布情况,初步探讨该化合物的抗肿瘤作用机制。结果异紫堇二酮对9种所选肿瘤细胞具有一定的生长抑制作用,IC50值在1.452×104～2.460×104 mol.L-1之间;流式细胞术检测结果显示异紫堇二酮可诱导A549细胞凋亡,使A549细胞阻滞于G0/G1期并随作用时间延长出现明显的凋亡峰;剂量为100和200 mg.kg-1.d-1时,异紫堇二酮对荷S180瘤和荷H22瘤小鼠的抑瘤率均大于40%,但剂量在200 mg.kg-1.d-1时,荷瘤小鼠的胸腺指数与脾脏指数相比较生理盐水组均有明显降低。结论异紫堇二酮具有一定的抗肿瘤活性,其主要是阻断肿瘤细胞由G0/G1期向S期转变来诱导肿瘤细胞死亡。     

KRIBB3, a novel microtubule inhibitor, induces mitotic arrest and apoptosis in human cancer cells

KRIBB3 (5-(5-ethyl-2-hydroxy-4-methoxyphenyl)-4-(4-methoxyphenyl) isoxazole) inhibited cancer cell growth in vitro and in vivo. Flow cytometry studies showed that KRIBB3 caused cell cycle arrest at the G(2)/M phase and subsequent apoptosis. This was confirmed as accumulation of Cyclin B1 and cleavage of poly(ADP-ribose) polymerase (PARP) were detected. While transient inhibition by KRIBB3 led to reversible mitotic arrest, prolonged exposure to KRIBB3-induced apoptosis. Co-immunoprecipitation experiments showed that KRIBB3 initially induced association of inhibitory Mad2 with p55CDC (mammalian homologue of CDC20), an activator of APC/C (anaphase-promoting complex/cyclosome), suggesting that the mitotic spindle checkpoint was activated by KRIBB3. However, the level of this inhibitory complex of Mad2 with p55CDC was gradually decreased 24 h after KRIBB3 treatment, and was hardly detectable after 48 h, indicating some slipping of the mitotic checkpoint. Consistent with these observations, KRIBB3 activated the mitotic spindle checkpoint by disrupting the microtubule cytoskeleton. KRIBB3 was proven to be a tubulin inhibitor using in vitro polymerization assays and in vivo indirect immunofluorescence staining. The temporal pattern of Bax activation by KRIBB3 was similar to PARP cleavage, suggesting that Bax is a mediator of KRIBB3-dependent apoptosis. Furthermore, when KRIBB3 was administered intraperitoneally into nude mice at 50 mg/kg or 100 mg/kg, it inhibited 49.5 or 70.3% of tumor growth, respectively. These results suggest that KRIBB3 is a good drug candidate for cancer therapy.     

抗坏血酸对人肝癌细胞增殖与再分化的作用

AIM: To examine the effects of ascorbic acid (AA) on hepatoma. METHODS: Choosing an all-trans tretinoin (Tre) as a positive control, cell growth, and cell redifferentiation tests by cell surface charges, biochemical changes, and cell growth in soft agar were measured. RESULTS: After being treated with AA 6 mmol.L-1, the growth curve and mitotic index of human hepatoma cells decreased remarkably, the cellular growth inhibitory rate amounted to 58.9%. The indices related with cell malignancy alleviated, such as cell surface charge obviously decreased, the electrophoresis rate dropped from 1.64 microns.s-1.V-1.cm-1 to 0.93, the average value of alpha-fetoprotein (alpha-FP) content decreased from 302 micrograms.g-1(protein) to 90, and gamma-glytamyl-transpeptidase (gamma-GT) activity from 0.81 U.g-1(protein) to 0.16. The index related with cell differentiation increased, such as the average level of tyrosine-alpha-ketoglutarate transminase activity increased from 10.3 micromol.g-1(protein) to 41.2, and the colonogenic potential decreased 94.4%. CONCLUSION: AA can inhibit human hepatoma cells proliferation, induce redifferentiation, and reverse its malignant phenotypic characteristics.     

In vitro cytogenetic assays for the detection of mitotic aneuploidy by particulate pollutants

The ability of particulate pollutants to act on the mitotic cell division process and to induce aneuploidy in V79 cell cultures has been investigated. Extracts of airborne particulates and particulate car exhaust caused mitotic arrest connected with a dose-dependent increase in the incidence of initial and full C-metaphases. Furthermore, numerical chromosome alterations such as hyperdiploidy and polyploidy in subsequent cell divisions were induced with increasing concentrations. These findings indicate that particulate pollutants from the ambient air and from car exhaust contain potent spindle poisons with the ability to produce mitotic aneuploidy in mammalian cells. The significance of these results is that induced aneuploidy by particulate pollutants represents a genetic and somatic risk to exposed populations.     

Detection of thyroid system-disrupting chemicals using in vitro and in vivo screening assays in Xenopus laevis.

We developed a thyroid hormone (TH) inducible primary screening assay for the identification and assessment of man-made chemicals that interfere with the TH-signalling pathway within target cells. The assay was developed in a Xenopus laevis cell line that was transduced with a self-inactivating (SIN) lentivirus vector (LV) containing a luciferase gene. The luciferase activation in this cell line was TH-specific: 3,3',5-L-triiodothyronine (T(3)) > 3,3'5-L-triiodothyroacetic acid (Triac) > 3,3',5-D-triiodothyronine (D-T(3)), > L-thyroxine (T(4)) > 3,3',5'-L-triiodothyronine (rT(3)). The application of the ligand-dependent luciferase assay for screening for thyroid system-disrupting chemicals revealed that three phthalates (dicyclohexyl phthalate, n-butylbenzyl phthalate, and di-n-butyl phthalate), two herbicides (ioxynil and pentachlorophenol) and a miticide (dicofol) had 3,3',5-L-triiodothyronine- T(3)- antagonist activity at concentrations ranging from 10(-6) to 10(-5) M. These chemicals also inhibited the expression of the endogenous primary T(3)-response TH nuclear receptor beta (TRbeta) gene. The inhibitory characteristics of these chemicals were similar for both assays performed, although the assay for T(3)-dependent activation of TRbeta gene was more sensitive than the luciferase assay. These results indicate that the luciferase assay was a rapid method with a small intra-assay variation for the primary screening of thyroid system-disrupting chemicals. Of the six chemicals, only n-butylbenzyl phthalate and pentachlorophenol exhibited T(3)-antagonist activity in an in vivo metamorphosis-based assay. It should be noted that chemicals elicited thyroid system-disrupting activity in the luciferase assay did not always interfere with the thyroid system in vivo.     

Cytogenetic variability of lymphocytes from phenotypically normal men: influence of age, smoking, season, and sample storage

A cytogenetic study was conducted on cultured lymphocytes from a group of 60 male volunteers to determine the baseline of chromosomal aberrations in nonchemical workers. Only males were included in the study to avoid any sex effects on the results. Blood samples were collected from each man every 13 w (quarterly) over a period of 12 m. A single batch of culture medium was used for the entire study. The influence of storing the blood samples prior to culture, donor's age, cigarette smoking, and seasonal variation on lymphocyte mitotic index and chromosomal aberration yield was analyzed. A significant decrease in mitotic activity was observed in cultures from samples stored for 3 d at room temperature (22 +/- 1 degree C). Storing of samples at refrigerator temperature (4 +/- 1 degree C) for up to 3 d prior to culture did not affect lymphocyte growth. Although the mitotic index was found to be inversely proportional to the age of the donors, a significant influence of age on total cytogenetic aberrations was not detectable. A group of 15 smokers appeared to have higher number of chromosomal aberrations; however, the difference in mean mitotic activity between lymphocytes of the two groups was not statistically significant. No detectable seasonal influence was found on any chromosomal aberration category except in the number of chromatid gaps. The mitotic indices of the first quarter cultures, on the other hand, showed significant differences from the other three quarters. The chromosomal aberration baseline of the group was not strikingly different from the ones reported by other investigators.(ABSTRACT TRUNCATED AT 250 WORDS)     

Cytotoxic effect of xanthotoxol (8-hydroxypsoralen) on TCTC cells in vitro.

The effect of xanthotoxol (8-hydroxypsoralen) on proliferation of TCTC cells in vitro has been studied. Xanthotoxol at concentrations of 5 to 50 micrograms/ml inhibited the growth of cells. In cultures with xanthotoxol, decreased amount of cell protein, mitotic index, and decreased ability to form a colony, were observed. Moreover, xanthotoxol disturbed mitoses elevating the number of mitotic cells in the telophase stage. An increase of giant and multinuclear cells was also found. On the basis of these results it can be concluded, that 8-hydroxypsoralen which in comparison with other psoralens is not sensitive to photostimulation, inhibits the cell proliferation anyway. This fact shows that the mechanism of the psoralens activity is to some extent independent from the photostimulation.     

Curcumin derivatives: Molecular basis of their anti-cancer activity

Curcumin, a phenolic compound from the plant Curcuma longa L., has shown a wide-spectrum of chemopreventive, antioxidant and antitumor properties. Although its promising chemotherapeutic activity, preclinical and clinical studies highlight Curcumin limited therapeutic application due to its instability in physiological conditions. To improve its stability and activity, many derivatives have been synthesized and studied, among which bis-DemethoxyCurcumin (bDMC) and diAcetylCurcumin (DAC). In this report, we show that both bDMC and DAC are more stable than Curcumin in physiological medium. To explore the mechanism of their chemotherapeutic effect, we studied their role in proliferation in the HCT116 human colon cancer cells. We correlated kinetic stability and cellular uptake data to their biological effects. Both bDMC and DAC impair correct spindles formation and induce a p53- and p21^CIP1/WAF1-independent mitotic arrest, which is more stable and long-lasting for bDMC. A subsequent p53/p21^CIP1/WAF1-dependent inhibition of G1 to S transition is triggered by Curcumin and DAC as a consequence of the mitotic slippage, preventing post-mitotic cells from re-entering the cell cycle. Conversely, the G1/S arrest induced by bDMC is a direct effect of the drug and concomitant to the mitotic block. Finally, we demonstrate that bDMC induces rapid DNA double-strand breaks, moving for its possible development in anti-cancer clinical applications.     

Rat epidermal keratinocyte organotypic culture (ROC) as a model for chemically induced skin irritation testing

The potential of rat epidermal keratinocyte (REK) organotypic culture (ROC) with proper stratum corneum barrier as a model for screening skin irritants was evaluated. The test chemicals were selected from ECETOC database (1995) and the observed in vitro irritation potential was compared to ECETOC in vivo primary irritation index (PII), to EU risk phrases, and to the harmonized OECD criteria. Chemicals were applied onto the stratum corneum surface of ROC for 30 min and samples were taken from the underlying medium at 4 and 8 h after exposure. Cell membrane integrity (determined by LDH assay) and pro-inflammatory effect (determined by IL-1alpha release) were verified at both time points and correlated to PII values. The best correlation (R(2) = 0.831) was seen with LDH leakage test. Based on obtained data, chemicals were classified according to criteria defined by EU and OECD. From 12 chemicals, only two were incorrectly classified according to OECD criteria when using LDH leakage and IL-1alpha release as irritation markers. At the end of experiment, chemical-treated ROC cultures were fixed and histological changes were assessed. Typical signs for irritation were lightly stained cytoplasm, condensed nuclei, cellular vacuolization, eosinophilic cytoplasms, and blebbing. These irritation effects of chemicals were graded visually into four classes (A-D). The extent of morphological perturbations of the cultures mostly correlated with PII. The present results indicate the validity of the ROC model in predicting skin irritation potential of chemicals and show that the use of set of irritation markers with different mechanistic responses gives more information on irritation than if only one marker was used.     

Evaluation of genotoxic effects of sodium propionate, calcium propionate and potassium propionate on the root meristem cells of Allium cepa

The effects of different treatments with food preservatives, sodium propionate (SP), calcium propionate (CP) and potassium propionate (PP), on the cytology and DNA content of Allium cepa were investigated. Five concentrations of these additives – 1000, 1500, 2000, 2500, and 3000 ppm – were applied for 24, 48, and 72 h. All concentrations of these chemicals showed an inhibitory effect on cell division in root-tips of A. cepa and caused a decrease in mitotic index values. Additionally, all treatments changed the frequency of mitotic phases when compared with the control groups. These compounds increased chromosome abnormalities in test material. Among these abnormalities were C-mitosis, anaphase bridges, micronuclei, binucleated cells, stickiness, laggards, and chromosome breaks. The nuclear DNA contents decreased when compared with control groups.     

Cytotoxicity/genotoxicity: The application of cell culture techniques to the measurement of marine sediment pollution

Fish cell cultures were used to determine whether in vitro aquatic animal cell culture systems were capable of detecting pollution in the marine environment. Cells derived from rainbow trout gonad (RTG-2) and bluegill fry tissues (BF-2) were used as model cell systems to measure cytotoxicity and genotoxicity following exposure to Puget Sound sediment extracts, benzo(a)pyrene and MNNG. Sediment was collected from several sites within Puget Sound known to be contaminated with compounds such as polycyclic aromatic hydrocarbons, polychlorinated biphenyls, chlorinated hydrocarbons and heavy metals. Each of the sediment samples was extracted with organic solvents and added to cultures of the two model cell systems in DMSO. Following exposure the cultures were evaluated for cell death, mitotic inhibition, stimulatory effects, and chromosomal damage. These cell cultures responded to the sediment extracts much as they did to known mutagenic/carcinogenic chemicals which were used as model compounds. Those sediments known to be contaminated, based on chemical analysis and historical use patterns were also found to be qualitatively and quantitatively more toxic than were sediments from areas which were relatively unaffected by human activity. The results show that in vitro cell systems are capable of detecting pollution in the marine environment and have the potential of being powerful tools in aquatic toxicology in conjunction with whole animals or as a method of screening materials prior to in vivo testing.