In vitro effects of fluor-hydroxyapatite, fluorapatite and hydroxyapatite on colony formation, DNA damage and mutagenicity

The number of biomaterials used in biomedical applications has rapidly increased in the past two decades. Fluorapatite (FA) is one of the inorganic constituents of bone or teeth used for hard-tissue repairs and replacements. Fluor-hydroxyapatite (FHA) is a new synthetically prepared composite that in its structure contains the same molecular concentration of OH(-) groups and F(-) ions. The aim of this experimental investigation was to evaluate cytotoxic, genotoxic and mutagenic effects of FHA and FA eluates on Chinese hamster V79 cells and to compare them with the effects of hydroxyapatite (HA) eluate. Cytotoxicity of the biomaterials tested was evaluated by use of the cell colony-formation assay and by direct counting of the cells in each colony. Genotoxicity was assessed by single-cell gel electrophoresis (comet assay) and mutagenicity was evaluated by the Hprt gene-mutation assay and in bacterial mutagenicity tests using Salmonella typhimurium TA100. The results show that the highest test concentrations of the biomaterials (100% and 75% eluates) induced very weak inhibition of colony growth (about 10%). On the other hand, the reduction of cell number per colony induced by these concentrations was in the range from 43% to 31%. The comet assay showed that biomaterials induced DNA breaks, which increased with increasing test concentrations in the order HA相似文献   

Comparative study of a new composite biomaterial fluor-hydroxyapatite on fibroblast cell line NIH-3T3 by direct test

The number of biomaterials used in biomedical applications has rapidly increased in the past two decades. Fluorapatite (FA) is one of the inorganic constituents of bone or teeth used for hard tissue repairs and replacements. Fluor-hydroxyapatite (FHA) is a new synthetically prepared composite that in its structure contains the same molecular concentration of OH⁻ groups and F⁻ ions. The aim of this experimental investigation was to use the embryonal mouse fibroblast cell line NIH-3T3 for comparative study of basal cytotoxicity of fluoridated biomaterials FHA and FA discs. Hydroxyapatite (HA) disc, high-density polyethylene as negative control and polyvinyl chloride (PVC) containing organotin stabilizer as positive control were used as standard biomaterials. The appropriateness of the use of NIH-3T3 cells and their sensitivity for tested biomaterials were evaluated on the basis of five cytotoxic end points: cell proliferation, cell morphology, lactate dehydrogenase (LDH) released, protein and DNA cell content. The basal cytotoxicity of FHA, FA and HA discs was measured by direct contact method. FHA composite, FA and HA demonstrated in cell line NIH-3T3 nearly similar basal cytotoxicity increasing with the time of treatment. After 72 h of biomaterials treatment, about 25% inhibition of cell number, unchanged morphology of dividing cells, 6.31–0.16% increase of released LDH, about 10% inhibition of cell protein content and about 20% inhibition of DNA content was found. On the other hand, from the growth rates it resulted that NIH-3T3 cells, affected by tested biomaterials, divided about 20% slowlier than the control (untreated cells). Using the linear regression analysis we found out that deviations in measurements of cytotoxicity by four methods were as follows: less than 10% for cell number, protein and DNA content methods and 12.4% for released LDH method. Based on a good correlation of the cytotoxicity of biomaterials obtained from all end points we could conclude that fibroblast NIH-3T3 cell line was appropriate for measuring the basal cytoxicity of tested biomaterials.     

Xanthomicrol is the main cytotoxic component of Dracocephalum kotschyii and a potential anti-cancer agent

Spinal-Z, a methanolic mixture of dried powdered seeds of Peganum harmala Linn. and leaf of Dracocephalum kotschyii Boiss. is an Iranian ethno-medical remedy. It has been used for the treatment of various types of cancer for many years. To evaluate the use of Spinal-Z in treatment of cancer, we examined its effects against a panel of malignant cell lines and tumors induced in mice. The in vitro antiproliferative activities of Spinal-Z, the seed extract of P. harmala and the leaf extract of D. kotschyii were determined using the MTT assay. The concentration of the agent required to inhibit cell growth by 50% (IC50) was estimated. In addition, the anti-tumor activities of the remedy and its constituents were investigated. Viability of cells treated with Spinal-Z and its components decreased in a dose dependent manner. Spinal-Z and its components showed cytotoxic effects against all cell lines tested. The leaf extract of D. kotschyii showed a greater preferential cytotoxic effect than the seed extract of P. harmala and Spinal-Z, on all cell lines tested. Harmine showed cytotoxicity against HL60 and K562 cell lines. This could explain the cytotoxic effect of P. harmala on these cells. The leaf extract of D. kotschyii was able to inhibit tumor proliferation in mice. The active ingredient in the leaf extract of D. kotschyii appears to be a flavone identified as xanthomicrol. Xanthomicrol was able to inhibit proliferation of a number of malignant cells. The cytotoxic effects of xanthomicrol were more selective towards malignant cells than doxorubicin.     

In vitro effects of fluor-hydroxyapatite, fluorapatite and hydroxyapatite on colony formation, DNA damage and mutagenicity

The number of biomaterials used in biomedical applications has rapidly increased in the past two decades. Fluorapatite (FA) is one of the inorganic constituents of bone or teeth used for hard-tissue repairs and replacements. Fluor-hydroxyapatite (FHA) is a new synthetically prepared composite that in its structure contains the same molecular concentration of OH⁻ groups and F⁻ ions. The aim of this experimental investigation was to evaluate cytotoxic, genotoxic and mutagenic effects of FHA and FA eluates on Chinese hamster V79 cells and to compare them with the effects of hydroxyapatite (HA) eluate. Cytotoxicity of the biomaterials tested was evaluated by use of the cell colony-formation assay and by direct counting of the cells in each colony. Genotoxicity was assessed by single-cell gel electrophoresis (comet assay) and mutagenicity was evaluated by the Hprt gene-mutation assay and in bacterial mutagenicity tests using Salmonella typhimurium TA100. The results show that the highest test concentrations of the biomaterials (100% and 75% eluates) induced very weak inhibition of colony growth (about 10%). On the other hand, the reduction of cell number per colony induced by these concentrations was in the range from 43% to 31%. The comet assay showed that biomaterials induced DNA breaks, which increased with increasing test concentrations in the order HA < FHA < FA. None of the biomaterials induced mutagenic effects compared with the positive control (N-methyl-N′-nitro-N-nitrosoguanidine), and DNA breakage was probably the reason for the inhibition of cell division in V79 cell colonies.     

藏药砂生槐种子提取物抑菌和细胞毒活性的初步研究

砂生槐是我国西藏高原一种特有植物,是一种极为宝贵的药用植物资源.我们首次从砂生槐种子中获得氯仿、95%乙醇、75%乙醇和水提取物,用琼脂扩散实验测定其抑菌活性和用MTT法测定其对肿瘤细胞的细胞毒效应,表明氯仿提取物和95%乙醇提取物具有广谱的抑菌活性,抑菌活性较强的是氯仿提取物.各种提取物显示出浓度依赖性的细胞毒效应,氯仿、95%乙醇、75%乙醇和水提取物对胃癌SGC-7901细胞系的LC50分别是4.5、1.4、1.9和41.7 mg/mL,抑制胃癌SGC-7901细胞增殖作用较强的成分是砂生槐种子95%乙醇提取物.这一发现为开发砂生槐这一宝贵植物资源的药用价值提供了重要的依据.     

华中五味子抗氧化和细胞毒活性研究

我们研究了华中五味子的根、茎、叶、果实乙醇提取物对脂性自由基DPPH及超氧阴离子的清除作用,同时观察了四种不同提取物在体外对肿瘤细胞株的细胞毒作用。我们发现,华中五味子根的乙醇提取物清除自由基活性和对肿瘤细胞杀伤作用比茎、叶和果实的提取物都强,研究结果提示华中五味子的根可以作为抗氧化剂的新来源。     

Cytotoxic and antiviral activities of Colombian medicinal plant extracts of the Euphorbia genus

Forty-seven plant extracts of 10 species of the genus Euphorbia (Euphorbiaceae) used by Colombian traditional healers for the treatment of ulcers, cancers, tumors, warts, and other diseases, were tested in vitro for their potential antitumour (antiproliferative and cytotoxic) and antiherpetic activity. To evaluate the capacity of the extracts to inhibit the lytic activity of herpes simplex virus type 2 (HSV-2) and the reduction of viability of infected or uninfected cell cultures, the end-point titration technique (EPTT) and the MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] colorimetric assay were used, respectively. The therapeutic index of the positive extracts for the antiviral activity was determined by calculating the ratio CC50 (50% cytotoxic concentration) over IC50 (50% inhibitory concentration of the viral effect). Five of the 47 extracts (11%) representing 3 out of 10 Euphorbia species (30%) exhibited antiherpetic action; the highest activity was found in the leaf/stem water-methanol extracts from E. cotinifolia and E. tirucalli. The therapeutic indexes of these two plant species were > 7.1; these extracts exhibited no cytotoxicity. Six extracts (13%) representing 4 plant species (40%) showed cytotoxic activity. The highest cytotoxicity was found in the dichloromethane extract obtained from E. cotinifolia leaves and the CC50 values for the most susceptible cell lines, HEp-2 and CHO, were 35.1 and 18.1 microgram/ml, respectively.     

淫羊藿苷促进宫颈癌TC-1细胞凋亡作用的研究

目的：研究淫羊藿苷体外对致宫颈癌TC-1细胞的增殖抑制及促凋亡作用。方法：利用细胞培养,用不同浓度的淫羊藿苷在一定的时间处理致宫颈癌TC-1细胞,光学显微镜直接观察药物对细胞的作用;MTT法检测淫羊藿苷对TC-1细胞的增殖抑制作用;Dapi核染色、Annexinv-FITC/PI流式细胞学检测细胞凋亡。结果：淫羊藿苷对TC-1细胞有显著的抑制作用,且呈时间、剂量依赖,20μg/ml作用72小时后,细胞抑制率达99%;DAPI核染色和流式细胞术检测可发现典型细胞凋亡特征。结论：淫羊藿苷对TC-1细胞增殖有抑制和促凋亡作用,并呈时间浓度依赖性。     

N⁴-[Alkyl-(hydroxyphosphono)phosphonate]-cytidine-new drugs covalently linking antimetabolites (5-FdU, araU or AZT) with bone-targeting bisphosphonates (alendronate or pamidronate)

Amino-bisphosphonates (alendronate, pamidronate) were covalently linked in a three step synthesis, with protected and triazolylated derivatives of therapeutically used nucleoside analogs (5-FdU, araC, AZT) by substitution of their triazolyl residue. From the deprotected and chromatographically purified reaction mixtures N?-[alkyl-(hydroxyphosphono) phosphonate]-cytidine combining two differently cytotoxic functions were obtained. This new family of bisphosphonates (BPs) contains as novelty an alkyl side chain with a cytotoxic nucleoside. The BPs moiety allows for a high binding to hydroxyapatite which is a prerequisite for bone targeting of the drugs. In vitro binding of 5-FdU-alendronate (5-FdU-ale) to hydroxyapatite showed a sixfold increased binding of these BPs as compared to 5-FdU. Exploratory cytotoxic properties of 5-FdU-ale were tested on a panel of human tumor cell lines resulting in growth inhibition ranging between 5% and 38%. The determination of IC??-concentrations of the conjugate in Lewis lung carcinoma and murine macrophages showed an incubation time dependent growth inhibition with higher sensitivity towards the tumor cells. We assume that the antimetabolite-BPs can be cleaved into different active metabolites that may exert cytotoxic and other therapeutic effects. However, the underlying mechanisms of these promising new antimetabolite-BPs conjugates remain to be evaluated in future experiments.     

DADS对人小细胞肺癌NCI-H446细胞系增殖抑制的研究

目的：研究二烯丙基二硫（diallyldisulfide,DADS）对人小细胞肺癌NCI．H446细胞增殖的抑制作用,并探讨其作用机制。方法：体外培养NCI-H446细胞,采用MTT、细胞计数实验方法检测DADS抑制NCI—H446细胞增殖;通过HE染色和AO—EB荧光染色方法,观察DADS处理后NCI—H446细胞的形态学改变。结果：MTT结果显示：DADS作用于NCI—H446细胞48h后,代谢MTT的能力明显降低,显示出较强的细胞毒性反应,IC50值介于20-40μg／ml之间。细胞计数结果表明：DADS作用于NCI—H446细胞后,随DADS浓度增加NCI—H446细胞倍增时间延长。HE染色显示：NCI—H446细胞经DADS处理24h后,与对照组相比,细胞体积变小,胞浆丰富,细胞核变小,染色变淡。AO-EB荧光染色显示：NCI-H446细胞经DADS处理24h后,与对照组相比,细胞皱缩、呈圆形,胞质黄色或橘红色,细胞核或细胞质内可见致密浓染的黄绿色或橘红色荧光,并可见橘红色碎片且随DADS浓度增加,随DADS浓度增加细胞密度逐渐减少。结论：DADS能抑制体外培养的NCI—H446细胞增殖,作用效果与药物浓度及作用时间相关。     

Biological studies of synthesized silver nanoparticles using <Emphasis Type="Italic">Prosopis farcta</Emphasis>

The evaluation of cytotoxic and apoptotic activities of silver nanoparticles (Ag-NPs) synthesized by aqueous extract of Prosopis farcta was investigated against lung (A549) and colon (HT-29) cell lines. The cytotoxic activity of nanoparticles was performed using MTT assay, while their apoptotic activity was tested using TUNEL method. The obtained results of MTT showed that the cell viability of A549 was dependent on the nanoparticles concentration and incubation time. Therefore, although the cytotoxic effect increased as the Ag-NPs concentration and incubation time heightened, yet the viability of HT-29 cells seems to be dependent only on the incubation time. The apoptotic results of the nanoparticles showed more than 50% of apoptosis on A549 and HT-29 cell lines, which in this case, HT-29 demonstrated 100% apoptosis at concentrations of more than 400 µg/ml. It seems that Ag-NPs synthesized using P. farcta extract can serve as anti-cancer agent in the treatment many cancers through creating or discovering new drug forms.     

Comparison of an antimetabolic assay and an antiproliferative assay, both using 3H-thymidine incorporation, to test drug sensitivity of human tumors

Two assays based on the inhibition of 3H-thymidine incorporation into DNA were used to measure either the antimetabolic or the antiproliferative effects of anticancer drugs. A direct comparison of the two assays was made with cell suspensions obtained from 11 ovarian cancers and 22 malignant melanomas. Drugs with different effects on cell cycle phases were tested by both assays, for a total of 53 drug comparisons. When the sensitivity indices specific for each system was used, a significant association (p less than 0.01) was noted between the two assays. The agreement of both assays in defining in vitro sensitivity or resistance was 100% for ovarian cancer. For melanoma, 97% of samples resistant to the antimetabolic assay were also resistant to the antiproliferative assay; whereas, only 45% of samples sensitive to the antimetabolic assay were sensitive to the antiproliferative assay.     

野生仙人掌多糖对肺鳞癌细胞SK-MES-1 生长的抑制作用

目的:探讨仙人掌多糖对体外肺鳞癌细胞(SK-MES-1)生长抑制作用。方法:体外培养SK-MES-1细胞,四甲基偶氮唑盐(MTT)法观察野生仙人掌多糖对其生长的抑制,计算最低抑制浓度及抑制率;观察不同浓度多糖对细胞形态的影响;SDS-PAGE凝胶电泳简析不同组别蛋白质表达的差异。结果:野生仙人掌多糖24 h和48 h对肿瘤细胞的最低抑制浓度和抑制率分别为0.0625 mg/ml.34.06%和0.0625 mg/ml 35.37%;不同组间蛋白质表达有差异。结论:野生仙人掌多糖对SK-MES-1肺鳞癌细胞有抑制作用。     

Do novel cement-type biomaterials reveal ion reactivity that affects cell viability in vitro?

Calcium phosphate bioceramics have been studied as bone filler materials for years and have become a component of many commercial products. It is widely known that surface-reactive biomaterials may cause changes in the concentration of crucial ions in the surrounding environment, thereby affecting cell metabolism and viability. The aim of this study was to produce five cement-type biomaterials and characterize their phase composition using X-ray diffraction method, and porosity and pore size distribution using mercury intrusion porosimeter. We then evaluated ion interactions of the novel biomaterials with the surrounding environment (culture medium). A commercially available bone substitute, HydroSet? (Stryker®), was used as a reference. MTT and NRU cytotoxicity tests were performed to assess the effect of changes in the concentration of crucial ions (calcium, magnesium, phosphate) on osteoblast metabolism and viability in vitro. Our study clearly indicated that various biomaterials demonstrated different ion reactivity and consequently may cause changes in ion concentration in the local environment. Critically low or high values of calcium, magnesium, and phosphate concentrations in the medium exerted cytotoxic effects on the cultured cells. Moreover, we discovered that the chemical composition of the culture medium had a substantial influence on ion interactions with biomaterials.     

毛筒壳科真菌次级代谢产物生物活性的评价

毛筒壳科Tubeufiaceae真菌具有产新结构、新活性次级代谢产物的潜力,目前对该科真菌次级代谢产物的研究较少。为了寻找具有生物活性的新化合物,有必要对毛筒壳科真菌次级代谢产物及其活性进行系统深入的研究。本文采用平板对峙法、生长速率法和MTT法,分别测定已分离得到的19株该科真菌活体菌株抑菌活性、发酵物抑菌活性以及发酵物粗提物对不同人体肿瘤细胞株增殖的抑制作用。通过平板对峙法,试验共筛选获得13株活性菌株,其中,红棕毛筒腔菌菌株Tubeufia rubra PF02-2对7种植物病原真菌有明显的抑菌效果,抑制率均高于60%且抑菌谱广。采用生长速率法,发现红棕毛筒腔菌菌株PF02-2经液体发酵后,发酵液对其中4种植物病原真菌仍有一定的抑制作用,且菌丝体部分的乙酸乙酯提取物对马铃薯早疫病病菌Alternaria solani（ZYB）的抑制效果最好。通过MTT法,发现发酵物粗提物对3种肿瘤细胞均具有不同程度的细胞毒活性,其中在300μg/mL时,剑叶莎毛筒腔菌菌株Tubeufia machaerinae ML03-2发酵液部分的乙酸乙酯提取物对人宫颈癌细胞株HeLa和人前列腺癌细胞株PC-3的抑制率（%）分别达到了98.92±0.15和97.86±0.18,在400μg/mL时,对人肝癌细胞株HEPG2的抑制率（%）达到了98.88±0.04;在500μg/mL时,明孢新旋卷孢菌菌株Neohelicosporium hyalosporum ML05-1菌丝体部分的乙酸乙酯提取物对人宫颈癌细胞株HeLa细胞的抑制率（%）为98.32±0.02,在600μg/mL时,对人肝癌细胞株HEPG2的抑制率（%）达到了97.62±0.20,在300μg/mL时,对人前列腺癌细胞株PC-3的抑制率（%）达到了98.91±0.02。该研究结果为开发利用毛筒壳科真菌提供了科学依据。     

Beta-carotene inhibits growth of human colon carcinoma cells in vitro by induction of apoptosis

Epidemiological studies suggest that beta-carotene is able to modulate the risk of cancer. A number of in vitro studies reported that beta-carotene inhibits the growth of cancer cells; however, so far little is known about the molecular mechanisms of the antiproliferative effect of beta-carotene. Here we have investigated the effects of two beta-carotene preparations, (i) beta-carotene dissolved in tetrahydrofuran (final concentration in cell culture medium: 0.5%) and (ii) beta-carotene incorporated in a water dispersible bead form, on cultured human colon carcinoma cells HT29. The treatment of cells with beta-carotene up to 30 microM for 72 h led to a significant increase in the cellular beta-carotene concentration and formation of retinol. Beta-Carotene showed only low cytotoxicity for confluent cells tested up to 30 microM, but at dietary relevant concentrations for the intestinal tract (10, 30 microM) beta-carotene was strongly cytotoxic for growing cells and induced apoptosis in HT29 cells as assessed by the Annexin-V assay (the maximal effect was observed 15 h after treatment with beta-carotene). Exposure of cells to retinol at concentrations yielding cellular retinol levels similar to those observed by beta-carotene treatment had no antiproliferative or cytotoxic effect. Furthermore, beta-carotene did not affect the activation of the extracellular signal-regulated kinases (ERK1 and ERK2) that are essential for cellular growth. In summary, beta-carotene can inhibit growth of human colon carcinoma cells in vitro by induction of apoptosis in proliferating cells.     

荔枝核离子交换树脂分离物对肝癌细胞体外增殖及凋亡的影响

为探讨荔枝核提取物对人肝癌细胞HepG-2增殖的影响,采用MTT法检测样品对HepG-2细胞的增殖抑制活性,选取活性最好的组分进行Hoechest 33258染色,检测其对HepG-2细胞的致凋亡能力;细胞凋亡率及细胞周期的改变采用PI染色和流式细胞术进行测定。结果表明,荔枝核提取物及纯化后的多个组分都显示出对HepG-2细胞的增殖抑制活性,其中组分L2．3效果最好,对HepG-2细胞有明显的剂量依赖性增殖抑制作用（P〈0．05）;倒置显微镜下可见经也．3处理72h后细胞形态明显变小变圆,正常存活的细胞随药物浓度增加而减少;Hoechest 33258染色后处理组HepG-2细胞染色质固缩,出现呈致密浓染蓝白色颗粒状荧光的凋亡小体;在100、50μg／mL浓度时,L2．3处理48h后HepG-2细胞凋亡率显著增高（P〈0．05）,G0／G1期细胞减少（P〈0．05）,S期细胞增多（P〈0．01）,G2／M期细胞减少。以上结果证明荔枝核提取物可通过诱导细胞凋亡,影响细胞周期分布抑制HepG-2细胞增殖。     

A polyphenolic compound rottlerin demonstrates significant in vitro cytotoxicity against human cancer cell lines: isolation and characterization from the fruits of Mallotus philippinensis

In vitro assay for cytotoxic activity of glands/hairs obtained from the fruits of Mallotus philippinensis has been carried out against 14 human cancer cell lines from nine different origins via 95% ethanolic, 50% ethanolic and aqueous extract at the concentration of 100 μg/ml. Results revealed that the 95% ethanolic extract showed highest in vitro cytotoxic effect against all the 14 human cancer cell lines. The fractions of the same extract i.e. 95% ethanolic were obtained and it was found that the significant cytotoxic potential was produced by the chloroform soluble fraction at 100 μg/ml as this fraction inhibited the growth of ten human cancer cell lines from seven different tissues. Further, the chromatographic analysis of the said fraction afforded a polyphenolic molecule rottlerin. This drug at the concentration of 1?×?10^?5M and 1?×?10^?4M suppressed the proliferation of eight human cancer cell lines from six different tissues and proved its exceptionally remarkable in vitro anticancer efficiency.     

Inhibition of tumour and non-tumour cell proliferation by pygidial gland secretions of four ground beetle species (Coleoptera: Carabidae)

Inhibition of the proliferation of human tumour cells and porcine non-tumour cells by the pygidial gland secretion released by adults of four ground beetle species was observed in this study. The sulphorhodamine B (SRB) assay was applied to establish the percentages of inhibition of the net growth of four human tumour cell lines and porcine liver primary non-tumour cells. The secretions of all tested ground beetle species were shown to have an antiproliferative effect on the tested cell lines. Special emphasis is put on the secretion of Abax parallelepipedus, which showed the highest antitumour potential and weakest inhibition of non-tumour cell proliferation. The antitumour and antiproliferative potential of the pygidial gland secretions of ground beetles is here demonstrated for the first time. It is suggested that certain organic acids are responsible for the action. Further investigation needs to be conducted in order to better understand the mechanisms governing the observed cytotoxic and antitumour activity.