DNA binding and biological studies of some novel water-soluble polymer-copper(II)-phenanthroline complexes

Some novel water-soluble polymer-copper(II)-phenanthroline complex samples, [Cu(phen)2(BPEI)]Cl(2).4H2O (phen=1,10-phenanthroline, BPEI=branched polyethyleneimine), with different degrees of copper complex content in the polymer chain have been prepared by ligand substitution method in water-ethanol medium and characterized by infrared, UV-visible, EPR spectral and elemental analysis methods. The binding of these complex samples with DNA has been investigated by electronic absorption spectroscopy, emission spectroscopy and gel retardation assay. Electrostatic interactions between DNA molecule and polymer-copper(II) complex molecule containing many high positive charges have been observed. Besides these ionic interactions, van der Waals interactions, hydrogen bonding and other partial intercalation binding modes may also exist in this system. The polymer-copper(II) complex with higher degree of copper complex content was screened for its antimicrobial activity and antitumor activity.     

Synthesis, characterization, and study on HeLa cells activity of a dinuclear complex [Cu4(phen)4(H2O)2]·(pyri)·3H2O

The complex [Cu4(phen)4(H2O)2]·(pyri)·3H2O(where phen=1,10-phenanthroline and pyri=3,5-pyridine dicarboxylic acid)has been synthesized and characterized. IR spectra, elemental analysis and X-ray single-crystal diffraction were carried out to determine the composition and crystal structure of the complex. The binding of the complex with HC-DNA (HeLa cells DNA, which was extracted by ourselves) was investigated by fluorescence spectrum. Gel electrophoresis assay demonstrates the ability of the complex to cleave the extracted HC-DNA. Additionally, the complex exhibited a significant cytotoxic specificity and cancer cell inhibitory rate. The apoptotic tests indicate that the complex have an apoptotic effect on HeLa cells.     

海洋天然活性小分子物质抗乙肝病毒效果筛选

目的　通过对从海洋生物提取的天然小分子物质进行体外抗乙肝病毒效果评价,希望能筛选出一些具有抗乙肝病毒(HBV)活性的物质。方法　采用HepG2.2.15细胞系作为抗HBV的筛选工具,用不同浓度的海洋小分子物质对其进行干预,测定小分子物质能否对细胞系分泌HBV产生影响。结果　本研究共评价了20种海洋天然小分子化合物,在对HBsAg、HBeAg和HBVDNA分泌的抑制作用方面,环（甘氨酸-L-脯氨酸）(H5）、环（4-羟基脯氨酸-苯丙氨酸）(H10）、环（L-2-羟基脯氨酸-苯丙氨酸）(H12)的治疗指数均大于2,其余17种化合物治疗指数均小于1。结论　海洋天然活性小分子物质环（甘氨酸-L-脯氨酸）、环（4-羟基脯氨酸-苯丙氨酸）、环（L-2-羟基脯氨酸-苯丙氨酸）三种化合物具有抗HBV活性,随着药物浓度的增加,它们对HBsAg、HBeAg及HBV DNA抑制作用也增加,呈明显剂量反应关系。     

Synergism between the toxicity of chlorophenols and iron complexes

Synergistic interactions could prove to be relevant when evaluating the toxicity of environmental pollutants in a complex mixture, especially when organic and inorganic substances co-occur at concentrations currently considered to be low-toxic or sublethal. Escherichia coli cells (SR-9 strain) were used as a model system for studying the cellular toxicity of environmental pollutants. Exposure of bacterial cells to a combination of pentachlorophenol (PCP) and a positively charged complex of iron or copper caused a dramatic inhibition of growth and an increase in cell death. Incubation of bacterial cells with PCP and either ferric-1,10-phenanthroline complex [Fe3+(OP)3]3+ (500 and 5 microM, respectively) or cupric-1,10-phenanthroline complex [Cu2+(OP)2]2+ (400 and 0.05 microM, respectively) showed two and four log units of cell death, respectively, in 30 min. In contrast, only minor amounts of cell death were observed with each component alone. Similar effects have been shown for other positively charged complexes of transition metals and for other biocides. The observed synergism was associated with the formation of novel noncharged and lipophilic ternary complexes, which contain PCP anions (or other polychlorinated anions) and the iron (or copper) complex. The ternary complexes demonstrated effective transport of their components into the cells.     

Synthesis, nucleic acid binding and cytotoxicity of polyethyleneimine-copper(II) complexes containing 1,10-phenanthroline and l-valine

The polymer-copper(II) complex samples, [Cu(phen)(l-Val) BPEI]Cl·H₂O, with varying degrees of coordination in the polymer chain, were prepared and characterized by elemental analysis and spectroscopic methods. The binding of these complex samples with both DNA and RNA has been investigated. The experimental results indicate that the polyethyleneimine-copper(II) complex samples bind with DNA and RNA mostly through surface binding; but hydrogen bonding and van der Waals interactions are also present. Evaluation of cytotoxic activity of a sample of polymer-copper(II) complex with higher degree of coordination against different cancer cell lines proved that the complex exhibited cytotoxic specificity and significant cancer cell inhibition rate.     

化疗药物对肝癌细胞株HepG2和HepG2.2.15表达TLR2、TLR4的影响

目的 观察氟尿嘧啶和顺铂对肝癌细胞株HepG2和HepG2.2.15表达Toll样受体2(TLR2)、Toll样受体4(TLR4)的影响.方法 用直接免疫荧光流式细胞术检测加入不同终浓度的氟尿嘧啶和顺铂后24、48和72 h HepG2和HepG2.2.15细胞表达TLR2和TLR4的平均荧光强度(MFI)及阳性细胞率.结果 HepG2.2.15细胞上TLR2和TLR4表达的MFI及阳性细胞率均明显高于HepG2(P＜0.01),但加入不同终浓度的氟尿嘧啶和顺铂后,HepG2和HepG2.2.15细胞表达TLR2和TLR4的MFI及阳性细胞率均基本无变化,仅在100、200 μg/ml的氟尿嘧啶和20μg/ml的顺铂作用72 h后,HepG2.2.15细胞表达TLR2的MFI低于空白对照组(P＜0.05).结论 体外实验显示在肝癌细胞中经典化疗药物氟尿嘧啶和顺铂不能激活TLR2和TLR4信号途径,要通过激活TLR2和TLR4信号途径而达到对抗肝癌细胞的作用,需要寻找其他的方式.     

Synthesis and in vitro antitumour activity evaluation of 1-aryl-1H,3H-thiazolo[4,3-b]quinazolines

A series of 1H,3H-thiazolo[4,3-b]quinazolines (2a-i) were synthesized and evaluated for their in vitro antitumour activity against ca. 60 human tumour cell lines. They exhibited moderate (2c, 2d, 2f and 2g) to strong (2a, 2b, 2e, 2h and 2i) cell-growth inhibition at a concentration of 10(-4) M, but weak activity at lower concentrations. Only 1-(2,6-dichlorophenyl)-1H,3H-thiazolo[4,3-b]quinazoline (2h) possesses a significant growth inhibitory activity on 22 cell lines at a concentration of 10(-5) M.     

Chemopreventive properties of trans-resveratrol against the cytotoxicity of chloroacetanilide herbicides in vitro

The beneficial effect of trans-resveratrol (RESV) on health is well documented. Our aim was to study the putative preventive effect of RESV on the cytotoxicity of frequently used herbicides (alachlor, acetochlor). Estrogen receptor positive (ER+) MCF-7 human mammary carcinoma, HepG2 (ER+) human hepatocellular carcinoma and VERO estrogen receptor negative (ER-) non-transformed monkey fibroblast cell lines were treated with alachlor and acetochlor (2-500 microg/ml) as toxic agents, and RESV (10 microM) as preventive agent. The MTT dye reduction assay was performed to test cytotoxicity, and flow cytometry to test cell proliferation and apoptosis. RESV is not cytotoxic in the concentration range of 1-100 microM on neither cell lines examined after 24 h, but cytotoxic on Vero and MCF-7 cells at 100 microM after 48h, and on all three cell lines after 72 h. On both ER+ cell lines a stimulation of viability occurs in the low concentration range (0.5-12.5 microM) as detected by the MTT assay. Cell cycle analysis of the culture shows a significant increase of S-phase cells at low concentrations of RESV (10-50 microM) and a decrease in the 100-200 microM concentration range. The ratio of apoptotic cells significantly increases after the administration of 50 microM RESV, depending on the incubation time. The cytotoxicity of 20-65 microg/ml alachlor and 10-65 microg/ml acetochlor was significantly decreased by the addition of 10 microM RESV in Vero ER- cells whereas no significant change was detected on ER+ cell lines MCF-7 and HepG2. These results show that RESV protects non-transformed ER- cells, but has no such effect on ER+ tumor cells.     

Synthesis and primary cytotoxicity evaluation of 3-[[(3-phenyl-4(3H)-quinazolinone-2-yl)mercaptoacetyl]hydrazono]-1H-2-indolinones

New esters (2b and 2c) and hydrazides (3b and 3c) were synthesized from 6-methyl/fluoro-3-phenyl-4(1H, 3H)-quinazolinone-2-thiones (1b and 1c). Subsequent treatment of 3-phenyl-4(3H)-quinazolinone-2-yl)mercaptoacetic acid hydrazides (3a-e) with 1H-indole-2,3-diones (4a-e) furnished the corresponding 3-[[(3-phenyl-4(3H)-quinazolinone-2-yl)mercaptoacetyl]hydrazono]-1H-2-indolinones (5a-u). The structures of new compounds were determined by analytical and spectral (IR, 1H-NMR, (13)C-NMR, EIMS) methods. Previously reported 3-[[(3-phenyl-4(3H)-quinazolinone-2-yl)mercaptoacetyl]hydrazono]-5-bromo-1H-2-indolinone 5v and compounds 5b, 5d and 5o chosen as prototypes were evaluated against the full panel of 60 human tumour cell lines at a minimum of five concentrations at tenfold dilutions in the National Cancer Institute in vitro primary cytotoxicity assay. Sulforhodamine B protein assay was used to estimate cell stability or growth. 3-[[(6-Chloro-3-phenyl-4(3H)-quinazolinone-2-yl)mercaptoacetyl]hydrazono]-5-fluoro-1H-2-indolinone 5o showed the most favourable cytotoxicity against a renal cancer cell line UO-31 (log(10)GI(50) value -6.68). Compound 5v was also tested against human immunodeficiency virus 1 (HIV-1). Compound 5v was confirmed moderately active against HIV-1.     

Synthesis and antitumour activity of 4-hydroxy-2-pyridone derivatives

4-hydroxy-2-pyridone derivatives 2 were prepared by reaction of 3-amino-3-dialkylaminopropenoates with bis(2,4, 6-trichlorophenyl)malonate. These compounds were further reacted with a set of aldehydes to give bis(pyridyl)methanes 3 and 4. The newly synthesized compounds 2, 3 and 4 were evaluated in vitro as antitumour agents against 60 human tumour cell lines. Some derivatives exhibit tumour growth inhibition activity. In particular, derivative 4g, the most active of the series, possesses significant activity on all cell lines at concentrations ranging from 1 x 10(-6) to 1 x 10(-5) M.     

Synthesis and primary cytotoxicity evaluation of new 5-nitroindole-2,3-dione derivatives

A new series of 5-nitro-1H-indole-2,3-dione-3-thiosemicarbazones (3a-k) obtained by condensation of 5-nitro-1H-indole-2,3-dione (1) with N-substituted-thiosemicarbazides (2a-k) were treated with morpholine or piperidine and formaldehyde to yield 1-morpholino/piperidinomethyl-5-nitroindole-2,3-dione-3-thiosemicarbazones (4a-m). The structures of all the compounds were determined by analytical and spectral (IR, 1H-NMR, EIMS) methods. Compounds 3b, 3c, 3f, 3k, 4a, 4c, 4f and 4l chosen as prototypes were evaluated in the National Cancer Institute's 3-cell line, one dose in vitro primary cytotoxicity assay. All the compounds that passed the criteria for activity in this assay were scheduled automatically for evaluation against the full panel of 60 human tumour cell lines at a minimum of five concentrations at 10-fold dilutions. Sulphorhodamine B (SRB) protein assay was used to estimate cell stability or growth. The most active compound was found to be 1-morpholinomethyl-5-nitroindole-2,3-dione-3-N-(chlorophenyl)thiosemicarbazone (4l). This compound demonstrated the most marked effects in the National Cancer Institute's 60 human tumour cell line in vitro screen on a non-small cell lung cancer cell line (HOP-62, log(10)GI(50) value <-8.00) and on leukaemia cell lines (HL-60(TB), log(10)GI(50) value -6.30; MOLT-4, log(10)GI(50) value -6.18).     

Qualitative and quantitative comparison of the cytotoxic and apoptotic potential of phytosterol oxidation products with their corresponding cholesterol oxidation products

Phytosterols contain an unsaturated ring structure and therefore are susceptible to oxidation under certain conditions. Whilst the cytotoxicity of the analogous cholesterol oxidation products (COP) has been well documented, the biological effects of phytosterol oxidation products (POP) have not yet been fully ascertained. The objective of the present study was to examine the cytotoxicity of beta-sitosterol oxides and their corresponding COP in a human monocytic cell line (U937), a colonic adenocarcinoma cell line (CaCo-2) and a hepatoma liver cell line (HepG2). 7beta-Hydroxysitosterol, 7-ketositosterol, sitosterol-3beta,5alpha,6beta-triol and a sitosterol-5alpha,6alpha-epoxide-sitosterol-5beta,6beta-epoxide (6:1) mixture were found to be cytotoxic to all three cell lines employed; the mode of cell death was by apoptosis in the U937 cell line and necrosis in the CaCo-2 and HepG2 cells. 7beta-Hydroxysitosterol was the only beta-sitosterol oxide to cause depletion in glutathione, indicating that POP-induced apoptosis may not be dependent on the generation of an oxidative stress. A further objective of this study was to assess the ability of the antioxidants alpha-tocopherol, gamma-tocopherol and beta-carotene to modulate POP-induced cytotoxicity in U937 cells. Whilst alpha/gamma-tocopherol protected against 7beta-hydroxycholesterol-induced apoptosis, they did not confer protection against 7beta-hydroxysitosterol- or 7-ketositosterol-induced toxicity, indicating that perhaps COP provoke different apoptotic pathways than POP. beta-Carotene did not protect against COP- or POP-induced toxicity. In general, results indicate that POP have qualitatively similar toxic effects to COP. However, higher concentrations of POP are required to elicit comparable levels of toxicity.     

Cytotoxic 2,6-bis(arylidene)cyclohexanones and related compounds

A number of 2-arylidenecyclohexanones 1, 2, 6-bis(arylidene)cyclohexanones 2 and related Mannich bases 3-5 were prepared. Various torsion angles as well as atomic charges on olefinic carbon atoms were determined by molecular modelling on all compounds. These molecules showed cytotoxicity towards murine P388 and L1210 cells as well as to human Molt 4/C8 and CEM T-lymphocytes. The average cytotoxicity of the dienones 2 was more than three times greater than was found with the monoarylidene analogues 1, and, in general, were slightly more cytotoxic than the Mannich bases 3-5. A number of the compounds displayed potency towards a panel of human tumour cell lines and most of the representative compounds in series 2-5 were selectively toxic to colon cancers and leukaemic cells.     

Cancer Cell Inhibiting Sea Cucumber (Holothuria leucospilota) Protein as a Novel Anti-Cancer Drug

Cancer remains the primary cause of death worldwide. To develop less toxic anti-cancer drugs to relieve the suffering and improve the survival of cancer patients is the major focus in the anti-cancer field. To this end, marine creatures are being extensively studied for their anti-cancer effects, since extracts from at least 10% of the marine organisms have been shown to possess anti-tumor activities. As a classic Chinese traditional medicine, sea cucumbers and compounds extracted from the sea cucumbers, such as polysaccharides and saponins, have recently been shown to exhibit anti-cancer, anti-inflammatory, and anti-oxidant effects. Holothuria leucospilota (H. leucospilota) is a tropical edible sea cucumber species that has been successfully cultivated and farmed in large scales, providing a readily available source of raw materials to support the development of novel marine anti-cancer drugs. However, very few studies have so far been performed on the biological activities of H. leucospilota. In this study, we first investigated the anti-cancer effect of H. leucospilota protein on three cancer cell lines (i.e., HepG2, A549, Panc02) and three normal cell lines (NIH-3T3, HaCaT, 16HBE). Our data showed that H. leucospilota protein decreased the cell viabilities of HepG2, A549, HaCaT, 16HBE in a concentration-dependent manner, while Panc02 and NIH-3T3 in a time- and concentration-dependent manner. We also found that the inhibitory effect of H. leucospilota protein (≥10 μg/mL) on cell viability is near or even superior to EPI, a clinical chemotherapeutic agent. In addition, our data also demonstrated that H. leucospilota protein significantly affected the cell cycle and induced apoptosis in the three cancer cell lines investigated; in comparison, it showed no effects on the normal cell lines (i.e., NIH-3T3, HaCaT and 16HBE). Finally, our results also showed that H. leucospilota protein exhibited the excellent performance in inhibiting cell immigrations. In conclusion, H. leucospilota protein targeted the cancer cell cycles and induced cancer cell apoptosis; its superiority to inhibit cancer cell migration compared with EPI, shows the potential as a promising anti-cancer drug.     

HBV基因瞬时和稳定转染HepG2细胞的TLR4表达

目的 观察HBV基因瞬时和稳定转染HepG2细胞后表达TLR4的变化.方法 采用免疫荧光流式细胞术检测TLR4在肝癌细胞株HepG2和HepG2.2.15上表达的平均荧光强度(MFI)及阳性细胞率,将前期试验获得的HBV全基因组,采用脂质体瞬时转染肝癌细胞株HepG2,采用免疫荧光流式细胞术检测TLR4在细胞上表达的MFI及阳性细胞率,台盼蓝计数检测细胞增殖活性,并分析其与细胞表达TLR4的关系.结果 HepG2.2.15细胞上TLR4表达的MFI及阳性细胞率均明显高于HepG2(均为P'<0.01),HepG2细胞于转染HBV DNA各剂量组48h后TLR4表达的MFI和阳性细胞率与无HBV DNA脂质体转染对照组相比均显著升高(均P'<0.01),并且MFI和阳性细胞率与转染剂量均呈正相关(均P<0.01),与细胞存活数均呈负相关(均P<0.01).结论 瞬时和稳定转染HBV的HepG2细胞,都存在细胞TLR4的上调,这种上调伴随着细胞存活的减少,并可能参与了急慢性乙型肝炎的免疫损伤.     

Flavonoids as aryl hydrocarbon receptor agonists/antagonists: effects of structure and cell context

Chemoprotective phytochemicals exhibit multiple activities and interact with several cellular receptors, including the aryl hydrocarbon (Ah) receptor (AhR). In this study we investigated the AhR agonist/antagonist activities of the following flavonoids: chrysin, phloretin, kaempferol, galangin, naringenin, genistein, quercetin, myricetin, luteolin, baicalein, daidzein, apigenin, and diosmin. We also investigated the AhR-dependent activities of cantharidin and emodin (in herbal extracts) in Ah-responsive MCF-7 human breast cells, HepG2 human liver cancer cells, and mouse Hepa-1 cells transiently or stably transfected with plasmids expressing a luciferase reporter gene linked to multiple copies of a consensus dioxin-responsive element. The AhR agonist activities of the compounds (1 and 10 micro M) were as high as 25% of the maximal response induced by 5 nM 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), and their potencies were dependent on cell context. Galangin, genistein, daidzein, and diosmin were active only in Hepa-1 cells, and cantharidin induced activity only in human HepG2 and MCF-7 cells. Western blot analysis confirmed that baicalein and emodin also induced CYP1A1 protein in the human cancer cell lines. The AhR antagonist activities of four compounds inactive as agonists in MCF-7 and HepG2 cells (kaempferol, quercetin, myricetin, and luteolin) were also investigated. Luteolin was an AhR antagonist in both cell lines, and the inhibitory effects of the other compound were dependent on cell context. These data suggest that dietary phytochemicals exhibit substantial cell context-dependent AhR agonist as well as antagonist activities. Moreover, because phytochemicals and other AhR-active compounds in food are present in the diet at relatively high concentrations, risk assessment of dietary toxic equivalents of TCDD and related compounds should also take into account AhR agonist/antagonist activities of phytochemicals.     

HBV基因瞬时和稳定转染HepG2细胞的TLR4表达

目的 观察HBV基因瞬时和稳定转染HepG2细胞后表达TLR4的变化.方法 采用免疫荧光流式细胞术检测TLR4在肝癌细胞株HepG2和HepG2.2.15上表达的平均荧光强度(MFI)及阳性细胞率,将前期试验获得的HBV全基因组,采用脂质体瞬时转染肝癌细胞株HepG2,采用免疫荧光流式细胞术检测TLR4在细胞上表达的MFI及阳性细胞率,台盼蓝计数检测细胞增殖活性,并分析其与细胞表达TLR4的关系.结果 HepG2.2.15细胞上TLR4表达的MFI及阳性细胞率均明显高于HepG2(均为P'<0.01),HepG2细胞于转染HBV DNA各剂量组48h后TLR4表达的MFI和阳性细胞率与无HBV DNA脂质体转染对照组相比均显著升高(均P'<0.01),并且MFI和阳性细胞率与转染剂量均呈正相关(均P<0.01),与细胞存活数均呈负相关(均P<0.01).结论 瞬时和稳定转染HBV的HepG2细胞,都存在细胞TLR4的上调,这种上调伴随着细胞存活的减少,并可能参与了急慢性乙型肝炎的免疫损伤.     

HBV基因瞬时和稳定转染HepG2细胞的TLR4表达

目的 观察HBV基因瞬时和稳定转染HepG2细胞后表达TLR4的变化.方法 采用免疫荧光流式细胞术检测TLR4在肝癌细胞株HepG2和HepG2.2.15上表达的平均荧光强度(MFI)及阳性细胞率,将前期试验获得的HBV全基因组,采用脂质体瞬时转染肝癌细胞株HepG2,采用免疫荧光流式细胞术检测TLR4在细胞上表达的MFI及阳性细胞率,台盼蓝计数检测细胞增殖活性,并分析其与细胞表达TLR4的关系.结果 HepG2.2.15细胞上TLR4表达的MFI及阳性细胞率均明显高于HepG2(均为P'<0.01),HepG2细胞于转染HBV DNA各剂量组48h后TLR4表达的MFI和阳性细胞率与无HBV DNA脂质体转染对照组相比均显著升高(均P'<0.01),并且MFI和阳性细胞率与转染剂量均呈正相关(均P<0.01),与细胞存活数均呈负相关(均P<0.01).结论 瞬时和稳定转染HBV的HepG2细胞,都存在细胞TLR4的上调,这种上调伴随着细胞存活的减少,并可能参与了急慢性乙型肝炎的免疫损伤.     

HBV基因瞬时和稳定转染HepG2细胞的TLR4表达

目的 观察HBV基因瞬时和稳定转染HepG2细胞后表达TLR4的变化.方法 采用免疫荧光流式细胞术检测TLR4在肝癌细胞株HepG2和HepG2.2.15上表达的平均荧光强度(MFI)及阳性细胞率,将前期试验获得的HBV全基因组,采用脂质体瞬时转染肝癌细胞株HepG2,采用免疫荧光流式细胞术检测TLR4在细胞上表达的MFI及阳性细胞率,台盼蓝计数检测细胞增殖活性,并分析其与细胞表达TLR4的关系.结果 HepG2.2.15细胞上TLR4表达的MFI及阳性细胞率均明显高于HepG2(均为P'<0.01),HepG2细胞于转染HBV DNA各剂量组48h后TLR4表达的MFI和阳性细胞率与无HBV DNA脂质体转染对照组相比均显著升高(均P'<0.01),并且MFI和阳性细胞率与转染剂量均呈正相关(均P<0.01),与细胞存活数均呈负相关(均P<0.01).结论 瞬时和稳定转染HBV的HepG2细胞,都存在细胞TLR4的上调,这种上调伴随着细胞存活的减少,并可能参与了急慢性乙型肝炎的免疫损伤.