Evaluation of the therapeutic efficacy of tripeptide tyroserleutide (YSL) for human hepatocarcinoma by in vivo hollow fiber assay

Summary  Tyroserleutide (YSL), extracted from spleen of pigs, is a tripeptide that has shown therapeutic efficacy in an experimental BEL-7402 human hepatocarcinoma model. The hollow fiber assay (HFA) is a solid tumor model for large-scale screening of potential anticancer compounds that minimizes expenditures of materials, time, and money. Tumor cells are cultivated within biocompatible, semipermeable hollow fibers, which are implanted in immunosuppressed mice. In this study, the HFA was used to investigate the therapeutic efficacy of YSL for human hepatocarcinoma. In vitro effects of YSL on human hepatocarcinoma cell lines BEL-7402, SMMC-7721, Hep3B, HepG2, and SK-HEP-1 were assayed by the MTS method. In vivo effects of YSL on the five human hepatocarcinoma cell lines were assayed by HFA. Mice implanted with tumor cells in hollow fibers were treated with YSL, and the effects of YSL on tumor cell populations were assessed by MTT assay. YSL significantly inhibited the proliferation of the five human hepatocarcinoma, both in vitro and in vivo (P < 0.05). The HFA is a rapid, accurate, and economical method for evaluating the inhibitory effects of drugs on different tumor cells in vivo. These results support the clinical application of YSL for treatment of human hepatocarcinoma. Xiao-lei Li and Jun-yan Liu contributed equally to this paper.     

应用中空纤维测定法评价藤甲酰苷对人癌细胞生长的抑制作用

目的应用中空纤维测定法评价藤甲酰苷（garcinia glycosides,GG）对8种人癌细胞的体内生长的抑制作用,通过裸鼠体内异位移植瘤实验,验证中空纤维测定法在抗肿瘤药效学研究中的可靠性。方法采用中空纤维测定法,将载有肿瘤细胞的中空纤维管植入NOD/SCID小鼠背部皮下,给药结束后取出中空纤维管,应用二苯基溴化四氮唑蓝（MTT）比色法检测GG对各种肿瘤细胞在体内的抑瘤率;选取HL-60及B16人癌细胞,异位移植于BALb/c裸鼠右下肢外侧皮下,以环磷酰胺（CTX）为阳性对照,各组连续给药10 d,于给药结束24 h后解剖小鼠,取出瘤块,计算CTX及GG的抑瘤率。结果应用中空纤维测定法及裸鼠体内异位移植瘤法测得GG高剂量组8 mg/（kg·d）、中剂量组4 mg/（kg·d）能明显抑制HL-60及B16人癌细胞在裸鼠体内的生长,实验测得结果与溶剂对照组比较,有显著性差异（P〈0.01）。结论应用中空纤维测定法测定样品GG对肿瘤细胞的生长抑制作用,实验结果与裸鼠体内异位移植瘤实验结果基本一致。该模型节约成本、效率提高、实验结果准确可靠,为GG的后续研究提供了指导和可靠证据。     

以K562细胞为靶点的抗肿瘤药物筛选模型的建立

目的：建立以白血病细胞系K562为靶点的高通量抗肿瘤药物筛选模型。方法：在96孔细胞培养板上,运用四唑氯化合物（MTS）和电子耦联剂（PMS）连用的方法,对K562细胞增殖情况进行检测。对在化合物影响下K562细胞增殖变化的检测条件进行了优化。结果：采用这一细胞水平的高通量抗肿瘤药物筛选模型,完成了800个小分子有机化合物的筛选,每个化合物的用量是500ng.11个化合物在浓度为5mg/L时可抑制细胞增殖达80％以上,其中9个通过多浓度复筛得到了确认。抑制活性最强的化合物的IC50为170nmol/L,共有7个化合物显示IC50低于10μmol/L。结论：采用K562细胞系进行高通量筛选是快速、经济、有效、实用的发现新型抗肿瘤药物的方法。     

Investigation into possible DNA damaging effects of ultrasound in occupationally exposed medical personnel--the alkaline comet assay study

In the present paper the possible DNA damaging effects of ultrasound in occupationally exposed medical personnel were investigated using the alkaline comet assay. The extent of DNA migration in peripheral blood leucocytes was measured. Parameters of the comet assay were studied in 30 medical workers occupationally exposed to ultrasound and in 30 corresponding unexposed control subjects. It was found that the subjects who were occupationally exposed to ultrasound for various periods of time showed a highly significant increase in levels of DNA damage compared with the control. The results obtained have confirmed the usefulness of the alkaline comet assay as a sensitive biodosimetric method, reflecting the current level of DNA damage and/or repair in peripheral blood leucocytes of ultrasound-exposed subjects. In spite of their limitations, the results of the present investigation indicate that individuals occupationally exposed to ultrasound may experience an increased genotoxic risk, emphasizing the need for more research into the nature and extent of the biological consequences to medical personnel working with ultrasonic equipment.     

DNA损伤剂与ADPRT介导的细胞NAD含量下降

本文观察了一系列已知致癌物及其非致癌性类同物对经β—萘黄酮诱导过的或未经诱导的人FL细胞中的作用,以验证有ADPRT介导的,即可被ADPRT抑制剂防止的细胞NAD含量下降作为判断终点来检测化学物质诱发的DNA损伤的可能性。实验表明在MFO诱发过的FL细胞中AFB_1(1～100μmol/L),B(a)P(10～1000μmol/L),芘(1～1000μmol/L),DMA(10～100μmol/L),2—AAF(100～1000μmol/L)及 EC(10～1000μmol/L)可引起剂量依存性ADPRT介导的细胞NAD含量下降,而蒽(0.1～1000μmol/L),4—AAF(0.1～1000μmol/L),IsoPC(1～100μmol/L),黄樟素0.1～100μmol/L)及 Cy(1～100μmol/L)则否。直接作用致癌物ProLac(1～100μmol/L)及其非致癌性类同物BuLac(1～1000μmol/L)在MFO未经诱导的FL细胞中皆不能诱发ADPRT介导的细胞NAD含量降低。与非程序性DNA合成试验结果相对比,上述结果表明这一以ADPRT介导的细胞NAD含量下降作为判断终点的新检测法是一个检测化学诱变剂/致癌物特异而价廉的方法,其特异性与灵敏度与UDS试验相仿。     

Enhancement of the predicted drug hepatotoxicity in gel entrapped hepatocytes within polysulfone-g-poly (ethylene glycol) modified hollow fiber

Collagen gel-based 3D cultures of hepatocytes have been proposed for evaluation of drug hepatotoxicity because of their more reliability than traditional monolayer culture. The collagen gel entrapment of hepatocytes in hollow fibers has been proven to well reflect the drug hepatotoxicity in vivo but was limited by adsorption of hydrophobic drugs onto hollow fibers. This study aimed to investigate the impact of hollow fibers on hepatocyte performance and drug hepatotoxicity. Polysulfone-g-poly (ethylene glycol) (PSf-g-PEG) hollow fiber was fabricated and applied for the first time to suppress the drug adsorption. Then, the impact of hollow fibers was evaluated by detecting the hepatotoxicity of eight selected drugs to gel entrapped hepatocytes within PSf and PSf-g-PEG hollow fibers, or without hollow fibers. The hepatocytes in PSf-g-PEG hollow fiber showed the highest sensitivity to drug hepatotoxicity, while those in PSf hollow fiber and cylindrical gel without hollow fiber underestimated the hepatotoxicity due to either drug adsorption or low hepatic functions. Therefore, the 3D culture of gel entrapped hepatocytes within PSf-g-PEG hollow fiber would be a promising tool for investigation of drug hepatotoxicity in vitro.     

Silencing of endo-exonuclease expression sensitizes mouse B16F10 melanoma cells to DNA damaging agents

Summary We previously identified an endo-exonuclease that is highly expressed in cancer cells and plays an important role in DSB repair mechanisms. A small molecular compound pentamidine, which specifically inhibited nuclease activity of the isolated endo-exonuclease from yeast as well as from mammalian cells, was capable of sensitizing tumor cells to DNA damaging agents. In this study, we investigated the effect of precisely silencing the endo-exonuclease expression by small interfering RNA (siRNA) upon treatment with a variety of DNA damaging agents in mouse B16F10 melanoma cells. A maximum of 3.6 to ∼4-fold reduction in endo-exonuclease mRNA expression was achieved, over a period of 48–72 h of post transfection with a concomitant reduction in protein expression (∼4–5 fold), resulting in a substantial reduction (∼45–50%) of the corresponding nuclease activity. Suppressed endo-exonuclease expression conferred significant decrease in cell survival, ranging from ∼30 to ∼50% cell killing, in presence of DNA damaging drugs methyl methane sulfonate (MMS), cisplatin, 5-fluoro uracil (5-FU) and gamma-irradiation but not at varying dosages of ultra violet (UV) radiation. The data strongly support a role for the endo-exonuclease in repairing DNA damages, induced by MMS, cisplatin, 5-FU and gamma irradiation but not by UV radiation. The results presented in this study suggest that the endo-exonuclease siRNA could be useful as a therapeutic tool in targeting the endo-exonuclease in cancer therapy.     

Extraction and determination of trace amounts of chlorpromazine in biological fluids using hollow fiber liquid phase microextraction followed by high-performance liquid chromatography

In the present work, hollow fiber liquid phase microextraction (HF-LPME) in conjunction with reversed-phase HPLC/UV was developed for extraction and determination of trace amounts of chlorpromazine in biological fluids. The drug was extracted from an 11 ml aqueous sample (source phase; SP) into an organic phase impregnated in the pores of the hollow fiber (membrane phase; MP) followed by the back-extraction into a second aqueous solution (receiving phase; RP) located in the lumen of the hollow fiber. The effects of several factors such as the nature of organic solvent, compositions of SP and RP solutions, extraction time, ionic strength and stirring rate on the extraction efficiency of the drug were examined and optimized. Under the optimal conditions, enrichment factor of 250, dynamic linear range of 1–500 μg l⁻¹, and limit of detection of 0.5 μg l⁻¹ were obtained for the drug. The percent relative intra-day and inter-day standard deviation (R.S.D.%) based on three replicate determinations were 6.7 and 10.3%, respectively. The method was applied to drug level monitoring in the biological fluids and satisfactory results were obtained.     

Comparative evaluation of a 5-day Hershberger assay utilizing mature male rats and a pubertal male assay for detection of flutamide's antiandrogenic activity.

A 5-day Hershberger assay utilizing mature male rats and a pubertal male assay were evaluated for the ability to detect antiandrogenic compounds such as flutamide, an androgen receptor antagonist. Six days after the operation, implantation with two silicon capsules containing testosterone (T) (30 mg/capsule) in castrated rats provided the ventral prostate and seminal vesicle weights as well as serum T and luteinizing hormone (LH) levels equivalent to those of the controls (non-castrated, non-implanted rats). Castrated rats implanted with two T-capsules (6 rats/dose) were treated by gavage for 5 days with vehicle (0.5% carboxymethylcellulose) or flutamide (0.15, 0.6, 2.5, or 10 mg/kg/day). Flutamide produced significant decreases in weights of the seminal vesicles and the levator ani plus bulbocavernosus muscles (> or =0.6 mg/kg/day) and ventral prostate (> or =2.5 mg/kg/day), and an increase in serum LH levels (> or =2.5 mg/kg/day), but no changes in serum T levels. When age-matched intact male rats were treated with 10-mg/kg/day flutamide, a significant increase in serum T levels was observed concomitant with a tendency of increased LH. The organ weights were also decreased; however, the changes were less than those in the castrated, T-implanted rats. Immature intact male rats (10 rats/dose) were treated for 20 days with flutamide (0, 0.15, 0.6, 2.5, or 10 mg/kg/day). Flutamide produced significant decreases in weights of the seminal vesicles, ventral prostate, and levator ani plus bulbocavernosus muscles at 2.5 and 10 mg/kg/day. Serum LH levels, but not T levels, were increased at 10 mg/kg/day. Statistical significance of some of these changes was not observed in the 6 animals/dose examined. Our findings support that the Hershberger assay, in the current conditions, is the most sensitive among the assays examined and a useful short-term screening method for the detection of antiandrogenic compounds.     

Use of the GTP��S ([35S]GTP��S and Eu-GTP��S) binding assay for analysis of ligand potency and efficacy at G protein-coupled receptors

In this review I consider assays for G protein-coupled receptor (GPCR) activity based on the binding of labelled analogues of GTPγS ([³⁵S]GTPγS or Eu-GTPγS) to G proteins in tissues (GTPγS binding assays). Such assays provide convenient measures of GPCR activity close to the receptor in the signalling cascade. In order to set up a GTPγS binding assay, the requirements of the assay must be considered. These are tissue source, GTPγS analogue, G protein, GDP, Mg²⁺/Na⁺ ions, saponin, incubation time. The assay, once optimized, can be used to generate concentration/response curves for GPCRs signalling via G_i/o proteins (or to other G proteins with a modified assay) and actions of agonists, inverse agonists and antagonists may, in principle, be assessed. For agonists and inverse agonists, data for the maximal agonist effect, the concentration of ligand giving a half-maximal response and the Hill coefficient may be derived. For antagonists, data for the equilibrium dissociation constant can be obtained. The mechanistic basis of the assay is considered. Although the assay can be used to profile ligands, under the conditions it is used, it may not be measuring the same event that determines GPCR action in cells.     

HIV-1前体蛋白早成熟化激活剂筛选模型的建立及应用

HIV-1蛋白酶 (PR) 活性的严格调控对于病毒的生存至关重要。在病毒蛋白表达及病毒颗粒装配过 程中, 处于病毒前体蛋白Gag-Pol中的蛋白酶必须以无活性状态存在, 避免前体蛋白Gag-Pol和Gag被提前酶切加工 (前体蛋白早成熟化)。干扰HIV-1蛋白酶活性的调控机制, 特异性的激活前体蛋白中的蛋白酶, 诱导前体蛋白早成熟化, 就可以直接抑制病毒的复制。根据这一设想, 运用生物发光共振能量转移技术, 建立细胞水平的HIV-1前体蛋白早成熟化激活剂筛选模型, 并通过3 000个化合物的试验性筛选对筛选模型进行评价。研究结果表明该筛选方法灵敏可靠, 特异性高, 重复性好 (Z' 因子为0.905)。     

A colorimetric assay for the measurement of the sensitivity of herpes simplex viruses to antiviral agents

A quantitative colorimetric method for measuring the inhibition of viral cytopathic effects has been adapted to the assay of antiviral compounds. Drug-treated, virus-infected cultures in microtiter plates were stained with the vital dye neutral red and the amount of dye incorporated was determined in a multichannel spectrophotometer. The technique required smaller volumes of reagents, was more easily automated than the standard plaque reduction assay and had good reproducibility. Standard conditions of 30 infectious units of challenge virus and 72-h incubation were judged to be optimal. Median inhibitory concentrations (ID50) for a number of compounds were approximately tenfold higher in the dye-uptake assay compared with the plaque reduction assay, possibly related to the higher multiplicity of infection required to give the desired level of cytopathic effect in the microtiter method.     

Unscheduled DNA synthesis in the testis,a secondary test for the evaluation of chemical mutagens

DNA damage induced by methylmethane sulfonate, cyclophosphamide, doxorubicin and procarbazine in male germ cells was assessed in rabbits by the demonstration of unscheduled DNA synthesis (UDS), in meiotic and postmeiotic phases of maturation. Immediately after treatment by the intravenous route tritiated thymidine was injected into both testicles. Subsequently, rabbits were ejaculated serially, sperm heads were isolated and assayed for radioactivity by liquid scintillation counting. Dose-dependent UDS was demonstrated in late spermatocytes and early spermatids. High doses of hycanthone also induced UDS, but isoniazid and metronidazole had no effect. The rabbit testis UDS test takes into account metabolic and pharmacokinetic aspects of the test substances and provides information about their penetration through the blood-testicular barrier. It is therefore useful for secondary evaluation of potential mutagens. UDS induced by procarbazine was abolished by simultaneous treatment with Ara-C. Thus, the test also recognizes substances that inhibit DNA repair synthesis.     

晚期糖基化交联产物裂解剂酶联免疫吸附测定筛选方法的建立

目的　建立一种快速、体外筛选晚期糖基化终产物 (AGE)交联结构裂解剂的方法。方法　以牛血清白蛋白 (BSA)与葡萄糖孵育形成AGE BSA ,并与包被于 96孔酶标板上的大鼠尾胶原蛋白交联制备AGE BSA 胶原交联结构 ,药物作用一定时间后 ,采用以抗BSA为抗体的ELISA方法检测BSA残留量 ,以此评价裂解剂的裂解强度。并对ELISA全程条件进行了优化。结果　BSA与葡萄糖孵育 3～ 4个月后 ,在Ex/Em( 395 / 4 6 0nm)下出现特异性荧光 ,SDS电泳显示 6 8.0ku的BSA已转化为分子量大于6 8.0ku蛋白质 ,表明已形成AGE BSA ;AGE BSA可与鼠尾胶原蛋白形成特异性的交联结构 ,最强特异性结合的包被量为每孔 0 .0 1～ 0 .1μg。最理想的封闭液为Pierce公司的SuperBlock封闭缓冲液。用新建立的ELISA方法对先导化合物苯基 4 ,5 二甲基噻唑氯嗡盐 (ALT 711)及新合成 10种裂解剂进行了体外裂解效应的评价 ,ALT 711结果与文献报道基本一致 ;本方法筛选的结果与高效液相以小分子二羰基化合物为底物的筛选方法结果趋势一致。结论此方法可用于体外筛选AGE交联裂解化合物 ,并具有高通量的特点     

Ionic liquid-based hollow fiber-supported liquid-phase microextraction enhanced electrically for the determination of neutral red

A method of ionic liquid-based hollow fiber liquid-phase microextraction enhanced electrically was successfully developed and applied to the extraction and determination of neutral red (NR) dye, which was selected as the model analyte. A room temperature ionic liquid, 1-octyl-3-methylimidazolium hexafluorophosphate ([C₈mim][PF₆]), was placed in the pores of a polytetrafluoethylene hollow fiber, which acts as a liquid membrane and the acceptor solution. The extraction parameters affecting the enrichment factor of NR, such as pH, extraction time, elution time, stirring rate, and the voltage were optimized. In addition, UV–Visible (UV–Vis) or electrochemiluminescence spectra were also determined. The extraction rate and capacity of NR could be improved significantly by cathodic polarization. Under the optimized extraction conditions (organic liquid microextraction phase [C₈mim][PF₆], pH 7, stirring rate 300 rpm, extraction time 20 minutes, ultrasonic-assisted elution time 3 minutes, voltage −70 V), the detection limit of 0.38 μg/L and linear correlation coefficient of r > 0.99 were obtained. The established method was successfully applied to the analysis of three soft drink samples, which were spiked with NR standards at the concentrations of 0.1, 1.0, and 5.0 mg/L, and satisfactory results were obtained.     

Application of DNA diffusion assay in earthworm coelomocytes

We have applied the DNA diffusion assay proposed by Singh (2000) Exp Cell Res 256:328-337, for quantitative estimation of apoptosis in earthworm coelomocytes, exposed to Chromium (VI) and cypermethrin as model toxicants in vitro. The DNA diffusion assay was originally described for mammalian cells. H2O2, Sodium ascorbate, and hyperthermia were used as positive controls in present study. Apoptosis such as DNA diffusion occurred in dose-dependent manner for Chromium (VI) and cypermethrin at very low concentration (1, 3, and 10 ppm for Chromium (VI) and 4, 8, and 16 ppm for cypermethrin). Three distinct patterns (apoptosis like DNA diffusion, necrosis, and normal) were observed in exposed and nonexposed cells. Present study is probably the first report of application of the DNA diffusion technique in earthworm coelomocytes. Findings of this study indicate that this assay has potential for use in invertebrate cells to differentiate between apoptosis and necrosis.     

中空纤维超滤法研究核苷类逆转录酶抑制药与DNA的结合率

目的:采用中空纤维超滤法研究核苷类逆转录酶抑制剂类药物与小牛胸腺DNA（ct-DNA）的相互结合率。方法:将中空纤维切成约15 cm小段,放入重蒸水中超声清洗15 s,晾干、备用。分别取阿德福韦（PMEA）、拉米夫定（LMVD）和恩曲他滨（FTC）与DNA摩尔比为1:1的混合液0.6 ml于一端封闭的细玻璃管中,玻璃管中再放入弯曲成U型的中空纤维,置离心机中离心(4 000 r·min^-1)60 min后,取出中空纤维,用微量进样器抽出中空纤维内液体约50μl,稀释至3.0 ml后分别测其最大紫外吸收。结果:PMEA、LMVD、FTC与DNA的平均结合率分别为40.7%、41.0%、38.6%。结论:确定了核苷类逆转录酶抑制剂类药物与DNA结合率的测定方法,可表征药物与DNA的结合强度。     

诺氟沙星薄膜衣片含量测定的不确定度评定

目的：对容量法测定诺氟沙星薄膜衣片含量的不确定度作出评定。方法：①建立数学模型，找出产生不确定度的各种成分；②估计这些成分的标准不确定度；③综合这些成分的标准不确定度；④计算扩展不确定度。结果：建立了容量法测定诺氟沙星薄膜衣片含量的数学模型，推导出不确定度的计算公式并计算各分量的不确定度，最后计算出合成不确定度。结论：本实验误差来源以标定0．1mol／L HClO4浓度和滴定管误差为主（B类），基准物质的误差可以忽略不计。     

In vitro and in vivo evaluation of ranitidine hydrochloride loaded hollow microspheres in rabbits

The objective of this investigation was to develop the hollow microspheres as a new dosage form of floating drug delivery systems with prolonged stomach retention time. Hollow microspheres containing ranitidine hydrochloride (RH) were prepared by a novel solvent diffusion-evaporation method using ethyl cellulose (EC) dissolved in a mixture of ethanol and ether (6:1.0, v/v). The yield and drug loading amount of hollow microspheres were 83.21±0.28% and 20.71±0.32%, respectively. The in vitro release profiles showed that the drug release rate decreased with increasing viscosity of EC and the diameter of hollow microspheres, while increased with the increase of RH/EC weight ratio. Hollow microspheres could prolong drug release time (approximately 24 h) and float over the simulate gastric fluid for more than 24 h. Pharmacokinetic analysis showed that the bioavailability from RH-hollow microspheres alone was about 3.0-times that of common RH gelatin capsules, and it was about 2.8-times that of the solid microspheres. These results demonstrated that RH hollow microspheres were capable of sustained delivery of the drug for longer period with increased bioavailability.     

Aptamer-mediated hollow MnO2 for targeting the delivery of sorafenib

Sorafenib (SRF) presents undesirable effects in clinical treatment, due to the lack of targeting, poor water solubility, and obvious side effects. In this study, we constructed a novel nanodrug carrier system for accurate and efficient delivery of SRF, improving its therapeutic effects and achieving tumor-specific imaging. The hollow mesoporous MnO₂ (H-MnO₂) nanoparticles equipped with target substance aptamers (APT) on the surface were used to load SRF for the first time. The resulting H-MnO₂-SRF-APT could specifically bound to glypican-3 (GPC3) receptors on the surface of hepatocellular carcinoma (HCC), rapidly undergoing subsequent degradation under decreased pH conditions in the tumor microenvironment (TME) and releasing the loaded SRF. In this process, Mn²⁺ ions were used for T₁-weighted magnetic resonance imaging simultaneously. The in vitro cell experiments indicated that H-MnO₂-SRF-APT showed much more effects on the inhibition in the proliferation of Huh7 and HepG2 HCC cells than that of the non-targeted H-MnO₂-SRF and free SRF. Besides, the in vivo results further confirmed that H-MnO₂-SRF-APT could effectively inhibit the growth of xenograft tumors Huh7 in the naked mouse with good biosafety. In conclusion, H-MnO₂-SRF-APT could significantly enhance the therapeutic effect of SRF and is expected to be a new way of diagnosis and treatment of HCC.