Antitumor activity of vinzolidine in the human tumor clonogenic assay and comparison with vinblastine

Summary Vinzolidine (VZL), a semisynthetic vinblastine (VLB) derivative, was tested against a variety of solid tumors in the human tumor clonogenic assay (HTCA). The emphasis was on continuous drug exposure because of the schedule-dependency of the vincas and long half-life of VZL. Of tumor types with more than ten samples tested, the percentage of cases exhibiting inhibition (50% or less of control) of tumor colony forming units (TCFU) was as follows: melanoma (48%), lung cancer (48%), breast cancer (40%), renal cancer (33%), and ovarian cancer (24%). In tumor types tested less frequently, inhibition of TCFU after continuous or one hour drug exposure was observed in 2/7 colon cancers, 1/3 pancreatic cancers and 3/4 gastric cancers. Paired analysis of tumors tested to both VZL and VLB demonstrated no significant difference in overall activity of these two vinca alkaloids.VZL appears to be a promising drug for clinical trials, with in vitro activity in melanoma, lung and breast cancers. More interesting is the suggestion of activity in gastrointestinal tumors, especially colon cancer which is generally resistant to drugs in the HTCA and in vivo.     

Evaluation of a new drug 7-N-(p-hydroxyphenyl)-mitomycin C [KW 2083] against carcinoma of the lung by the human tumor clonogenic assay

Summary KW 2083, which is a new mitomycin C derivative currently under clinical investigation, was tested for its antitumor effect on the growth of human carcinoma of the lung by using an in vitro clonogenic assay system. The in vitro results in this study were as follows: 1) We succeeded in producing clonal growth at a high rate in all histologic types of carcinoma of the lung in the assay system. That is, 61 of 81 specimens (75%) of either primary or metastatic tumors gave adequate growth for chemosensitivity testing. The in vitro responses to vindesine, adriamycin, mitomycin C and melphalan (substituted for cyclophosphamide in vitro), as standard anticancer drugs for chemotherapy of carcinoma of the lung were 18, 23, 18 and 19%, respectively, which are in good general agreement with the clinical responses to these drugs reported previously. Therefore, the clonogenic assay system might prove to be a very effective tool for an in vitro phase II study of new drugs. 2) The rate of response to KW 2083 tested simultaneously in 51 cancer specimens was 22%, which was superior to that of mitomycin C. These results indicate that KW 2083 might be more useful than mitomycin C in clinical practice.     

微量液体克隆形成法体外检测三尖杉酯碱、高三尖杉酯碱对CFU—GM及L—CFU的细胞毒作用

基于琼脂半固体培养法和极限稀释微孔池培养法,建立了一种新的体外药敏检测体系——微量液体克隆形成法。并应用该体系检测了三尖杉酯碱(H)和高三尖杉酯碱(HH)对CFU—GM及L—CFU的细胞毒作用。结果提示:24h及168h作用法时,HH对CFU—GM以及L—CFU的细胞毒作用均大于H,H、HH对L—CFO的细胞毒作用选择性均大于CFU—GM,但HH的细胞毒作用选择性不及H;H、HH的TDI(Time—schedule Depne—dence Index),IC_(90-24h)÷IC_(-90-168h)分别为68、60,说明两药物均为细胞周期时相特异性药物;并对H、HH之间可能存在的同系物间的交叉耐药性作了探讨。     

In vitro antitumor effect of recombinant human tumor necrotizing factor on cultured human cancer cell lines and freshly isolated lung cancer cells by the human tumor clonogenic assay

Summary In vitro antitumor effects of human recombinant tumor necrotizing factor (rH-TNF) were examined against nine lung cancer cell lines including six non small and three small cell lung cancer, four stomach cancer cell lines and 30 freshly isolated lung cancer cell samples by the human tumor clonogenic assay. rH-TNF did not show any inhibitory effect on the colony formations of lung and stomach cancer cell lines, except for PC10 established from squamous cell carcinoma even at the high concentration. The overall response rate of fresh material was 11.5%. The colony formations of only two materials from 20 patients without prior chemotherapy were significantly suppressed by rH-TNF in vitro. Three specimens of adenocarcinoma exhibited more than 70% decrease in colony number by treating with 100 and 1000 u/ml of rH-TNF resulting in the response rate of 15.8% (3/19). From these results, it can be concluded that rH-TNF has modest direct cytotoxic effect on lung cancer, and additional study against adenocarcinoma of the lung might be warranted.     

MTT法测定肿瘤细胞药物敏感性与临床用药

目的:研究MTT法测定肿瘤细胞药物敏感性与临床用药之间的关系.方法:将119例7类肿瘤标本(52例膀胱癌、19例卵巢癌、14例宫颈癌、12例子宫内膜癌、12例肺癌、5例胃癌、5例肠癌)的药敏结果进行统计分析,并与临床实际用药进行比较,评价其疗效.结果:肿瘤细胞体外药敏存在着明显的个体差异,而其平均抑制率结果与临床用药情况基本相符.结论:肿瘤细胞体外药敏试验结果可以为临床个体化治疗提供参考.     

Evaluation of the genotoxic and antigenotoxic potential of Baccharis dracunculifolia extract on V79 cells by the comet assay

Baccharis dracunculifolia (Asteraceae), the main botanical source of green propolis, is a shrub of the Brazilian ‘cerrado’. In folk medicine it is used as an anti‐inflammatory agent, mainly for the treatment of gastrointestinal diseases. The aim of the present study was to evaluate the genotoxic and antigenotoxic effects of B. dracunculifolia ethyl acetate extract (Bd‐EAE) on Chinese hamster lung fibroblasts (V79 cells) by the comet assay. Methyl methanesulfonate (MMS; 200 μM ) was used as an inducer of DNA damage. Genotoxicity was evaluated using four different concentrations of Bd‐EAE: 12.5, 25.0, 50.0 and 100.0 μg ml^?1. Antigenotoxicity was assessed before, simultaneously, and after treatment with the mutagen. The results showed a significant increase in the frequency of DNA damage in cultures treated with 50.0 and 100.0 μg ml^?1 Bd‐EAE. Regarding its antigenotoxic potential, Bd‐EAE reduced the frequency of DNA damage induced by MMS. However, this chemopreventive activity depended on the concentrations and treatment regimens used. The antioxidant activity of phenolic components present in Bd‐EAE may contribute to reduce the alkylation damage induced by MMS. In conclusion, our findings confirmed the chemopreventive activity of Bd‐EAE and showed that this effect occurs under different mechanism. Copyright © 2009 John Wiley & Sons, Ltd.     

Evaluation of the role of P-glycoprotein in the uptake of paroxetine, clozapine, phenytoin and carbamazapine by bovine retinal endothelial cells

Expression of the drug transport proteins, including P-glycoprotein (Pgp), in the brain vascular endothelium represents a challenge for the effective delivery of drugs for the treatment of several central nervous system (CNS) disorders including depression, schizophrenia and epilepsy. It has been hypothesized that Pgp plays a major role in drug efflux at the blood-brain barrier, and may be an underlying factor in the variable responses of patients to CNS drugs. However, the role of Pgp in the transport of many CNS drugs has not been directly demonstrated. To explore the role of Pgp in drug transport across an endothelial cell barrier derived from the central nervous system, the expression and activity of Pgp in bovine retinal endothelial cells (BRECs) and the effects of representative CNS drugs on Pgp activity were examined. Significant Pgp expression in BRECs was demonstrated by western analyses, and expression was increased by treatment of the cells with hydrocortisone. Intracellular accumulation of the well-characterized Pgp-substrate Taxol was markedly increased by the non-selective transporter inhibitor verapamil and the Pgp-selective antagonist PGP-4008, demonstrating that Pgp is active in these endothelial cells. In contrast, neither verapamil nor PGP-4008 affected the intracellular accumulation of [3H]paroxetine, [14C]phenytoin, [3H]clozapine or [14C]carbamazapine, indicating that these drugs are not substrates for Pgp. Paroxetine, clozapine and phenytoin were shown to be Pgp inhibitors, while carbamazapine did not inhibit Pgp at any concentration tested. These results indicate that Pgp is not likely to modulate patient responses to these drugs.     

Evaluation of the peripapillary retinal nerve fiber layer and ganglion cell-inner plexiform layer measurements in patients with iron deficiency anemia with optical coherence tomography

Objective: To evaluate the thickness of the peripapillary retinal fiber layer (RNFL) and macula ganglion cell-inner plexiform layer (GCL+) using optical coherence tomography (OCT) in patients with iron deficiency (ID) anemia.

Methods: This study included 73 eyes of 39 patients with ID anemia and 68 eyes of 34 age- and sex-matched healthy subjects. The measurements included the peripapillary RNFL thicknesses as average, 4 quadrant and 12 clock-hour (CH) based and macula GCL+ thicknesses as average and 6 quadrant based. All measurements were completed with Cirrus HD-OCT and the results were compared between the groups.

Results: A total of 73 eyes of 39 patients with ID anemia and 68 eyes of 34 healthy subjects were included to the study. Regarding peripapillary RNFL thicknesses of the study and control patients, the values of average and quadrants revealed no significant differences between the groups. In CH sectors comparison, peripapillary RNFL thicknesses were significantly decreased only in CH4 (68.7?±?14.5?μm in study versus 72.0?±?13.4?μm in control patients, p?=?0.049) and CH5 (93.4?±?20.0μm in study versus 102.2?±?20.1?μm in control patients, p?=?0.01) sectors. All measured quadrants were statistically similar, when macula GCL+ thicknesses were compared between the groups. When the correlations between peripapillary RNFL and macula GCL+ thicknesses and serum hemoglobin and ferritin levels of study and control patients were calculated, the only statistically significant parameter was the correlation of peripapillary RNFL thickness in CH10 sector with serum ferritin level (p?=?0.032, Spearman correlation coefficient: 0.369).

Conclusion: The study revealed that peripapillary RNFL is thinner in nasal-inferior quadrant in patients with ID anemia. The measurements of macula GCL+ thicknesses were similar between the groups. Analyzing the retinal layers using OCT may provide valuable information in neurodegenerative events.     

红细胞在不同pH下致敏对头孢他啶针剂的IgM和IgG抗体检测结果的影响

目的研究在用微柱凝胶免疫法检测人血中头孢他啶针剂的IgM和IgG抗体时,红细胞在不同pH下被药物致敏后对检测结果的影响。方法 O型红细胞在苯巴比妥缓冲液（pH=7.0,8.0,9.6）中用头孢他啶于室温下孵育2小时后作为致敏后的红细胞,以致敏后的红细胞（pH=9.6）为初试抗原,测试600份待检血清,根据检测结果选出10份血清样品,阳性3份,弱阳性3份,阴性4份,再分别用经pH8.0及pH7.0时致敏的红细胞检测,比较结果的异同。结果红细胞在不同pH下致敏对4份阴性结果的血清和3份阳性结果的血清都没影响,但3份弱阳性标本在pH8.0时一份转阴,在pH7.0时3份都转阴。结论用微柱凝胶免疫技术检测人血中头孢他啶IgM和IgG抗体时,红细胞致敏的pH小于9.6可能出现假阴性结果。     

Evaluation of the 20-day pubertal female assay in Sprague-Dawley rats treated with DES, tamoxifen, testosterone, and flutamide.

The Endocrine Disrupter Screening and Testing Advisory Committee (EDSTAC) has recommended the rodent pubertal female assay as a Tier I test to detect potential endocrine disrupters (EDs). This assay is designed to screen estrogenic activity in immature rats exposed to chemicals during sexual maturation. The aim of this study was to evaluate whether this assay can detect the EDs with effects brought about through various mechanisms. Immature Sprague-Dawley female rats (21 days of age) were dosed daily for 20 days by oral gavage (DES, tamoxifen, and flutamide) or sc injection (testosterone). The mean age at vaginal opening (VO) was 32.3 +/- 0.5 days in control rats. Although VO was unaffected by DES at doses of 0.2 and 1.0 microg/kg, a high dose of DES (5.0 microg/kg) significantly advanced the age at VO to 24 days. Both tamoxifen (50 and 200 microg/kg) and flutamide (25 mg/kg) also significantly accelerated VO to 27.8 +/- 0.5, 25.1 +/- 0.1, and 26.1 +/- 0.1, respectively. However, testosterone dose-dependently delayed VO (exposure to 1.0 mg/kg extended VO to 37.3 +/- 0.8 days, and VO did not occur in 2 of 10 animals by the time of necropsy at 41 days of age). Estrous cyclicity was monitored in rats from VO to necropsy. Irregular cycles were observed in the groups treated with DES (5.0 microg/kg), tamoxifen (200 microg/kg), testosterone (1.0 mg/kg), and flutamide (25 mg/kg). High dose of DES showed a persistent estrus state throughout the entire observation period. In addition, the number of days in diestrus was increased by tamoxifen (200 microg/kg) and flutamide (25 mg/kg) treatments. Significant decreases in ovarian weight were observed in 5.0 microg/kg DES (64% of control), 25 mg/kg flutamide (76% of control), and 200 microg/kg tamoxifen (47% of control). Testosterone also significantly decreased the ovarian weights in all treatment groups. Uterine weights were also decreased significantly at high doses of tamoxifen (200 microg/kg, 39% of control) or testosterone (1.0 mg/kg, 47% of control). In hormone analysis, tamoxifen significantly increased serum E(2) levels at 50 microg/kg. The mean serum levels of TSH were significantly increased in tamoxifen (10 and 50 microg/kg), testosterone (0.2 mg/kg), and flutamide (1.0 and 25 mg/kg) treatment groups compared with the control. However, serum T(4) levels were significantly reduced by testosterone. Furthermore, serum T(3) levels were significantly increased in DES, tamoxifen (10 and 50 microg/kg), testosterone (1.0 mg/kg), and flutamide (1.0 and 5 mg/kg). Our data demonstrate that the rodent pubertal female assay is useful for identifying potential EDs having not only estrogenic/antiestrogenic but also androgenic/antiandrogenic activities. However, further validation study is necessary to identify chemicals that operate through other action mechanisms, including steroid biosynthesis inhibitors and thyroid inhibitors. Moreover, additional data on other compounds with weak endocrine disrupting activity will be required to further characterize the sensitivity of the female pubertal assay.     

DNA alkylation lesions and their repair in human cells: modification of the comet assay with 3-methyladenine DNA glycosylase (AlkD)

3-Methyladenine DNA glycosylase (AlkD) belongs to a new family of DNA glycosylases; it initiates repair of cytotoxic and promutagenic alkylated bases (its main substrates being 3-methyladenine and 7-methylguanine). The modification of the comet assay (single cell gel electrophoresis) using AlkD enzyme thus allows assessment of specific DNA alkylation lesions. The resulting baseless sugars are alkali-labile, and under the conditions of the alkaline comet assay they appear as DNA strand breaks. The alkylating agent methyl methanesulfonate (MMS) was used to induce alkylation lesions and to optimize conditions for the modified comet assay method with AlkD on human lymphoblastoid (TK6) cells. We also studied cellular and in vitro DNA repair of alkylated bases in DNA in TK6 cells after treatment with MMS. Results from cellular repair indicate that 50% of DNA alkylation is repaired in the first 60 min. The in vitro repair assay shows that while AlkD recognises most alkylation lesions after 60 min, a cell extract from TK6 cells recognises most of the MMS-induced DNA adducts already in the first 15 min of incubation, with maximum detection of lesions after 60 min’ incubation. Additionally, we tested the in vitro repair capacity of human lymphocyte extracts from 5 individuals and found them to be able to incise DNA alkylations in the same range as AlkD. The modification of the comet assay with AlkD can be useful for in vitro and in vivo genotoxicity studies to detect alkylation damage and repair and also for human biomonitoring and molecular epidemiology studies.