Imaging of differential protease expression in breast cancers for detection of aggressive tumor phenotypes

PURPOSE: To determine if different expression levels of tumor cathepsin-B activity in well differentiated and undifferentiated breast cancers could be revealed in vivo with optical imaging. MATERIALS AND METHODS: A well differentiated human breast cancer (BT20, n = 8) and a highly invasive metastatic human breast cancer (DU4475, n = 8) were implanted orthotopically in athymic nude mice. Tumor-bearing animals were examined in vivo with near-infrared fluorescence (NIRF) imaging 24 hours after intravenous injection of an enzyme-sensing imaging probe. Immunohistochemistry, Western blotting (on cells and whole tumor samples), and correlative fluorescence microscopy were performed. RESULTS: Both types of breast cancers activated the NIRF probe so that tumors became readily detectable. However, in tumors of equal size, there was a 1.5-fold higher fluorescence signal in the highly invasive breast cancer (861 arbitrary units +/- 88) compared with the well differentiated lesion (566 arbitrary units +/- 36, P <.01). Western blotting confirmed a higher cathepsin-B protein content in the highly invasive breast cancer (DU4475) of about 1.4-fold (whole tumor samples) to 1.7-fold (cells). Immunohistochemistry and fluorescence microscopy findings confirmed the imaging findings. CONCLUSION: Cathepsin-B enzyme activity can be determined in vivo with NIRF optical imaging, while differences in tumoral expression may correlate with tumor aggressiveness.     

Irradiation and Various Cytotoxic Drugs Enhance Tyrosine Phosphorylation and β1-Integrin Clustering in Human A549 Lung Cancer Cells in a Substratum-Dependent Manner in Vitro

BACKGROUND AND PURPOSE: Interactions of cells with a substratum, especially extracellular matrix proteins, initiate clustering of integrin receptors in the cell membrane. This process represents the initial step for the activation of signaling pathways regulating survival, proliferation, differentiation, adhesion, and migration, and could, furthermore, be important for cellular resistance mediating mechanisms against radiation or cytotoxic drugs. The lack of data elucidating the impact of irradiation or cytotoxic drugs on this important phenomenon led to this study on human A549 lung cancer cells in vitro. MATERIAL AND METHODS: The human lung carcinoma cell line A549 grown on polystyrene or fibronectin (FN) was irradiated with 0-8 Gy or treated with cisplatin (0.1-50 microM), paclitaxel (0.1-50 nM), or mitomycin (0.1-50 microM). Colony formation assays, immunofluorescence staining in combination with activation of integrin clustering using anti-beta(1)-integrin antibodies (K20), and Western blotting for tyrosine phosphorylation under treatment of cells with the IC(50) for irradiation (2 Gy; IC(50) = 2.2 Gy), cisplatin (2 microM), paclitaxel (5 nM), or mitomycin (7 microM) were performed. RESULTS: Attachment of cells to FN resulted in a significantly reduced radio- and chemosensitivity compared to polystyrene. The clustering of beta(1)-integrins examined by immunofluorescence staining was only stimulated by irradiation, cisplatin, paclitaxel, or mitomycin in case of cell attachment to FN. By contrast, tyrosine phosphorylation, as one of the major events following beta(1)-integrin clustering, showed a 3.7-fold, FN-related enhancement, and treatment of cells with the IC(50) of radiation, cisplatin, paclitaxel, or mitomycin showed a substratum-dependent induction. CONCLUSION: For the first time, a strong influence of irradiation and a variety of cytotoxic drugs on the clustering of beta(1)-integrins could be shown. This event is a prerequisite for tyrosine phosphorylation and, thus, the activation of cellular mechanisms regulating survival, proliferation, and adhesion. These data are not only important for the understanding of cellular resistance against cytotoxic agents but, furthermore, for tumor progression, anchorage-independent cell growth, and, possibly, the optimization of radiochemotherapeutic strategies.     

2004 Dr. Gary J. Becker Young Investigator Award: Relative thermosensitivity of cytotoxic drugs used in transcatheter arterial chemoembolization

PURPOSE: Large hepatocellular carcinoma tumors are being treated increasingly with a combination of transcatheter arterial chemoembolization (TACE) and radiofrequency (RF) ablation. However, the high temperatures reached during RF ablation may reduce the cytotoxic effects of antineoplastic agents, but this has not been studied. Therefore, in the present study, the relative thermosensitivity of cytotoxic drugs commonly used in TACE was studied.MATERIALS AND METHODS: The relative cytotoxic effects of cisplatin, doxorubicin HCl, and mitomycin on the growth of human colon HT29 and lung A549 adenocarcinoma cells before and after heating each drug in solution was determined from the standpoints of different durations of exposure (15, 30, 60, 90, and 120 minutes) at a fixed temperature (120 degrees C) and exposure to different temperatures (60 degrees C, 80 degrees C, 100 degrees C, and 120 degrees C) for a fixed period of time (2 hours). After 72 hours of exposure of the cells to each drug, relative cell growth inhibition was assessed by MTT assay, and 50% inhibitory concentration (IC(50)) values were calculated for each cytotoxic agent. Finally, the heat-dependent degradation of mitomycin and doxorubicin was analyzed with use of tandem electrospray mass spectrometry. RESULTS: The relative cytotoxic activities (shown by cell growth inhibition and IC(50) values) of cisplatin, doxorubicin, and mitomycin heated to 120 degrees C for 2 hours decreased by factors of 1.35 (range, 1-1.75), 9.5 (range, 8.5-10.5), and 7.05 (range, 3.5-12), respectively. The cytotoxic activities of doxorubicin and mitomycin continued to decrease with incremental increases in temperature. Similarly, with incremental increases in the duration of exposure to heat, the cytotoxic activities of doxorubicin and mitomycin decreased. Mass spectrometric analysis of residual drug content showed that a 2-hour exposure to a temperature of 120 degrees C caused doxorubicin and mitomycin to degrade by 95% and 84%, respectively. CONCLUSIONS: The cytotoxicity of cisplatin is not affected by heat. The cytotoxicities of doxorubicin and mitomycin are reduced by high temperature and duration of exposure to heat. Although degradation of cytotoxicity starts at 60 degrees C and after 30 minutes of exposure to heat, statistically significant changes are encountered at 100 degrees C and after 90 minutes of exposure.     

Uptake of 99mTc-MIBI and 99mTc-tetrofosmin into malignant versus nonmalignant breast cell lines.

The kinetics and cellular uptake of 99mTc-2-hexakis 2-methoxyiso-butyl-isonitrile (MIBI) and 99mTc-1 ,2-bis[bis(2-ethoxyethyl)phosphino]ethane (tetrofosmin) into malignant versus nonmalignant human breast cell lines were investigated and compared. METHODS: At specific intervals after incubation at 37 degrees C and 22 degrees C with 99mTc-MIBI or 99mTc-tetrofosmin, the uptake characteristics of radiotracers into human adenocarcinoma breast cell lines MCF-7 and SK-BR-3 and human breast, nontumor cell line HBL-100 were assessed. RESULTS: The uptake of 99mTc-MIBI and 99mTc-tetrofosmin was lower at an incubation temperature of 22 degrees C than that at 37 degrees C in the 3 cell lines. In MCF-7 and in SK-BR-3 cells the uptake of 99mTc-MIBI was significantly higher than the uptake of 99mTc-tetrofosmin. The uptake of 99mTc-MIBI was significantly higher into MCF-7 and SK-BR-3 cells than that into HBL-100 cells. In comparison with HBL-100 cells, uptake of 99mTc-tetrofosmin into SK-BR-3 cells was significantly higher, whereas uptake into MCF-7 cells was similar. CONCLUSION: In vitro data suggest that 99mTc-MIBI may be a better tracer than 99mTc-tetrofosmin for discrimination between malignant and nonmalignant breast disease.     

Fibronectin and laminin increase resistance to ionizing radiation and the cytotoxic drug Ukrain in human tumour and normal cells in vitro

PURPOSE: Cell-extracellular matrix (ECM) interactions are thought to mediate drug and radiation resistance. Dependence of cell survival, beta1-integrin expression and cell cycling on the ECM proteins and beta1-integrin ligands fibronectin (FN) and laminin (LA) were examined in malignant and normal cells exposed to the cytotoxic drug Ukrain plus/minus irradiation. MATERIALS AND METHODS: Human A549 lung cancer and MDAMB231 (MDA231) breast cancer cells and normal fibroblasts (HSF1) grown on FN, LA, bovine serum albumin (BSA) or polystyrene were treated with Ukrain (1 microg ml(-1), 24 h) plus/minus irradiation (2-8 Gy) and the effects studied using colony formation assays, flow cytometry (beta1-integrin, DNA analysis) and adhesion assays. RESULTS: FN and LA reduced the cytotoxic effect of single Ukrain treatment compared with polystyrene and BSA. FN and LA also abolished Ukrain-dependent radiosensitization in A549 cells and decreased the radiosensitivity of MDA231 and HSF1 cells. Single Ukrain exposure on polystyrene significantly reduced beta1-integrin expression and promoted G2-phase accumulation of A549 cells. In contrast, Ukrain-treated MDA231 and HSF1 cells showed elevated beta1-integrin expression and no Ukrain-specific cell cycle effect. Under Ukrain-radiation exposure, irradiation, FN or LA abolished Ukrain-mediated reduction of beta1-integrin expression and G2-phase accumulation in A549 cells, whereas in MDA231 cells and fibroblasts beta1-integrin expression and cell cycle distribution were stabilized. Cell adhesion to FN or LA was significantly impaired (A549) or improved (MDA231, HSF1) upon Ukrain treatment. CONCLUSIONS: The data corroborate the findings of other groups that cell adhesion-mediated resistance to either single or combined drug and radiation exposure is tightly correlated to specific ECM proteins. By demonstrating a strong modulatory impact of FN and LA on the radiosensitivity-modifying activity of the drug Ukrain, the set findings are also highly important for the assessment of drug and radiation effects within in vitro cytotoxicity studies. The data give the first mechanistic insights into specific FN- and LA-modulated cellular resistance mechanisms as well as into the important role for beta1-integrins using the unique cytotoxic and radiosensitivity-modifying drug Ukrain.     

五种竹红菌素衍生物对A549细胞的光动力效应研究

目的比较五种新型竹红菌素衍生物分别为竹红菌素乙素（hypocrellin,HB）的二位ω-氨基磺酸衍生物THB、3HB和4HB,及十七位ω-氨基磺酸衍生物3SB和4SB对体外培养的人肺腺癌上皮细胞（A549）的光动力（photodynamic therapy,PDT）效应,筛选光动力活性和安全性较好的竹红菌素衍生物。方法 （1）杀伤效应。将0.94 nmol/ml的5种新型竹红菌素衍生物和HB分别与A549细胞孵育4 h后,分别以波长630和532 nm激光照射,功率密度20 mW/cm2,照射时间1 000 s,能量密度20 J/cm2,照光后继续避光孵育24 h后采用MTT法测定细胞存活率。（2）安全系数。分别以波长532和630 nm激光照射,以血卟啉（hematoporph-yrin derivative,HpD）为对照光敏剂,研究17-4-amino-1-butane-sulfonic acid-hypocrellin B（4SB）对A549细胞的光动力效应及和暗毒性,并比较安全系数（暗毒性IC50/光毒性IC50）。结果 （1）杀伤效应。五种竹红菌素衍生物中,4SB在630和532 nm激光照射下对A549的光动力杀伤作用强于其它衍生物,接近HB。（2）安全系数。波长532 nm激光照射,4SB的光毒性分别为103.86和84.16 ng/ml是HpD 960.14 ng/ml的10.53和11.4倍,但前两者之间差异无显著意义（P〉0.05）;波长630 nm激光照射下,4SB光毒性的IC50为50.7 ng/ml,HpDIC50为1 069.88 ng/ml,暗毒性HpD、4SB分别为7.84、21.93μg/ml,安全系数4SB（432.5）〉HpD（7.3）。HpD在532和630 nm两波长下的光毒性IC50差异无显著意义（P〉0.05）,而4SB在532和630 nm两波长下的光毒性差异有显著意义（P〈0.05）。结论 5种衍生物可能成为有价值的光敏剂,值得进一步深入研究。     

Novel piperazine-substituted silicon phthalocyanines exert anti-cancer effects against breast cancer cells

BackgroundPhotodynamic therapy (PDT) is one of the effective methods that can be used in cancer treatment. In this study, we aimed to investigate the PDT-mediated anti-cancer effects of newly synthesized piperazine-substituted silicon phthalocyanine molecules on breast cancer cells.MethodsThe compounds were analyzed by different spectroscopic techniques (FT-IR, UV–vis, ¹H NMR, ¹³C NMR, MS) and the absorbance characteristics were determined. The cytotoxic effects of silicon phthalocyanines on MDA-MB-231 breast cancer cells and non-tumorigenic MCF-10A cells were evaluated using MTT assay. Detection of apoptotic populations was performed by Annexin V/7AAD assay. H₂DCFDA dye was used to analyze intracellular reactive oxygen species. The clonogenic activity and cellular motility were analyzed by colony formation assay and in vitro scratch assay, respectively. Caspase-3, PARP1, and cleaved-PARP1 protein levels were analyzed by western blot studies.ResultsPiperazine-substituted silicon phthalocyanines caused high levels of cytotoxic effects and apoptotic cell population in MDA-MB-231 cells, while low levels of cytotoxic effects were observed in MCF-10A cells. Following PDT, intense ROS formation was detected in MDA-MB-231 cells. Colony-forming capacity and cellular motility of MDA-MB-231 cells were highly restricted following PDT, whereas these effects were observed at lower levels in MCF-10A cells. Silicon phthalocyanines caused different effects on cleaved-PARP1 expressions of MDA-MB-231 and MCF-10A cells.ConclusionThese results suggest that piperazine-substituted silicon phthalocyanines can exert selective anti-cancer effects on breast cancer cells and activate cellular death through different molecular pathways. Hence, we believe that they may be used as effective photosensitizer agents in the future.     

Fibronectin and laminin increase resistance to ionizing radiation and the cytotoxic drug Ukrain® in human tumour and normal cells in vitro

Purpose: Cell–extracellular matrix (ECM) interactions are thought to mediate drug and radiation resistance. Dependence of cell survival, β1‐integrin expression and cell cycling on the ECM proteins and β1‐integrin ligands fibronectin (FN) and laminin (LA) were examined in malignant and normal cells exposed to the cytotoxic drug Ukrain plus/minus irradiation.

Materials and methods: Human A549 lung cancer and MDAMB231 (MDA231) breast cancer cells and normal fibroblasts (HSF1) grown on FN, LA, bovine serum albumin (BSA) or polystyrene were treated with Ukrain (1?µg?ml^?1, 24?h) plus/minus irradiation (2–8?Gy) and the effects studied using colony formation assays, flow cytometry (β1‐integrin, DNA analysis) and adhesion assays.

Results: FN and LA reduced the cytotoxic effect of single Ukrain treatment compared with polystyrene and BSA. FN and LA also abolished Ukrain‐dependent radiosensitization in A549 cells and decreased the radiosensitivity of MDA231 and HSF1 cells. Single Ukrain exposure on polystyrene significantly reduced β1‐integrin expression and promoted G2‐phase accumulation of A549 cells. In contrast, Ukrain‐treated MDA231 and HSF1 cells showed elevated β1‐integrin expression and no Ukrain‐specific cell cycle effect. Under Ukrain‐radiation exposure, irradiation, FN or LA abolished Ukrain‐mediated reduction of β1‐integrin expression and G2‐phase accumulation in A549 cells, whereas in MDA231 cells and fibroblasts β1‐integrin expression and cell cycle distribution were stabilized. Cell adhesion to FN or LA was significantly impaired (A549) or improved (MDA231, HSF1) upon Ukrain treatment.

Conclusions: The data corroborate the findings of other groups that cell adhesion‐mediated resistance to either single or combined drug and radiation exposure is tightly correlated to specific ECM proteins. By demonstrating a strong modulatory impact of FN and LA on the radiosensitivity‐modifying activity of the drug Ukrain, these findings are also highly important for the assessment of drug and radiation effects within in vitro cytotoxicity studies. The data give the first mechanistic insights into specific FN‐ and LA‐modulated cellular resistance mechanisms as well as into the important role for β1‐integrins using the unique cytotoxic and radiosensitivity‐modifying drug Ukrain.     

Radiosensitizing effect of epothilone?B on human epithelial cancer cells

Background

A combined modality treatment employing radiation and chemotherapy plays a central role in the management of solid tumors. In our study, we examined the cytotoxic and radiosensitive effect of the microtubule stabilizer epothilone?B on two human epithelial tumor cell lines in vitro and its influence on the microtubule assembly.

Methods

Cancer cells were treated with epothilone?B in proliferation assays and in combination with radiation in colony-forming assays. For the analysis of ionizing radiation-induced DNA damage and the influence of the drug on its repair a ??H2AX foci assay was used. To determine the effect of epothilone?B on the microtubule assembly in cells and on purified tubulin, immunofluorescence staining and tubulin polymerization assay, respectively, were conducted.

Results

Epothilone?B induced a concentration- and application-dependent antiproliferative effect on the cells, with IC₅₀?values in the low nanomolar range. Colony forming assays showed a synergistic radiosensitive effect on both cell lines which was dependent on incubation time and applied concentration of epothilone?B. The ??H2AX assays demonstrated that ionizing radiation combined with the drug resulted in a concentration-dependent increase in the number of double-strand breaks and suggested a reduction in DNA repair capacity. Epothilone?B produced enhanced microtubule bundling and abnormal spindle formation as revealed by immunofluorescence microscopy and caused microtubule formation from purified tubulin.

Conclusion

The results of this study showed that epothilone?B displays cytotoxic antitumor activity at low nanomolar concentrations and also enhances the radiation response in the tumor cells tested; this may be induced by a reduced DNA repair capacity triggered by epothilone?B. It was also demonstrated that epothilone?B in fact targets microtubules in a more effective manner than paclitaxel.     

奥美拉唑三联10日疗法治疗幽门螺杆菌感染疗效观察

幽门螺杆菌（Ｈｐ）相关性十二指肠球部溃疡（ＤＵ）患者８３例，随机分为２组，口服不同剂量的奥美拉唑、克拉霉素和羟氨苄青霉素１０日。疗程结束后４周复查Ｈｐ、ＤＵ愈合和观察组织炎症细胞浸润情况。结果：２组间的Ｈｐ根除率分别为８８．６％和９２．３％；溃疡愈合总有效率为９７．７％和１００％，均无统计学差异。而Ｈｐ根除后胃窦粘膜组织炎症细胞浸润程度和活动度，均比治疗前明显减轻。结果表明：奥美拉唑、克拉霉素和羟     

三氧化二砷逆转乳腺癌细胞株MCF-7/ADM多药耐药的研究

目的探讨三氧化二砷（As2O3）体外逆转人乳腺癌细胞多药耐药性的作用及机制。方法采用MTT法检测As2O3的细胞毒作用和处理前后耐药细胞对化疗药物的敏感性,用流式细胞仪检测细胞内阿霉素浓度,通过RT-RCR检测MDR1基因的表达。结果 As2O3在0.25mg/L以下剂量时对MCF-7和MCF-7/ADM耐药细胞株的抑制率均小于15%,半数抑制率（IC50）分别为1.01m/L和1.28mg/L,无细胞毒剂量0.2mg/L的As2O3能部分逆转MCF-7/ADM细胞对阿霉素的耐药性。同时无细胞毒剂量0.2mg/L的As2O3能使MCF-7/ADM细胞内阿霉素浓度明显增加,MDR1表达下降。结论 As2O3具有体外部分逆转人乳腺癌细胞多药耐药性的作用,可能与增加细胞内药物积累、下调MDR1表达有关。     

Development of 111In-labeled tumor-associated antigen peptides for monitoring dendritic-cell-based vaccination

Dendritic cells (DC) are professional antigen-presenting cells capable of inducing potent immune responses. In our ongoing clinical trials, human leukocyte antigen (HLA)-A2.1+ melanoma patients are vaccinated with mature DC, presenting tumor-derived peptides in major histocompatibility complexes (MHC) to naive T cells. Previously, we have shown that both intradermally and intranodally injected (111)In-labeled mature DC migrate to draining lymph nodes. However, little is known about the fate of the MHC-peptide complex after injection of these peptide-loaded DC. The aim of the present study was to develop radiolabeled, tumor-derived peptides to monitor their binding to MHC Class I. METHODS: The HLA-A2.1 binding peptide gp100:154-162mod (gp100:154m) was conjugated with diethylenetriamine pentaacetic acid (DTPA) either at the N-terminus (alpha-DTPA-gp100:154m) or at the epsilon amino group of the Lys(154) residue (epsilon-DTPA-gp100:154m) and labeled with (111)In. RESULTS: The maximum specific activity for both peptides was 13 GBq/micromol. The IC50 of the alpha-[(111)In]DTPA-gp100:154m peptide was >75 microM. The IC50 of the (111)In-labeled epsilon-DTPA-gp100:154m was 3 microM, similar to the unconjugated peptide. MHC binding studies showed specific binding of the epsilon-[(111)In]DTPA-gp100:154m peptide to the JY cells at 4 degrees C. Interestingly, no specific binding was observed for the alpha-[(111)In]DTPA-gp100:154m peptide. In contrast to the alpha-[(111)In]DTPA-gp100:154m peptide, the epsilon-[(111)In]DTPA-gp100:154m peptide was recognized by cytotoxic T cells. CONCLUSION: When DTPA was conjugated to the epsilon NH2 group of the Lys(154) residue, MHC binding of the peptide was preserved and could still be recognized by cytotoxic T cells. These studies allow the noninvasive determination of the behavior of MHC-peptide complexes on DC in vivo.     

Intracellular volume and apparent diffusion constants of perfused cancer cell cultures,as measured by NMR

Diffusion NMR spectroscopy was used to study intracellular volume and apparent water diffusion constants in different cell lines (DU145, human prostate cancer; AT3, rat prostate cancer; MCF-7, human breast cancer; RIF-1, mouse fibrosacroma). The cells were grown on various matrices (collagen sponge, collagen beads, polystyrene beads) which enabled continuous growth in perfused high density cell culture suitable for NMR studies. In perfused cell systems, the attenuation of the water signal versus the squared gradient strength was fitted by the sum of two decaying exponentials. For the slowly decaying component the apparent water diffusion constant at 37°C was 0.22 (±0.02) × 10^?9 s/m² for all cell lines at diffusion times > 100 ms. It continuously increased up to 0.47(±0.05) × 10^?9 s/m² when the diffusion time was decreased to 8 ms, indicating restricted diffusion. No significant effect of the matrices was observed. The fractional volume of the slow component as determined from the biexponential diffusion curve correlated with the relative intracellular volume, as obtained from the cell density in the sample and the cell size as measured by light microsocopy. Therefore, this simple NMR approach can be used to determine intracellular volume in perfused cell cultures suitable for NMR studies. Using this information in combination with spectroscopic data, changes in intracellular metabolite concentration can be detected even when the cellular volume is changing during the experiment. The apparent diffusion constant for the fast diffusing component varied with growth matrix, cell density and cell type and also showed the typical characteristics of restricted diffusion (increase of apparent diffusion constant with time).     

Quantitative two‐dimensional HRMAS 1H‐NMR spectroscopy‐based metabolite profiling of human cancer cell lines and response to chemotherapy

NMR spectroscopy‐based metabolomics still needs development in quantification procedures. A method was designed for quantitative two‐dimensional high resolution magic angle spinning (HRMAS) proton‐NMR spectroscopy‐based metabolite profiling of intact cells. It uses referencing of metabolite‐related NMR signals to protein‐related NMR signals and yields straightforward and automatable metabolite profiling. The method enables exploitation of only two‐dimensionally visible metabolites and combination of one‐ and two‐dimensional spectra, thus providing an appreciable number of screened metabolites. With this procedure, 32 intracellular metabolites were attributed and quantified in human normal fibroblasts and tumor cells. The phenotype of several tumor cell lines (MCF7, PC3, 143B, and HepG2) was characterized by high levels of glutathione in cell lines with the higher proliferation rate, high levels of creatine, low levels of free amino acids, increased levels of phospholipid derivatives (mostly phosphocholine), and lower lactate content in cell lines with the higher proliferation rate. Other metabolites such as fatty acids differed widely among tumor cell lines. The response of tumor cell lines to chemotherapy also was evaluated by differential metabolite profiling, bringing insights into drug cytotoxicity and tumor cell adaptive mechanisms. The method may prove widely applicable to tumor cell phenotyping. Magn Reson Med 63:1172–1183, 2010. © 2010 Wiley‐Liss, Inc.     

Colorectal tumor cells treated with 5-FU, oxaliplatin, irinotecan, and cetuximab exhibit changes in 18F-FDG incorporation corresponding to hexokinase activity and glucose transport

The purpose of this study was to determine therapy-induced changes in 18F-FDG incorporation at the colorectal tumor cell level in response to conventional and novel chemotherapy agents and examine how these changes relate to factors involved in 18F-FDG incorporation. METHODS: SW620 cells were treated with inhibitory concentration of 50% (IC50) doses (determined by MTT) of 5-fluorouracil (5-FU), oxaliplatin, and irinotecan; HCT-8 cells were treated with IC50 doses of irinotecan, cetuximab, and irinotecan plus cetuximab. 18F-FDG incorporation, glucose transport, hexokinase (HK) activity, adenosine triphosphate (ATP) content, annexin V binding, and cell cycle distribution were determined after 24-, 48-, and 72-h treatments. Eight-hour treatments with and without subsequent incubation in drug-free medium were also examined. A clonogenic assay was used to determine the tumor-forming ability of treated cells. RESULTS: Apoptosis was evident in SW620 cells, especially after treatment with irinotecan and 5-FU. 18F-FDG incorporation was increased in SW620 cells after 24- or 48-h treatments with some agents and in HCT-8 cells after irinotecan treatment but was decreased in all 72-h treatments or cell-line combinations including cetuximab. Treatment of SW620 cells for 8 h followed by 64 h in drug-free medium also resulted in decreased 18F-FDG incorporation. Decreased 18F-FDG incorporation broadly corresponded to glucose transport in HCT-8 cells and to HK activity in SW620 cells. Inhibition of glucose transport decreased 18F-FDG incorporation into HCT-8 but not into SW620 cells. ATP levels were decreased by oxaliplatin treatment and increased at 48 or 72 h after irinotecan treatment. CONCLUSION: 18F-FDG incorporation is modulated by therapy-induced changes in both glucose transport and HK activity depending on the tumor cell. Colorectal cells treated with IC50 doses of cetuximab also exhibit decreased 18F-FDG.     

Steady-state blood volume measurements in experimental tumors with different angiogenic burdens a study in mice

PURPOSE: To experimentally validate the effectiveness of magnetic resonance (MR) imaging enhanced with long-circulating iron oxide for measurement of vascular volume fractions (VVFs) as indicators of angiogenesis in different experimental tumor models. MATERIALS AND METHODS: Tumors with differing degrees of angiogenesis-9L rodent gliosarcoma, DU4475 human mammary adenocarcinoma, HT1080 human fibrosarcoma, and EOMA hemangioendothelioma--were implanted in nude mice. Tumoral VVFs were measured at submillimeter voxel resolutions by using 1.5-T MR imaging. A technetium-labeled intravascular radiotracer was injected into a subset of the animals to validate the MR imaging measurements. Microvessel density and vascular endothelial growth factor (VEGF) also were measured. Statistical analysis was performed with analysis of variance. RESULTS: High-resolution multisection MR maps of tumor blood volume were obtained in all tumor models. Mean tumoral VVF differed significantly among the different tumors: 2.1% +/- 0.3 (standard error of mean) for 9L gliosarcoma, 3.1% +/- 0.4 for DU4475 mammary adenocarcinoma, 5.5% +/- 0.8 for HT1080 fibrosarcoma, and 6.6% +/- 0.9 for EOMA hemangioendothelioma (P <.01). There was a strong correlation between the MR imaging and radiotracer measurements. There was considerable intra- and intertumoral heterogeneity among the VVFs. MR imaging measurements were in accordance with conventional measurements of angiogenesis, such as microvessel density count and VEGF. CONCLUSION: Measurements of tumoral VVF at high-resolution MR imaging with long-circulating iron oxide are feasible and correlate with angiogenic burden in experimental tumor models.     

抗SARS冠状病毒药物细胞筛选模型的建立及应用

目的：建立抗SARS冠状病毒(SARS-CoV)药物细胞筛选模型，应用于抗SARS-CoV药物的筛选，为SARS的防治奠定基础。方法：将一定数量的细胞接种96孔板，根据药物的作用机制采用不同的给药途径，以观察到的细胞病变(cytopathic effect，CPE)为指标，按照：Reed-Muench法，计算药物的细胞半数中毒浓度(TD50)和抑制细胞半数病变的有效浓度(IC50)。结果与结论：Vero-E6细胞接种SARS-CoV后24h即可出现CPE，且CPE明显，便于观察，可作为抗SARS病毒药物细胞筛选模型。依此模型筛选了3类87种药物，确认6种药物在Veto-E6细胞上具有抑制SARS病毒的作用。该模型的建立也为其他的抗病毒药物的筛选提供了技术平台。     

EGFR抑制剂C225对人前列腺癌细胞的放射增敏作用

目的 探讨表皮生长因子抑制剂C225对人前列腺癌细胞株(DU145) 放射敏感性的影响。方法 实验组用表皮生长因子抑制剂C225(100 nmol/L)处理后,⁶⁰Co γ射线(吸收剂量率1.953 Gy/min)对体外培养的DU145细胞进行0、2、4、6 和8 Gy 照射,用MTT法、集落形成法检测细胞增殖和细胞存活率;用单击多靶模型拟合剂量存活曲线;用流式细胞仪检测细胞周期和凋亡。结果 C225对照射后DU145细胞增殖有明显抑制作用。C225组的放射生物学参数值(D₀、D_q、N、SF₂)较对照组低。C225组和对照组的RBE 比值为1.39。C225组处于S期的细胞比例降低,出现明显的G₀/G₁期阻滞。C225组细胞的早期凋亡率在照射剂量达4 Gy后明显高于对照组(t=-14.55,P＜0.05)。结论 表皮生长因子抑制剂C225通过抑制细胞增殖、G₀/G₁ 期阻滞和诱导凋亡来增加人前列腺癌细胞放射敏感性。     

双氢青蒿素联合吉非替尼抑制肺腺癌细胞周期和迁移能力的体外研究

 目的 探究双氢青蒿素(dihydroartemisinin, DHA)联合吉非替尼(gefitinib, GEF)对肺腺癌细胞系的细胞周期和迁移能力的影响。方法 10 μM DHA和(或)50 μM GEF处理肺腺癌细胞系A549和SPC-A-1,四唑盐(methyl thiazolyl tetrazolim, MTT)比色法检测细胞增殖,流式细胞术检测细胞周期,划痕实验检测细胞迁移,蛋白质印记法(western blot, WB)检测单独或联合应用DHA和GEF对细胞周期和迁移相关蛋白表达的影响。结果 MTT实验结果显示联合应用DHA和GEF(Com组)后,A549和SPC-A-1的增殖能力显著低于单药组和对照(NC)组,且具有时间依赖性(P＜0.05);碘化丙啶(propidium iodide, PI)单染流式细胞术检测细胞阻滞于G₀/G₁期;WB显示与NC组和单药组相比,Com组细胞周期蛋白依赖性蛋白激酶4(cyclin dependent kinase 4,CDK4)和细胞周期素D1(Cyclin D1)表达显著下降;划痕实验中Com组细胞融合率显著低于NC组和单药组,并伴随基质金属蛋白酶(matrix metalloproteinase, MMP)2和MMP9表达下降,差异均具有统计学意义(P＜0.05)。结论 DHA联合GEF可以显著抑制肺腺癌细胞的增殖和迁移,并阻滞细胞周期。     

Indomethacin lowers the threshold thermal exposure for hyperthermic radiosensitization and heat-shock inhibition of ionizing radiation-induced activation of NF-κB

Purpose : It is well established that salicylate and several other non-steroidal anti-inflammatory agents (NSAID), including indomethacin, can activate the heat-shock response, albeit at high concentrations. This is significant since heat shock significantly alters the cellular cytotoxic response to ionizing radiation (IR). It was previously shown that heat shock, as well as NSAIDs, inhibits IR-induced activation of NF- κB and that NF- κB protects against IR-induced cytotoxicity. Hence, it is hypothesized that pretreatment with indomethacin before heating will lower the temperature and heating times required to inhibit the activation of NF- κB and induce significant hyperthermic radiosensitization. Materials and methods : Experiments were performed in HeLa cell lines and the DNA-binding activity was determined by EMSA. Cellular radiosensitivity was determined by clonogenic assay. Results : HeLa cells pretreated with indomethacin showed a decrease in the temperature-time combination necessary to inhibit IR-induction of NF- κB DNA binding. In addition, clonogenic cell survival assays using identical conditions showed an indomethacin dose-dependent enhancement of hyperthermic radiosensitization. Thus, similar concentrations of indomethacin both lowered the threshold thermal exposure to inhibit activation of NF- κB DNA-binding and increased the sensitivity of tumour cells to hyperthermic radiosensitization-induced cytotoxicity. In HeLa cells treated with N - α -tosylphenylalanyl-chloromethyl ketone (TPCK), a serine protease inhibitor that blocks activation of NF- κB, an increase in radiosensitivity was observed. Interestingly, no additional cell killing was observed when heat shock was added to cells treated with TPCK before IR, suggesting a possible common cytotoxic pathway. Conclusions : The results demonstrate that indomethacin lowers the temperature-time conbination necessary to induce several physiological processes associated with the heat-shock response. Furthermore, NSAID may be potential adjuvants in improving the clinical effectiveness of hyperthermia in radiation therapy.