5-Fluorouracil modulation of radiosensitivity in cultured human carcinoma cells

We evaluated conventional pulse exposure versus continuous exposure models of 5-fluorouracil (5-FU) radiosensitization in HT-29 (human colon adenocarcinoma) and DU-145 (human prostate cancer adenocarcinoma) cell lines. Cell survival following treatment with drug and/or radiation was determined by colony formation assays. Radiation was delivered either by itself, approximately midway through a 1-hr exposure to 5-FU (10 micrograms/ml), or at various times following initiation of exposure to 5-FU (0.5 microgram/ml) present throughout the entire period of incubation. Drug concentrations were selected to approximate those achieved in vivo in humans. HT-29 cells showed a plating efficiency of 87% and similar cytotoxicity (survival reduced to 0.57-0.71) for all 5-FU conditions. The Do's of the radiation survival curves were not different for 1 hr of 5-FU exposure versus radiation alone. However, continuous exposure conditions demonstrated statistically significantly different Do's from radiation alone and pulse 5-FU exposure. DU-145 cells displayed a plating efficiency of 17% and cytotoxicities of 0.10-0.91 for the 5-FU conditions. DU-145 cells showed different radiation 5-FU interactions: 5-FU produced statistically significant changes in Do well as the differences between cell lines insofar as their radiosensitization by 5-FU underscore the caution required in extrapolating these radiobiologic models to the clinical setting.     

Differential activation of ERKs to focal adhesions by PKC epsilon is required for PMA-induced adhesion and migration of human glioma cells.

Protein kinase C (PKC) is a family of serine/threonine kinases involved in the transduction of a variety of signals. There is increasing evidence to indicate that specific PKC isoforms are involved in the regulation of distinct cellular processes. In glioma cells, PKC alpha was found to be a critical regulator of proliferation and cell cycle progression, while PKC epsilon was found to regulate adhesion and migration. Herein, we report that specific PKC isoforms are able to differentially activate extracellular-signal regulated kinase (ERK) in distinct cellular locations: while PKC alpha induces the activation of nuclear ERK, PKC epsilon induces the activation of ERK at focal adhesions. Inhibition of the ERK pathway completely abolished the PKC-induced integrin-mediated adhesion and migration. Thus, we present the first evidence that PKC epsilon is able to activate ERK at focal adhesions to mediate glioma cell adhesion and motility, providing a molecular mechanism to explain the different biological functions of PKC alpha and epsilon in glioma cells.     

The tumor invasion inhibitor dihydromotuporamine C activates RHO, remodels stress fibers and focal adhesions, and stimulates sodium-proton exchange

The motuporamines are macrocyclic alkaloids that inhibit tumor cell invasion by an, as yet, unknown mechanism. A structure-activity study recently identified dihydromotuporamine C (dhMotC) as a highly active and readily synthesized analogue. Here, we show that dhMotC causes subtle cytoskeletal alterations in highly invasive MDA231 breast tumor cells that include an increase in the thickness and number of cytoplasmic actin stress fibers. Experiments with serum-starved Swiss 3T3 fibroblasts showed that micromolar concentrations of dhMotC that inhibit tumor cell invasion induce the formation of new stress fibers and large focal adhesion complexes that are dispersed around the entire cell periphery. dhMotC treatment of Swiss 3T3 cells also initiates a strong, long-lived activation of the small GTP-binding protein Rho, and it stimulates Rho kinase-dependent sodium-proton exchanger activity. Liposome-mediated cell loading of C3 exoenzyme prevents dhMotC-mediated Rho activation and stress fiber formation in 3T3 cells. C3 exoenzyme loading also reestablishes elongated MDA231 breast tumor cell invasion in the presence of dhMotC. Taken together, these results indicate that the ability to activate Rho is one important determinant of the anti-invasive activity of dhMotC.     

Rapid loss of stress fibers in Chinese hamster ovary cells after hyperthermia

This study was initiated to characterize the effect of hyperthermia (45 degrees) on the distribution of actin stress fibers in Chinese hamster ovary cells using rhodamine-conjugated phalloidin, a probe specific for F-actin. Fluorescent microscopy revealed a rapid loss of stress fibers after immersion in a 45 degrees water bath. After 5-min immersion at 45 degrees, approximately 90% of the cells analyzed did not contain observable stress fibers. Stress fibers were visible after incubation of cells at 37 degrees after heating. The recovery of the appearance of the stress fibers occurred as protein synthesis resumed, and addition of protein synthesis inhibitors following heat treatment blocked the reappearance of these structures. These results support the hypothesis that cytoskeletal components may be a target of hyperthermia, explaining the pleotropic biological effects of heat and, in particular, heat radiosensitization.     

葡萄糖诱导的体外培养人脐静脉内皮细胞膜中葡糖基磷脂酰肌醇锚式补体活化膜调节物(CD59,CD55)的下调

研究表明糖尿病血管病变发生的主要原因是内皮细胞功能异常,高血糖又是加速内皮功能紊乱的重要原因之一。糖尿病患者中还可发现补体系统活性增加,导致糖尿病患者肾脏血管壁、肾小球及系膜区补体分子沉积;血浆中可溶性非细胞裂解的C5b-9的浓度及代表内皮功能紊乱的von Willebrand因子浓度增加。补体介导的自体细胞损伤有以下几种调节因子:衰败加速因子(DAF、CD55)、膜共因子蛋白(MCP、CD46)、C3b受体、唾液酸和膜溶解反应抑制物(MIRL、CD59)等。     

Biallelic loss of FAM46C triggers tumor growth with concomitant activation of Akt signaling in multiple myeloma cells

Loss of heterozygosity or mutation of the family with sequence similarity 46, member C (FAM46C) gene on chromosome band 1p12 is associated with shorter overall survival of patients with multiple myeloma (MM). In this study, using human MM cell lines (KMS‐11, OCI‐My5, and ANBL‐6), we generated FAM46C^?/? cell clones and examined the effect of disruption of FAM46C on cell survival and cellular signaling. Cell proliferation assays showed increased clonogenicity of FAM46C^?/? KMS‐11 cells compared to WT cells. Xenograft experiments showed significantly shorter overall survival of mice harboring the FAM46C^?/? cell‐derived tumors than mice with the FAM46C^WT cell‐derived tumors. Notably, levels of phosphorylated Akt and its substrates increased both in vitro and in vivo in the FAM46C^?/? cells compared to WT cells. In addition, caspase activities decreased in the FAM46C^?/? cells. Results of gene set enrichment analysis showed that loss of FAM46C significantly activated serum‐responsive genes while inactivating phosphatase and tensin homolog (PTEN)‐related genes. Mechanistically, loss of FAM46C decreased the PTEN activity, number of apoptotic cells, and caspase activities. PF‐04691502, a selective PI3K inhibitor, suppressed the augmented phosphorylation of Akt and its substrate FoxO3a. Treatment with afuresertib (a specific Akt inhibitor) in combination with bortezomib additively decreased FAM46C^?/? MM cell survival. Collectively, this study is the first to report that loss of FAM46C triggers the concomitant activation of the PI3K‐Akt signaling pathway, which might be a therapeutic target for MM with abnormalities in the FAM46C gene.     

Photofrin and light induces microtubule depolymerization in cultured human endothelial cells.

Endothelial cells were cultured from human umbilical veins and incubated with Photofrin (1 microgram/ml). Cells were then exposed to light, and cytoplasmic microtubule (MT) status was monitored by immunofluorescence microscopy using alpha-tubulin antibody. As early as 15 min following irradiation, a light dose-dependent depolymerization of MT was observed. At sublethal light doses, this effect was transient, with MT repolymerizing within 2-3 h. Cellular ATP levels were monitored to determine whether diminished ATP levels were correlated with MT depolymerization. No correlation was found, since ATP levels remained at a constant value near 50% of unirradiated controls during a time interval in which transient MT depolymerization was observed. Cell viability was monitored by trypan blue exclusion. Transient MT depolymerization occurred at photodynamic doses that produced essentially no decrease in cell viability, while at higher doses, irreversible MT depolymerization was observed prior to loss of viability. Since MT are unstable at intracellular calcium levels greater than 1 microM, we postulate that MT depolymerization results from increases in intracellular calcium caused by photodynamic insult. MT are important in maintaining cell shape. Disruption of MT in endothelial cells due to photodynamic therapy could result in or contribute to exposure of the thrombogenic subendothelium or could alter vascular permeability in the treatment area.     

Interaction between human cancer cells and cultured murine endothelial cells, and its relationship with metastatic potential

The hematogenous metastasis of cancer consists of a multistep process. It is surmised that a number of interactions between cancer and endothelial cells occur, with cell adhesion molecules playing certain roles in this process. The authors conducted an investigation on the interaction between human cancer cells and cultured murine endothelial cells (F-2 cells) in vitro, and on its relationship with the metastatic activity of cancer cells in vivo. A correlation was found between the degree of expression of carbohydrate antigens on the cell surface and adhesion of cancer cells to F-2 cells. Five of 13 examined cell lines showed liver metastasis after inoculation to the spleen of nude mice. These cell lines showed not only a strong binding activity to F-2 cells but implantation in F-2 cells in vitro was also observed. These findings suggest that adhesion to, and implantation in endothelial cells are necessary for the induction of distant metastasis. Treatment with antibodies against carbohydrate antigens inhibited the formation of liver metastasis in nude mice. It is possible that strategies to interfere with the function of cell adhesion molecules may be formulated to result in the decreased distant metastasis of cancer.     

Celecoxib induces anoikis in human colon carcinoma cells associated with the deregulation of focal adhesions and nuclear translocation of p130Cas

Celecoxib, a selective cyclooxygenase-2 (COX-2) inhibitor, is effective as chemopreventive against colon cancer and it is the only nonsteoroidal antiinflammatory drug approved by the FDA for adjuvant therapy in patients with familial adenomatous polyposis. It is also being evaluated, within Phase II and III clinical trials, in combination with standard chemotherapy to treat sporadic colorectal cancer. Nevertheless, its antitumor mechanism of action is still not fully understood. In this study, we have evaluated the in vitro growth inhibitory effect of celecoxib in colon carcinoma cells and analyzed its mechanism of action. We report that the deregulation of the focal adhesion assembly protein Crk-associated substrate 130 kDa (p130Cas) by celecoxib plays a relevant role in the cytotoxic effect of this drug. Thus, celecoxib induces the proteolysis of p130Cas and the nuclear translocation of the 31 kDa generated fragment leading to apoptosis. Furthermore, overexpression of wild-type p130Cas reverts, in part, the growth inhibitory effect of celecoxib. In contrast, FAK and AKT do not appear to be involved in this activity. Our data suggest, for the first time, that the antitumor mechanism of action of celecoxib includes the induction of anoikis, an effect that is not related to COX-2 inhibition. Besides providing new insights into the antitumor effect of celecoxib, this novel mechanism of action holds potential relevance in drug development. Indeed, our results open the possibility to develop new celecoxib derivatives that induce anoikis without COX-2 inhibition so as to avoid the cardiovascular toxicity recently described for the COX-2 inhibitors.     

NF-kappaB dependent activation of human endothelial cells treated with soluble products derived from human lymphomas

Nuclear factor of the Immunoglobulin Kappa chain of B cells (NF-kappaB) activation is an early event during cytokine-mediated endothelial activation related to increased adhesion of leucocytes. We report that soluble products secreted by two human lymphomas activate NF-kappaB, and increase the ability of endothelial cells to adhere U937 cells in vitro. Analysis of the tumor-derived products revealed the absence of tumor necrosis factor-alpha and interleukin-1beta. Interference of NF-kappaB activation prevented the increase in U937 cell adhesion, suggesting a potential role for endothelial NF-kappaB activation in the establishment of physical interactions between the vascular endothelium and tumor cells.     

Photosensitized release of von Willebrand factor from cultured human endothelial cells.

Cultured endothelial cells from the human umbilical vein were incubated with low concentrations (1 microgram/ml) of the photosensitizer Photofrin II. Following a sublethal light exposure, a light dose-dependent release of von Willebrand factor (vWf) into the culture medium was observed. Analysis of the multimeric composition of the released protein indicated that it originated from the intracellular pool of large vWf multimers stored in the Weibel-Palade bodies. This release was detected as early as 1 h postirradiation. Release was inhibited at low temperature and was dependent upon the presence of extracellular calcium. Photosensitization resulted in an influx of calcium whose time course paralleled vWf release from the cells. Since vWf mediates platelet adhesion to the vascular subendothelium, it is possible that its photochemically stimulated release in vivo could contribute to platelet thrombus formation observed in tissue following photodynamic therapy.     

The topology of plasminogen binding and activation on the surface of human breast cancer cells

The urokinase-dependent activation of plasminogen by breast cancer cells plays an important role in metastasis. We have previously shown that the metastatic breast cancer cell line MDA-MB-231 over-expresses urokinase and binds and efficiently activates plasminogen at the cell surface compared to non-metastatic cells. The aim of this study was to further characterise plasminogen binding and determine the topology of cell surface-bound plasminogen in terms of its potential for activation. The lysine-dependent binding of plasminogen at 4 degrees C to MDA-MB-231 cells was stable and resulted in an activation-susceptible conformation of plasminogen. Topologically, a fraction of bound plasminogen was co-localised with urokinase on the surfaces of MDA-MB-231 cells where it could be activated to plasmin. At 37 degrees C plasmin was rapidly lost from the cell surface. Apart from actin, other candidate plasminogen receptors were either not expressed or did not co-localise with plasminogen at the cell surface. Thus, based on co-localisation with urokinase, plasminogen binding is partitioned into two functional pools on the surface of MDA-MB-231 cells. In conclusion, these results shed further light on the functional organisation of the plasminogen activation cascade on the surface of a metastatic cancer cell.     

Intracellular activation and cytotoxic action of RS-1541 against cultured human tumor cells

RS-1541, an acyl-derivative of rhizoxin (Fig. 1), is a potent antitumor compound. This agent showed cytotoxicity in vitro on some cultured human tumor cells, although it was less potent than rhizoxin. Rhizoxin exhibited antitumor effects by inhibiting the polymerization of tubulin, whereas RS-1541 did not inhibit tubulin polymerization in vitro. However, cell cycle analysis in vivo showed that the two agents had the same mode of action. The cytotoxicity of RS-1541 was enhanced when the initial cell density of the cells was increased. The cytotoxicity was also enhanced when the membrane fraction of St-4 cells, which were the most sensitive to RS-1541 among the cell lines tested, was added to the target cells. When St-4 cells were incubated with [¹⁴C]-RS-1541, significant amounts of [¹⁴C]-rhizoxin were produced within the cells. Further fractionation of the crude membrane showed that the activity that enhanced the cytotoxicity of RS-1541 (RS-1541-enhancing activity) belonged to the mitochondrial-lysosomal fraction, not to the microsomal fraction. Both the enhancing activity and the activity that converting [¹⁴C]-RS-1541 to [¹⁴C]-rhizoxin (RS-1541-converting activity) were inhibited by treatment with chloroquine, an inhibitor of lysosomal function. Cholesterol esterase derived fromCandida cylindracea had RS-1541-enhancingand-converting activities. These data suggest that RS-1541 exerts its cytotoxic action after being converted to rhizoxin within the cells by a lysosomal enzyme such as cholesterol esterase.Abbreviations DMSO Dimethylsulfoxide - PBS(-) Ca²⁺ Mg²⁺-free phosphate-buffered saline - HCO60 hydrogenated castor oil polyethylene glycor ether - DMA dimethylacetamide - RSB reticulocyte standard buffer, consisting of 10mM NaCl, 1.5 mM MgCl₂, and 10 mM TRIS-HCl, (pH 7.4) - TLC thin-layer chromatography - ara-C 1--D-arabinofuranosylcytosine - LDL low-density lipoprotein     

Protection against radiation-induced oxidative stress in cultured human epithelial cells by treatment with antioxidant agents

PURPOSE: To evaluate the protective effects of antioxidant agents against space radiation-induced oxidative stress in cultured human epithelial cells. METHODS AND MATERIALS: The effects of selected concentrations of N-acetylcysteine, ascorbic acid, sodium ascorbate, co-enzyme Q10, alpha-lipoic acid, l-selenomethionine, and vitamin E succinate on radiation-induced oxidative stress were evaluated in MCF10 human breast epithelial cells exposed to radiation with X-rays, gamma-rays, protons, or high mass, high atomic number, and high energy particles using a dichlorofluorescein assay. RESULTS: The results demonstrated that these antioxidants are effective in protecting against radiation-induced oxidative stress and complete or nearly complete protection was achieved by treating the cells with a combination of these agents before and during the radiation exposure. CONCLUSION: The combination of antioxidants evaluated in this study is likely be a promising countermeasure for protection against space radiation-induced adverse biologic effects.     

Mechanisms of platelet activation by cultured human cancer cells and cells freshly isolated from tumor tissues

We studied the effects on platelet function of cells isolated from freshly dissociated human tumor tissues (11 breast carcinomas, 9 colon carcinomas and 1 lymph node metastasis from melanoma) obtained at surgery as compared with cultured human tumor cells: namely, human melanoma 1402 cell line derived from a primary tumor and two lines derived from lymph node metastases (ME 7110/2 and Me 665/1) as well as a human hepatoma cell line (Hep G2). The three melanoma cell lines activated platelets by producing ADP, as evidenced by the inhibitory effect of apyrase and by the direct measurement of the agonist in the supernatants of tumor cell suspensions; this production was much greater by the cells derived from metastases than by the cells derived from the primary tumor. On the other hand, aggregation induced by Hep G2 hepatoma cells was unaffected by apyrase and was inhibited by hirudin or concanavalin A, suggesting that the cells aggregate platelets by producing thrombin, probably through tissue factor activity of the cells themselves. Cells isolated from 16 of the 21 human tumor tissues possessed a potent platelet-aggregating effect, which was not inhibited by apyrase, hirudin or concanavalin A, but was virtually abolished by the cysteine protease inhibitors iodoacetic acid or p-hydroxymercuri-phenylsulfonate. Collectively, our data demonstrate that cells isolated from freshly dissociated tumor tissues activate platelets through tumor-associated cysteine proteinases rather than by the ADP- or thrombin-dependent mechanisms characteristic of cultured human tumor cell lines.     

Synaptopodin-2 induces assembly of peripheral actin bundles and immature focal adhesions to promote lamellipodia formation and prostate cancer cell migration

Synaptopodin-2 (Synpo2), an actin-binding protein and invasive cancer biomarker, induces formation of complex stress fiber networks in the cell body and promotes PC3 prostate cancer cell migration in response to serum stimulation. The role of these actin networks in enhanced cancer cell migration is unknown. Using time-course analysis and live cell imaging of mock- and Synpo2-transduced PC3 cells, we now show that Synpo2 induces assembly of actin fibers near the cell periphery and Arp2/3-dependent lamellipodia formation. Lamellipodia formed in a non-directional manner or repeatedly changed direction, explaining the enhanced chemokinetic activity of PC3 cells in response to serum stimulation. Myosin contraction promotes retrograde flow of the Synpo2-associated actin filaments at the leading edge and their merger with actin networks in the cell body. Enhanced PC3 cell migration correlates with Synpo2-induced formation of lamellipodia and immature focal adhesions (FAs), but is not dependent on myosin contraction or FA maturation. The previously reported correlation between Synpo2-induced stress fiber assembly and enhanced PC3 cell migration therefore reflects the role of Synpo2 as a newly identified regulator of actin bundle formation and nascent FA assembly near the leading cell edge.     

Downregulation of urokinase plasminogen activator receptor expression inhibits Erk signalling with concomitant suppression of invasiveness due to loss of uPAR-beta1 integrin complex in colon cancer cells

Cancer invasion is regulated by cell surface proteinases and adhesion molecules. Interaction between specific cell surface molecules such as urokinase plasminogen activator receptor (uPAR) and integrins is crucial for tumour invasion and metastasis. In this study, we examined whether uPAR and beta1 integrin form a functional complex to mediate signalling required for tumour invasion. We assessed the expression of uPAR/beta1 integrin complex, Erk signalling pathway, adhesion, uPA and matrix metalloproteinase (MMP) expression, migration/invasion and matrix degradation in a colon cancer cell line in which uPAR expression was modified. Antisense inhibition of the cell surface expression of uPAR by 50% in human colon carcinoma HCT116 cells (A/S) suppressed Erk-MAP kinase activity by two-fold. Urokinase plasminogen activator receptor antisense treatment of HCT116 cells was associated with a 1.3-fold inhibition of adhesion, approximately four-fold suppression of HMW-uPA secretion and inhibition of pro-MMP-9 secretion. At a functional level, uPAR antisense resulted in a four-fold decline in migration/invasion and abatement of plasmin-mediated matrix degradation. In empty vector-transfected cells (mock), uPA strongly elevated basal Erk activation. In contrast, in A/S cells, uPA induction of Erk activation was not observed. Urokinase plasminogen activator receptor associated with beta1 integrin in mock-transfected cells. Disruption of uPAR-beta1 integrin complex in mock-transfected cells with a specific peptide (P25) inhibited uPA-mediated Erk-MAP kinase pathway and inhibited migration/invasion and plasmin-dependent matrix degradation through suppression of pro-MMP-9/MMP-2 expression. This novel paradigm of uPAR-integrin signalling may afford opportunities for alternative therapeutic strategies for the treatment of cancer.