CFL1 expression levels as a prognostic and drug resistance marker in nonsmall cell lung cancer

BACKGROUND:

Nonsmall cell lung cancer (NSCLC) is the major determinant of overall cancer mortality worldwide. Despite progress in molecular research, current treatments offer limited benefits. Because NSCLC generates early metastasis, and this behavior requires great cell motility, herein the authors assessed the potential value of CFL1 gene (main member of the invasion/metastasis pathway) as a prognostic and predictive NSCLC biomarker.

METHODS:

Metadata analysis of tumor tissue microarray was applied to examine expression of CFL1 in archival lung cancer samples from 111 patients, and its clinicopathologic significance was investigated. The robustness of the finding was validated using another independent data set. Finally, the authors assayed in vitro the role of CFL1 levels in tumor invasiveness and drug resistance using 6 human NSCLC cell lines with different basal degrees of CFL1 gene expression.

RESULTS:

CFL1 levels in biopsies discriminate between good and bad prognosis at early tumor stages (IA, IB, and IIA/B), where high CFL1 levels are correlated with lower overall survival rate (P < .0001). Biomarker performance was further analyzed by immunohistochemistry, hazard ratio (P < .001), and receiver‐operating characteristic curve (area = 0.787; P < .001). High CFL1 mRNA levels and protein content are positively correlated with cellular invasiveness (determined by Matrigel Invasion Chamber System) and resistance (2‐fold increase in drug 50% growth inhibition dose) against a list of 22 alkylating agents. Hierarchical clustering analysis of the CFL1 gene network had the same robustness for stratified NSCLC patients.

CONCLUSIONS:

This study indicates that the CFL1 gene and its functional gene network can be used as prognostic biomarkers for NSCLC and could also guide chemotherapeutic interventions. Cancer 2010. © 2010 American Cancer Society.     

Inhibition of the myeloperoxidase-H2O2-Cl- system of neutrophils by indomethacin and other non-steroidal anti-inflammatory drugs.

The results presented herein demonstrate that the non-steroidal anti-inflammatory drug (NSAID) indomethacin is a strong inhibitor of the formation of HOCl by murine neutrophils (50% inhibition at 15 microM). Addition of 40 microM indomethacin to activated neutrophils caused 80% inhibition of HOCl formation throughout a 60-min time course while slightly increasing the levels of O2- and H2O2 produced. Comparable degrees of inhibition were achieved when the cells were stimulated with phorbol myristate acetate and with opsonized zymosan. Control experiments indicated that the drug did not act by scavenging HOCl. Direct inhibition of the chlorinating activity of myeloperoxidase (MPO) was confirmed using highly purified human enzyme in vitro. Kinetic analysis of the mechanism of inhibition showed that the drug was competitive with respect to Cl- and uncompetitive with respect to H2O2, showing a Ki of 37 microM. In contrast to its inhibition of the oxidation of Cl- by MPO, indomethacin had no effect on the peroxidative activity of the enzyme (oxidation of 4-aminoantipyrene), nor did it inhibit the activity of several other enzymes involved in H2O2 metabolism, including horseradish peroxidase, catalase, xanthine oxidase, and superoxide dismutase. Finally, it was found that inhibition of HOCl formation was a shared but non-uniform property of many NSAIDs; piroxicam, salicylate, sulindac, ibuprofen, and aspirin were all inhibitory but at widely different concentrations [Ki(app) values of 0.05, 0.18, 0.18, greater than 1, and 3 mM respectively] that correlated only partially with their therapeutic dose range. The results encourage further studies into the possibility that inhibition of HOCl formation may constitute an additional mechanism whereby NSAIDs reduce tissue destruction in chronically inflamed tissues.     

Activated neutrophils induce prolonged DNA damage in neighboring cells

We have measured the capacity of highly-purified, paraffin oil-elicitedneutrophils to induce DNA single-strand breaks in a newly establishedplasmacytoma cell line, RIMPC 2304, which was induced by a retroviruscontaining the c-myc and V-Ha-ras oncogenes. This cell lineeffectively repairs DNA damage induced by -irradiation. DNAdamage induced by neutrophils was correlated with the oxidativeburst of the neutrophils. The levels of superoxide anion, H₂O₂and HOCl produced after stimulation of the neutrophils (6 x10⁵/cm³) with the tumor promoter phorbol myristate acetate (100nM) were 33.8 µM, 12.8µM and 1.7 µM respectivelyin 15 mm, and 98 µM, 20 µM and 8.7 µM respectivelyin 90 mm.The results of alkaline elution experiments revealedthat when the same concentration of neutrophils was co-incubatedfor15 min in serum-free medium with an equal number of radioactivelylabeled RIMPC 2304 cells, the latter incurred a level of damagethat approximated that caused by 300 rad equivalents of -irradiationor by a 1-min treatment with 20 µM H₂O₂ at 37C. Damagefrom neutrophils was coincident with the oxidative burst; itwas induced rapidly (within 5 min) but remained high for morethan 90 min. The level of damage achieved was dependent uponthe ratio of neutrophils: target cells and was clearly detectableat ratios as low as 0.25:1. Induction of single-strand breakswas completely inhibited by catalase and partially inhibitedby superoxide dismutase, mannitol, and reduced glutathione butnot by Na azide. Addition of the non-steroidal anti-inflammatorydrug indomethacin either enhanced (at 50 µM) or had noeffect (at 2 µM) on the damage detected. Finally, repairof strand breaks induced by neutrophils was significantly slower(half time 10 min) than that observed for repair of similarlevels of damage induced by H₂O₂ or -irradlatlon (half-times3 min, each). The results indicate that neutrophils cause prolongedDNA damage in neighboring cells. Moreover, they indicate thatalthough H₂O₂ produced in the oxidative burst is an essentialmediator of the damage observed, additional reactive oxygenintermediates including the superoxide anion are also implicated.The data are discussed in relation to the possible role of neutrophilsin chronic inflammation and in pristane-induced plasmacytomaformation in mice.     

The FDA's assessment of follow-on protein products: a historical perspective

The scientific and regulatory issues that are associated with the possible introduction of 'follow-on' versions of protein drug products are the topic of considerable debate at present. Because of the differences between protein drug products and small-molecule drugs, the development of follow-on versions of protein products presents more complex scientific challenges than those presented by the development of generic versions of small-molecule drugs. Here, with a view to illustrating the Food and Drug Administration's (FDA's) scientific reasoning and experience in this area, we discuss past examples of the FDA's actions involving the evaluation of various types of follow-on and second-generation protein products and within-product manufacturing changes. The FDA believes its evaluation of the safety and effectiveness of follow-on protein products will evolve as scientific and technological advances in product characterization and manufacturing continue to reduce some of the complexity and uncertainty that are inherent in the manufacturing of protein products.     

Stimulation of interleukin-6 and prostaglandin E2 secretion from peritoneal macrophages by polymers of albumin

Interleukin-6 (IL-6) is a multifunctional cytokine that is elevated in vivo during acute infection, chronic inflammation, and some hematopoietic malignancies. To understand how IL-6 becomes elevated in vivo, it is important to identify factors that can stimulate its secretion from effector cells. We found that commercial preparations of bovine serum albumin (BSA) stimulated murine macrophages to secrete high levels of IL-6. In fact, BSA was at least as potent as bacterial lipopolysaccharide (LPS) in stimulating IL-6 production. Stimulation was clearly visible at concentrations as low as 20 micrograms/mL and reached saturation at 0.5 to 1 mg/mL albumin, at which concentration 1.1 x 10(6) oil-elicited macrophages produced 6,000 +/- 700 B9 units of IL-6 in an overnight incubation. Prostaglandin E2 production was induced by the same concentrations of BSA. Both resident and oil- elicited peritoneal cells were responsive to the albumin. The stimulatory activity did not derive from contamination of the protein with Escherichia coli LPS; when compared directly with LPS, the response to BSA was more rapid, had a higher amplitude, and was not inhibitable by polymyxin B. In addition, macrophages isolated from C3H/HeJ mice, which have an inherited defect in their ability to respond to LPS, secreted IL-6 in response to BSA but not to LPS. The stimulatory activity was stable to heat, mild acid, and reduction/alkylation and copurified with albumin on Cibachron Blue agarose (Sigma, St Louis, MO) and anti-albumin immunoaffinity chromatography. Comparison of different sources and preparations of albumin showed differences in the levels of IL-6-inducing activity; three different lots of commercial fatty acid-free BSA and one lot of polymer-enhanced BSA stimulated IL-6 secretion by more than 100-fold over basal levels whereas other preparations showed more limited activity. A sample of BSA that was active in vitro caused a marked elevation of IL-6 when injected into BALB/c mice, thus demonstrating inflammatory activity in vivo. When the albumin preparations were fractionated by ion exchange and gel filtration chromatography and then analyzed by sodium dodecyl sulfate-gel electrophoresis and Western blot immunoassay, it was found that the IL-6-inducing activity resided in high molecular weight polymers of albumin. The ability of albumin polymers to stimulate IL-6 production represents a novel mechanism for modulation of this cytokine.     

Quantification and significance of protein oxidation in biological samples

Protein oxidation is defined here as the covalent modification of a protein induced either directly by reactive oxygen species or indirectly by reaction with secondary by-products of oxidative stress. Oxidative modification of proteins can be induced experimentally by a wide array of prooxidant agents and occurs in vivo during aging and in certain disease conditions. Oxidative changes to proteins can lead to diverse functional consequences, such as inhibition of enzymatic and binding activities, increased susceptibility to aggregation and proteolysis, increased or decreased uptake by cells, and altered immunogenicity. There are numerous types of protein oxidative modification and these can be measured with a variety of methods. Protein oxidation serves as a useful marker for assessing oxidative stress in vivo. There are both advantages and disadvantages to using proteins for this purpose compared to lipids and DNA. Finally, it is important to monitor the degree of oxidative modification of therapeutic proteins manufactured for commercial use. This review will examine various aspects of protein oxidation, with emphasis on using proteins as markers of oxidative stress in biological samples.     

Elevation of interleukin-6 in response to a chronic inflammatory stimulus in mice: inhibition by indomethacin.

Intraperitoneal (i.p.) injection of a mineral oil such as pristane induces a chronic inflammatory response in mice. This is characterized by a large influx of macrophages and other inflammatory cells into the peritoneal cavity for months after injection of the oil. By using the B9 cell bioassay, it was found that injection of pristane caused a marked and prolonged elevation of interleukin-6 (IL-6) levels in the peritoneal cavities of the mice. IL-6 was undetectable (less than 15 U/mL) in the peritoneal fluids of unprimed mice and during the first week after injecting pristane. From 4 to 20 weeks, the concentration of IL-6 increased to an apparent plateau with concentrations ranging from 200 to 2,000 U/mL. Increasing the dose of pristane did not substantially increase the peritoneal levels of IL-6 established at 20 weeks after pristane treatment. At later times (by day 250), the level decreased to 263 +/- 217 U/mL. However, mice that developed plasma cell tumors around day 300 showed high levels of IL-6 in the ascites fluid (650 to 2,400 U/mL). Serum levels of IL-6 were also elevated in pristane-primed mice but were substantially lower than those found in the peritoneal cavity. Chronic administration of the nonsteroidal anti-inflammatory drug indomethacin decreased the levels of IL-6 by 75% to 80%. Experiments performed in vitro showed that pristane-elicited macrophages secreted low levels of IL-6 constitutively and high levels of IL-6 in the presence of lipopolysaccharide. Both IL-6 and prostaglandin E2 production were inhibited by addition of indomethacin to macrophage cultures in vitro. Treatment of mice with pristane may provide a model system for studying the inflammatory pathways that control IL-6 levels in vivo. The relevance of these results to elucidation of the role of IL-6 in plasma cell tumorigenesis is discussed.     

Oxidative stress interferes with cancer chemotherapy: inhibition of lymphoma cell apoptosis and phagocytosis

Many antineoplastic drugs kill tumor cells by inducing apoptosis. This highly controlled mechanism of cell death is thought to be physiologically advantageous because apoptotic cells are removed by phagocytosis before they lose their permeability barrier, thus preventing induction of an inflammatory response to the dying cells. In contrast, necrotic cells lyse and release their contents into the extracellular space, thus inducing inflammation. In this report, we examine the effects of oxidative stress on chemotherapy-induced cell killing. We find that H(2)O(2) inhibits the ability of 4 different chemotherapy drugs (VP-16, doxorubicin, cisplatin, and AraC) to induce apoptosis in human Burkitt lymphoma cells. H(2)O(2) shifts the form of cell death from apoptosis to pyknosis/necrosis, which occurs after a significant delay compared with chemotherapy-induced apoptosis. It can also lower the degree of cell killing by these drugs. These effects of H(2)O(2) can be prevented by the antioxidant agents Desferal, Tempol, and dimethylsulfoxide. Phagocytosis by monocyte-derived macrophages of VP-16-treated lymphoma cells is also inhibited by H(2)O(2). Cells killed with H(2)O(2) (with or without VP-16) do ultimately undergo phagocytosis, but this occurs only after they have lost their permeability barrier. Thus, membrane-intact apoptotic cells are recognized and phagocytosed by monocyte-derived macrophages, but membrane-intact pyknotic/necrotic cells are not. The results suggest that chemotherapy-induced apoptosis and phagocytosis of cancer cells may be enhanced by including certain antioxidant agents in the treatment protocol.