The cardenolide UNBS1450 is able to deactivate nuclear factor KB–mediated cytoprotective effects in human non–small cell lung cancer cells

Non–small cell lung cancers (NSCLC) are associated with very dismal prognoses, and adjuvant chemotherapy, including irinotecan, taxanes, platin, and Vinca alkaloid derivatives, offers patients only slight clinical benefits. Part of the chemoresistance of NSCLCs results from the constitutive or anticancer drug-induced activation of the nuclear factor-KB (NF-KB) signaling pathways. The present study shows that human A549 NSCLC cells display highly activated cytoprotective NF-KB signaling pathways. UNBS1450, which is a cardenolide belonging to the same class of chemicals as ouabain and digitoxin, affected the expression and activation status of different constituents of the NF-KB pathways in these A549 tumor cells. The modifications brought about by UNBS1450 led to a decrease in both the DNA-binding capacity of the p65 subunit and the NF-KB transcriptional activity. Using the 3-(4,5-dimethylthiazol-2yl)-dephenyltetrazolium bromide colorimetric assay, we observed in vitro that UNBS1450 was as potent as taxol and SN38 (the active metabolite of irinotecan) in reducing the overall growth levels of the human A549 NSCLC cell line, and was more efficient than platin derivatives, including cisplatin, carboplatin, and oxaliplatin. The chronic in vivo i.p. and p.o. UNBS1450 treatments of human A549 orthotopic xenografts metastasizing to the brains and the livers of immunodeficient mice had a number of significant therapeutic effects on this very aggressive model. [Mol Cancer Ther 2006;5(2):391–9] Introduction Because adjuvant chemotherapy (including camptothecin, taxane, platin, and Vinca alkaloid derivatives) has only a limited therapeutic effect, non–small cell lung cancers (NSCLC) are the leading cause of death from cancer in most developed countries, and are characterized by an overall 5-year survival rate as low as 15% (1, 2). Most tumor cells (including NSCLC) are naturally resistant not only to apoptotic-related cell death (type I programmed cell death), but are also resistant to nonapoptotic types such as necrosis, autophagy (type II programmed cell death), senescence, mitotic catastrophe, and paraptosis (3–5). In addition to mechanisms enabling them to resist cell death, NSCLCs are also able to resist various cytotoxic insults because they possess a large set of intracellular signaling pathways that counteract chemotherapeutic insults including the constitutive activation of the phosphatidylinositide3-kinase (6), Akt (7, 8), and the nuclear factor-nB (NF-nB) signaling pathways (8, 9), all of which are interlinked (8, 10). In fact, the constitutive or drug-induced activation of the NF-nB signaling cascade constitutes one of the major pathways by which tumor cells escape cytotoxic insults (11–13). NF-nB is a collective designation for a family of highly regulated dimeric transcription factors regulating the expression of genes encoding cytokines and chemokines, factors involved in tumor promotion/proliferation, angiogenesis, and a plethora of antiapoptotic proteins (11–13). Virtually all vertebrate cells express at least one of the five Rel/NF-nB members, p50/p105 (NF-nB1), p52/100 (NFnB2), c-Rel, p65 (RelA), and RelB, which are assembled into homodimers and heterodimers binding a common DNA sequence motif known as the nB (11–13). The most commonly encountered dimer in mammalian cells is the p65/p50 dimer (11–13). In most normal cells, Rel/NF-nB dimers are retained in the cytoplasm as an inactive complex by means of direct binding with specific inhibitors, i.e., I-nB proteins (11–13). Various signals can lead to the phosphorylation and the subsequent ubiquitin proteasome – mediated degradation of the I-nB proteins with the resultant translocation of the active Rel/NF-nB complex into the nucleus (11–13). In contrast, a large number of tumor cells display constitutively high levels of nuclear NF-nB activity due to the hyperactivation of the NF-nB signaling pathways or to inactivating mutations in the regulatory I-nB subunits (11–13). Experimental data involving NF-nB inhibition/deactivation as an important new approach in the treatment of various malignancies have shown that the transcriptional activation of genes associated with cell proliferation, angiogenesis, metastasis, and the suppression of apoptosis seems to lie at the heart Received 9/13/05; revised 10/31/05; accepted 12/1/05. Grant support: ‘‘Fonds de la Recherche Scientifique Médicale’’ (Belgium), the ‘‘Fonds Yvonne Boël’’ (Brussels, Belgium), and the ‘‘Région de Bruxelles-Capitale’’ (Brussels, Belgium). R. Kiss is a Director of Research with the ‘‘Fonds National de la Recherche Scientifique’’ (Belgium). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Requests for reprints: Robert Kiss, Laboratory of Toxicology, Institute of Pharmacy (CP 205/1), Université Libre de Bruxelles, Campus Plaine, Boulevard du Triomphe, 1050 Brussels, Belgium. Phone: 32-47762-2083; Fax: 32-2332-5335. E-mail: [email protected] Copyright C 2006 American Association for Cancer Research. doi:10.1158/1535-7163.MCT-05-0367 391 Mol Cancer Ther 2006;5(2). February 2006 Research. on January 6, 2018. © 2006 American Association for Cancer mct.aacrjournals.org Downloaded from of the ability of NF-nB to promote oncogenesis and resistance to cancer therapy (13). In our present study, we investigated whether a cardenolide (such as ouabain and digitoxin, belonging to the so-called group of cardiotonic steroids; refs. 14, 15) could deactivate a constitutively activated NF-nB signaling pathway in the experimental A549 NSCLC model. In vivo orthotopic xenografts of human A549 NSCLC cells developing in the lungs of immunodeficient mice clearly metastasize into the brains and livers of the host mice and display a broad panel of mechanisms enabling them to resist chemotherapeutic insults including cyclooxygenase-2, prostaglandin E synthetase, ornithine decarboxylase, the lung-related resistance protein, and glutathione S-transferase-a, -A, and -k (16). The present study clearly shows that A549 NSCLC cells also display a constitutively activated NF-nB signaling pathway. We recently showed that in vivo, various compounds used to treat patients with NSCLC (including taxol, irinotecan, and oxaliplatin) are of very limited therapeutic value in the case of the orthotopic A549 NSCLC model (16). We decided to use a cardenolide in order to try to deactivate the NF-nB signaling pathway in A549 tumor cells because (a) this type of drug [including digitoxin (17), oleandrin (18), and ouabain (19)] has previously been shown to interfere with the NF-nB pathway, and (b) cardenolides have a number of antitumor effects on lung cancers both in vitro (20) and in vivo (21). The antitumor effects of cardenolides have also been reported for prostate (22, 23) and breast (24) cancers even at the clinical level (25). UNBS1450, the cardenolide used in the present work, is a hemisynthetic derivative of a novel compound (UNBS1244) that we identified in an African plant, Calotropis procera (26). Figure 1 illustrates the chemical structure of UNBS1450 and UNBS1244 in comparison with ouabain. Cardenolides bind to the sodium pump, i.e., Na/KATPase, which is an integral membrane protein found in the cells of all higher eukaryotes and is responsible for translocating sodium and potassium ions across cell membranes with ATP as the driving force (14, 15, 27). Sodium pump activity can be decreased or even inhibited by one class of chemicals only, i.e., the cardiotonic steroids, to which cardenolides belong (27). In addition to pumping ions, the sodium pump interacts with neighboring membrane proteins and organizes cytosolic cascades of signaling proteins to send messages to various intracellular organelles (27, 28). The signaling pathways rapidly elicited by the interaction of cardiotonic steroids with the sodium pump are independent of changes in intracellular Na and K concentrations (27, 28). We have already shown that UNBS1450 reduces the sodium pump activity more markedly than ouabain and digitoxin, and at the same time, is much better tolerated in vivo than either ouabain or digitoxin (26). Materials andMethods Compounds The drugs were purchased as follows: taxol (Paclitaxel; S.A. Bristol-Myers Squibb, Brussels, Belgium), irinotecan (Campto, Aventis, Brussels, Belgium), SN-38 (7-ethyl10-hydroxycamptothecin; Aventis), oxaliplatin (Oxaliplatin; Inter-Chemical, Ltd., Shen Zhen, China), cisplatin (Platinol; S.A. Bristol-Myers Squibb), carboplatin (Paraplatin; S.A. Bristol-Myers Squibb). The UNBS1450 was obtained by means of chemical hemisynthesis, as detailed elsewhere (26). Cell Lines The A549 (ATCC code CCL-185) cell line was obtained from the American Type Culture Collection (Manassas, VA) and was maintained in MEM supplemented with 5% fetal bovine serum, in a mixture of 0.6 mg/mL glutamine (Life Technologies-Invitrogen SA, Merelbeke, Belgium), 200 IU/mL penicillin (Life Technologies-Invitrogen), 200 IU/ mL streptomycin (Life Technologies-Invitrogen), and 0.1 mg/mL gentamicin (Life Technologies-Invitrogen). The trypsin-EDTA, the fetal bovine serum, and the cell culture media and their supplements were obtained from Life Technologies-Invitrogen. The fetal bovine serum was heatinactivated for 1 hour at 56jC. The cells were incubated at 37jC, in sealed (airtight) Falcon plastic dishes (Nunc, Invitrogen SA, Merelbeke, Belgium) in a 5% CO2 atmosphere. In vitro Overall Growth Determination Overall cell growth was assessed by means of the colorimetric 3-(4,5-dimethylthiazol-2yl)-dephenyltetrazolium bromide (Sigma, Bornem, Belgium) assay, as detailed elsewhere (26). The tumor cells were incubated for 72 hours in the presence (or absence control) of the various drugs. The drug concentrations ranged between 10 9 and 10 5 mol/L (with a half-log concentration increases). The experiments were carried out in sextuplicate. Figure 1. Comparison of the chemical structures of one well-known cardenolide, i.e., ouabain (A), with 200-oxovoruscharin (UNBS1244; B) and its hemisynthetic derivative, UNBS1450 (B). Cardenolide Treatment of Experimental Human NSCLCs 392 Mol Cancer Ther 2006;5(2). February 2006 Research. on January 6, 2018. © 2006 American Association for Cancer mct.aacrjournals.org Downloaded from Western Blotting Analyses Cell extracts were prepared by the lysis of subconfluent A549 cells in the SDS-PAGE loading buffer [1 SDS sample buffer, 62.5 mmol/L Tris-HCl (pH 6.8 at 25jC), 2% w/v SDS, 10% glycerol, 50 mmol/L DTT, 0.01% w/v bromophenol blue]. The A549 cell lysates were loaded onto a denaturing polyacrylamide gel (5–12%) and blotted onto a Polyscreen-polyvinylidene difluoride membrane (NEN Life Science Products, Boston, MA). The proteins analyzed (see below) were immunodetected by specific affinity-purified primary antibodies (in TBS containing 0.1% Tween 20 and 5% fat-free dry milk powder or bovine serum albumin) in conjunction with a secondary antibody, in the form of IgG conjugated with horseradish peroxidase. Control experiments included the omission of the incubation step with the primary antibodies (negative control). Equal loading was verified after the Ponceau red coloration of the membranes. The integrity and quantity of the extracts was assessed by means of tubulin immunoblotting. The proteins submitted to Western blotting analyses were detected by means of primary antibodies provided by (a) CST Technologies (Westburg, Leusden, the Netherlands): I-nBa (1:1,000), phospho-I-nBa (1:500), ubiquitin (1:500); (b) AbCam (Cambridge, United Kingdom): cdc34 (1:500), tubulin (1:3,000); (c) BD Transduction Laboratories (Erembodegem, Belgium): p65 (1:500); and (d) Santa Cruz (Tebu-Bio, Boechout, Belgium): I-nBh (1:100). The secondary antibodies used were obtained as follows: anti-mouse and anti-goat IgG from Pierce (PerbioScience, Erembodegem, Belgium), antirabbit from NEN Life Science Products, and anti-rat from AbCam. Blots were developed using the Pierce Supersignal Chemiluminescence system (PerbioScience). NF-KBDNABinding Assay NF-nB DNA binding activity was assessed with the Trans-AM NF-nB family transcription factor assay kits (Active Motif Europe, Rixensart, Belgium) developed by Renard et al. (29) as a new sensitive assay to estimate the amount of activated NF-nB in whole-cell protein extracts. This ELISA-like test measures the level of the active form of NF-nB contained in cell extracts specifically able to bind to an oligonucleotide containing the NF-nB consensus site (5V-GGGACTTTCC-3V) attached to a 96-well plate (29). Whole cell lysates were prepared and 10 Ag extracts were added to the 96-well plates. The binding of NF-nB to the DNA was visualized by incubation with anti-p50, antip52, anti-p65/Rel-A, and anti-Rel-B antibodies that specifically target activated NF-nB, followed by a secondary antibody conjugated with horseradish peroxidase. Antibody binding was determined as absorbance values at 450 nm. Luciferase ReporterAssay Although a luciferase reporter plasmid construct (pNFnB-Luc) containing five NF-nB binding sites was purchased from Stratagene (Amsterdam, the Netherlands), the plasmid construct containing three NF-nB (p3NF-nB-Luc) binding sites was kindly provided by Dr. Corinne Grangette (Laboratoire de Bactériologie des Ecosystèmes, Institut Pasteur, Lille, France). Clones of A549 tumor cells stably containing the NF-nB-Luc constructs were obtained by cotransfection with the NF-nB-Luc plasmid and pSV2neo (30) in a molecular ratio of 10:1. The A549 tumor cells were plated 24 hours after transfection and the isolated clones were selected by cultivating the A549 cells in MEM supplemented with 700 Ag/mL of the neomycin analogue G418 (Geneticin, Life Technologies-Invitrogen). Selected A549 clones were analyzed for luciferase activity by the luciferase assay system (Promega, Leiden, the Netherlands). Briefly, 10 A549 cells were plated in 96-well plates and, 24 hours after plating (see Results), the cells were cultivated in MEM supplemented with 10 nmol/L UNBS1450 for various experimental times. The cells were rinsed with PBS, lysed in 20 AL of lysis buffer (Promega), and frozen at 20jC. The A549 cell lysates were tested for luciferase activity with 40 AL of luciferase reagent on a TD-20/20 luminometer (Turner Designs, Promega) with an integration time of 30 seconds. In vivo Orthotopic Grafting of A549 NSCLC Human Cells The potential in vivo UNBS1450-related therapeutic effects were determined on human A549 orthotopic xenografts by administering UNBS1450 chronically by i.p. or p.o. injections thrice a week for 4 consecutive weeks and at different concentrations of the maximum tolerated dose (MTD) varying from (MTD)/32 to (MTD)/8 (see Results). The MTD for UNBS1450 was determined by administering it acutely (i.e., in one single i.p. or p.o. dose) to healthy mice (i.e., not grafted with tumors). The survival periods and the weights of the animals were recorded for up to 28 days after the injection of the compound. Seven different doses of UNBS1450 (i.e., 2.5, 5, 10, 20, 40, 80, 120, and 160 mg/kg) were used to determinate the MTD index. The MTD index is defined as the concentration killing at least one mouse in a group of three after a minimum of 28 days. Acute UNBS1450 MTDs via the i.p. and the p.o. route were 120 and >160 mg/kg, respectively. As detailed elsewhere (16), orthotopic A549 xenografts were obtained by grafting 2 10 human A549 cells through the thorax into the left-hand-side of the lungs of nude mice. All the orthotopic grafts were done under anesthesia [saline; Rompun (Bayer, Leverkusen, Germany); Imalgene (Merial, Lyon, France); 5/1/1, vol/vol/vol]. Highly reproducible tumor developments (100%) were obtained from the A549 tumor grafting in each experiment. The end point of the A549-related experiments was the recording of the survival periods of each of the A549NSCLC–bearing nude mice. For ethical reasons, each animal was submitted to euthanasia (in a CO2 atmosphere) when it had lost 20% of its weight as compared with its weight at the time of the tumor graft. As detailed elsewhere, autopsies and histology were done on each mouse to confirm the presence of the tumor development (16). All the in vivo experiments described in the current study were done on the basis of authorization no. LA1230509 of the Animal Ethics Committee of the Belgian Federal Department of Health, Nutritional Safety, and the Environment. Molecular Cancer Therapeutics 393 Mol Cancer Ther 2006;5(2). February 2006 Research. on January 6, 2018. © 2006 American Association for Cancer mct.aacrjournals.org Downloaded from Statistical Analyses Survival analysis was done by using Kaplan-Meier curves and the Gehan generalized Wilcoxon test. All the statistical analyses were carried out using Statistica software (Statsoft, Tulsa, OK). Results HumanA549NSCLCCellsDisplayConstitutivelyHigh Levels of NF-KBActivity Figure 2A shows that A549 tumor cells (black columns) display basal RelA/p65 DNA-binding activity as high as in the tumor necrosis factor-a–stimulated Raji cells (gray columns) used as the positive control in the colorimetric NF-nB DNA binding assay (29) employed here. High basal levels of NF-nB activity were also observed in stably transfected clones of A549 cells containing three (Fig. 2B) or five (Fig. 2C) NF-nB binding sites in luciferase reporter constructs. The high basal levels of NF-nB activity in A549 tumor cells were further outlined by the fact that TNF-a, one of the most potent NF-nB inducers, less than doubled the NF-nB activity in these A549 cells, even when used at 100 ng/mL (Fig. 2B and C). UNBS1450 Affects the Expression and Activation Status of NF-KB in Human A549 NSCLCCells UNBS1450-mediated effects in connection with expression and phosphorylation status of the different constituents of the NF-nB pathways in the A549 tumor cells were characterized at a 10 nmol/L dose, which, in its turn, constitutes the IC50 value in terms of the concentration that reduces the overall growth rate of the A549 cell line by 50% after 3 days of treatment (see below). This dose is at least 100 times lower than the doses reported in other studies whose purpose is to characterize the influences of various cardenolides on the NF-nB signaling pathways (18, 19). The treatment of A549 tumor cells with 10 nmol/L UNBS1450 for 24 hours did not trigger off any cell death processes, a point which was determined by means of flow cytometry (data not shown). We chose to analyze the effects of UNBS1450 over periods as long as 24 hours because we were interested in the ability of this compound to deactivate constitutively activated NF-nB activity and not in its ability to counteract the rapid NF-nB activation resulting from proinflammatory stimulation which occurs within minutes. Although UNBS1450 (10 nmol/L) was the major factor in inducing the accumulation of I-nBh in the A549 tumor cells (Fig. 3A), it seemed to leave the levels of expression of I-nBa unchanged (Fig. 3B). In contrast, UNBS1450 decreased I-nBa phosphorylation between 4 and 8 hours following its addition to the A549 tumor cell culture medium (Fig. 3C). UNBS1450 (10 nmol/L) also decreased the levels of expression of cdc34 (Fig. 3D). cdc34 is a ubiquitin-conjugating enzyme (E2) involved in I-nBa ubiquitination and its subsequent proteasomal degradation (31). In addition, 10 nmol/L UNBS1450 also led to a decrease in the accumulation of the hTrCP protein in the A549 tumor cells (data not shown). hTrCP is a ubiquitin ligase (E3) that forms a complex with Skip1 and Cullin1; this complex is involved in I-nBa ubiquitination (32). Furthermore, 10 nmol/L of UNBS1450 induced an increase in the accumulation of ubiquitinylated proteins in the A549 tumor cells by f8 to 10 hours following its addition to the culture medium of these cells (Fig. 3E). Treatment with MG132, a proteasome inhibitor, was used as a control because proteasome inhibition also results in the accumulation of ubiquitinylated proteins, a feature observed here in the case of MG132 (Fig. 3E). Figure 2. Characterization of the basal NF-nB activity in untreated A549 cells. A, characterization by means of the Trans-AM NF-nB family transcription factor assay kit, of the NF-nB DNA binding activity (p50, p52, p65/Rel-A, and Rel-B subunits) in untreated A549 cells (black columns) in comparison with TNF-a-treated Raji lymphoblast-like cell extracts (positive control; gray columns ) arbitrarily normalized to 100%. Columns, mean percentages of NF-nB binding activity (Y axis); bars, FSE. Each experiment was carried out in triplicate. B and C, characterization of the NF-nB-dependent luciferase reporter gene expression. A549 tumor cell clones stably transfected with luciferase reporter construct containing either three (B) or five (C) NF-nB binding sites were left untreated (Ct ) or treated with 100 ng/mL TNF-a for 6 h. The results are expressed as NF-nB reported luciferase activity (Y axis, a.u.). Each sample was assessed thrice. Columns, means; bars, FSE. Cardenolide Treatment of Experimental Human NSCLCs 394 Mol Cancer Ther 2006;5(2). February 2006 Research. on January 6, 2018. © 2006 American Association for Cancer mct.aacrjournals.org Downloaded from UNBS1450 Decreased the DNA Binding Capacity of the p65 Subunit UNBS1450 (10 nmol/L) decreased the levels of the p65/ RelA subunit protein accumulation (Fig. 4A). A colorimetric NF-nB DNA-binding assay with cell lysates from untreated and UNBS1450-treated A549 cells at two doses (10 and 100 nmol/L) further revealed that the UNBS1450 treatment of the A549 tumor cells resulted in a doseand time-dependent decrease in p65 DNA-binding activity (Fig. 4B). UNBS1450 Decreased the NF-KB Transcriptional Activity The data in support of an UNBS1450-induced downregulation of the NF-nB activity presented in Fig. 3 and in Figs. 4A and B were further confirmed by the use of luciferase reporter constructs containing three and five NF-nB binding sites stably transfected into A549 tumor cells (Fig. 4C). Indeed, 10 nmol/L UNBS1450 induced marked time-dependent decreases in the NF-nB reported luciferase activity in the four different A549 tumor cell clones used in the present study. In vitro Antitumor Effects of UNBS1450 on the Overall Growth of Human A549 NSCLCCells UNBS1450 displayed in vitro antigrowth activities with respect to the A549 tumor cell populations, similar to those effects observed with taxol or SN38, the active metabolite of irinotecan, but its effects were significantly more pronounced than those observed with various platin derivatives (Fig. 5A). In vivo Antitumor Effects of UNBS1450 in A549 Orthotopic Xenografts UNBS1244, the novel cardenolide that we identified in C. procera , had an acute MTD level of 10 mg/kg when injected i.p. in healthy mice. This value is similar to those observed with respect to ouabain (5 mg/kg) and digitoxin (10 mg/kg; ref. 26). In sharp contrast, whereas both UNBS1244 and UNBS1450 showed in vitro IC50 values of f10 nmol/L with respect to the mean inhibition of the overall growth levels of 57 human cancer cell lines (26), UNBS1450 showed an acute i.p. MTD value of 120 mg/kg. Thus, although UNBS1450 is much better tolerated in vivo than its mother compound, UNBS1244, the in vitro antiproliferative effects of both compounds are identical. Orthotopic xenografts of human A549 NSCLCs into nude mice are resistant to taxol and oxaliplatin, and only weakly sensitive to irinotecan (16). However, UNBS1450 markedly increased the survival of A549 NSCLC orthotopic xenograft-bearing mice when given i.p. chronically (12 times) at 10 or 20 mg/kg (Fig. 5B). There are no statistically significant differences between the 10 and the 20 mg/kg dose schedules. Chronic i.p. administrations of UNBS1450 at 5 mg/kg also contributed weak, but nevertheless significant, increases in these survival periods (Fig. 5B). At higher doses, i.e., 40 mg/kg, chronic i.p. treatment was associated with slight toxic effects, i.e., a transient loss of weight in the A549 xenograft-bearing mice, but without the deaths that occurred with a chronic i.p. treatment of 80 mg/ kg (data not shown). The experiments illustrated in Fig. 5B were reproduced 6 months later with identical results (data not shown). Furthermore, when chronically administered 12 times p.o. at 80 mg/kg, UNBS1450 was able to bring about a beneficial therapeutic effect in the case of the orthotopic A549 model (Fig. 5C). One can wonder what happened to A549 orthotopic xenografts in vivo following UNBS1450 treatment. Based on the in vitro data reported above and on the data relying on an in vivo experiment carried out with the s.c. NCI-H727 NSCLC xenograft model, we hypothesize that UNBS1450 decreased the growth rates of both the primary xenografts and brain and liver metastases. Indeed, we treated s.c. NCIH727 NSCLC xenografts with UNBS1450 exactly as we did here with respect to the A549 NSCLC orthotopic xenograft model: we observed that UNBS1450 decreased by f50% Figure 3. Characterization by means of Western blotting analyses of the 10 nmol/L UNBS1450–mediated effects for a period between 45 min and 12 to 20 h on the expression and phosphorylation levels of the NF-nB inhibitory proteins I-nBh (A) and I-nBa (B and C). D, the effect of 10 nmol/L UNBS1450 treatment on cdc34 protein expression over a period of 20 h. E, the effect of 10 nmol/L UNBS1450 treatment on the accumulation of ubiquitinylated proteins in A549 tumor cells, with 50 Amol/L MG132 (a proteasome inhibitor) chosen as a reference. Molecular Cancer Therapeutics 395 Mol Cancer Ther 2006;5(2). February 2006 Research. on January 6, 2018. © 2006 American Association for Cancer mct.aacrjournals.org Downloaded from the growth rates of the NCI-H727 xenografts at the end of the experiment (data not shown). Experiments are under way in order to characterize the effects of UNBS1450 on the growth rates of brain and liver metastases associated with the orthotopic grafting of the A549 NSCLC xenografts.