Growth inhibition of non-small-cell lung carcinoma by BN/GRP antagonist is linked with suppression of K-Ras, COX-2, and pAkt
- Florian Hohla*,†,
- Andrew V. Schally*,‡,§,¶,
- Celia A. Kanashiro*,‖,
- Stefan Buchholz*,
- Benjamin Baker*,
- Chandrika Kannadka*,
- Angelika Moder†,
- Elmar Aigner†,
- Christian Datz†, and
- Gabor Halmos*,§,**
- *Veterans Affairs Medical Center and Tulane University School of Medicine, New Orleans, LA 70112;
- ‡Veterans Affairs Medical Center and South Florida Veterans Affairs Foundation for Research and Education, Miami, FL 33125; and
- §Departments of Pathology and Medicine, Division of Haematology and Oncology, University of Miami Miller School of Medicine, Miami, FL 33101
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Contributed by Andrew V. Schally, October 5, 2007 (received for review September 12, 2007)
Abstract
Bombesin (BN) or gastrin-releasing peptide (GRP) can stimulate the growth of neoplasms such as breast cancer and small-cell lung carcinoma (SCLC). Antagonists of BN/GRP have been shown to inhibit these cancers. We evaluated whether antagonists of BN/GRP can suppress the growth of human non-SCLC (NSCLC) xenografted into nude mice. The effect of the administration of BN/GRP antagonist RC-3940-II on the growth of H460 and A549 NSCLC cell lines orthotopically xenografted into the intrapulmonary interstitium was examined. Protein levels of K-Ras, COX-2, Akt/pAkt, WT p53, Erk1/2, and lung resistance-related protein (LRP) in tumors were analyzed by Western blot analaysis, and receptors for BN/GRP were investigated by radioligand-binding studies. The effect of RC-3940-II on the proliferation of H460 and A549 cells in vitro was assessed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assays. High-affinity receptors for BN/GRP were found on tumors. Treatment with RC-3940-II significantly (P < 0.001) inhibited growth of H460 and A549 NSCLC xenografts by 30–50% and led to an improved performance status, compared with controls. In H460 NSCLC, the antitumor effect was associated with a significant (P < 0.001) reduction in protein levels of K-Ras, COX-2, pAkt, and pERK1/2 and with a major augmentation in the expression of WT p53, compared with controls. In A549 NSCLC, pAkt and LRP were significantly down-regulated. Our findings demonstrate the efficacy of BN/GRP antagonist RC-3940-II for the treatment of NSCLC. The suppression of K-Ras, COX-2, pAkt, and LRP, as well as the up-regulation of WT p53 might contribute to the antitumor action of BN/GRP antagonists.
Lung carcinoma is the leading cause of cancer-related deaths in both men and women worldwide (1). Non-small-cell lung cancers (NSCLC) represent 75–80% of all lung neoplasms. Many types of tumor cells are considered to be under control of specific growth factors and neuropeptides that act by endocrine or autocrine/paracrine mechanisms to stimulate their proliferation and decrease apoptosis (2). Gastrin-releasing peptide (GRP) is a member of the bombesin (BN)-like peptide family and functions as a gastrointestinal hormone and neurotransmitter (2). Cuttita et al. discovered that BN/GRP can act as an autocrine growth factor for SCLC (3). Subsequently, it was shown that BN/GRP also stimulates cell proliferation of other neoplasms, such as breast cancer and NSCLC (2, 4). Several subtypes of receptors for BN/GRP are present in NSCLC cell lines, including A549 and H460 (4, 5). In an endeavor to develop a new class of anticancer agents, various antagonistic analogues of BN/GRP were synthesized in our institute (2, 6). The antagonists of BN/GRP, such as RC-3095 and RC-3940-II, have been previously shown to be effective against a variety of cancers in vitro and in vivo, but so far not against NSCLC (2, 7). The antitumor effect of these antagonists in breast cancers and SCLC was associated with a significant reduction in the expression of GRP-receptors (GRP-Rs) and EGF-receptors (EGF-Rs) on cell membrane; diminished mRNA levels for GRP-R, ErbB-2/Her-2, c-jun, and c-fos oncogenes; and a decrease of mutant p53 protein production (8–10).
Recent studies suggest that modern chemotherapeutics may be approaching the limits of their clinical efficacy: The 5-year survival rate for NSCLC has leveled at 15% (11) because NSCLCs possess various cell mechanisms lead to cellular survival and chemoresistance. Among these mechanisms, mutant Ras, COX-2, constitutive active Akt, deregulation of tumor suppressor genes including p53, as well as expression of the lung resistance-related protein (LRP) might be the limiting factors in reducing the efficacy of current therapeutic regimes.
Ras proteins (H-, N-, and K-Ras) are involved in many aspects of cell growth, mediating mitogenic and differentiation signals and apoptotic signals. K-Ras point mutations, which occur in 10–30% of lung adenocarcinomas, cause constitutive activation of the protein product p21ras, which results in an excessive activation of its downstream pathways (mainly Raf/MEK/ERK1/2 and PI3K/Akt) involved in proliferative and survival signals triggered by Ras (12, 13).
In addition to the up-regulation of K-Ras, recent evidence suggests a potential role of COX-2 in the development of some lung cancers (14). Two isoforms of COX have been described: a constitutively expressed enzyme COX-1 present in most cell lines, and an inducible form, COX-2, expressed in response to cytokines, tumor promoters, and growth factors (15). Tumor cells with elevated COX-2 levels are angiogenic, invasive and suppressive of host immunity, and highly resistant to apoptosis (16).
Akt is a cytosolic signal transduction protein kinase that plays an important role in cell survival pathways (17). To date, three isoforms of Akt have been identified: Akt1, Akt2, and Akt3 (17). Induction of Akt activity is primarily dependent on the PI3K pathway. For full activation, Akt must be phosphorylated at two sites, the first within the activation loop (T-308) and the second within the C terminus (S-473) (17). In addition to the activation by receptor tyrosine kinase (RTK), G protein-coupled receptors (GPCRs), and K-Ras, Akt also can be activated by many forms of cellular stress, such as those observed under treatment with chemotherapeutic substances (17). Once active, Akt controls cellular functions such as apoptosis, cell cycle, gene transcription, and protein synthesis through the phosphorylation of downstream substrates (17).
p53 is the most extensively studied tumor suppressor gene in human cancer. The p53 gene product is involved in multiple pivotal cellular processes as a potent transcriptional regulator, and one of its most important roles is the regulation of apoptosis (18).
LRP, a member of the membrane transporter proteins, was originally identified in a multidrug-resistant cell line of lung cancer. Classical multidrug resistance (MDR) is mediated by drug efflux mechanisms. LRP is a member of the membrane transporter proteins not belonging to the ATP-binding superfamily of transporter proteins such as P-glycoprotein and MDR-associated protein (19). It has been reported that the LRP expression is inversely correlated with responses to chemotherapy in patients with NSCLC (20).
Although antagonists of BN/GRP have been studied in a wide range of solid tumors, including SCLC, little is known about their effectiveness against NSCLC. Thus, in the present study, we tested the ability of BN/GRP antagonist RC-3940-II to arrest the growth of human NSCLC H460 and A549 in an orthotopic lung model, which is clinically more relevant than s.c. models. In an attempt to shed more light on its mechanisms of action, we examined the effects of RC-3940-II on the expression of K- Ras/Erk1/2, COX-2, Akt/pAkt, WT p53, and LRP in orthotopic xenografts of H460 and A549 NSCLC.
Results
Analysis of BN/GRP Receptors in H460 and A549 Tumors.
The presence of BN/GRP receptors was assessed by radioligand-binding assays in control tumors of both models investigated. In the membranes of H460 tumors, radiolabeled [Tyr4]BN was bound to a single class of specific, high-affinity (K d = 1.29 ± 0.08 nM) binding sites with a mean maximal binding capacity (B max) of 563.43 ± 21.6 fmol/mg membrane protein. Binding studies also demonstrated the presence of a single class of specific, high-affinity (K d = 7.45 ± 0.63 nM) binding sites for BN/GRP in the membrane preparation of A549 NSCLC tumors with a mean Bmax of 798.4 ± 48.7 fmol/mg membrane protein.
Inhibition of Orthotopic Tumor Growth by BN/GRP Antagonist RC-3940-II.
To study the effect of BN/GRP antagonist RC-3940-II against human NSCLC cells H460 and A549 growing in an orthotopic environment, we used a model in which tumor cells were surgically introduced into the pulmonary interstitium. Treatment with BN/GRP antagonists RC-3940-II at doses of 10 μg/day s.c. was initiated in the H460 group on day 4 and in the A549 group on day 21 for 21 days. The mice were killed under deep anesthesia, and the lungs were removed, checked for tumor growth, and weighed. Tumor growth was observed in all animals (take rate 100%). Therapy with RC-3940-II caused a significant reduction in the mean lung weight because of a decrease in the growth of both H460 and A549 tumors (Table 1 and Fig. 1). Untreated H460 and A549 tumors are shown in Fig. 1 a and c, respectively. Treated H460 tumors can be seen in Fig. 1 b and A549 tumors in Fig. 1 d. Daily treatment with RC-3940-II reduced the mean lung weight by 53% and 30% in animals bearing H460 and A549 NSCLC tumors, respectively (Table 1).
Effect of therapy with BN antagonist RC-3940-II on the lung weight (lung block) and body weights (BW) of nude mice orthotopically xenografted with H460 or A549 human NSCLC
Orthotopic growth of 10 μg/day untreated and treated (RC-3940-II) human NSCLC H460 (a and b) and A549 (c and d) xenografts. H460 xenografts grew more extensively, whereas A549 xenografts showed more nodular growth. The tumors are marked with arrows.
H460 tumors (Fig. 1 a and b) showed a different growth pattern, compared with A549 xenografts (Fig. 1 c and d). H460 xenografts grew faster and more extensively and metastasized more often to the contralateral side. In untreated animals (Fig. 1 a), virtually no residual lung parenchyma was visible, leading to symptoms of dyspnea such as elevated breathing rates. Although there were no significant differences in body weight among the groups before the start of treatment, untreated control mice showed a significant (P < 0.001) reduction in body weight, compared with mice treated with RC-3940 II (Table 1). A549 xenografts grew more slowly and showed nodular growth (Fig. 1 c and d). Untreated control animals showed significant weight loss (P < 0.001), compared with mice in the treatment group (Table 1).
Effect of Antagonist of BN/GRP RC-3940-II on the Proliferation of Human H460 and A549 NSCLC.
H460 and A549 NSCLC cells cultured in vitro were exposed to various concentrations of BN/GRP antagonist RC-3940-II, and the effect on cell growth was followed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay (data not shown). RC-3940-II at 0.1–10 μM did not affect the growth of H460 and A549 NSCLC cells in vitro.
Effect of Antagonist of BN/GRP RC-3940-II on the Protein Expression of K-Ras, Erk1/2, pErk1/2, COX-2, Akt/pAkt, and p53 in Orthotopic Xenografts of H460.
Significant immunoreactive bands to polyclonal antibodies to COX-2, Akt1/2, and Phospho-Akt (Ser 473), LRP, and monoclonal antibodies to COX-2, K-Ras, Erk1/2, pErk1/2 (Thr 202/Tyr 204), and WT p53 were detected with specific antisera (Fig. 2 a). The bands were submitted to densitometric analysis and normalized to β-actin levels. The levels of expression of these proteins are shown as a percentage respective of control, which were accepted as 100% (Table 2). Treatment with RC-3940-II induced significant decreases in the expression of K-Ras (73%), pErk (29%), COX-2 (85%), and pAkt (85%) and an increase in the protein level of WT p53 to 227%, compared with controls (Table 2). No significant inhibitory effect on the expression of Akt1/2 protein was detected after treatment with RC-3940-II.
Western blot for protein expression of K-Ras, COX-2, Akt1/2, pAkt (S-473), Erk1/2, pErk1/2, and WT p53 in orthotopic xenografts of human NSCLC H460 (a), and Akt1/2, pAkt (S-473), and LRP expression in orthotopic xenografts of human NSCLC A549 (b). Experiments were repeated at least three times. Protein levels were normalized to 42 kDa of β-actin protein and are expressed as the percentage of control values, as shown in Table 2.
Protein levels of K-Ras, Cox-2, Akt1/2, pAkt, p53, Erk1/2, and pErk1/2 in orthotopic xenografts of H460 human NSCLC and of Akt, pAkt, and LRP in A549 human NSCLC after treatment with 10 μg/day BN antagonist RC-3940-II
Effect of Antagonist of BN/GRP RC-3940-II on the Protein Expression of Akt/pAkt and LRP in Orthotopic Xenografts of A549.
Protein expression of Akt1/2 and Phospho-Akt (Ser 473) and LRP was assessed by Western blot analysis in xenograft of A549 NSCLC (Fig. 2 b). The levels of expression of this protein are shown as a percentage of control (Table 2), with the controls being accepted as 100%. Treatment with RC-3940-II decreased the expression of LRP by 70% and pAkt by 80%, compared with untreated controls.
Discussion
Although progress has been made in the past 10 years in the management of patients with NSCLC, the 5-year relative survival rates have not improved substantially (11). Thus, new approaches to the treatment of lung cancer are mandatory. BN/GRP antagonists synthesized in our laboratory have already been shown to be effective against a wide range of solid tumors, including SCLC; breast, ovarian, and prostatic cancers; renal cell carcinomas; and brain tumors (2, 7, 21) Although pseudononapeptide BN/GRP antagonists are rapidly eliminated from the blood stream after a single administration, EGF-Rs remain down-regulated for many hours (2, 22). This finding explains how daily single injections of the antagonists can maintain tumor growth inhibition (2, 22).
An antiproliferative effect of BN/GRP antagonists against NSCLC has not been demonstrated previously (2, 7). Because GRP also was postulated to be an autocrine growth factor for NSCLC (23) and because many NSCLC cell lines express receptors for BN/GRP (4, 5), the aim of this study was to demonstrate the efficacy of BN/GRP antagonist RC-3940-II in the treatment of human NSCLC.
We used NSCLC lines H460 and A549 in an orthotopic model, which is clinically more relevant than s.c. models. The organ microenvironment influences the phenotype of tumor cells and is essential for optimal growth of tumors, as originally enunciated by Paget's seed-and-soil hypothesis and confirmed by others (24). We selected human NSCLC lines H460 and A549 because these cells rapidly form tumors after xenografting into nude mice and express receptors for BN/GRP subtypes, such as GRP-R, NMB-R, and BRS-3 (4). The presence of receptors for BN/GRP was confirmed by radioreceptor assays. Furthermore, NSCLC H460 expresses high levels of mutant K-Ras, COX-2, pAkt, and WT p53, whereas LRP is up-regulated in the chemoresistant NSCLC cell line A549 (25, 26).
After 3 weeks of treatment with RC-3940-II, the mean lung weights of animals inoculated with human NSCLC H460 or A549 were significantly decreased by 53% and 30%, respectively, compared with controls. In accordance with this reduction in lung weight, which was because of the inhibition of tumor cell growth, treated animals showed a much better performance status, demonstrated no body weight loss, and lacked symptoms of dyspnea.
It has been described that BN receptors interact with p21ras proteins in plasma membranes from rat pancreatic acinar cells (27). Thus, we evaluated the protein expression of mutant K-Ras p21ras, which is expressed in NSCLC H460, but not in NSCLC A549 (25). We could show that treatment with RC-3940-II decreased p21ras in xenografts of H460 NSCLC ≤73%, compared with controls. It has been shown previously that treatment with BN is followed by a down-regulation of calmodulin and consecutive up-regulation of K-Ras in Swiss 3T3 cells (28). Thus, a possible mechanism involved in the down-regulation of protein expression of mutant K-Ras could be the up-regulation of calmodulin by antagonists of BN. Meta-analysis of recent literature showed that Ras gene alteration and/or protein overexpression is a prognostic factor for poor survival of patients with NSCLC in univariate analysis (29). Thus, the Ras family of genes has been identified as a potential target for therapeutic intervention with farnesyltransferase inhibitors (FTIs) (30). A recent phase II study reported clinical activity of the FTI Lonafarnib in combination with paclitaxel in patients with taxane-refractory-resistant metastatic NSCLC (31). Inhibition of K-Ras activity was shown to enhance radiosensitivity (32) and probably could overcome resistance to treatment with single-agent EGF-R inhibitors because patients with K-Ras mutations showed poorer clinical outcomes when treated with EGFR-inhibitor erlotinib and chemotherapy than patients lacking oncogenic Ras (33).
To study the functional effect of this observation, we tried to investigate the downstream MAPK/Erk cascade activation. We could show that treatment with RC-3940-II diminished protein levels of pERK 1/2 in xenografts of H460 NSCLC ≤30%, compared with controls.
COX-2, a marker of poor prognosis in stage I NSCLC, also is a promising target in treating NSCLC (34). Inhibition of COX-2 by selective COX-2 inhibitors enhances the response to chemotherapeutic regimens and leads to a prolonged progression-free survival when used in combination with radiotherapy (35, 36). As can be seen in Table 2, treatment of orthotopic xenografts of NSCLC H460 with BN antagonist RC-3940-II caused a significant down-regulation (≤85%) of the protein expression of COX-2. Previously, it has been shown that BN stimulates COX-2 expression in intestinal epithelial cells, in part, through a Ca2+/MAPK/AP-1-dependent signaling pathway (37). Recently, we could show that the antiproliferative action of BN/GRP antagonist RC-3940-II is associated with a down-regulation of expression levels of MAPK (38). The decrease of COX-2 levels also could be attributed to the down-regulation of K-Ras because this oncogene has been previously implicated in the positive regulation of COX-2 (39).
Akt is probably the best characterized kinase known to promote cellular survival downstream of growth factor activation. Among activators of the PI3K/Akt pathway are RTKs such as the EGF-R and oncogenic Ras (17). Cumulative evidence suggests a new and potentially central role for EGF-R as a convergence point for input from a diverse number of signaling pathways, including that of several GPCRs. Thus, many of the growth-promoting effects of GPCR stimulation are mediated through activation of receptor tyrosine kinases, a mechanism called transactivation (40). Transactivation of the EGF-R has already been described for BN in head and neck squamous cancer cells (41). It has been shown that BN antagonists down-regulate the levels and mRNA expression of EGF-R in SCLC and breast cancer models (10, 22), and it was recently found that the inhibition of H-460 and A549 NSCLC tumors by RC-3940-II is associated with a major down-regulation of EGFR as well as HER-2, HER-3, and HER-4 protein levels (42). Thus, negative regulation of RTKs, inhibition of transactivation of the EGF-R, and impaired levels of K-Ras might contribute to the decrease in pAkt levels of ≤85% after treatment with BN/GRP antagonist RC-3940-II, as found in our study.
We demonstrated previously that treatment with BN/GRP antagonist RC-3940-II is followed by a significant decrease in protein expression of mutant p53 of DMS-153 human SCLC xenografts (8). However, H460 NSCLC expresses WT p53 (25). An immunohistochemical study revealed that up-regulation of WT p53 in human NSCLC is a favorable prognostic factor and is associated with a significantly longer survival (43). Rescue of p53-induced apoptosis by survival factors has been associated with the activation of AKT kinase (44). AKT can phosphorylate and activate MDM2, thereby enhancing the degradation of p53 (45, 46) Thus, up-regulation of WT p53 ≤227% after treatment with RC-3940-II, as observed in our study, could be explained by down-regulation of the pAkt/MDM2 pathway. In addition to its interplay in the regulation of apoptosis, WT p53 has been shown to directly affect Bax and BCl-2 expression. Thus, WT p53 is able to mediate repression of the BCL-2 gene and the transactivation of Bax (47). Therefore, up-regulation of WT p53 could be responsible for the increase in the Bax/BCL-2 ratio in xenografts of H460 NSCLC after treatment with BN/GRP antagonist RC-3940-II (42).
Resistance to chemotherapeutic agents is a major problem in the treatment of patients with NSCLC. Classical MDR is mediated by drug efflux mechanisms. LRP is a member of the group of membrane transporter proteins not belonging to the ATP-binding cassette superfamily of transporter proteins, such as p-glycoprotein and MDR-associated protein (19). Although the exact function of LRP has not been established, evidence suggests that it has a role in detoxification processes (48). LRP also has been reported to correlate with resistance to cisplatin in NSCLC cell lines (49). In our study, BN/GRP antagonist RC-3940-II inhibited LRP expression in A549 human NSCLC xenografts ≤70%, compared with controls.
It has been reported that chemotherapeutic substances enhance prosurvival signals, including pAkt and COX-2 (50–52). Thus, a reduction in LRP and pAkt and COX-2 as seen in our study could improve the therapeutic efficacy in combination with chemotherapeutic agents.
Despite a clear suppressive action of BN antagonist RC-3940-II in vivo, we could not show any inhibitory effects of RC-3940-II on growth of human NSCLC H460 and A549 in vitro. However, RC-3940-II has been shown previously to down-regulate protein expression of VEGF and decrease vessel density of human experimental breast cancers (53). Alterations of K-Ras, COX-2, Akt/pAkt, and p53 have been implicated in tumor neoangiogenesis (54). Thus, the antiproliferative effect of RC-3940-II also could be because of interaction with neovascularization, which is vital for tumor growth (55). Thus, the treatment with BN antagonist RC-3095, which is related to RC-3940-II, significantly inhibited neoangiogenesis in renal cell carcinomas (55).
Taken together, our work shows that BN/GRP antagonist RC-3940-II inhibits the growth of H460 and A549 human NSCLC cell lines xenografted orthotopically into nude mice. This inhibition of proliferation in vivo was associated with a marked down-regulation of K-Ras/pErk, COX-2, pAkt, and LRP and an up-regulation of WT p53. This favorable profile of activity of RC-3940-II could offer a new multimodality approach against NSCLC, especially in combination with current chemotherapeutic agents. Our work suggests that antagonists of BN/GRP inhibit growth of some human NSCLC cell lines and merit to be considered for further experimental studies aimed at confirmation and extension of these findings.
Materials and Methods
Peptides and Reagents.
BN/GRP antagonist RC-3940-II, with the structure of [Hca6, Leu13 ψ(CH2N)-Tac14]BN (6–14), based on the BN sequence and containing a reduced peptide bond at its COOH terminus originally synthesized in our laboratory (2, 21), was made and provided by Zentaris AG. Matrigel (phenol red-free) was obtained from BD Biosciences. For daily injection, the compounds were dissolved in 0.1% DMSO in 10% aqueous propylene glycol solution.
Cell Line and Animals.
Human NSCLC H460 and A549 cell lines were obtained from American Type Culture Collection. H460 was grown in RPMI supplemented with 1 μM sodium pyruvate, and A549 was grown in F-12K medium. Trypan blue staining was used to assess cell viability, and only single-cell suspensions of viability >90% were used for the intrapulmonary injection.
Five- to six-week-old female athymic nude mice (Ncr nu/nu) were obtained from the National Cancer Institute. The animals were housed in laminar air-flow cabinets under pathogen-free conditions with a 12-h light/12-h dark schedule and fed autoclaved standard chow and water ad libitum.
The surgery procedure for the orthotopic xenograft model as reported by Doki et al. (56) was used with modification. Under deep anesthesia with isoflurane, a 5-mm skin incision in the left chest was made ≈4 mm (tail side) from the scapula. Fat and muscle were separated from costal bones. On observing left lung motion through the pleura, a 27-gauge needle attached to a 50-μl Hamilton syringe was directly inserted through the sixth intercostal space into the lung to a depth of 3 mm. Human H460 (5 × 105) and A549 (1 × 106) cells suspended in 20 μl of Hanks' balanced salt solution containing Matrigel (1:1) were injected into the lung parenchyma. After the injection, a cotton-tipped applicator was pressed on the injection site to stop any bleeding, and the skin incision was closed with a surgical skin clip. A pilot study showed that all animals (n = 20) developed lung cancer with macroscopically visible tumor nodes within 4 days after intrapulmonary injection of H460 cells, whereas animals orthotopically xenografted with A549 cells developed detectable tumors within 3 weeks. Thus, after 4 days for H460 and after 21 days for A549, mice were randomized and treated with vehicle or RC-3940-II at the dose of 10 μg/day per animal. Body weight was measured weekly. There was no significant intergroup difference in body weight at the beginning of the treatment, as shown in Table 1. After 21 days of treatment, when control mice started to become moribund with symptoms of weight loss and elevated breathing rates, animals were killed under deep anesthesia by exsanguination of the inferior vena cava. After removal of the heart, the whole lung block was weighed. One-half of the macroscopical tumor was fixed in 10% buffered formalin for histological examination, and the other half was snap-frozen and stored at −70°C until further analyses. Histopathological examination of each specimen was undertaken to confirm the presence of cancer with minimal admixed nonmalignant tissue (<20%) before Western blotting and receptor-binding studies.
All experiments were reviewed by the institutional animal care and use committee and were performed in accordance with institutional guidelines for animal care.
Cell Proliferation Determination (MTT Assay).
For MTT assay, H460 and A549 human NSCLC cell lines (2,500 cells per well) were seeded in serum-free and serum-reduced growth medium (1% FCS) and grown overnight. After 24 h, BN/GRP antagonist RC-3940-II or DMSO only (0.1% vol/vol) was added, and the incubation was continued for 72 h. Cell growth was determined by MTT assay as described in ref. 57.
Receptor-Binding Assays.
Radioligand-binding assays for receptors of BN/GRP in H460 and A549 NSCLC samples were performed as described in ref. 10. Binding characteristics of receptors for BN/GRP were determined by in vitro ligand competition assays based on the binding of radiolabeled [Tyr4]BN to tumor membrane fractions (10).
Western Blotting Assays.
Orthotopic H460 and A549 tumors were homogenized, and 20-μg amounts of protein from each sample were separated by SDS/7.5–15% PAGE Tris·HCl Criterion Precasted Gels (Bio-Rad) depending on the molecular weight of the selected protein. The membranes were incubated for 3–5 h at room temperature in 5% nonfat dry milk in TBS-Tween, followed by incubation with the specific polyclonal antisera (1:1,000) to COX-2 (C-20), Akt1/2 (N-19), Phospho-Akt (Ser 473), and LRP and monoclonal antibodies (1:1,000) to COX-2 (29), K-Ras (F234), Erk1/2, pErk1/2 (Thr 202/Tyr 204), and p53 (DO-1). All antibodies were from Santa Cruz Biotechnology, except the Phospho-Akt (Ser 473) Erk1/2, pErk1/2 (Thr 202/Tyr 204), and LRP, which were purchased from Cell Signaling Technology. The blots were probed at 4°C overnight with the specific antisera, and the signal for the immunoreactive proteins was developed with peroxidase-conjugated secondary antibodies (Santa Cruz Biotechnology) and visualized by exposure to the chemiluminescence substrate (Amersham Biosciences). The protein bands were quantified by normalizing the signals of different proteins to β-actin signal (1:2,000; Santa Cruz Biotechnology) by using the Kodak EDAS 290 imaging system with 1D Image Analysis Software.
Statistical Analyses.
SigmaStat Software (Jandel Scientific) was used for the statistical analysis of data. Results are presented as means ± SE and were evaluated by one-way ANOVA test.
Acknowledgments
We thank Dr. J. Varga for suggestions. This work was supported by the Medical Research Service of the Veterans Affairs Department, a grant from Zentaris GmbH to Tulane University (to A.V.S.), and the Foundation Propter Homines Fürstentum Liechtenstein 9490 Vaduz (C.D.).
Footnotes
- ¶To whom correspondence should be addressed. E-mail: andrew.schally{at}va.gov
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Author contributions: F.H., A.V.S., A.M., and C.D. designed research; F.H., C.A.K., S.B., B.B., C.K., A.M., E.A., C.D., and G.H. performed research; A.V.S. contributed new reagents/analytic tools; F.H., A.V.S., S.B., C.D., and G.H. analyzed data; and F.H. and A.V.S. wrote the paper.
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↵ †Present address: Department of Internal Medicine, Hospital Oberndorf, 5110 Oberndorf, Austria.
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↵ ‖Present address: Department of Physiology, State University of Health Sciences of Alagoas, 57010 Maceio, Brazil.
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↵**Present address: Department of Biopharmacy, School of Pharmacy, University of Debrecen, H-4032, Debrecen, Hungary.
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Conflict of interest statement: Tulane University has a patent on BN/GRP antagonists, and A.V.S. is one of the inventors. However, this study is purely academic, and A.V.S. is now affiliated with the University of Miami.
- © 2007 by The National Academy of Sciences of the USA