Skip to main content
NCBI home page
NIHPA Author Manuscripts logoLink to NIHPA Author Manuscripts
. Author manuscript; available in PMC: 2014 Dec 15.
Published in final edited form as: Biochem Pharmacol. 2013 Oct 4;86(12):1664–1672. doi: 10.1016/j.bcp.2013.09.022

Novel Compound 1, 3-bis (3, 5-dichlorophenyl) urea Inhibits Lung Cancer Progression

Sharad S Singhal 1, James Figarola 1, Jyotsana Singhal 1, Lokesh Nagaprashantha 1, Samuel Rahbar 1, Sanjay Awasthi 1
PMCID: PMC4186798  NIHMSID: NIHMS535378  PMID: 24099794

Abstract

The successful clinical management of lung cancer is limited by frequent loss-of-function mutations in p53 which cooperates with chronic oxidant-stress induced adaptations in mercapturic acid pathway (MAP) which in turn regulates critical intracellular signaling cascades that determine therapeutic refractoriness. Hence, we investigated the anti-cancer effects and mechanisms of action of a novel compound called 1, 3 bis (3, 5-dichlorophenyl) urea (COH-SR4) in lung cancer. Treatment with COH-SR4 effectively inhibited the survival and clonogenic potential along with inducing apoptosis in lung cancer cells. COH-SR4 treatment caused the inhibition of GST activity and G0/G1 cell cycle arrest and inhibited the expression of cell cycle regulatory proteins CDK2, CDK4, cyclin A, cyclin B1, cyclin E1, and p27. The COH-SR4 activated AMPK pathway and knock-down of AMPK partially reversed the cytotoxic effects of COH-SR4 in lung cancer. COH-SR4 treatment lead to regression of established xenografts of H358 lung cancer cells without any overt toxicity. The histopathology of resected tumor sections revealed an increase in pAMPK, a decrease in the nuclear proliferative marker Ki67 and angiogenesis marker CD31. Western-blot analyses of resected tumor lysates revealed a decrease in pAkt and anti-apoptotic protein Bcl2 along with an increase in pAMPK, pro-apoptotic protein Bax and cleaved PARP levels. Importantly, COH-SR4 lead to decrease in the mesenchymal marker vimentin and increase in the normal epithelial marker E-cadherin. The results from our in-vitro and in-vivo studies reveal that COH-SR4 represents a novel candidate with strong mechanistic relevance to target aggressive and drug-resistant lung tumors.

Keywords: Lung cancer, SR4, AMPK, tumor xenografts

1. Introduction

Lung cancer remains the most prominent cause of cancer mortality in the developed world [1]. According to recent estimates in 2012, there will be 226, 000 new cases and 160, 000 deaths yearly from lung cancer in the United States. A dynamic interplay between host and environmental factors is responsible for the development, progression and the acquisition of drug-resistance in lung cancer. The major risk factor for the development of lung cancer is tobacco smoking and about 90% of all lung cancer patients are current or previous smokers indicating tobacco smoke and associated oxidative-stress in both incidence and progression of lung tumors [2–4]. Also, a significant fraction of the remaining patients have other risk factors, such as passive smoking, certain genetic factors or exposure to other environmental pathogens [4–6]. This calls for development of novel agents for the treatment of small-cell and non-small cell lung cancers [7, 8].

The chronic oxidant-stress induced by tobacco smoking as well as oxidative-stress prevalent in lung tumors leads to up-regulation of the cellular defense pathways that enhance detoxification of toxic products of lipid peroxidation resulting from oxidant-stress. Mercapturic acid pathway (MAP) represents a central axis of the detoxification of toxic end-products of lipid peroxidation [9]. The products of lipid peroxidation like 4-hydroxynonenal (4-HNE) which are formed due to oxidative-stress are conjugated by cellular glutathione S transferases (GSTs) leading to formation of glutathione-conjugate of 4-HNE (GS-HNE) which is rapidly effluxed by the MAP transporter RLIP76, thereby simultaneously preventing the cellular cytotoxicity and feedback product inhibition of GST [10]. Our previous studies have revealed that small molecule inhibitors of RLIP76 and RLIP76 targeted antibody are effective choices for targeting lung cancer progression and drug-resistance, which reinforces the mechanistic significance of these first two rate limiting steps in MAP [11].

The loss of tumor suppressor p53 has not only been implicated as an early molecular event in the development of lung cancer in smokers, but also enables the acquisition of drug-resistant and metastatic phenotypes [12]. Activating mutations in intracellular signal transduction pathways like KRAS G12V lead to constitutive activation of proliferative signals which further enhance the aggressive behavior of p53 null lung tumors [13, 14]. The onset of epithelial-to-mesenchymal transition that accompanies the malignant transformation of normal cells is associated with increased expression of fibronectin and vimentin [15]. Tumor cells also modulate the intracellular energy sensor pathways mediated by the AMPK pathway. Suppression of AMPK activation allows for tumor cell survival in energy depleted conditions [16]. Thus, novel agents that can collectively target the critical nodes of adaptations to oxidative-stress, low energy status and enhanced proliferative signaling in lung cancer cells would immensely contribute to the development of more effective therapies for lung cancer. Our previous studies have identified dichlorophenyl urea compound as an active compound effective in a panel of cancer cell lines and xenografts, including leukemia and melanoma [17, 18]. Hence, we are presenting a study of the effect of COH-SR4 on critical signaling proteins in lung cancer.

2. Materials and methods

2.1 Reagents

Terminal deoxynucleotidyl-transferase deoxyuridine triphosphate nick-end labeling (TUNEL) fluorescence and avidin/biotin complex (ABC) detection kits were purchased from Promega (Madison, WI) and Vector (Burlingame, CA), respectively. MTT, horseradish-peroxidase (HRP)-conjugated anti-mouse, and anti-rabbit secondary antibodies were procured from Sigma (St. Louis, MO). AMPK and scrambled siRNA were obtained from Invitrogen (San Diego, CA). XTT proliferation assay kit was purchased from American Type Culture Collection (ATCC, Manassas, VA). PARP, Bcl2, β-actin, vimentin, fibronectin, Bim, Akt, pAkt (S473), GAPDH, Ki67, and CD31 antibodies were purchased from Santa Cruz Biotechnology (Columbus, OH) and Cell Signaling Technologies (Danvers, MA). Antibodies against CDK2, CDK4, cyclin A, cyclin B1, cyclin E1, p27Kip1, AMPK, pAMPK (T172), ACC, pACC (S79), Raptor, p-Raptor (S792), TSC2, and pTSC2 (S1387) were purchased from Cell Signaling Technology (Danvers, MA, USA). Universal Mycoplasma Detection Kit was procured from ATCC (Manassas, VA).

2.2 Synthesis of COH-SR4

The 1,3-bis(3,5-dichlorophenyl)urea compound “COH-SR4” was synthesized according to a previously validated protocol by Dr. Christopher Lincoln, Director of Chemical GMP Synthesis Facility at Beckman Research Institute, City of Hope [17]. Briefly, 3,5-dichlorophenyl isocyanate (1.21g (96%), 6.17 mmol) was added portion-wise to a stirring solution of 3,5-dichloroaniline (1.00g (98%), 6.33 mol) in dichloromethane (15 mL) under N2. After 19 h at ambient temperature, the entire reaction mixture was filtered and the filter cake was washed with dichloromethane (2 × 10 mL). The solid was dried in-vacuo to obtain 1, 3-bis (3,5-dichlorophenyl) urea (1.78g, 82%) as a white crystalline solid. 1H-NMR (400 MHz, DMSO-d6) δ 9.35 (s, 2H), 7.53 (d, J = 1.8 Hz, 4H), 7.17 (t, J = 1.8 Hz, 3H); 13C NMR (100 MHz, DMSO-d6) δ 152.0, 141.8, 134.1, 121.3, 116.7; HRMS-ESI (m/z (%)) 348.9278 (100), 346.9310 (73), 350.9255 (48), 349.9323 (10), 352.9239 (8), 347.9357 (7), 351.9303 (4).

2.3 Cell Lines and Cultures

The H1417, H1618, H520 and H358 lung cancer cell lines were purchased from the ATCC. Normal human aortic vascular smooth muscle cells (HAVSMC) was kindly authenticated and donated by Dr. Paul Boor, UTMB, Galveston, TX. All cells were cultured at 37 °C in a humidified atmosphere of 5 % CO2 in the appropriate medium: RPMI-1640 (H1417, H1618, H520 and H358) and DMEM (HAVSMC) medium supplemented with 10 % heat-inactivated FBS and 1% penicillin/streptomycin (P/S) solution [18–20]. The cells were immediately expanded and frozen after being obtained from ATCC and restarted every 3 to 4 months from a frozen vial of the same batch of cells and no additional authentication was done on these cells. All cells lines were free of Mycoplasma infection tested by Universal Mycoplasma Detection kit.

2.4 Cell survival (MTT) assay

Cell density measurements were performed using a hemocytometer to count reproductive cells resistant to staining with trypan blue. Approximately 20,000 cells were plated into each well of 96-well flat-bottomed micro-titer plates. After 24 h incubation at 37 °C, medium containing COH-SR4 (ranging 0–100 µM) was added to the cells. After 48 h incubation, 20 µL of 5 mg/mL MTT were introduced to each well and incubated for 2 h. The plates were centrifuged and medium was decanted. Cells were subsequently dissolved in 100 µL dimethyl-sulfoxide with gentle shaking for 2 h at room temperature, followed by measurement of optical density at 570 nm [18–20].

2.5 Colony forming assay

Cell survival was also evaluated using a standard colony-forming assay. 1×105 cells / mL were incubated with COH-SR4 (1.5 µM) for 24 h, and aliquots of 50 or 100 µL were added to 60-mm size Petri dishes containing 4 mL culture medium. After 7 days, adherent colonies were fixed, stained with 0.5% methylene blue for 30 min, and colonies were counted using the Innotech Alpha Imager HP [21].

2.6 Cell proliferation assays (XTT assay)

The effects of SR4 on cell viability were assessed in quadruplicate samples using the 2,3-bis (2-methoxy-4-nitro-5-sulfophenly)-5-[(phenylamino) carbonyl]-2H-tetrazolium hydroxide (XTT) assay. Approximately 20,000 cells were seeded and incubated in 96-well, flat-bottomed plates in 10% FBS-supplemented culture medium 24 h before drug treatment. After 24 h incubation at 37 °C, cells were then exposed to the indicated concentrations of drug at 37 °C in 5% CO2 for 48 h. The medium was removed and replaced with 100 µl fresh medium containing 50 µl of the activated-XTT solution to each well, and the cells were further cultured in the CO2 incubator at 37 °C for 4 h. Absorbance was determined on a plate reader at 475 nm.

2.7 Western blotting

Cell or tissue proteins were extracted with cell lysis buffer (Cell Signaling Technology) and protein concentration was determined using the DC Protein Assay kit (Bio-Rad, Hercules, CA, USA). Equal amount of proteins (~40 µg) were loaded onto 4–15% Criterion TGX gels (Bio-Rad, Hercules, CA), resolved by SDS-PAGE electrophoresis, and then transferred onto nitrocellulose membranes for immunoblotting. Membranes were blocked with 5% skimmed milk in Tris-buffered saline containing 0.05% Tween 20 before incubation overnight at 4 °C with desired primary antibodies. Immuno-reactive proteins were visualized by peroxidase-labeled secondary antibodies and ECL system (Western Lightning Chemiluminescence Reagent, Perkin-Elmer, MA, USA). Equal loading of proteins was confirmed by stripping and re-probing the membranes with either β-actin or GAPDH antibodies. Band intensities were quantified using a densitometer (Quantity One, Bio-Rad, Hercules, CA).

2.8 TUNEL Apoptosis assay

For TUNEL assay, 1×105 cells were grown on the cover-slips for ~12 h followed by treatment with COH-SR4 (1.5 µM) for 24 h. Apoptosis was determined by the labeling of DNA fragments with TUNEL assay using Promega fluorescence apoptosis detection system [21, 22].

2.9 Flow cytometry analysis of cell cycle regulation

2 × 105 cells were treated with COH-SR4 (0–5 µM) for 18 h at 37 °C. After treatment, floating and adherent cells were collected, washed with PBS, and fixed with 70 % ethanol. On the day of flow analysis, cell suspensions were centrifuged; counted and equal numbers of cells were resuspended in 500 µL PBS in flow cytometry tubes. Cells were then incubated with 2.5 µL of RNase (stock 20 mg/ml) at 37 °C for 30 min after which they were treated with 10 µL of propidium iodide (stock 1mg/mL) solution and then incubated at room temperature for 30 min in the dark. The stained cells were analyzed using the Beckman Coulter Cytomics FC500, Flow Cytometry Analyzer. Results were processed using CXP2.2 analysis software from Beckman Coulter.

2.10 In vivo xenograft studies

Hsd: Athymic nude nu/nu mice were obtained from Harlan, Indianapolis, IN. All animal experiments were carried out in accordance with a protocol approved by the Institutional Animal Care and Use Committee (IACUC). Twelve 10-weeks-old mice were divided into two groups of 6 animals (treated with corn oil (vehicle), and COH-SR4 compound 4 mg / kg b.w.). All animals were injected with 2 × 106 H358 cells suspensions in 100 µL of PBS, subcutaneously into one flank of each mouse. At the same time, animals were randomized treatment groups as well as control groups (Fig 4). Treatment was started 10 days after the implantation to see palpable tumor growth. Treatment consisted of 0.1 mg of COH-SR4/mice in 200 µL corn oil by oral gavage alternate day. Control groups were treated with 200 µL corn oil by oral gavage alternate day. Animals were examined daily for signs of tumor growth. Tumors were measured in two dimensions using calipers. Photographs of animals were taken at day 1, day 10, day 14, day 18, day 30, and day 60 after subcutaneous injection, are shown for all groups. Photographs of tumors were also taken at day 60.

Figure 4. Effect of oral administration of COH-SR4 on progression of lung cancer xenografts in mice.

Figure 4

Open in a new tab

Hsd: Athymic nude nu/nu mice were obtained from Harlan, Indianapolis, IN. Twelve 10-weeks-old mice were divided into two groups of 6 animals (treated with corn oil (vehicle), and COH-SR4 4 mg / kg b.w.). All animals were injected with 2 × 106 H358 cells suspensions in 100 µL of PBS, subcutaneously into one flank of each mouse. Treatment was started 10 days after the implantation to see palpable tumor growth. Treatment consisted of 0.1 mg of COH-SR4/mice in 200 µL corn oil by oral-gavage alternate day. Control groups were treated with 200 µL corn oil by oral-gavage alternate day. Animals were examined daily for signs of tumor growth and body weights were recorded (panel A). Weights and photographs of tumors were taken at day 60 for comparing the change in final tumor weight between COH-SR4 treated and control groups (panel B). Tumors were measured in two dimensions using calipers and time-course analysis of tumor regression was performed during the study (panel C). Photographs of animals were also taken at day 1, 10, 14, 18, 30, and 60, after subcutaneous injection, are shown for all groups (panel D).

2.11 Histopathological examination of tumors for angiogenic, proliferative and differentiation markers

Control and COH-SR4 treated H358 lung cancer bearing mice tumor sections were used for histopathologic analyses. Immuno-histochemistry analyses were performed for Ki67, CD31, E-cadherin, and pAMPK expressions. Immuno-reactivity is evident as a dark brown stain, whereas non-reactive areas display only the background color. Sections were counterstained with Hematoxylin (blue). Photomicrographs at 40× magnification were acquired using Olympus DP 72 microscope and were processed with DP2-BSW software. Percent staining was determined by measuring positive immuno-reactivity per unit area. The intensity of antigen staining was quantified by digital image analysis using Image Pro plus 6.3 software.

2.12 Statistical Analyses

All data were evaluated with a two-tailed unpaired student’s t test are expressed as the mean ± SD. The statistical significance of differences between control and treatment groups was determined by ANOVA followed by multiple comparison tests. Changes in tumor size and body weight during the course of the experiments were visualized by scatter plot. Differences were considered statistically significant when the p value was less than 0.05.

3. Results

3.1 Impact of COH-SR4 treatment in-vitro on lung cancer cell survival, clonogenic potential and apoptosis

The dichlorophenyl urea compound COH-SR4 was synthesized by the Drug Discovery Core Facility within City of Hope’s Comprehensive Cancer Center [17]. The structure of COH-SR4 is represented in Figure 1A. The effect of COH-SR4 was first examined in a NCI panel of cancer cell lines, the results of which indicated good activity of COH-SR4 towards lung cancer (Fig 1B). We confirmed activity of COH-SR4 in a selected panel of lung cancer cell lines after 48 h treatment using MTT assay. The COH-SR4 treatment exerted a significant inhibitory effect on the survival of lung cancer cells [IC50: H1417 cells-1.2 ± 0.2 µM, H1618 cells- 1.5 ± 0.2 µM, H358 cells-2.1 ± 0.2 µM and H520 cells-2.4 ± 0.3 µM]. One of the striking observations was that COH-SR4, at concentrations effective in inhibiting the survival of lung cancer cell lines, did not exert any significant cytotoxicity in normal HAVSMC (Fig 1C). Following initial screening for the anticancer activity of COH-SR4, we further studied the effect of COH-SR4 on clonogenic potential. The COH-SR4 (1.5 µM) treatment resulted in 39 ± 8 %, 48 ± 9 %, 47 ± 7% and 54 ± 5% colony formation in H1417, H1618, H358 and H520 lung cancer cells, respectively. In accordance with MTT assay, the 1.5 µM COH-SR4 treatment did not result in significant inhibition of colony formation in normal HAVSMC (Fig 1D). We further investigated the effect of COH-SR4 on induction of apoptosis. The 1.5 µM COH-SR4 treatment for 24 h induced apoptosis in all the lung cancer cell lines as determined by TUNEL assay (Fig 1E). These studies revealing the COH-SR4 induced anti-proliferative and pro-apoptotic effect in lung cancer cells were promising and lead to further in-vitro and in-vivo characterization of the anticancer effects of COH-SR4.

Figure 1. Anti-proliferative and pro-apoptotic effects of COH-SR4 in lung cancer.

Figure 1

Open in a new tab

The chemical structure of 1,3-bis(3,5-dichlorophenyl)urea compound also called “COH-SR4” (panel A). Dose-dependent growth inhibition of various lung cancer cell lines by COH-SR4 (NIH/NCI DTP60 screening data) (panel B). Drug-sensitivity assays were performed by MTT assay using COH-SR4 at 48 h post-treatment to determine IC50. Values are presented as mean ± SD from two separate determinations with eight replicates each (n= 16) (panel C). Colony-forming assay was performed and the colonies were counted using Innotech Alpha Imager HP. * p < 0.001 compared with control (n=3, panel D). For TUNEL apoptosis assay, cells were grown on cover-slips and treated with 1.5 µM COH-SR4 for 24 h. TUNEL assay was performed using Promega fluorescence detection kit and examined using Zeiss LSM 510 META laser-scanning fluorescence microscope with filters 520 and 620 nm. Photographs taken at identical exposure at × 40 magnification are presented. Apoptotic cells showed green fluorescence (panel E).

3.2 Effect of COH-SR4 on cell cycle progression and GST activity in lung cancer

The treatment with COH-SR4 induced G0/G1 phase arrest in a concentration dependent manner thereby providing corroborative evidence for the anti-proliferative and pro-apoptotic effects of COH-SR4 in lung cancer (Fig 2A). In the context of the elevation of GSTs in lung cancer [23], we further analyzed the impact of COH-SR4 on the catalytic activity of GSTs towards 1-chloro 2, 4-dinitro benzene (CDNB), a model substrate used for GST activity [24]. The COH-SR4 treatment inhibited the total GST activity to a significant extent in the lung cancer cells (Fig 2B). GSTs are a class of phase II detoxifying enzymes, which regulate detoxification of administered chemotherapy drugs for further efflux out of cells by transport proteins. The GST inhibition leads to accumulation of toxic end-products of lipid peroxidation due to decreased efflux of GS-HNE which also reinforces the GST inhibition by feedback inhibition [25]. The over-expression of GSTs is a common phenomenon associated with malignant progression of many cancers including lung cancer, melanomas skin and prostate cancers [23, 26, 27]. Previous studies have shown that the glutathione-conjugate transport by the MAP transporter RLIP76 is essential for the clathrin-dependent ligand-receptor endocytosis (CDE) which in turn regulates the activation of intracellular signaling cascades [10, 28], we further investigated the effect of COH-SR4 on downstream signaling proteins of significance for cellular proliferation and survival.

Figure 2. Effect of COH-SR4 on cell cycle progression in lung cancer.

Figure 2

Open in a new tab

Inhibitory effect of COH-SR4 on cell cycle distribution was determined by fluorescence activated cell sorting (FACS) analysis (panel A). GST activity towards 1-chloro 2,4-dinitro benzene (CDNB) and its inhibition by COH-SR4 was performed in 28000×g crude supernatant prepared from H1417, H1618, H520 and H358 cells. Human liver purified GST was used as a control (inset). The inhibitory effect of COH-SR4 on GST was studied at a fixed concentration of GSH and CDNB (1 mM each) and varying concentrations of inhibitor. The enzymes were pre-incubated with the inhibitor for 5 min at 37 °C prior to the addition of the substrates (panel B). The experiment was repeated three times and similar results were obtained.

Western-blot results of cell cycle regulator proteins confirmed that COH-SR4 induces cell cycle arrest in H358 and H520 lung cancer cells. After 24 h treatment, COH-SR4 decreased the protein levels of CDK2, CDK4, cyclin A, cyclin B1 and cyclin E1. In addition, the protein level of p27, a potent CDK inhibitor of cyclin E- and cyclin A-CDK2 complexes involved in G1 arrest, was up-regulated by COH-SR4 (Fig 3A). Based on these results, COH-SR4 treatment modulated the level of proteins active during S and G2 phases of the cell cycle, confirming the results of FACS analysis indicating G1 arrest induced by COH-SR4.

Figure 3. Western-blot analyses and cell survival consequent to AMPK knock-down.

Figure 3

Open in a new tab

Western-blot analyses revealing the effect of COH-SR4 on cell cycle proteins (panel A), AMPK pathway (panel B) and expression of AMPK and ACC following siRNA mediated knock-down of AMPK (panel C). Cell survival determined by XTT assay showing the effect of COH-SR4 following siRNA mediated knock-down of AMPK (panel D). Numbers below the blots represent the fold change in the levels of proteins after SR4 treatment as compared to control.

AMPK, a known transducer of signaling generated from cellular energy depletion, has been implicated to be activated consequent to oxidant-stress and inhibit mTOR pathway through downstream activation of TSC2 [29]. COH-SR4 treatment lead to increased levels of pAMPK (T172) along with an increase in the activation of down-stream pACC (S79), pRaptor (S792) and pTSC2 (S1387) without significant change in the targeted protein levels of AMPK pathway (Fig 3B). As AMPK is a critical regulator of both tumor cell energy and response to oxidant-stress, we knocked-down AMPK by siRNA and assessed the impact on SR4-induced cytotoxicity. The knock-down of AMPK lead to significant reversal in the cytotoxicity of COH-SR4 in both H358 and H520 cells (Fig 3C and 3D). Thus, the in-vitro analyses of critical signaling proteins of cell proliferation provided supportive mechanistic evidence for the anti-cancer effects of COH-SR4 in lung cancer cells. Hence, we further tested the efficacy of COH-SR4 in-vivo in mouse xenografts models of lung cancer.

3.3 Anti-tumor effect of COH-SR4 in-vivo on lung cancer progression

Hsd: Athymic nude nu/nu mice were used for the oral administration of COH-SR4 on lung cancer progression in in-vivo xenograft model. All animals were subcutaneously injected with 2 × 106 H358 cells in 100 µL of PBS into one flank of each mouse. Treatment was started 10 days after the implantation of cells. Treatment consisted of 0.1 mg (4 mg/kg b.w.) of COH-SR4/mice in 200 µL corn oil by oral gavage alternate day. Control groups were treated with 200 µL corn oil by oral gavage alternate day. The 0.1 mg of COH-SR4 treatment was well tolerated by the mice and did not result in significant change in animal body weight or any signs of overt toxicity (Fig 4A). Animals were examined daily for signs of tumor growth. Tumors were measured in two dimensions using calipers. The COH-SR4 treatment lead to significant reduction in the tumor burdens in the treated groups [2.02 ± 0.3 g vs. 0.77 ± 0.2g in control and COH-SR4 treated groups, respectively, on day 60] (Fig 4B). The COH-SR4 treatment revealed a substantial inhibition of tumor progression as evident by a time course analyses of the tumor sizes at different points (Fig 4C). Photographs of animals were taken at day 1, day 10, day 14, day 18, day 30 and day 60 after subcutaneous injection, are shown for all groups (Fig 4D). We also assessed the absorption of orally administered COH-SR4 in mice. HPLC analysis of 4 mg/kg COH-SR4 treated mice serum revealed that COH-SR4 is effectively absorbed after oral dosage and it reaches a serum concentration of 1.1± 0.3 µM.

3.4 Histopathological examination of control and COH-SR4 treated tumors

Following the in-vivo animal studies; the histopathological examination of paraffin-embedded tumor xenograft sections by H&E staining revealed that COH-SR4 reduces the number of tumor blood-vessels and restores the normal morphology when compared to controls. COH-SR4 treatment decreased the levels of proliferation marker Ki67, and angiogenesis marker CD31 as revealed by ABC staining. COH-SR4 treatments lead to increase in the levels of pAMPK and normal differentiation marker E-cadherin, which provides corroborative evidence for the induction of anti-tumor effects using in-vivo models of lung cancer (Fig 5A).

Figure 5. Histopathologic and Western-blot analyses of resected tumors after COH-SR4 treatment.

Figure 5

Open in a new tab

Control and COH-SR4 treated tumor sections were used for histopathologic analyses. Immuno-histochemical analyses for Ki67 (marker of cellular proliferation), CD31 (angiogenesis marker), E-cadherin (tumor suppressor) and pAMPK (cellular regulator of lipid and glucose metabolism) expressions were performed from tumors in mice of control and COH-SR4 treated groups. Arrows represent the area for positive staining for an antigen. Bars represent means with 95% confidence intervals (n = 5); Asterisks denote statistically significant differences (p<0.001) compared with control by two-sided Student’s t test. Statistical significance of difference was determined following image analyses as described in methods section (panel A). Western-blot analyses of signaling proteins in tumor tissue lysates in control and COH-SR4 treated experimental groups. GAPDH was used as internal control. The bar diagrams represent the fold change in the levels of proteins as compared to controls as determined by densitometry. Dotted line represents no significant change as observed with control (panel B).

3.5 Effect of COH-SR4 on tumor signaling pathways in lung cancer

The effect of COH-SR4 on signaling pathways of relevance to lung cancer progression was further analyzed by Western-blots of lysates from resected tumors. The COH-SR4 treated groups had high levels of the cleaved-PARP compared to untreated controls, which reinforces the finding of apoptotic effect as observed in-vitro by TUNEL assay. Akt is a critical signaling protein that transduces the proliferative signals from upstream integrins and growth factor receptors [30]. The COH-SR4 treatments lead to an increase in the levels of PARP cleavage along with decreasing the levels of pAkt (S473). The activated AMPK transduces signals through mTOR pathway [31]. In accordance with decreased levels of pAkt (S473), the levels of pP70S6K were decreased in COH-SR4 treated groups compared to controls. The cellular levels of vimentin and fibronectin determine the extent of migration and proliferation in lung cancer cells [32, 33]. We also analyzed SR4 treated tumor tissue lysates to test the impact of long term SR4 treatment in vivo on GST expression and activity. Western blot analyses of tumor tissues revealed a decrease in the levels of GSTπ in SR4 treated groups compared to controls. The total GST activity as measured by activity towards CDNB as a substrate, in SR4 treated tumor tissue lysates was lower compared to controls [control, 0.32 ± 0.03 U/mg protein; SR4 treated, 0.21 ± 0.05 U/mg protein; (n = 3)]. COH-SR4 treatment leads to decrease in the expression of vimentin and fibronectin which are associated with invasive progression of lung cancer. COH-SR4 treated groups had an enhanced expression of pro-apoptotic protein Bax along with a parallel decrease in the levels of anti-apoptotic protein Bcl2. Importantly, the expression of cell cycle regulatory proteins CDK4, which is a critical determinant of KRAS G12V induced lung tumor formation, and Cyclin B1 were also decreased following COH-SR4 treatment [34] Also, the normal epithelial marker E-cadherin showed increased expression following COH-SR4 treatment. In accordance with the in-vitro Western-blot analyses and in-vivo histopathological examination, the levels of pAMPK (T172) were enhanced in COH-SR4 treated groups compared to controls in tumor tissue lysates (Fig 5B).

4. Discussion

The management of lung cancer continues to represent a major clinical challenge [35]. Advances in the understanding of the molecular and histologic profiles of different subsets of lung cancers have been paralled by the development of novel effective therapies. The present study on the anticancer effects of a novel dichlorophenyl urea compound “COH-SR4” holds great future promise for further development in lung cancer therapy.

Our initial findings on the ability of COH-SR4 to inhibit the growth of a panel of lung cancer cell lines was further corroborated by assessment of the inhibition of clonogenic potential, angiogenesis, cell proliferation and cellular energy metabolism. In addition, we observed a decrease of mesenchymal and antiapoptotic markers, accompanied by an increase in physiologic epithelial markers and proapoptotic proteins in the COH-SR4 treated cells. The early progression of lung cancers following transformation has been associated with the process of epithelial-to-mesenchymal transition [32, 36].

The oxidative-stress and the nicotine exposure associated with tobacco smoke are known to enhance the expression of fibronectin, a component of lung interstitium [37–39]. In our study, we observed a significant down-regulation of fibronectin and vimentin expression, a mesenchymal marker, whilst the physiologic epithelial marker E-cadherin was up-regulated when the cancer cells were exposed to COH-SR4. Along with a profound reduction of Ki67, we observed a decreased angiogenic response in COH-SR4 treated animals compared to controls. This was reflected by a decrease in the number of blood vessels as determined by gross H& E staining as well as in the angiogenesis marker CD31. These findings are of potential significance as angiogenesis is for long a wide field of study and clinical application in the treatment of lung cancer [40].

The ability of COH-SR4 to inhibit GST activity and impact multiple downstream signaling cascades is highly significant in targeting both the progression and therapy resistance of lung cancer. Our Western-blot analyses on resected tumor tissue lysates revealed COH-SR4 induced changes on tumorigenic intracellular signaling transduction pathways, including apoptotic pathway proteins like Bcl2 and Bax (Fig 5B). The expression of pro-apoptotic protein Bax was increased where as the levels of anti-apoptotic Bcl2 were decreased in COH-SR4 treated tumors. In accordance with in-vitro studies, there was an increase in the levels of PARP cleavage, confirming the proapoptotic effect of COH-SR4 in lung cancer.

The oxidative-stress and enhanced metabolic stress due to mismatch between the rates of proliferation and angiogenesis in tumors result in increased levels of AMP. Normally, enhanced AMP/ATP ratio leads to activation of AMPK, which in turn inhibits mTOR [16]. But, in tumor cells, the activation of AMPK is suppressed in spite of high AMP levels [41]. There was an increase in the phosphorylation of AMPK (T172) in COH-SR4 treated tumors as revealed by both histopathological and Western-blot analyses. Also, the levels of pP70S6K were decreased in COH-SR4 treated mice. It has been shown that CDK4, an interphase kinase, is specifically essential for the development of lung tumors in KRAS G12V mutant mice whereas the other interphase kinases CDK2 and CDK6 are dispensable for the oncogenic transformation of lung epithelium in KRAS G12V mutant mice [34].

After studying the cell survival and apoptotic effects of SR4 in a panel of lung cancer cells, we chose H520 and H358 non-small cell lung cancer cells for further investigation of molecular effectors that mediate the anti-cancer activity of SR4 in aggressive lung cancers. GSTs are known to promote aggressive lung cancer growth [23]. The H520 lung cancer cells have very high GST activity compared to many other lung cancer cells [42]. The H358 cells carry a homozygous deletion of p53, a relevant tumor suppressor, along with the constitutively activating KRAS G12V mutation which signals through MAPK pathway which also involves CDK4 [43, 44]. Hence, the results from our in vitro studies and murine xenograft experiments, using the H358 and H520 lung cancer cell lines, provide significant credence to the ability of COH-SR4 to inhibit the progression of lung cancers and regulate critical signaling pathways that are of significance to aggressive lung tumors.

Importantly, the oral administration of COH-SR4 (4 mg/ kg b.w.) for 8 weeks not only lead to significant tumor inhibition was also extremely well tolerated without significant changes in the mice body weight or overt toxicity. Interestingly, the expression of fibronectin, which is associated with tobacco smoking and precancerous conditions like chronic COPD, was decreased in COH-SR4 treated groups compared to controls [33]. Fibronectin signals through its ligand α5β1 integrin. The expression of α5β1 integrin is increased in lung cancer and has been linked to the progression of lung cancers [45]. The integrins signal through PI3K/Akt pathways which regulate critical downstream signaling mediated by Ras/Raf/Mek/Erk MAPK and mTOR pathways [46].

Oxidative stress also leads to up-regulation of the enzymes that catalyze the products of lipid peroxidation, a major process that connects oxidative-stress with its biological effects on cells. The cellular levels of 4-HNE, a major toxic end-product of lipid peroxidation, significantly determine the sensitivity of cells to oxidative-stress-induced toxicity [25]. The cancer cells acclimatize to enhanced levels of oxidative-stress by up-regulating the expression of GSTs which catalyze 4-HNE to form glutathione-adducts (GS-HNE) [25, 37] (Fig 6). An SR4-glutatione-conjugate (GS-SR4) was detected in a reaction mixture containing SR4, GSH and GSTπ at [M+H] = 613, along with multiple de-chlorinated species, consistent a catalytic activity of GST towards SR4 to form glutathione-conjugates (unpublished observations). However, the poor solubility of SR4 in aqueous solutions precluded detailed kinetic characterization of this catalytic activity. In this context, the inhibition of GST activity in lung cancer cells by COH-SR4, and perhaps also product inhibition by GS-SR4 represents a mechanistically significant finding as it is directly associated with decrease substrate availability to MAP enzyme RLIP76, which regulates clathrin-dependent endocytosis of ligand-receptor complexes, and downstream signaling. In summary, the novel dichlorophenyl compound “COH-SR4” induces effective anti-proliferative and pro-apoptotic effects as confirmed by both in-vitro and in-vivo studies. The ability of COH-SR4 to regulate important nodes of oxidative, metabolic and proliferative signaling of specific significance to the tumor biology of lung cancers provide a broad and encompassing mechanistic evidence for the multi-targeted potential of the compound. Hence, COH-SR4 holds great promise as a novel candidate drug for targeting progression and drug-resistance in lung cancers.

Figure 6. Mechanisms of action of COH-SR4 on critical signaling proteins regulating lung cancer survival and progression.

Figure 6

Open in a new tab

Tumors up-regulate detoxification enzymes like GSTs, which play a vital role in buffering the toxic effects of oxidative-stress by catalyzing the glutathione-conjugation of the products of lipid peroxidation like 4-HNE in the initial step of mercapturic acid pathway (MAP). The anticancer compound COH-SR4 inhibits the GST activity resulting in decreased metabolic flux. This results in increased intracellular concentration of toxic products of lipid peroxidation. The efflux of glutathione-conjugates by RLIP76 is essential for the clathrin-dependent ligand-receptor endocytosis (CDE) and downstream activation of signaling cascades [10]. AMPK forms a critical node of transducing oxidant and energy deficiency signals in tumors. Also, the presence of oncogenes like KRAS G12V in lung cancers and suppression of energy sensor proteins like AMPK in spite of high AMP and low ATP levels as seen in tumors, in contrast to normal cells where AMPK is activated when AMP levels are high, together enhance the rate of proliferation and survival of lung cancer cells. Hence, the ability of COH-SR4 to regulate the critical nodes of lung cancer signaling downstream of oxidative-stress and CDE like levels of pACC, pTSC2, raptor, mTOR, p70S6K along with regulating the expression of fibronectin, vimentin, E-cadherin and Ki67, represents a promising spectrum of causes for the anti-cancer effects of COH-SR4. Green arrow: up-regulation following COH-SR4 treatment; Red arrow: down-regulation following COH-SR4 treatment; Blue arrow: normal signal transduction.

Acknowledgements

This work was supported by the National Institutes of Health grant (CA 77495), and the funds from Perricone Family Foundation, Los Angeles, CA. Funding from Department’s Chair (Prof. Arthur Riggs) and Beckman Research Institute of the City of Hope is also acknowledged.

The abbreviations used are

ACC

acetyl Co-A carboxylase

AMPK

AMP-activated protein kinase

COH-SR4

1,3-bis (3,5-dichlorophenyl) urea “City of Hope compound"

GST

glutathione S-transferase

TSC2

tuberous sclerosis complex 2 protein

Footnotes

Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

References

  • 1.Howlader N, Noone AM, Krapcho M, Neyman N, Aminou R, editors. SEER Cancer Statistics Review, 1975–2008. Bethesda, MD: National Cancer Institute; 2011. http://seer.cancer.gov/csr/ 1975_2008/, based on November 2010 SEER data submission, posted to the SEER web site. [Google Scholar]
  • 2.Wild P, Gonzalez M, Bourgkard E, et al. Occupationalrisk factors have to be considered in the definition of high-risk lung cancer populations. Br J Cancer. 2012;106:1346–1352. doi: 10.1038/bjc.2012.75. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 3.Tsurutani J, Castillo SS, Brognard J, et al. Tobacco components stimulate Akt-dependent proliferation and NFkappaB-dependent survival in lung cancer cells. Carcinogenesis. 2005;26:1182–1195. doi: 10.1093/carcin/bgi072. [DOI] [PubMed] [Google Scholar]
  • 4.Freedman ND, Leitzmann MF, Hollenbeck AR, Schatzkin A, Abnet CC. Cigarette smoking and subsequent risk of lung cancer in men and women: analysis of a prospective cohort study. Lancet Oncol. 2008;9:649–656. doi: 10.1016/S1470-2045(08)70154-2. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 5.Darby S, Hill D, Auvinen A, et al. Radon in homes and risk of lung cancer: collaborative analysis of individual data from 13 European case-control studies. BMJ. 2005;330:223. doi: 10.1136/bmj.38308.477650.63. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 6.Heintz NH, Janssen-Heininger YM, Mossman BT. Asbestos, lung cancers, and mesotheliomas: from molecular approaches to targeting tumor survival pathways. Am J Respir Cell Mol Biol. 2010;42:133–139. doi: 10.1165/rcmb.2009-0206TR. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 7.Maemondo M, Inoue A, Kobayashi K, et al. Gefitinib or chemotherapy for non-small-cell lung cancer with mutated EGFR. N Engl J Med. 2010;362:2380–2388. doi: 10.1056/NEJMoa0909530. [DOI] [PubMed] [Google Scholar]
  • 8.Schiller JH, Larson T, Ou SH, et al. Efficacy and safety of axitinib in patients with advanced non-small-cell lung cancer: results from a phase II study. J Clin Oncol. 2009;27:3836–3841. doi: 10.1200/JCO.2008.20.8355. [DOI] [PubMed] [Google Scholar]
  • 9.Marchand DH, Abdel-Monem MM. Glutathione S-transferases catalyzed conjugation of 1,4-disubstituted butanes with glutathione in vitro. Biochem Biophys Res Commun. 1985;128:360–367. doi: 10.1016/0006-291x(85)91687-0. [DOI] [PubMed] [Google Scholar]
  • 10.Awasthi S, Singhal SS, Sharma R, Zimniak P, Awasthi YC. Transport of glutathione-conjugates and chemotherapeutic drugs by RLIP76: a novel link between G-protein and tyrosine-kinase signaling and drug-resistance. Int J Cancer. 2003;106:635–646. doi: 10.1002/ijc.11260. [DOI] [PubMed] [Google Scholar]
  • 11.Singhal SS, Yadav S, Singhal J, Zajac E, Awasthi YC, Awasthi S. Depletion of RLIP76 sensitizes lung cancer cells to doxorubicin. Biochem Pharmacol. 2005;70:481–488. doi: 10.1016/j.bcp.2005.05.005. [DOI] [PubMed] [Google Scholar]
  • 12.Takagi Y, Osada H, Kuroishi T, et al. P53 Mutations in Non-Small-Cell Lung Cancers Occurring in Individuals without a Past History of Active Smoking. Br J Cancer. 1998;77:1568–1572. doi: 10.1038/bjc.1998.258. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 13.Johnson L, Mercer K, Greenbaum D, et al. Somatic activation of the K-ras oncogene causes early onset lung cancer in mice. Nature. 2001;410:1111–1116. doi: 10.1038/35074129. [DOI] [PubMed] [Google Scholar]
  • 14.Lee SH, Lee SJ, Jung YS, et al. Blocking of p53-Snail binding, promoted by oncogenic K-Ras, recovers p53 expression and function. Neoplasia. 2009;11:22–31. doi: 10.1593/neo.81006. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 15.Prudkin L, Liu DD, Ozburn NC, et al. Epithelial-to-mesenchymal transition in the development and progression of adenocarcinoma and squamous cell carcinoma of the lung. Mod Pathol. 2009;22:668–678. doi: 10.1038/modpathol.2009.19. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 16.Carretero J, Medina PP, Blanco R, et al. Dysfunctional AMPK activity, signalling through mTOR and survival in response to energetic stress in LKB1-deficient lung cancer. Oncogene. 2007;26:1616–1625. doi: 10.1038/sj.onc.1209951. [DOI] [PubMed] [Google Scholar]
  • 17.Figarola JL, Weng Y, Lincoln C, Horne D, Rahbar S. Novel dichlorophenyl urea compounds inhibit proliferation of human leukemia HL-60 cells by inducing cell cycle arrest, differentiation and apoptosis. Invest New Drugs. 2012;30:1413–1425. doi: 10.1007/s10637-011-9711-8. [DOI] [PubMed] [Google Scholar]
  • 18.Singhal SS, Figarola J, Singhal J, et al. 1,3-Bis(3,5-dichlorophenyl) urea compound 'COH-SR4' inhibits proliferation and activates apoptosis in melanoma. Biochem Pharmacol. 2012;84:1419–1427. doi: 10.1016/j.bcp.2012.08.020. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 19.Yadav A, Janaratne T, Krishnan A, et al. Regression of lung cancer by hypoxia sensitizing ruthenium polypyridyl complexes. Mol Cancer Ther. 2013;12:643–653. doi: 10.1158/1535-7163.MCT-12-1130. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 20.Singhal SS, Sehrawat A, Sahu M, et al. RLIP76 transports sunitinib and sorafenib and mediates drug resistance in kidney cancer. Int J Cancer. 2010;126:1327–1338. doi: 10.1002/ijc.24767. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 21.Singhal SS, Yadav S, Drake K, Singhal J, Awasthi S. Hsf-1 and POB1 induce drug sensitivity and apoptosis by inhibiting Ralbp1. J Biol Chem. 2008;283:19714–19729. doi: 10.1074/jbc.M708703200. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 22.Singhal J, Singhal SS, Yadav S, et al. RLIP76 in defense of radiation poisoning. Int J Radiat Oncol Biol Phys. 2008;72:553–561. doi: 10.1016/j.ijrobp.2008.06.1497. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 23.Ritchie KJ, Henderson CJ, Wang XJ, et al. Glutathione transferase pi plays a critical role in the development of lung carcinogenesis following exposure to tobacco-related carcinogens and urethane. Cancer Res. 2007;67:9248–9257. doi: 10.1158/0008-5472.CAN-07-1764. [DOI] [PubMed] [Google Scholar]
  • 24.Singhal SS, Saxena M, Ahmad H, Awasthi S, Haque AK, Awasthi YC. Glutathione S-transferases of human lung: Characterization and evaluation of the protective role of the alpha-class isozymes against lipid peroxidation. Arch Biochem Biophys. 1992;299:232–241. doi: 10.1016/0003-9861(92)90269-3. [DOI] [PubMed] [Google Scholar]
  • 25.Awasthi YC, Sharma R, Sharma A, et al. Self-regulatory role of 4-hydroxynonenal in signaling for stress-induced programmed cell death. Free Radic Biol Med. 2008;45:111–118. doi: 10.1016/j.freeradbiomed.2008.04.007. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 26.Moral A, Palou J, Lafuente A, et al. Immuno-histochemical study of alpha, mu and pi class glutathione S transferase expression in malignant melanoma. MMM group. Multidisciplinary malignant melanoma group. Br J Dermatol. 1997;136:345–350. [PubMed] [Google Scholar]
  • 27.Trachte AL, Suthers SE, Lerner MR, et al. Increased expression of alpha-1-antitrypsin, glutathione S-transferase pi and vascular endothelial growth factor in human pancreatic adenocarcinoma. Am J Surg. 2002;184:642–647. doi: 10.1016/s0002-9610(02)01105-4. [DOI] [PubMed] [Google Scholar]
  • 28.Singhal SS, Wickramarachchi D, Yadav S, et al. Glutathione-conjugate transport by RLIP76 is required for clathrin-dependent endocytosis and chemical carcinogenesis. Mol Cancer Ther. 2011;10:16–28. doi: 10.1158/1535-7163.MCT-10-0699. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 29.Hardie DG. Minireview: The AMP-activated protein kinase cascade: The key sensor of cellular energy status. Endocrinology. 2003;144:5179–5183. doi: 10.1210/en.2003-0982. [DOI] [PubMed] [Google Scholar]
  • 30.Tang JM, He QY, Guo RX, Chang XJ. Phosphorylated Akt overexpression and loss of PTEN expression in non-small cell lung cancer confers poor prognosis. Lung Cancer. 2006;51:181–191. doi: 10.1016/j.lungcan.2005.10.003. [DOI] [PubMed] [Google Scholar]
  • 31.Gadgeel SM, Wozniak A. Preclinical rationale for PI3K/Akt/mTOR pathway inhibitors as therapy for epidermal growth factor receptor inhibitor-resistant non-small-cell lung cancer. Clin Lung Cancer. 2013;S1525:7304. doi: 10.1016/j.cllc.2012.12.001. [DOI] [PubMed] [Google Scholar]
  • 32.Mendez MG, Kojim S, Goldman RD. Vimentin induces changes in cell shape, motility, and adhesion during the epithelial to mesenchymal transition. FASEB J. 2010;24:1838–1851. doi: 10.1096/fj.09-151639. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 33.Jia D, Yan M, Wang X, et al. Development of a highly metastatic model that reveals a crucial role of fibronectin in lung cancer cell migration and invasion. BMC Cancer. 2010;10:364. doi: 10.1186/1471-2407-10-364. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 34.Puyol M, Martin A, Dubus P, et al. A synthetic lethal interaction between K-Ras oncogenes and Cdk4 unveils a therapeutic strategy for non-small cell lung carcinoma. Cancer. Cell. 2010;18:63–73. doi: 10.1016/j.ccr.2010.05.025. [DOI] [PubMed] [Google Scholar]
  • 35.Rusch V, Klimstra D, Venkatraman E, et al. Aberrant p53 expression predicts clinical resistance to cisplatin-based chemotherapy in locally advanced non-small cell lung cancer. Cancer Res. 1995;55:5038–5042. [PubMed] [Google Scholar]
  • 36.Heinrich EL, Walser TC, Krysan K, et al. The inflammatory tumor microenvironment, epithelial mesenchymal transition and lung carcinogenesis. Cancer Microenviron. 2012;5:5–18. doi: 10.1007/s12307-011-0089-0. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 37.Nakajima T, Elovaara E, Anttila S, et al. Expression and polymorphism of glutathione S-transferase in human lungs: risk factors in smoking-related lung cancer. Carcinogenesis. 1995;16:707–711. doi: 10.1093/carcin/16.4.707. [DOI] [PubMed] [Google Scholar]
  • 38.Thomson S, Petti F, Sujka-Kwok I, et al. A systems view of epithelial-mesenchymal transition signaling states. Clin Exp Metastasis. 2011;28:137–155. doi: 10.1007/s10585-010-9367-3. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 39.Cano A, Perez-Moreno MA, Rodrigo I, et al. The transcription factor snail controls epithelial-mesenchymal transitions by repressing E-cadherin expression. Nat Cell Biol. 2000;2:76–83. doi: 10.1038/35000025. [DOI] [PubMed] [Google Scholar]
  • 40.Aggarwal C, Somaiah N, Simon G. Antiangiogenic agents in the management of non-small cell lung cancer: Where do we stand now and where are we headed? Cancer Biol Ther. 2012;13:247–263. doi: 10.4161/cbt.13.5.19594. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 41.Vignot S, Faivre S, Aguirre D, Raymond E. mTOR-targeted therapy of cancer with rapamycin derivatives. Ann Oncol. 2005;16:525–537. doi: 10.1093/annonc/mdi113. [DOI] [PubMed] [Google Scholar]
  • 42.Carmichael J, Mitchell JB, Friedman N, Gazdar AF, Russo A. Glutathione and related enzyme activity in human lung cancer cell lines. Br J Cancer. 1988;58:437–440. doi: 10.1038/bjc.1988.236. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 43.Ling YH, Zou Y, Perez-Soler R. Induction of senescence-like phenotype and loss of paclitaxel sensitivity after wild-type p53 gene transfection of p53-null human non-small cell lung cancer H358 cells. Anticancer Res. 2000;20:693–702. [PubMed] [Google Scholar]
  • 44.Yoon YK, Kim HP, Han SW, Oh do Y, Im SA, Bang YJ. KRAS mutant lung cancer cells are differentially responsive to MEK inhibitor due to AKT or STAT3 activation: implication for combinatorial approach. Mol Carcinog. 2010;49:353–362. doi: 10.1002/mc.20607. [DOI] [PubMed] [Google Scholar]
  • 45.Roman J, Ritzenthaler JD, Roser-Page S, Sun X, Han S. Alpha5beta1-integrin expression is essential for tumor progression in experimental lung cancer. Am J Respir Cell Mol. Biol. 2010;43:684–691. doi: 10.1165/rcmb.2009-0375OC. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 46.Cabodi S, del Pilar Camacho-Leal M, Di Stefano P, Defilippi P. Integrin signalling adaptors: not only figurants in the cancer story. Nat Rev Cancer. 2010;10:858–870. doi: 10.1038/nrc2967. [DOI] [PubMed] [Google Scholar]

RESOURCES