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Carcinogenesis Advance Access originally published online on January 6, 2006
Carcinogenesis 2006 27(7):1369-1376; doi:10.1093/carcin/bgi328
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© The Author 2006. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oxfordjournals.org

Molecular targets of the chemopreventive agent 1,4-phenylenebis (methylene)-selenocyanate in human non-small cell lung cancer

Karam El-Bayoumy *, Arunangshu Das, Bhagavathi Narayanan 1, Narayanan Narayanan 1, Emerich S. Fiala 1, Dhimant Desai, Chinthalapally V. Rao 2, Shantu Amin and Raghu Sinha

Penn State College of Medicine, 500 University Drive, Hershey, PA 17033, USA, 1 New York University School of Medicine, 57 Old Forge Road, Tuxedo, NY 10987, USA and 2 The University of Oklahoma Health Sciences Center, 975 NE 10th Street, Oklahoma City, OK 73104, USA

* To whom correspondence and reprint requests should be addressed. Email: Kelbayoumy{at}aol.com


   Abstract
Top
 Abstract
Introduction
 Materials and methods
 Results and discussion
 References
 
Clinical chemoprevention trials of lung cancer have been somewhat disappointing and the development of highly effective chemopreventive agents is urgently needed. We previously showed that the organoselenium 1,4-phenylenebis(methylene)selenocyanate (p-XSC) is a potent chemopreventive agent in numerous preclinical animal models including a lung tumor model that employs carcinogens found in tobacco smoke. The goal of this study is to define molecular targets that will be highly promising in the design of future chemoprevention trials of non-small cell lung cancer (NSCLC), which is by far the most common type of lung cancer cases. In the present investigation, we showed that p-XSC at several doses (2.5, 5, 10 and 20 µM) including physiological levels (2.5–5.0 µM) of selenium is capable of inhibiting cell growth in a dose-dependent manner and inducing apoptosis in three NSCLC cells (NCI-H460, NCI-1299 and A549). To clarify the mechanism involved at the molecular level, we focused only on NCI-460 cells and examined the effects of p-XSC on markers that are known to be critical in the development of NSCLC. Using western blot analysis, we showed that p-XSC reduced the expression of cyclooxygenase-2 (COX-2) and phospholipase A2 (PLA2); although p-XSC inhibited both Akt and p-Akt but its effect was not significant. Using cDNA microarray approach (3800 genes per array) we found that p-XSC upregulates 22 genes by ≥2-fold while downregulates 13 genes by ≤0.5-fold; these altered genes include transcriptional factors, growth factors and those involved in xenobiotic metabolism as well as pro- and anti-apoptotic genes. Expression of selected genes was confirmed by RT–PCR; p-XSC reduced the levels of COX-2, PLA2, NF-{kappa}B and Cyclin D1 but enhanced the levels of glutathione peroxidase-5. Collectively, the results of this study showed that p-XSC alters several molecular markers in a manner that can account for its inhibitory effect of cell growth and induction of apoptosis; therefore, p-XSC may be considered a promising candidate for clinical chemoprevention of NSCLC.

Abbreviations: COX-2, cyclooxygenase-2; GPX, glutathione peroxidase; NDUFS3, NADH dehydrogenase (ubiquinone)Fe-S protein 3; NF-{kappa}B, Nuclear factor-{kappa}B; NNK, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone; NSCLC, non-small cell lung cancer; PI3K, phosphatidylinositol 3-kinase; PLA2, phospholipase A2; p-XSC, 1,4-phenylenebis(methylene)selenocyanate; RT–PCR, Reverse transcription polymerase chain reaction; SCLC, small cell lung cancer


   Introduction
Top
 Abstract
 Introduction
Materials and methods
 Results and discussion
 References
 
Lung cancer is the leading cause of mortality in men and women. In the USA, smoking contributes to >90% of all deaths from lung cancer in men and to ~80% of all deaths from lung cancer in women (1–4). Two main types of lung cancer, namely small cell lung cancer (SCLC) and non-small cell lung cancer (NSCLC), account for 90% of all lung cancers, while the less common types of lung cancer, such as carcinoid, mucoepidermoid and malignant mesothelioma, together account for ~10% (5). NSCLC is by far the most common type, occurring in 75–80% of all lung cancer cases. Despite surgery with intent to cure, and utilizing the best available therapeutic approaches, <10% of all lung cancer patients survive 5 years (1,2,6). To reduce the lung cancer epidemic is to quit smoking; this has been, and continues to be, the ultimate goal of preventive medicine (7–9).

Dietary interventions or the use of chemopreventive agents can provide complementary approaches for cancer prevention in lowering the risk in ex-smokers and, possibly, in active smokers (10,11). Clinical chemoprevention trials of lung cancer using vitamins have been unsuccessful (12–15). However, it appears that the use of selenium, in the form of selenized yeast, provides some protection against lung cancer in people whose baseline plasma selenium is ≤106 ng/ml prior to supplementation (16–18). Selenomethionine accounts for 60–80% of the selenium content in the selenized yeast (19,20). Since lung cancer chemoprevention intervention trials have been somewhat disappointing, our current research efforts are aimed at defining a molecular staging system that is highly promising in cancer chemoprevention and chemotherapy rather than existing histologic staging that has not changed for the past 30 years.

Toward this end, we demonstrated that the organoselenium 1,4-phenylenebis(methylene)selenocyanate (p-XSC) inhibited cell growth in vitro using human lung adenocarcinoma (NCI-H460) cells and the data support the hypothesis that levels of cyclooxygenase-2 (COX-2) expression determine the extent of human lung tumor growth in athymic mice (21). Inhibition of cell growth and induction of apoptosis are considered major cellular alterations that can account for cancer chemoprevention by selenium compounds (22,23). In addition to COX-2, it is of paramount importance to identify other molecular targets that may be involved in cell growth inhibition, apoptosis induction and inherent to lung tumorigenesis; such information will provide the foundation for future cancer chemoprevention intervention trial. Therefore, in this study, we examined the effect of p-XSC at several doses including physiological levels (blood levels range between 2.5 and 5.0 µM in people supplemented with selenium-enriched yeast at 200–400 µg/day) (16,20,24) on cell growth and apoptosis using three NSCLC cell lines (NCI-H460, NCI-H1299 and A549). However, only NCI-H460 cells were used to further examine by western blot analysis the effects of p-XSC on certain molecular markers that are known to be involved in the development of lung cancer (25). Using a cDNA microarray approach, we determined the effects of p-XSC on global gene expression using NCI-H460 cell line; the expression of representative genes was confirmed by RT–PCR.


   Materials and methods
Top
 Abstract
 Introduction
 Materials and methods
Results and discussion
 References
 
Treatment of NSCLC cells with p-XSC
Human NSCLC lung cancer cell lines (NCI-H460, NCI-H1299 and A549) obtained from American Type Culture Collection (Rockville, MD) were grown in RPMI 1640 containing 10% fetal bovine serum (FBS) in a humidified environment at 37°C with 5% CO2. p-XSC was synthesized according to the methods described previously (26), and a stock was prepared in dimethyl sulfoxide (DMSO). The cells were treated with various concentrations of p-XSC in the cell culture medium for 24 h. Control cultures were treated with DMSO alone (not exceeding 0.1% of volume) and were processed similarly.

MTT assay
NCI-H460, NCI-H1299 and A549 cells (10 000/well) were grown in a 96-well plate for 24 h and then treated with different doses of p-XSC for 24 h followed by the MTT (Methylthiazoletetrazolium) treatment (50 µg/100 µl) to the cells in each well for 4 h at 37°C as described previously (27). MTT was aspirated and 100 µl of DMSO was added to each well and absorbance at 570 nm was read in a plate reader. Each treatment and corresponding control was carried out in triplicate. Mean of three values are taken and assay is presented as percent of control.

Apoptosis assay
H460, H1299 and A549 cells (50 000/well) were grown for 24 h in a 24-well plate and then treated with varying doses of p-XSC for 24 h. The assay was carried out using a cell death detection enzyme-linked immunosorbent assay (ELISA) kit from Roche Diagnostics (Indianapolis, IN) and absorbance was read at 410 nm in a plate reader. The assay was performed in triplicate and data are presented as percent of control.

Western blot analysis
NCI-H460 cells were treated with p-XSC (5. 10 and 20 µM) for 24 h, harvested by scrapping and washed with phosphate-buffered saline. Cellular proteins were isolated with cell lysis buffer containing 20 mM Tris (pH 7.5), 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1% Triton X-100, 2.5 mM sodium pyrophosphate, 1 mM ß-glycerophosphate, 1 mM Na3VO4, 1 µg/ml leupeptin and freshly added 1 mM PMSF (Cell Signaling, Beverly, MA). Equal amounts of protein (30 µg) were separated on 10% SDS–PAGE gels and transferred to nitrocellulose membranes. Antibodies used for western blots were COX-2 from Cayman Chemicals (Ann Arbor, MI), PLA2 (phospholipase A2) and actin from Santa Cruz (Santa Cruz, CA), Akt and p-Akt from Cell Signaling Technology (Beverly, MA) and NADH dehydrogenase (ubiquinone)Fe-S protein 3 (NDUFS3) from Molecular Probes (Invitrogen, Carlsbad, CA). Band expressions were developed using ECL reagents from Amersham (Piscataway, NJ). Densitometric analysis of the protein bands was performed with the LabWorks Analysis Software (UVP, Upland, CA).

RNA isolation and cDNA labeling
Total RNA was extracted from untreated NCI-H460 cells and from those treated with p-XSC (10 µM) for 24 h using Trizol reagent, followed by Qiagen columns (Invitrogen Life Technologies, Carlsbad, CA and Qiagen, Valencia, CA). RNA was extracted from three independent experiments. Preparations of cDNA, labeling and hybridization were performed as described previously (28). Cyanine dye Cy3 was used for cDNA from untreated control and Cy5 was used to label the cDNA from treatment. The labeled probes were washed with 70 and 95% ethanol, respectively, and were stored at –20°C.

DNA microarray hybridization
Atlas Glass Human 3.8 I microarrays containing 3800 genes per array were purchased from BD Bioscience Clontech (Palo Alto, CA). The human DNA microarrays used in this study consisted of more than 40 diverse functional categories of genes. Hybridizations of the microarray with the probes were carried out overnight at 65°C using a hybridization chamber as described in our earlier report (28).

Scanning and data analysis
Scanning of the hybridized microarray slides were carried out using the Scan Array Express-HT Microarray Scanner, a confocal-scanning instrument that contains two lasers which excite cyanine dyes at appropriate wavelengths, 635 nm (Cy5) and 532 nm (Cy3), respectively. High-resolution (10 micron pixel size) photo multiplier tubes that detect flurochrome emission employing Scan Array Express Pro Software (PE/Packard Biosciences, Boston, MA) was used to analyze the images and to extract the data sets into a Microsoft Excel spreadsheet. The data sets consist of signal and background intensity, standard deviation of signal and background intensity, and ratio of media and/or mean or total intensity including flags. Data mining and data analysis were carried out using a Genespring bioinformatics software package (Silicon Genetics, Redwood City, CA) as described previously (28).

RT–PCR
COX-2, nuclear factor-kappaB (NF-{kappa}B; p105), PLA2, glutathione peroxidase-5 (GPX5), Cyclin D1, NDUFS3 (30 kDa) and house keeping gene GAPDH (Glyceraldehyde-3-phosphate dehydrogenase) were analyzed in p-XSC-treated cells by semi-quantitative RT–PCR. Briefly, total RNA isolated from p-XSC-treated cells was reverse transcribed using the first strand cDNA synthesis kit (SuperArray Bioscience, Frederick, MD). The entire RT reaction was used as template for PCR. Specific sequence primers were customized in a profiling kit format and PCR was performed in an Eppendorf Mastercycler gradient after denaturation (94°C for 5 min), 30 cycles of (94°C for 30 s; 50°C for 30 s; and 72°C for 45 s). The following products were obtained after running the PCR on 1% agarose gels, COX-2: 472 bp; NF-{kappa}B1: 433 bp; PLA2: 512 bp; GPX5: 531/414 bp; Cyclin D1: 551 bp; NDUFS3: 482 bp and GAPDH: 399 bp. The bands for respective products were quantified using LabWorks Analysis Software (UVP) and the densities were normalized with GAPDH.


   Results and discussion
Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
References
 
Basic research and clinical chemoprevention trials support the protective role of selenium in cancer prevention including lung cancer but the mechanism based on the molecular level remains to be fully defined. Numerous studies conducted in our laboratory showed that p-XSC inhibits chemically induced carcinogenesis in several target organs (lung, colon, mammary and oral cavity) (22,26,27). We also showed that p-XSC inhibits both phases of carcinogenesis (initiation and post-initiation) as well as inhibits lung tumor growth in nude mice (21,22). In preclinical investigations, we demonstrated that selenium in the form of p-XSC, but not selenized yeast, inhibited the tobacco-specific nitrosamine (NNK) induced lung tumors in A/J mouse (29). We also showed that p-XSC inhibits NNK-induced DNA methylation (29) and DNA oxidative damage in mouse lung; the latter was assessed by measuring 8-hydroxy-2'-deoxyguanosine (30). These results demonstrate that p-XSC acts at earlier stage (e.g. reduce genotoxicity) of the carcinogenesis process. Epidemiological studies and clinical trials, as well as preclinical investigations, demonstrate the importance of COX-2 as a critical target for chemoprevention of lung cancer (31,32). In our previous report, we demonstrated that p-XSC inhibited the growth of human lung tumor and COX-2 expression in vitro and in vivo in nude mice (21).

The present study is aimed at determining the effects of p-XSC on cancer cell growth and apoptosis as well as molecular markers that may be critical targets in the etiology and prevention of lung cancer. Consistent with our previous investigation (21), we have shown that p-XSC significantly inhibits cell growth of NCI-H460 cells (Figure 1A); its effect is not unique to NCI-H460 cells but it exerts a similar growth inhibition effect on the other NSCLC cell lines (Figure 1B and C). Selenomethionine and Se-methylselenocysteine (components of selenized yeast) had no effect on cell growth at doses equal to those of p-XSC (data not shown). Collectively, the effect of selenium compounds on cell growth appears to depend on both dose and form (structure) of selenium compounds. Figure 2 shows the inductive effects of p-XSC on apoptosis using the three cell lines. Similarly, the effect of selenium compounds on apoptosis is dependent on the form as well as the dose (22). As shown in Figure 2 and contrary to our expectation, p-XSC at the highest dose (20 µM) caused lower levels of apoptotic cell death than that observed at 10 µM. It is possible that toxicity of p-XSC at the highest dose may cause cell death by necrosis rather than by apoptosis.


Figure 1
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  		Fig. 1. The effect of p-XSC on cell growth using NSCLC: NCI-H460 (A), NCI-H1299 (B) and A549 (C) as determined by MTT assay. Percentage of control was quantitated from three independent experiments performed in triplicate. Data are presented as mean ± SE. *, statistically significant (P < 0.05) as compared with untreated controls.

 

Figure 2
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  		Fig. 2. The effect of p-XSC on the induction of apoptosis using NSCLC: NCI-H460 (A), NCI-H1299 (B) and A549 (C) as determined by cell-death-ELISA. Percentages of apoptotic cells were quantitated from three independent experiments. Data are presented as mean ± SE. *, statistically significant (P < 0.05) as compared with untreated controls.

 
In the second phase of this study, we focused only on NCI-H460 cells to further examine the effect of p-XSC on several molecular targets that are known to be critical in the development of NSCLC including COX-2. The inhibition of COX-2 expression (Figure 3A) at all selenium doses employed was evident but the effect was not dose-dependent. In addition to COX-2, NSCLC cells have constitutively high expression of cytosolic phospholipase A2 (PLA2) (33). Significant inhibition of PLA2 (Figure 3B) was observed only at the highest dose of p-XSC (20 µM). It has also been shown that phosphatidylinositol 3-kinase (PI3K)/Akt is active in NSCLC cells and promotes cellular survival and resistance to chemotherapy or radiation (34). The results reported by West et al. (35) support the hypothesis that maintenance of Akt activity is necessary for survival of preneoplastic, as well as transformed lung epithelial cells, and suggest that inhibition of the Akt pathway might be a useful approach to arrest tumorigenesis. A recent report by Yang et al. (36) provides strong evidence for pharmacologically targeting Akt for anticancer drug discovery. It appears that p-XSC inhibits both Akt and p-Akt expression but its effect was not significant (Figure 3C and D). It is of particular significance that the tobacco-specific nitrosamine NNK, which is known to induce adenomas and adenocarcinomas in the lung of laboratory animals (37), is also capable of Akt activation in a dose- and time-dependent manner in primary human lung epithelial cells of both large and small airway origin (38). Collectively, literature data suggest that rapid Akt activation may play a role in tobacco-induced lung cancer. Future studies will determine whether the inhibition of NNK-induced lung tumors by p-XSC in A/J mouse in vivo (29) is due, in part, to inhibition of Akt.


Figure 3
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  		Fig. 3. The effect of p-XSC, using western blot analysis on COX-2 (A), PLA2 (B), AkT (C), p-AkT (D) in NCI-H460 cells. Experiment was conducted in triplicate and each bar represents an average of three densitometric measurements. Representative western blot for each biomarker is shown. *, statistically significant (P < 0.05) as compared with untreated controls.

 
The ‘Omics’ approaches (39) including genomics have provided useful tools to study molecular mechanisms of carcinogenesis and cancer chemoprevention (40). Using a cDNA microarray approach, we examined the effects of p-XSC at 10 µM on global gene expression using NCI-H460 cells; this approach is highly useful in the formulation of new hypotheses that can be tested in future clinical chemoprevention trials. The results are shown in Figure 4 as a scatter plot that represents the gene expression patterns based on the magnitude of change in the intensity of color (Cy5/Cy3) ratio. An in-depth analysis of microarray data was performed in order to obtain an overall gene expression pattern on specific cluster of genes that was modulated by p-XSC. Biochemical functions of the genes in the expression profiles are diverse and include oncogenes, transcriptional factors, genes involved in xenobiotic metabolism, pro- and anti-apoptotic genes, and growth factors (Table I). We focused on genes whose expression was altered by ≥2-fold and ≤0.5-fold by p-XSC (those genes labeled in bold letters, Table I). The results showed that 22 genes were upregulated while 13 genes were downregulated by p-XSC.


Figure 4
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  		Fig. 4. Scatter plot for gene expression following cDNA microarray analysis. The ratio (Cy5:Cy3) of genes that have ≥2-fold expression are considered up-regulated and those with ≤0.5-fold expression are considered down-regulated. Of the 3800 genes 35 genes (0.92%) were differentially expressed in p-XSC-treated cells (Table I).

 

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  		Table I. Effects of p-XSC (10 µM) on gene expression in human lung adenocarcinoma cells

 
The RT–PCR assay was performed on selected genes, known to be important in the development of lung cancer, to further confirm the microarray results. We have calculated the mean + SE (n = 3) for each of the molecular targets. Significant changes were only observed in the expressions of cyclin D1 and PLA2. The results, however, are presented (Figure 5) as fold difference as compared with untreated control. The results showed that p-XSC reduced the levels of COX-2, NF-{kappa}B (p105), PLA2 and cyclin D1 but enhanced the levels of GPX5. However, it has no effect on NDUFS3. The results based on western blot analysis are consistent with RT–PCR in respect to the effect of p-XSC on levels of COX-2 and PLA2 expression. Cyclin D1 gene amplification and overexpression have been shown to be overexpressed in a wide variety of tumors including lung tumors induced by NNK (41,42). Altered expression of Cyclin D1 is an early event in NSCLC development and is required to advance cells from G1 to S-phase of the cell cycle (43). Cyclin D1 is also known to be regulated by NF-{kappa}B (44). There are several factors described in the literature that can activate NF-{kappa}B-related gene expression by initiating degradation of the cytoplasmic complex (NF-{kappa}B–I{kappa}B) followed by subsequent translocation of NF-{kappa}B into the nucleus where it activates gene expression (45).


Figure 5
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  		Fig. 5. The effect of p-XSC on expression of selected genes by semi-quantitative RT–PCR in NCI-H460 cells. Experiment was conducted in triplicate and a representative RT–PCR with fold difference as compared with control for each treatment is shown.

 
Using laser capture microdissection and microarray expression analysis of lung adenocarcinoma, Miura et al. (46) reported that 27 genes were differentially expressed between 5-year or more survivors and non-survivors. As an example, NADH dehydrogenase (ubiquinone) Fe-S protein 3 (NDUFS3, 30 kDa) was highly expressed in survivors and low in non-survivors (46). Our results based on cDNA analysis indicated that p-XSC upregulates NDUFS3, but this was not supported by either RT–PCR or western Blot analysis (data not shown). GPX5, a selenium-independent isozyme but with activity toward hydrogen peroxide and organic peroxides and, thus, may act as an antioxidant. On the basis of RT–PCR analysis (Figure 5), the induction of GPX5 by p-XSC as demonstrated in this study suggests that perhaps an earlier step in the carcinogenesis process, one dependent on oxidant stress, might be the target of p-XSC action. The induction of GPX5 in NSCLC by p-XSC is considered a novel finding and could be highly significant since the expression of this gene has been shown to be lower in smokers than in non-smokers (46). Collectively, the results of this study showed that p-XSC alters several molecular markers known to be critical in the development of lung cancer, in a manner that can account for its inhibitory effect on cell growth and induction of apoptosis. In summary, p-XSC may be considered a promising candidate for the chemoprevention of NSCLC.


   Acknowledgments
 
The authors would like to thank Dr Venkat S. Malisetty for assisting with the cell cultures. This work is supported by the National Cancer Institute Grant PO1-CA70972.

Conflict of Interest Statement: None declared.


   References
Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 

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Received September 20, 2005; revised October 30, 2005; accepted December 20, 2005.


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K.-M. Chen, T. E. Spratt, B. A. Stanley, D. A. De Cotiis, M. C. Bewley, J. M. Flanagan, D. Desai, A. Das, E. S. Fiala, S. Amin, et al.
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