Abstract
Lung cancer is the leading cause of cancer-related deaths and new lung cancer cases are continuously emerging around the globe; however, treatment of lung cancer remains unsatisfactory. Demethoxycurcumin (DMC) has been shown to exert cytotoxic effects in human cancer cells via induction of apoptosis. However, the effects of DMC on genetic mechanisms associated with these actions have not been yet elucidated. Human lung cancer NCI-H460 cells were incubated with or without 35 μM of DMC for 24 h and total RNA was extracted for cDNA synthesis labeling and microarray hybridization, followed by fluor-labeled cDNA hybridization on chip. Expression Console software with default Robust Multichip Analysis (RMA) parameters were used for detecting and quantitating the localized concentrations of fluorescent molecules. The GeneGo software was used for investigating key genes involved and their possible interaction pathways. Genes associated with DNA damage and repair, cell-cycle check point and apoptosis could be altered by DMC; in particular, 144 genes were found up-regulated and 179 genes down-regulated in NCI-H460 cells after exposure to DMC. In general, DMC-altered genes may offer information to understand the cytotoxic mechanism of this agent at the genetic level since gene alterations can be useful biomarkers or targets for the diagnosis and treatment of human lung cancer in the future.
Lung cancer, the leading cause of malignancy-related death globally and is one of the most aggressive human cancers worldwide. Lung cancer most commonly gives rise to metastases in the brain (1) and peripheral tissues before being diagnosed in many clinical cases (2). In lung cancer, about 80% of the cases are non-small cell lung cancer (NSCLC) (3) and early detection is possible (4). The major treatment options for lung cancer are surgical resection, chemotherapy and radiation therapy; however, the survival rate remains very low (5) and serious side-effects impairing quality of life have been registered (6). Currently, numerous studies have focused on personalized modes of therapy for assessing the efficacy and safety of agents used as molecular targets (7). Thus, many investigators are searching for new drugs from natural products to improve the disadvantages of the current treatments, especially in lung cancer.
Curcumin, a component of turmeric, is derived from the rhizome of Curcuma longa. Curcumin, acting as a chemoprotective agent (8-10), has been shown to inhibit cancer cell proliferation, induce cell apoptosis in many cancer cell lines (11-15) and modulate multiple cell signaling pathways, including apoptosis, proliferation, angiogenesis and inflammation (16). Demethoxycurcumin (DMC) is a synthetic curcumin analogue with increased metabolic stability compared to curcumin (17). DMC has been reported to induce apoptosis in human HCT116 colon cancer cells (17), human renal carcinoma Caki cells (18) and in human breast carcinoma MCF7 cells (19). It has been reported that DMC inhibits NO production, inducible NO synthase expression and NF-kB activation in RAW264.7 macrophages activated with LPS (20). Furthermore, DMC induces heme oxygenase-1 expression through Nrf2 activation in RAW264.7 macrophages (21). However, the effects of DMC on human lung cancer cells have not yet been studied. We, thus, investigated the role and action of DMC on gene expression in vitro using the human lung cancer NCI-H460 cell line.
It is well-known that genome's instability, recognized to be a hallmark of most cancers, can cause genetic aberrations as genetic mutations play an important role for oncogenes (22). Typical examples are offered by the p53 mutation and dysfunction found in over 50% of all types of human cancers (23) and the high incidence of oncogenic K-RAS mutations found in human lung adenocarcinomas and carcinogen-induced animal models (24). Thus, finding which gene is affected by certain anticancer drugs, is necessary for exploring the molecular mechanism of these drugs' function. Although DMC has been shown to induce cell apoptosis in various cancer types, there is no report to show how this agent affects gene expression in human lung cancer cells. To answer this question, we used cDNA microarrays to investigate the altered gene expression in NCI-H460 cells and our results indicated that DMC could act on the expression of some genes associated with DNA damage, cell cycle and apoptosis. These findings may, thus, form the basis for future studies about anticancer development by DMC.
Materials and Methods
Chemicals and reagents. Demethoxycurcumin (DMC) and dimethyl sulfoxide (DMSO) were obtained from Sigma Chemical Co. (St. Louis, MO, USA). Culture medium RPMI-1640, fetal bovine serum, L-glutamine, penicillin and streptomycin were obtained from Gibco BRL (Grand Island, NY, USA). DMC was dissolved in DMSO and stocked at −20°C.
Lung cancer cells. The NCI-H460 human non-small cell lung cancer (NSCLC) cell line was obtained from the Food Industry Research and Development Institute (Hsinchu, Taiwan) and cultured in RPMI-1640 medium containing 10% fetal bovine serum, 1% L-glutamine, 100 units/ml of penicillin G and 100 microgram/ml of streptomycin at 37°C in a humidified atmosphere of a 5% CO2 and 95% air. Cells were split every 4 days to maintain exponential growth before the experiments.
cDNA microarray assay for gene expression in NCI-H460 cells after exposure to DMC. NCI-H460 cells (1×105 cells/ml) were maintained in RPMI-1640 medium in a 12-well plate for 24 h. Cells were treated with or without 35 μM of DMC for 48 h and then harvested for extracting the total RNAs by using the Qiagen RNeasy Mini Kit (Qiagen, Inc., Valencia, CA, USA), as described previously (25). Quantitavely isolated total RNA from control and DMC treated cells was used for cDNA synthesis, labeling and microarray hybridization, followed by flour-labeled cDNA hybridizing of the cell complements on the chip (Affymetrix GeneChip Human Gene 1.0 ST array; Affymetrix, Santa Clara, CA, USA). Finally, the resulting localized concentrations of fluorescent molecules on the chip were detected and quantitated (Asia BioInnovations Corporation, Taipei, Taiwan). The resulting data were further analyzed by Expression Console software (Affymetrix) with default Robust Multichip Analysis (RMA) parameters (25-27). Down- or up-regulated gene expressions at least at two-fold change by DMC were recorded and identified.
Statistical analysis. Data are representative of three seperate assays. Differences between control and DMC-treated groups were presented for up to a 2-fold-change. +, up-regulation; −, down-regulation.
Results
DMC up- and down-regulated gene expression in NCI-H460 cells. Table I indicates that several genes were up-regulated after treatment with 35 μM of DMC. In particular, 2 genes were up-regulated over 4-fold, one being the TIPARP (TCDD-inducible poly(ADP-ribose) polymerase) gene, associated with apoptosis; 13 genes were over from 3- to 4-fold, like, for instance, CCNE2 (cyclin E2), associated with cell cycle and 129 genes were up-regulated from 2- to 3-fold, with ERCC6L (excision repair cross-complementing rodent repair deficiency, complementation group 6-like), TP53INP1 (tumor protein p53 inducible nuclear protein 1) and BRCC3 (BRCA1/BRCA2-containing complex, subunit 3) associated with DNA damage and repair, TUBB4B (tubulin, beta 4B class IVb), CCNE1 (cyclin E1), CDC6 (cell division cycle 6 homolog (S. cerevisiae), MAP9 (microtubule-associated protein 9) and CDC25A [cell division cycle 25 homolog A (S. pombe)], associated with cell cycle distribution and CARD6 (caspase recruitment domain family, member 6), associated with cell apoptosis, to name a few.
Table II shows that the expression of 28 genes was down-regulated over 4-fold, such as DDIT3 (DNA-damage-inducible transcript 3), associated with DNA damage, CHL1 [cell adhesion molecule with homology to L1CAM (close homolog of L1)] associated with cell apoptosis; expression of expression of 33 genes was decreased 3- to 4-fold, such as CLK1 (CDC-like kinase 1) associated with cell cycle, PDCD1LG2 (programmed cell death 1 ligand 2), associated with cell apoptosis. The expression of 118 genes was decreased 2- to 3-fold, such as ERCC3 (excision repair cross-complementing rodent repair deficiency, complementation group 3), DDIT4 (DNA-damage-inducible transcript 4) associated with DNA damage and repair, CCNL (cyclin L2), TUBB4B (tubulin, beta 4B class IVb), CCNE1 (cyclin E1), CDC6 (cell division cycle 6 homolog (S. cerevisiae)) and CCPG1 (cell cycle progression 1; DYX1C1-CCPG1) readthrough (non-protein coding) associated with cell cycle, MCL1 [myeloid cell leukemia sequence 1 (BCL2-related)], HYOU1 (hypoxia up-regulated 1), SLC25A37 [solute carrier family 25 (mitochondrial iron transporter) member 37)] and TP53INP1 (tumor protein p53 inducible nuclear protein 1).
Representative genes of NCI-H460 cells that were up-regulated by DMC treatment.
Representative genes of NCI-H460 cells those were down-regulated by DMC treatment.
The top scored (by the number of pathways) network from GeneGo 02. Thick cyan lines were used as fragments of canonical pathways. Red-colored circles represent up-regulated genes and blue-colored circles show down-regulated genes. The ‘checkerboard’ color indicates mixed expressions for the genes between files or between multiple tags for the same gene.
GeneGo analysis by the number of pathway networks involved for the top scored gene expression alterations on DMC-treated NCI-H460 cells. After cDNA microarray analysis, all samples were further processed by using the GeneGo system to enrich the analysis of significant genes in the context of pathways. The results shown in Figures 1, 2 and 3 map the processes in possible signal outcomes. Red-colored (up-regulation) and blue-colored circles (down-regulation) represent different intensities indicating various enhancing or inhibiting effects in NCI-H460 cell after DMC treatment.
Discussion
In the present study, we demonstrated that several genes involved in DNA damage and repair were up-regulated as was case with the TP53INP1 (tumor protein p53 inducible nuclear protein 1) gene that was increased 2.18-fold; it is well known that, after DNA damage, p53 protein expression is increased (28). Other examples include ERCC6L and BRCC3 (BRCA1/BRCA2-containing complex, subunit 3) that were up-regulated by 2.85- and 2.13-fold, respectively. These two genes have been reported to be involved in cell responses concerning repair of DNA damage for maintaining survival (29, 30). Similarly, CCNE2 showed a 3.32-fold increase, and the associated with cell-cycle distribution genes CCNE1, CDC6, MAP9 and CDC25A exhibited a 2.49-, 2.31-, 2.14- and 2.02-fold up-regulation, respectively (Table I). Also, Table I shows that TIPARP gene expression was increased by 5.44-fold; it is well known that cells under apoptosis may go through an increase of poly(ADP-ribose) polymerase levels (31). Moreover, the associated with cell apoptosis gene CARD6 was up-regulated by 2.28-fold. In general, it is well-documented that some anticancer drugs induce cancer cell apoptosis through the activation of the caspase pathway, especially the caspase-8, -9 and -3 (32).
The second scored (by the number of pathways) network from GeneGo 02. Thick cyan lines were used as fragments of canonical pathways. Red-colored circles represent up-regulated genes and blue-colored circles show down-regulated genes. The ‘checkerboard’ color indicates mixed expressions for the genes between files or between multiple tags for the same gene.
Table II shows that DMC down-regulated the gene levels of several genes like, DDIT3 and DDIT4 that were suppressed by 5.18- and 2.92-fold, respectively; DDIT3 has been reported to be involved in DNA damage and repair mechanisms (33). DMC also inhibited the levels of genes associated with cell cycle like CLK1 by 3.04-fold, CCNL1 by 2.02-fold, TUBE1 (tubulin, epsilon 1) by 2.45-fold, CHL1 by 6.82-fold and CCPG1 by 2.47-fold.
It is also well-known that anticancer drugs induce cancer cell apoptosis through the arrest of cell cycle at the G0/G1 or G2/M phase (34, 35). Indeed, DMC inhibited the levels of genes associated with cell apoptosis, like CHL1 by 6.82-fold, PDCD1LG2 by 3.15-fold, MCL1 by 2.05-fold and SLC25A37 by 2.46-fold.
Conclusion
Tables I and II indicate that numerous genes that are associated with DNA damage and repair, cell-cycle check point and cell apoptosis in NCI-H460 cells after exposure to DMC can be up- or down-regulated. These results were further confirmed by using the GeneGo analysis program, as depicted in Figures 1, 2 and 3, showing their possible signaling complex interactions. The noted changes provide information for understanding the cytotoxic action of DMC at the genetic level. Gene alterations may be proven as useful biomarkers or targets for the diagnosis and treatment of human lung cancer in the future. However, further studies are necessary in order to expand or append our current knowledge.
The third scored (by the number of pathways) network from GeneGo 02. Thick cyan lines were used as fragments of canonical pathways. Red-colored circles represent up-regulated genes and blue-colored circles show down-regulated genes. The ‘checkerboard’ color indicates mixed expressions for the genes between files or between multiple tags for the same gene.
Acknowledgements
This work was supported by grant CMU 101-AWARD-03(1/2) from China Medical University, Taichung, Taiwan, R.O.C.
- Received October 15, 2014.
- Revision received November 26, 2014.
- Accepted November 28, 2014.
- Copyright © 2015 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved
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