in vivo 29: 83-94 (2015) Demethoxycurcumin Alters Gene Expression Associated with DNA Damage, Cell Cycle and Apoptosis in Human Lung Cancer NCI-H460 Cells In Vitro YANG-CHING KO1, SHU-CHUN HSU2, HSIN-CHUNG LIU2, YUNG-TING HSIAO2, TE-CHUN HSIA3,4, SU-TSO YANG5,6, WU-HUEI HSU7,8 and JING-GUNG CHUNG2,9 1Institute of Clinical Medical Science, China Medical University, Taichung, Taiwan, R.O.C.; Departments of 2Biological Science and Technology and 7Internal Medicine, 6School of Chinese Medicine, China Medical University, Taichung, Taiwan, R.O.C.; 3Gradualted Institute of Chinese Medical Science, China Medical University, Taichung, Taiwan, R.O.C.; Departments of 4Internal Medicine and 5Radiology, China Medical University Hospital, Taichung, Taiwan, R.O.C.; 8Department of Internal Medicine, China Medical University, Taichung, Taiwan, R.O.C.; 9Department of Biotechnology, Asia University, Taichung, Taiwan, R.O.C. Abstract. Lung cancer is the leading cause of cancer- altered genes may offer information to understand the related deaths and new lung cancer cases are continuously cytotoxic mechanism of this agent at the genetic level since emerging around the globe; however, treatment of lung gene alterations can be useful biomarkers or targets for the cancer remains unsatisfactory. Demethoxycurcumin (DMC) diagnosis and treatment of human lung cancer in the future. has been shown to exert cytotoxic effects in human cancer cells via induction of apoptosis. However, the effects of DMC Lung cancer, the leading cause of malignancy-related death on genetic mechanisms associated with these actions have globally and is one of the most aggressive human cancers not been yet elucidated. Human lung cancer NCI-H460 cells worldwide. Lung cancer most commonly gives rise to were incubated with or without 35 μM of DMC for 24 h and metastases in the brain (1) and peripheral tissues before total RNA was extracted for cDNA synthesis labeling and being diagnosed in many clinical cases (2). In lung cancer, microarray hybridization, followed by fluor-labeled cDNA about 80% of the cases are non-small cell lung cancer hybridization on chip. Expression Console software with (NSCLC) (3) and early detection is possible (4). The major default Robust Multichip Analysis (RMA) parameters were treatment options for lung cancer are surgical resection, used for detecting and quantitating the localized chemotherapy and radiation therapy; however, the survival concentrations of fluorescent molecules. The GeneGo rate remains very low (5) and serious side-effects impairing software was used for investigating key genes involved and quality of life have been registered (6). Currently, numerous their possible interaction pathways. Genes associated with studies have focused on personalized modes of therapy for DNA damage and repair, cell-cycle check point and assessing the efficacy and safety of agents used as molecular apoptosis could be altered by DMC; in particular, 144 genes targets (7). Thus, many investigators are searching for new were found up-regulated and 179 genes down-regulated in drugs from natural products to improve the disadvantages of NCI-H460 cells after exposure to DMC. In general, DMC- the current treatments, especially in lung cancer. Curcumin, a component of turmeric, is derived from the rhizome of Curcuma longa. Curcumin, acting as a Correspondence to: Jing-Gung Chung, Ph.D., Department of chemoprotective agent (8-10), has been shown to inhibit Biological Science and Technology, China Medical University, No 91, cancer cell proliferation, induce cell apoptosis in many Hsueh-Shih Road, Taichung 404, Taiwan. Tel: +886 422053366 (ext. cancer cell lines (11-15) and modulate multiple cell signaling 8000), Fax: +886 422053764, e-mail: [email protected] and pathways, including apoptosis, proliferation, angiogenesis Wu-Huei Hsu, Department of Internal Medicine, China Medical and inflammation (16). Demethoxycurcumin (DMC) is a University, Taichung, Taiwan. Tel: +886 422053366 (ext. 3483), Fax: synthetic curcumin analogue with increased metabolic +886 422038883, e-mail: [email protected] stability compared to curcumin (17). DMC has been reported Key Words: Demethoxycurcumin (DMC), cDNA microarray, DNA to induce apoptosis in human HCT116 colon cancer cells damage, cell cycle, apoptosis, NCI-H460 cells. (17), human renal carcinoma Caki cells (18) and in human 0258-851X/2015 $2.00+.40 83 in vivo 29: 83-94 (2015) breast carcinoma MCF7 cells (19). It has been reported that hybridization, followed by flour-labeled cDNA hybridizing of the DMC inhibits NO production, inducible NO synthase cell complements on the chip (Affymetrix GeneChip Human Gene expression and NF-kB activation in RAW264.7 macrophages 1.0 ST array; Affymetrix, Santa Clara, CA, USA). Finally, the resulting localized concentrations of fluorescent molecules on the activated with LPS (20). Furthermore, DMC induces heme chip were detected and quantitated (Asia BioInnovations oxygenase-1 expression through Nrf2 activation in Corporation, Taipei, Taiwan). The resulting data were further RAW264.7 macrophages (21). However, the effects of DMC analyzed by Expression Console software (Affymetrix) with default on human lung cancer cells have not yet been studied. We, Robust Multichip Analysis (RMA) parameters (25-27). Down- or thus, investigated the role and action of DMC on gene up-regulated gene expressions at least at two-fold change by DMC expression in vitro using the human lung cancer NCI-H460 were recorded and identified. cell line. Statistical analysis. Data are representative of three seperate assays. It is well-known that genome’s instability, recognized to Differences between control and DMC-treated groups were presented be a hallmark of most cancers, can cause genetic aberrations for up to a 2-fold-change. +, up-regulation; –, down-regulation. as genetic mutations play an important role for oncogenes (22). Typical examples are offered by the p53 mutation and Results dysfunction found in over 50% of all types of human cancers (23) and the high incidence of oncogenic K-RAS mutations DMC up- and down-regulated gene expression in NCI-H460 found in human lung adenocarcinomas and carcinogen- cells. Table I indicates that several genes were up-regulated induced animal models (24). Thus, finding which gene is after treatment with 35 μΜ of DMC. In particular, 2 genes affected by certain anticancer drugs, is necessary for were up-regulated over 4-fold, one being the TIPARP exploring the molecular mechanism of these drugs’ function. (TCDD-inducible poly(ADP-ribose) polymerase) gene, Although DMC has been shown to induce cell apoptosis in associated with apoptosis; 13 genes were over from 3- to 4- various cancer types, there is no report to show how this fold, like, for instance, CCNE2 (cyclin E2), associated with agent affects gene expression in human lung cancer cells. To cell cycle and 129 genes were up-regulated from 2- to 3-fold, answer this question, we used cDNA microarrays to with ERCC6L (excision repair cross-complementing rodent investigate the altered gene expression in NCI-H460 cells repair deficiency, complementation group 6-like), TP53INP1 and our results indicated that DMC could act on the (tumor protein p53 inducible nuclear protein 1) and BRCC3 expression of some genes associated with DNA damage, cell (BRCA1/BRCA2-containing complex, subunit 3) associated cycle and apoptosis. These findings may, thus, form the basis with DNA damage and repair, TUBB4B (tubulin, beta 4B for future studies about anticancer development by DMC. class IVb), CCNE1 (cyclin E1), CDC6 (cell division cycle 6 homolog (S. cerevisiae), MAP9 (microtubule-associated Materials and Methods protein 9) and CDC25A [cell division cycle 25 homolog A (S. pombe)], associated with cell cycle distribution and Chemicals and reagents. Demethoxycurcumin (DMC) and dimethyl CARD6 (caspase recruitment domain family, member 6), sulfoxide (DMSO) were obtained from Sigma Chemical Co. (St. associated with cell apoptosis, to name a few. Louis, MO, USA). Culture medium RPMI-1640, fetal bovine serum, Table II shows that the expression of 28 genes was down- L-glutamine, penicillin and streptomycin were obtained from Gibco regulated over 4-fold, such as DDIT3 (DNA-damage- BRL (Grand Island, NY, USA). DMC was dissolved in DMSO and stocked at –20˚C. inducible transcript 3), associated with DNA damage, CHL1 [cell adhesion molecule with homology to L1CAM (close Lung cancer cells. The NCI-H460 human non-small cell lung cancer homolog of L1)] associated with cell apoptosis; expression (NSCLC) cell line was obtained from the Food Industry Research of expression of 33 genes was decreased 3- to 4-fold, such and Development Institute (Hsinchu, Taiwan) and cultured in RPMI- as CLK1 (CDC-like kinase 1) associated with cell cycle, 1640 medium containing 10% fetal bovine serum, 1% L-glutamine, PDCD1LG2 (programmed cell death 1 ligand 2), associated 100 units/ml of penicillin G and 100 microgram/ml of streptomycin with cell apoptosis. The expression of 118 genes was 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 decreased 2- to 3-fold, such as ERCC3 (excision repair experiments. cross-complementing rodent repair deficiency, complementation group 3), DDIT4 (DNA-damage-inducible cDNA microarray assay for gene expression in NCI-H460 cells after transcript 4) associated with DNA damage and repair, CCNL exposure to DMC. NCI-H460 cells (1×105 cells/ml) were (cyclin L2), TUBB4B