Abstract
Background/Aim: Lung cancer is one of the most common malignancies and a predominant cause of cancer-related death. It can metastasize in almost all organs, and currently, while new cases are increasing, treatment is still insufficient. Bisdemethoxycurcumin (BDMC), one of the components of turmeric, has been known to possess biological activities. However, the effects of BDMC on the genetic level remain unclear. Materials and Methods: Human lung cancer NCI-H460 cells were treated with 35 μM BDMC for 24 h and cells were harvested for total RNA extraction. The purified RNA was used for cDNA synthesis, labeling, microarray hybridization, and flour-labeled cDNA on-chip hybridization. The expression Console software (Affymetrix) with default RNA parameters was used to detect and quantitate concentrations of fluorescent molecules. The key genes involved and their possible interaction pathways were analyzed by the GeneGo software. Results: Seven genes, such as CCNE2 (cyclin E), associated with cell cycle, were over 4-fold overexpressed, 22 genes, such as ERCC6L (excision repair cross-complementing rodent repair deficiency, complementation group 6-like) associated with DNA damage and repair, were from 3- to 4-fold overexpressed and 266, such as cell division cycle, S-phase associated kinase and associated with cell death, genes were from 2- to 3-fold overexpressed. Conclusion: BDMC induced changes in gene expression that may reveal cytotoxic information on the genetic level while presenting novel biomarkers or targets for treatment of human lung cancer in the future.
Lung cancer is one of the major causes of death due to cancer worldwide and early metastatic dissemination and resistance to therapy are causal factors giving a median survival of less than 12 months (1). Recently, in lung cancer patients, new improvements have focused on chemotherapy and molecular-targeted therapy, however, the outcome of patients remains unsatisfactory (2). Non small-cell lung cancer (NSCLC) accounts for 85% of lung cancer cases (3, 4) and is also the main cause of cancer-related death worldwide (5) and the overall 5-year survival rate of NSCLC patients remains lower than 15% (6). Thus, new therapeutic strategies for NSCLC are urgently needed. Many current investigations are focusing on anticancer drugs originating from natural products. Furthermore, in psychiatric medicine, herbals and phytochemicals have been recognized to be of great interest, as complementary and/or alternative therapies (7).
Turmeric, a ground rhizome of Curcuma longa, is widely used in Asian traditional medicine for wound healing, inflammatory conditions, and blood purification (8, 9). In traditional Chinese medicine, turmeric has a long history of treating diseases associated with abdominal pain (10). Bisdemethoxycurcumin (BDMC), one of the components of turmeric, has been shown to possess anti-inflammatory and anti-proliferative activities (11), anti-metastasis potency via the differentially down-regulation of ECM degradation enzymes (12) and to induce rapid DNA double-strand breaks in the human colon cancer HCT116 cells (13). BDMC inhibited cell invasion and motility and modulated MMP-3 expression in human invasive breast carcinoma cells (14). BDMC decreased inducible nitric oxide synthase (iNOS) expression and nitric oxide (NO) production (15) and inhibited the Wnt/beta-catenin pathway (16). Furthermore, the dual activity of BDMC on topoisomerases-IIα (TOP2A) has been used in novel therapeutic strategies in order to induce apoptosis in cancer cells (17).
Existing studies included patients treated with targeted-agents based on specific molecular alterations of neoplastic cells (18, 19). Thus, molecular alterations in cancer cells are called to attention for aiding the development of specific targeted drugs. Moreover, in clinical practice, predictive biomarkers have been assessed and increased the availability of anticancer drugs corresponding to specific molecular alterations (20). In the past decade, the identification of specific predictive and/or prognostic molecular alterations for NSCLC and colorectal carcinoma (CRC) have been the center of attention (21, 22). Currently, the development of targeted-therapeutics for NSCLC treatment aim at defining genetic abnormalities in NSCLC such as mutations in EGFR or a fusion of the EML4, while the ALK gene has been recognized as a target for first-line therapies in NSCLC (23-25).
Although BDMC has been shown to induce cell death in NSCLC cells, the exact genes affected by BDMC remain unknown. Thus, we investigated altered gene expression in NCI-H460 cells after exposure of cells to BDMC and results indicated that affected genes are involved in apoptosis pathways.
Materials and Methods
Chemicals and reagents. DMSO was obtained from Sigma Chemical Co. (St. Louis, MO, USA). Culture medium RPMI-1640, fetal bovine serum (FBS), 1% L-glutamine, 100 U/ml penicillin and 100 μg/ml streptomycin were obtained from Gibco BRL (Grand Island, NY, USA). BDMC was dissolved in DMSO and stored at −20°C before use in experiments.
Human lung cancer cells. NCI-H460 human non-small cell lung cancer cells were obtained from the Food Industry Research and Development Institute (Hsinchu, Taiwan). The cells were cultured at 37°C in a humidified atmosphere of 95% air and 5% CO2 air in RPMI-1640 medium supplemented with 10% (FBS), 1% L-glutamine and 1% penicillin/streptomycin (Invitrogen, Carlsbad, CA, USA). Cultured NCI-H460 cells were split every 4 days for maintaining exponential growth and were harvested with 0.025% trypsin and 0.52 mM EDTA in phosphate buffer saline (PBS), plated at required cell numbers and allowed to adhere for 24 h before BDMC treatment.
cDNA Microarray assay for gene expression in NCI-H460 cells after exposure to BDMC. NCI-H460 cells at a density of 5×105 cells/ml were maintained on 12-well plates with RPMI-1640 medium for 24 h and then incubated with or without 35 μM of BDMC for 24 h. At the end of incubation, cells were collected from each treatment and total RNA was extracted by using the Qiagen RNeasy Mini Kit (Qiagen, Inc, Valencia, CA, USA), as described previously (26) and individually quantitated and used for cDNA synthesis, labeling and microarray hybridization, followed by flour-labeled cDNA hybridizing their complements on the chip (Affymetrix GeneChip Human Gene 1.0 ST array, Affymetrix, Santa Clara, CA, USA) (26). The resulting localized concentrations of fluorescent molecules on the chip were detected and quantified (Asia BioInnovations Corporation, Taipei, Taiwan) and data were further analyzed by the Expression Console software (Affymetrix) with default RMA parameters (26, 27). Data are representative of three separate assays.
Statistical analysis. All results were presented as mean±SD of three independent experiments. Significant differences between BDMC-treated and -untreated groups were considered if at least a 2-fold change was recorded. +, Up-regulation; −, down-regulation.
Results
BDMC induced both up-regulation and down-regulation of gene expression in NCI-H460 cells. After NCI-H460 cells were treated with or without 35 μM of BDMC for 48 h, they were extracted for total RNA from each treatment. Isolated total RNA was quantified, followed by cDNA microarray analysis and results are shown in Tables I and II. Table I shows that 7 genes were over 4-fold up-regulated, such as CCNE2 (cyclin E), 22 genes were from over 3- to 4-fold such as ERCC6L (excision repair cross-complementing rodent repair deficiency, complementation group 6-like), and 266 genes were over from 2- to 3-fold up-regulated, such as cell division cycle 6 homolog (CDC6), cell division cycle associated 5 (CDCA5), cell division cycle 25 homolog A (CDC25A) and cell division cycle associated 7-like (CDCA7L) associated with cell division, S-phase kinase-associated protein 2, E3 ubiquitin protein ligase (SKP2), Cdk5 and Abl enzyme substrate 2 (CABLES2), Cdk5 and Abl enzyme substrate 1 (CABLES1) and cyclin E1 (CCNE1).
Table II indicates that 41 genes were over 4-fold down-regulated such as DDIT3 associated with DNA damage; 57 genes were down-regulated from 3- to 4 -fold such as DDIT4 associated with DNA damage, CCPG1 associated with cell cycle and 255 genes were down-regulated from 2 to 3 folds such as growth arrest and DNA-damage-inducible, alpha (GADD45A), DNA-damage-inducible transcript 4 (DDIT4), DNA-damage-inducible transcript 3 (DDIT3), cyclin-dependent kinase 17 (CDK17), CDC-like kinase 1 (CLK1), and cell cycle progression 1, DYX1C1-CCPG1 read through (CCPG1) associated with cell cycle, tumor necrosis factor receptor superfamily, member 19 (TNFRSF19), early growth response 1 (EGR1), programmed cell death 1 ligand 2 (PDCD1LG2), and ATP-binding cassette, sub-family C (CFTR/MRP), member 3 (ABCC3) and member 9 (ABCC9), intercellular adhesion molecule 1 (ICAM1), Ras association (RalGDS/AF-6) domain family (N-terminal) member 8 (RASSF8), Rho guanine nucleotide exchange factor 10 (ARHGEF10) and cell adhesion molecule with homology to L1CAM (close homolog of L1) (CHL1) associated with cell migration.
BDMC affected the gene expression score measured by GeneGo analysis program in NCI-H460 cells by the number of pathway networks. After total mRNA was isolated from BDMC-treated or -untreated cells and used for cDNA microarrays, the analysis was followed with further processing by GeneGo and the results are shown in Figures 1, 2 and 3. Figure 1 indicates that BDMC affected associated gene expression with cell cycle that starts from DNA replication in the early S phase. For example, BDMC up-regulates cyclin E and CDC6 but down-regulates CDK2 and CDC7, leading to affect the start of DNA replication in NCI-H460 cells. Figure 2 indicates that BDMC affected associated gene expression in development with TGF-beta-dependent induction of EMT via SMADs. For example, TGF-beta receptor type 1 and II were up-regulated by BDMC but SNAIL, SIP1 and MMP-2 were down-regulated affecting cell motility, adhesion and epithelial-to-mesenchymal transition in NCI-H460 cells. Figure 3 indicates that BDMC affected gene expression, which associates with targets of tissue factor signaling in cancer. BDMC affected angiogenesis, inhibition of apoptosis, cell migration and invasion, leading to tumor progression.
Discussion
Several reports have shown that BDMC induces cytotoxic effects in human cancer cells in vitro, however, there is no available information on how BDMC affects gene expression and associated signaling pathways in human lung cancer cells. In the present study, we examined whether BDMC induced up- or down-regulation of genes associated with cell cycle, DNA replication, cell survival, cell migration and invasion and tumor progression in NCI H460 cells.
Table I indicates that ERCC6L, a gene associated with DNA damage and repair, was increased by 3.58-fold, four genes CDC6, CDCA5, CDC25A and CDCA7L associated with cell division, were increased by 2.98-, 2.74-, 2.62- and 2.22-fold, respectively, three genes SKP2, CABLES2 and CABLES1 associated with cell cycle, were increased by 2.51, 2.39, and 2.17 times respectively, three genes CARD6, ATP6V0D1 and CASP8AP2, associated with cell death, were found increased by 2.23-, 2.13- and 2.07-times, respectively.
Table II demonstrates that BDMC suppressed expression of numerous genes associated with DNA damage, cell cycle, cell survival and cell migration and invasion. In particular, three genes DDIT3, DDIT4 and GADD45A associated with DNA damage, were decreased by 4.51-, 2.99- and 2.04-times, respectively, three genes CDK17, CLK1 and CCPG1, associated with cell cycle, were found decreased by 2.03-, 2.48- and 2.62-times, respectively, five genes TNFRSF19, EGR1, PDCD1LG2, ABCC3 and ABCC9 associated with cell death, were decreased 2.09-, 2.31-, 2.50-, 2.95- and 3.97-fold, respectively, four genes ICAM1, RASSF8, ARHGEF10 and CHL1, associated with cell migration, were decreased 2.02-, 2.07-, 2.20-, and 5.47-fold, respectively.
It is well-documented that cell-cycle regulation is the key mechanism of cancer cell growth (28, 29). Results from Figure 1 show that BDMC up-regulated cyclin E and CDC6, however, down-regulated CDK2 and CDC7 affecting the start of DNA replication in NCI-H460 cells, and in turn affecting S phase. It has been reported that cyclin E/Cdk2 complex is involved in G1-S transition and is also associated with initiation of DNA synthesis. Furthermore, the cyclin A/Cdk2 complex is associated with the initiation of DNA synthesis and is also associated with the progression to S phase (30). It has also been reported that increase in Cdc7 and/or Dbf4 can arrest cells in G1 phase, or slow down S-phase progression when cells are already in S phase (31). Furthermore, it has also been reported that Cdc6 and Mcm proteins are required to establish pre-replicative complex and the activities of Cdks and of Cdc7 kinase for triggering the G1-S transition (32).
Results from Figure 2 demonstrate that BDMC up-regulated the transforming growth factor-beta (TGF-β) receptor type 1 and II, however down-regulated SNAIL, SIP1 and MMP-2 that were associated with cell motility, adhesion and epithelial-to-mesenchymal transition in NCI-H460 cells. It is well-known that TGF-β ligands play an important role in cell proliferation, extracellular matrix production, cell motility and apoptosis (33). It has also been reported that in the mammary gland of transgenic mice, if there was overexpression of active TGF-β1 or an activated type I TGF-β receptor (TβRI), it can lead to accelerated metastases derived from Neu-induced mammary tumors (34, 35).
The zinc-finger transcription factors Snail and SIP1 (Smad interacting protein 1) have been demonstrated to repress transcription of the E-cadherin (E-cad) gene by binding to E-boxes (CACCTG sequence) on the E-cad promoter (36, 37). E-cad plays a major role in the establishment and maintenance of intercellular adhesion, cell polarity, and tissue architecture (38, 39). Furthermore, matrix metalloproteinase (MMP-1, MMP-2, MMP-7), and MT1-MMP expressions are strongly upregulated by Snail (40). Results from Figure 3 indicate that BDMC affects angiogenesis, inhibition of apoptosis, cell migration and invasion, leading to tumor progression. Thus, further studies are required to expand or append our current findings and possible understanding of them. In the present study, the genes affected by BDMC in vitro may offer certain insight on the cytotoxic mechanism of BDMC in the genetic level, which in turn may provide potentially useful biomarkers or targets for diagnosis and treatment of human lung cancer.
Acknowledgements
This work was supported by grant DMR-101-051 from China Medical University Hospital, Taichung, Taiwan.
Footnotes
↵* These authors contributed equally to the study.
- Received July 22, 2015.
- Revision received September 14, 2015.
- Accepted September 17, 2015.
- Copyright © 2015 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved