Abstract
α-phellandrene (α-PA) is a cyclic monoterpene, present in natural plants such as Schinus molle L. α-PA promotes immune responses in mice in vivo. However, there is no available information on whether α-PA affects gene expression in leukemia cells. The present study determined effects of α-PA on expression levels of genes associated with DNA damage, cell cycle and apoptotic cell death in mouse leukemia WEHI-3 cells. WEHI-3 cells were treated with 10 μM α-PA for 24 h, cells were harvested and total RNA was extracted, and gene expression was analyzed by cDNA microarray. Results indicated that α-PA up-regulated 10 genes 4-fold, 13 by over 3-fold and 175 by over 2-fold; 21 genes were down-regulated by over 4-fold, 26 genes by over 3-fold and expression of 204 genes was altered by at leas 2-fold compared with the untreated control cells. DNA damage-associated genes such as DNA damage-inducer transcript 4 and DNA fragmentation factor were up-regulated by 4-fold and over 2-fold, respectively; cell-cycle check point genes such as cyclin G2 and cyclin-dependent kinases inhibitor 2D and IA (p21) were up-regulated by over 3-fold and over 2-fold, respectively; apoptosis-associated genes such as BCL2/adenovirus EIB interacting protein 3, XIAP-associated factor 1, BCL2 modifying factor, caspase-8 and FADD-like apoptosis regulator were over 2-fold up-regulated. Furthermore, DNA damage-associated gene TATA box binding protein was over 4-fold down-regulated, and D19Ertd652c (DNA segment) over 2-fold down-regulated; cell cycle-associated gene cyclin E2 was over 2-fold down-regulated; apoptosis associated gene growth arrest-specific 5 was over 9-fold down-regulated, Gm5426 (ATP synthase) was over 3-fold down-regulated, and death box polypeptide 33 was over 2-fold down-regulated. Based on these observations, α-PA altered gene expression in WEHI-3 cells in vitro.
Leukemia, a myeloproliferative disease (abnormal growth of phenotypically immature leukocytes), is the second most common type of cancer in children (1). The incidence of leukemia in humans is increasing worldwide. Leukemia can be divided into acute myeloid leukemia (AML) and chronic myeloid leukemia (CML). In AML, Paired box 5 (PAX5) expression selectively clusters with t(8;21) and it likely explains a peculiar biological feature of this subset of myeloid leukemias (2). In CML, NM23-H1 gene expression may inhibit K562 cell proliferation and migration and suggests that NM23-H1 may be a cancer-suppressor gene and play a significant role in inhibiting the survival of CML cells (3). Currently, treatment for leukemia is unsatisfactory. Therapies with increased efficacy and decreased toxicity are, therefore, required. There is increasing interest in identifying compounds from natural products.
α-Phellandrene (5-isopropyl-2-methyl-1,3-cyclohexadiene; α-PA) is a cyclic monoterpene and is a component in essential oil of natural plants such as Schinus molle L., Schinus terebinthifolius Raddi (4), and Zingiber officinale Roscoe (5). Recently, we were the first to show that α-PA stimulated immune responses by increasing macrophage phagocytosis and inducing natural killer cell cytotoxic effects in normal Balb/c mice in vivo (6). The detection of leukemia-associated gene expression (up- or down-regulation) is essential for diagnosis and therapy. Thus, in the present study, we investigated the effects of α-PA on gene expression of WEHI-3 cells in vitro and found that α-PA altered expression of cell cycle-, DNA damage- and apoptosis-associated genes.
Materials and Methods
Chemicals and reagents. α-PA, propidium iodide (PI) and dimethyl sulfoxide (DMSO) were purchased from Sigma Chemical Co. (St. Louis, MO, USA). RPMI-1640 medium, glutamine, fetal bovine serum (FBS) and penicillin-streptomycin, trypsin-EDTA were purchased from Invitrogen (Carlsbad, CA, USA).
Cell culture. The mouse myelomonocytic WEHI-3 cell line was purchased from the Food Industry Research and Development Institute (Hsinchu, Taiwan). Cells were grown in RPMI-1640 medium supplemented with 10% (v/v) fetal bovine serum (FBS), 100 Units/ml penicillin, 100 μg/ml streptomycin and 2 mM L-glutamine (Sigma-Aldrich, St. Louis, MO, USA). The cells were maintained in a humidified incubator with 5% CO2 at 37°C, and the culture medium was changed every two days (7).
cDNA microarray assay in WEHI-3 cells treated with α-PA. WEHI-3 cells were plated at a density of 5×105 cells/ml in 24-well plates containing RPMI-1640 medium with 10% (v/v) FBS and 2 mM L-glutamine, 100 Units/ml penicillin and 100 μg/ml streptomycin for 24 h. After this procedure, cells were treated with 10 μM α-PA or vehicle for 48 h and then harvested and centrifuged at 1500 × g for 5 min then washed twice with PBS. Total RNA was isolated using a Qiagen RNeasy Mini Kit (Qiagen, Valencia, CA, USA) and used for cDNA synthesis and labeling, microarray hybridization. In turn, this was followed by fluorescent-labeled cDNA hybridization (Affymetrix GeneChip Human Gene 1.0 ST array; Affymetrix, Santa Clara, CA, USA) on the chip as described previously (8). Fluorescence of the samples was quantitated by Asia BioInnovations Corporation (Taipei, Taiwan) and the data were analyzed using Expression Console software (Affymetrix) with default RNA parameters. For gene expression changes, a 1.8-fold difference in hybridization intensity/average differences was considered significantly different compared with the control group (8-11).
Statistical analysis. All assays were carried out in triplicate, and the results are presented as the means±SD. Genes regulated by α-PA by at least a 2-fold change were recorded. Data are representative of three separate assays.
Results
cDNA microarray analysis for α-PA-induced changes in expression of genes associated with DNA damage, cell cycle, and apoptosis in mouse myelomonocytic WEHI-3 cells. WEHI-3 cells were treated with or without 10 μM α-PA for 48 h, then were harvested and total RNA from each treatment was isolated and then cDNA microarray analysis was performed. The results from microarray analysis are shown in Tables I and II, which indicate that 10 genes were 4-fold up-regulated, 13 were over 3-fold up-regulated and 175 were over 2-fold up-regulated; moreover, Table II indicates 21 genes over 4-fold down-regulated, 26 genes over 3-fold down-regulated and expression of 204 genes was altered by at least 2-fold compared to the untreated control cells.
Our results showed that genes associated with DNA damage such as DNA damage inducer transcript-4 was up-regulated 4-fold and DNA fragmentation factor was up-regulated over 2-fold. Genes associated with the cell cycle (cell check point genes), such as cyclin G2, were up-regulated over 3-fold, and cyclin-dependent kinase inhibitor 2D and IA (p21) were up-regulated over 2-fold. Apoptosis-associated genes such as BCL2/adenovirus EIB interacting protein 3, associated factor 3, BCL2-modifying factor, caspase-8 and like apoptosis regulator were up-regulated over 2-fold (Table I). Data in Table II shows that DNA damage-associated genes such as TATA box binding proteins were down-regulated over 4-fold and DNA segment down-regulated over 2-fold. Cell cycle-associated genes such as cyclin F2 were down-regulated over 2-fold, apoptosis-associated genes such as growth arrest-specific 5 were over 9-fold down-regulated, ATP synthase were over 3-fold down-regulated, Death box polypeptide 33 was over 2-fold down-regulated (Table II).
The top alterations in gene expression scored by the number of pathway networks from GeneGo analysis program (Taichung, Taiwain) from α-PA treated WEHI-3 cells for 48 h can be seen in Figures 1, 2 and 3. Experimental data are mapped on the processes and shown as red (up regulation) and blue (down regulation) circles of different intensity. These genes may also be involved in DNA damage, cell cycle arrest and apoptosis-associated responses in α-PA-treated WEHI-3 cells.
Discussion
α-PA is present in plants such as Schinus molle L., Schinus terebinthifolius Raddi. (4) and Zingiber officinale Roscoe (5). It has been used as a spice and perfume but its biological function is scarcely reported. We found that α-PA can induce apoptosis of murine leukemia WEHI-3 cells (data not shown). We previously showed that α-PA induced immune responses in normal mice (6), however, its exact effects on cells are still unclear. Thus, in the present study, we are the first to show that α-PA affects gene expression in WEHI-3 cells.
Numerous studies have shown that cancer development, progression, and response to therapeutic agents are associated with stromal cells, matrix proteins, and secreted molecules that participate in the tumor microenvironment (12-14). Furthermore, a better understanding of the tumor microenvironment may assist in the development of cell culture and media. Under suitable in vivo conditions, then we may suggest that tumor microenvironment could provide a more accurate prediction of patient response to the therapy agents. Agents affect cells in culture media, including DNA damage, cell-cycle arrest and induction of apoptosis. In our earlier studies, we observed the cytotoxic effects of α-PA on WEHI-3 cells (data not shown), thus, we further investigated whether α-PA affects expression of genes associated with DNA damage, cell-cycle arrest and apoptosis in WEHI-3 cells. Tables I and II show that α-PA up-regulated or down-regulated some genes associated with DNA damage, cell cycle and apoptosis in WEHI-3 cells.
In order to understand the associated changes in gene expression, Figures 1, 2 and 3 were obtained from GeneGo Process Networks and include about 110 cellular and molecular processes whose content is defined and annotated by GeneGo. Each process represents a preset network of protein interactions characteristic for the process. Experimental data are mapped on the processes and shown as up-regulation (in red) and down regulation (in blue) circles of different intensity. The relative intensity corresponds to the expression value. Based on those observations, our findings provided important possible molecular mechanisms of how α-PA affects gene expression and possibly signaling pathways in WEHI-3 cells and they may also provide characteristics for further investigations.
Acknowledgements
This work was supported by grant CMU101-ASIA-09 from China Medical University, Taichung, Taiwan.
- Received April 3, 2014.
- Revision received June 6, 2014.
- Accepted June 9, 2014.
- Copyright© 2014 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved