ANTICANCER RESEARCH 34: 4161-4180 (2014) α-Phellandrene Alters Expression of Genes Associated with DNA Damage, Cell Cycle, and Apoptosis in Murine Leukemia WEHI-3 Cells JEN-JYH LIN1,2, CHIEN-CHIH YU3, KUNG-WEN LU1, SHU-JEN CHANG3, FU-SHUN YU4, CHING-LUNG LIAO1, JAUNG-GENG LIN1 and JING-GUNG CHUNG5,6 1Graduate Institute of Chinese Medicine, 3School of Pharmacy, Departments of 4Dentistry, and 5Biological Science and Technology, China Medical University, Taichung, Taiwan, R.O.C.; 2Division of Cardiology, China Medical University Hospital, Taichung, Taiwan, R.O.C.; 6Department of Biotechnology, Asia University, Taichung, Taiwan, R.O.C. Abstract. α-phellandrene (α-PA) is a cyclic monoterpene, regulated. Furthermore, DNA damage-associated gene TATA present in natural plants such as Schinus molle L. α-PA box binding protein was over 4-fold down-regulated, and promotes immune responses in mice in vivo. However, there D19Ertd652c (DNA segment) over 2-fold down-regulated; is no available information on whether α-PA affects gene cell cycle-associated gene cyclin E2 was over 2-fold down- expression in leukemia cells. The present study determined regulated; apoptosis associated gene growth arrest-specific effects of α-PA on expression levels of genes associated with 5 was over 9-fold down-regulated, Gm5426 (ATP synthase) DNA damage, cell cycle and apoptotic cell death in mouse was over 3-fold down-regulated, and death box polypeptide leukemia WEHI-3 cells. WEHI-3 cells were treated with 10 33 was over 2-fold down-regulated. Based on these μM α-PA for 24 h, cells were harvested and total RNA was observations, α-PA altered gene expression in WEHI-3 cells extracted, and gene expression was analyzed by cDNA in vitro. microarray. Results indicated that α-PA up-regulated 10 genes 4-fold, 13 by over 3-fold and 175 by over 2-fold; 21 Leukemia, a myeloproliferative disease (abnormal growth of genes were down-regulated by over 4-fold, 26 genes by over phenotypically immature leukocytes), is the second most 3-fold and expression of 204 genes was altered by at leas common type of cancer in children (1). The incidence of 2-fold compared with the untreated control cells. DNA leukemia in humans is increasing worldwide. Leukemia can damage-associated genes such as DNA damage-inducer be divided into acute myeloid leukemia (AML) and chronic transcript 4 and DNA fragmentation factor were up- myeloid leukemia (CML). In AML, Paired box 5 (PAX5) regulated by 4-fold and over 2-fold, respectively; cell-cycle expression selectively clusters with t(8;21) and it likely check point genes such as cyclin G2 and cyclin-dependent explains a peculiar biological feature of this subset of kinases inhibitor 2D and IA (p21) were up-regulated by over myeloid leukemias (2). In CML, NM23-H1 gene expression 3-fold and over 2-fold, respectively; apoptosis-associated may inhibit K562 cell proliferation and migration and genes such as BCL2/adenovirus EIB interacting protein 3, suggests that NM23-H1 may be a cancer-suppressor gene XIAP-associated factor 1, BCL2 modifying factor, caspase- and play a significant role in inhibiting the survival of CML 8 and FADD-like apoptosis regulator were over 2-fold up- cells (3). Currently, treatment for leukemia is unsatisfactory. Therapies with increased efficacy and decreased toxicity are, therefore, required. There is increasing interest in identifying Correspondence to: Dr. Jing-Gung Chung, Department of compounds from natural products. Biological Science and Technology, China Medical University, No α-Phellandrene (5-isopropyl-2-methyl-1,3-cyclohexadiene; 91, Hsueh-Shih Road, Taichung 40402, Taiwan, ROC. Tel: +886 α-PA) is a cyclic monoterpene and is a component in 422053366 ext 2161, Fax: +886 422053764, e-mail: essential oil of natural plants such as Schinus molle L., [email protected] and Jaung-Geng Lin, Graduate Institute Schinus terebinthifolius Raddi (4), and Zingiber officinale of Chinese Medicine, China Medical University, No 91, Hsueh-Shih Roscoe (5). Recently, we were the first to show that α-PA Road, Taichung 40402, Taiwan, R.O.C. Tel: +886 422053366 ext stimulated immune responses by increasing macrophage 3113, Fax: +886 422053764, e-mail: [email protected] phagocytosis and inducing natural killer cell cytotoxic effects Key Words: α-Phellandrene, cDNA microarray, cell cycle, DNA in normal Balb/c mice in vivo (6). The detection of leukemia- damage, apoptosis, WEHI-3 leukemia cells. associated gene expression (up- or down-regulation) is 0250-7005/2014 $2.00+.40 4161 ANTICANCER RESEARCH 34: 4161-4180 (2014) essential for diagnosis and therapy. Thus, in the present genes over 4-fold down-regulated, 26 genes over 3-fold study, we investigated the effects of α-PA on gene expression down-regulated and expression of 204 genes was altered by at of WEHI-3 cells in vitro and found that α-PA altered least 2-fold compared to the untreated control cells. expression of cell cycle-, DNA damage- and apoptosis- Our results showed that genes associated with DNA associated genes. damage such as DNA damage inducer transcript-4 was up- regulated 4-fold and DNA fragmentation factor was up- Materials and Methods regulated over 2-fold. Genes associated with the cell cycle (cell check point genes), such as cyclin G2, were up- Chemicals and reagents. α-PA, propidium iodide (PI) and dimethyl regulated over 3-fold, and cyclin-dependent kinase inhibitor sulfoxide (DMSO) were purchased from Sigma Chemical Co. (St. 2D and IA (p21) were up-regulated over 2-fold. Apoptosis- Louis, MO, USA). RPMI-1640 medium, glutamine, fetal bovine associated genes such as BCL2/adenovirus EIB interacting serum (FBS) and penicillin-streptomycin, trypsin-EDTA were purchased from Invitrogen (Carlsbad, CA, USA). protein 3, associated factor 3, BCL2-modifying factor, caspase-8 and like apoptosis regulator were up-regulated Cell culture. The mouse myelomonocytic WEHI-3 cell line was over 2-fold (Table I). Data in Table II shows that DNA purchased from the Food Industry Research and Development damage-associated genes such as TATA box binding proteins Institute (Hsinchu, Taiwan). Cells were grown in RPMI-1640 were down-regulated over 4-fold and DNA segment down- medium supplemented with 10% (v/v) fetal bovine serum (FBS), regulated over 2-fold. Cell cycle-associated genes such as 100 Units/ml penicillin, 100 μg/ml streptomycin and 2 mM L- cyclin F2 were down-regulated over 2-fold, apoptosis- glutamine (Sigma-Aldrich, St. Louis, MO, USA). The cells were associated genes such as growth arrest-specific 5 were over maintained in a humidified incubator with 5% CO2 at 37˚C, and the culture medium was changed every two days (7). 9-fold down-regulated, ATP synthase were over 3-fold down- cDNA microarray assay in WEHI-3 cells treated with α-PA. regulated, Death box polypeptide 33 was over 2-fold down- WEHI-3 cells were plated at a density of 5×105 cells/ml in 24-well regulated (Table II). plates containing RPMI-1640 medium with 10% (v/v) FBS and 2 mM The top alterations in gene expression scored by the L-glutamine, 100 Units/ml penicillin and 100 μg/ml streptomycin for number of pathway networks from GeneGo analysis program 24 h. After this procedure, cells were treated with 10 μM α-PA or (Taichung, Taiwain) from α-PA treated WEHI-3 cells for 48 h 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 can be seen in Figures 1-3. Experimental data are mapped on Qiagen RNeasy Mini Kit (Qiagen, Valencia, CA, USA) and used for the processes and shown as red (up regulation) and blue cDNA synthesis and labeling, microarray hybridization. In turn, this (down regulation) circles of different intensity. These genes was followed by fluorescent-labeled cDNA hybridization (Affymetrix may also be involved in DNA damage, cell cycle arrest and GeneChip Human Gene 1.0 ST array; Affymetrix, Santa Clara, CA, apoptosis-associated responses in α-PA-treated WEHI-3 cells. USA) on the chip as described previously (8). Fluorescence of the samples was quantitated by Asia BioInnovations Corporation (Taipei, Discussion 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 α-PA is present in plants such as Schinus molle L., Schinus intensity/average differences was considered significantly different terebinthifolius Raddi. (4) and Zingiber officinale Roscoe (5). compared with the control group (8-11). It has been used as a spice and perfume but its biological function is scarcely reported. We found that α-PA can induce Statistical analysis. All assays were carried out in triplicate, and the apoptosis of murine leukemia WEHI-3 cells (data not shown). results are presented as the means±SD. Genes regulated by α-PA by We previously showed that α-PA induced immune responses at least a 2-fold change were recorded. Data are representative of three separate assays. in normal mice (6), however, its exact effects on cells are still unclear. Thus, in the present study, we are the first to show Results that α-PA affects gene expression in WEHI-3 cells. Numerous studies have shown that cancer development, cDNA microarray analysis for α-PA-induced changes in progression, and response to therapeutic agents are associated expression of genes associated with DNA damage, cell cycle, with stromal cells, matrix proteins, and secreted molecules that and apoptosis in mouse myelomonocytic WEHI-3 cells. participate in the tumor microenvironment (12-14). Furthermore, WEHI-3 cells were treated with or without 10 μM α-PA for a better understanding of the tumor microenvironment may assist 48 h, then were harvested and total RNA from each treatment in the development of cell culture and media.