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Induction of Necrosis in Human Liver Tumor Cells by αPhellandrene a
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Shu-Ling Hsieh , Yi-Chen Li , Weng-Cheng Chang , Jing-Gung Chung , Lan-Chi Hsieh & ChihChung Wu
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Department of Seafood Sciences, National Kaohsiung Marine University, Kaohsiung, Taiwan
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Graduate Institute of Medical Sciences, Chang Jung Christian University, Tainan, Taiwan
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Department of Biological Science and Technology, China Medical University, Taichung, Taiwan d
Department of Dietetics, Kaohsiung Municipal United Hospital, Kaohsiung, Taiwan
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Department of Nutrition and Health Sciences, Chang Jung Christian University, Tainan, Taiwan Published online: 31 Jul 2014.
To cite this article: Shu-Ling Hsieh, Yi-Chen Li, Weng-Cheng Chang, Jing-Gung Chung, Lan-Chi Hsieh & Chih-Chung Wu (2014) Induction of Necrosis in Human Liver Tumor Cells by α-Phellandrene, Nutrition and Cancer, 66:6, 970-979, DOI: 10.1080/01635581.2014.936946 To link to this article: http://dx.doi.org/10.1080/01635581.2014.936946
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Nutrition and Cancer, 66(6), 970–979 Copyright Ó 2014, Taylor & Francis Group, LLC ISSN: 0163-5581 print / 1532-7914 online DOI: 10.1080/01635581.2014.936946
Induction of Necrosis in Human Liver Tumor Cells by a-Phellandrene Shu-Ling Hsieh Department of Seafood Sciences, National Kaohsiung Marine University, Kaohsiung, Taiwan
Yi-Chen Li and Weng-Cheng Chang Graduate Institute of Medical Sciences, Chang Jung Christian University, Tainan, Taiwan
Jing-Gung Chung Downloaded by [Michigan State University] at 13:48 16 February 2015
Department of Biological Science and Technology, China Medical University, Taichung, Taiwan
Lan-Chi Hsieh Department of Dietetics, Kaohsiung Municipal United Hospital, Kaohsiung, Taiwan
Chih-Chung Wu Department of Nutrition and Health Sciences, Chang Jung Christian University, Tainan, Taiwan
a-Phellandrene (a-PA) is a component of dietary spices and herbs. The effect of a-PA on anticancer is unclear. This study aims to investigate the effects of a-PA on liver tumor cell death fate. Human liver tumor (J5) cells were incubated with a-PA and analyzed for cell cycle distribution, expression of Bax, Bcl-2, poly (ADP-ribose) polymerase (PARP) protein, and caspase-3 activity of J5 cells, and levels of nitric oxide (NO) production, lactate dehydrogenase (LDH) leakage, and ATP depletion were also analyzed in this study. Results found that a-PA significantly (P < 0.05) decreased the cell viability of J5 cells after 24-h treatment. The cell cycle distribution, Bax, Bcl-2, PARP protein levels, and caspase-3 activity of J5 cells did not change for 24 h after treatment with 30 mM a-PA. Reactive oxygen species levels significantly increased, mitochondrial membrane potential levels significantly decreased when J5 cells were treated with 30 mM a-PA for 24 h (P < 0.05). Thirty mM a-PA significantly (P < 0.05) increased the necrotic cell number, NO production, LDH leakage, and ATP depletion after 24 h of incubation. These results suggest that a-PA induced J5 cell necrosis but not apoptosis, and a-PAinduced necrosis possibly involved ATP depletion.
Submitted 13 June 2013; accepted in final from 10 March 2014. Address correspondence to Chih-Chung Wu, Department of Nutrition and Health Sciences, Chang Jung Christian University, No. 1, Changda Road, Guiren Dist. Tainan 71101, Taiwan. Tel: 886-62785123 ext. 3304. Fax: 886-7-3640634. E-mail: [email protected]. edu.tw Color versions of one or more of the figures in the article can be found online at http://www.tandfonline.com/hnuc.
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INTRODUCTION a-Phellandrene (a-PA; 5-isopropyl-2-methyl-1,3-cyclohexadiene) is a compound from the essential oil of many dietary species and herbs, such as the essential oil of pepper (ex. Schinus molle L. and S. terebinthifolius Raddi.), salvadora (Solanum erianthum D.), artemisia (Artemisia feddei), and ginger (Zingiber officinale Roscoe). These essential oils have antibacterial, lipid-lowering, and anticancer functions (1–5). However, a-PA-related research in cancer prevention and application is limited. Hepatocellular carcinoma (HCC) is the sixth most common malignancy worldwide. It is the third most common cause of death from cancer, after lung and stomach cancers. More than 600,000 people die of HCC each year worldwide. HCC is also one of the major causes of death from cancer in several regions of Africa and Asia (6). Typical treatment approaches for liver cancer include surgery, radiotherapy, chemotherapy, and transplantation (7), but therapeutic efficacies are not satisfactory. From a chemopreventive view, use of phytochemicals and/or chemotherapeutic agents to induce cell death may be one of the best strategies for killing tumor cells. As currently understood, cell death can be divided into two models: nonprogrammed cell death, also known as necrosis, and programmed cell death, also known as apoptosis and autophagy (8). Necrosis is triggered via many unfavorable chemical or physical conditions such as hypothermia, hypoxia, radiation, low pH, and cell trauma. Necrosis will cause cellular
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damage and disfunction of cell membrane. Necrotic cells exhibit swelling and swollen organelles, leading to membrane rupture and cracking, and cells may eventually cause inflammation due to this phenomenon (9,10). Programmed cell death is usually triggered when cells respond to environmental stimuli. Programmed cell death can be divided into the first type of death program (Type I cell death), also known as apoptosis, and a second type of program (Type II cell death), also known as autophagy (11–13). If we can promote tumor cell death, whether nonprogrammed or programmed cell death, it would be quite useful for cancer prevention and therapy. The aim of this study was to determine if a-PA has cytotoxic effects, if such an effect involves necrotic or apoptotic cell death in human liver tumor cells. To understand whether or not the effect of a-PA on the death of J5 cells is through an apoptotic pathway, the cell cycle distribution and protein expressions of Bax, Bcl-2 and poly (ADP-ribose) polymerase (PARP) and caspase-3 activity were investigated. On the other hand, levels of necrotic cell number, reactive oxygen species (ROS), the mitochondrial membrane potential (MMP), and ATP and LDH leakage after J5 cells were treated with a-PA were also investigated. It will be helpful to understand whether the effect of a-PA on the death of J5 cells is through the necrosis pathway. These experiments will be help determine how a-PA induces human liver tumor cell death fate, and its potential effects on chemoprevention and/or cancer therapy.
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All a-PA was diluted in dimethyl sulfoxide (DMSO). Cells treated with DMSO alone were regarded as the control.
Cell Viability Examination After various time intervals treated by various concentrations of a-PA, the J5 cells (2.5 £ 106 cell/plate) were suspended by trypsin and washed by 2 mL phosphate buffer saline (PBS) containing 9 mM Na2HPO4, 140 mM NaCl, and 1 mM NaH2PO4 (pH 7.4). After centrifuging 300 £ g for 5 min and removing supernatant, cells were stained by 0.5 mL PI (containing 40 mg/mL PI) and analyzed on a flow cytometry (Becton-Dickinson, San Jose, CA) equipped with an argon ion laser at 488 nm. Then the viable cell number in total analyzed cells was determined (14).
Chemical and Reagents a-PA and propidium iodide (PI) was obtained from Sigma (St. Louis, MO). DCFH-DA and DiOC6 were purchased from Molecular Probes (Invitrogen, Carlsbad, CA). Annexin VFluos staining Kit was obtained from Roche Diagnostics GmbH Co. (Mannheim, Germany). The ATP detection kit was part of the Luminescence ATP Detection Assay of the ATPliteTM kit (PerkinElmer, Waltham, MA).
Cell Cycle Distribution and Cell Death Analysis J5 cells were treated with various concentrations of a-PA for various time intervals. The cell cycle distribution analysis was performed on total cells. Cells were washed in PBS then treated with trypsin and harvested after removal of trypsin. After centrifugation (300 £ g, 10 min), the pelleted cells were fixed in 70% ethanol overnight at 20 C. The cells were centrifuged, washed with PBS, and resuspended in PBS containing 40 mg/mL PI and 0.1 mg/mL RNase (Sigma, St. Louis, MO). After 30 min at 37 C, 1 £ 105 cells were analyzed on a flow cytometry (Becton-Dickinson, San Jose, CA) equipped with an argon ion laser at 488 nm. Then the cell cycle was determined. In addition, necrosis was examined, and rates were analyzed by flow cytometry using an Annexin V-Fluos staining Kit. In this experimental, Annexin-V-FLUOS and Annexin-V-Alexa 568 serves as a fluorescent probe for apoptotic cells. They will not bind normal, intact cells. PI and BOBO-1 are excluded from apoptotic and normal cells but are taken up by necrotic cells (14,15).
Cell Culture and a-PA Treatment The human liver cancer (J5) cells, also known as a human hepatocellular carcinoma (HCC) HepJ5 or J5 cells, was obtained from the Food Industry Research and Development Institute (Hsinchu, Taiwan) (14). J5 cells (2.5 £ 106) were plated on 60-mm plastic tissue culture dishes (Becton Dickinson Labware, Franklin Lakes, NJ) and incubated at 37 C (in humidified 5% CO2 and 95% air at 1 atm) in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum, 1% penicillin and streptomycin (100 U/ml), and 1% L-glutamine (0.33%, w/v). After being plated for 24 h, cells were treated with various concentrations (0, 10, 30, or 50 mM) of a-PA for various times (0, 6, 12, 24, or 48 h).
Caspase-3 Activity Assay To measure the enzymatic activity of caspase-3, J5 cells were grown and treated with a-PA (30 mM). Cells were harvested by centrifugation and the mediums were removed. Then, 50 mL of 10 mM substrate solution (PhiPhilux is a unique class of substrates for caspase-3) was added to cell pellet (1 £ 105 cells per sample). Cells then were incubated at 37 C for 60 min. Then the cells were washed once by adding 1 mL of ice cold PBS and resuspended in fresh 1 mL. Cells were analyzed with a flow cytometry (Becton Dickinson FACS Calibur) equipped with an argonion laser at 488 nm wavelength. Then the caspase-3 activity was determined and analyzed (16).
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Cytosolic Bax, Bcl-2, and PARP Protein Expression Analysis Cytosolic Bax, Bcl-2, and PARP expressions were determined by sodium dodecylsulfate polyacrylamide gel electrophoresis and immunoblot assay. J5 cells were treated with 30 mM of a-PA for 0, 6, 12, and 24 h. Total proteins were collected and prepared from cells. Protein concentrations of cells were assayed by the method of Lowry et al. (17). Equal amounts of cytosol proteins were subjected to 10% polyacrylamide gels. Following electrophoresis, proteins separated on SDS–polyacrylamide gels were transferred to polyvinyldiene difluoride membranes. To block the nonspecific binding, membranes were incubated at 4 C overnight with 5% skim milk, followed by a 30 min incubation at 37 C with a antiserum containing antibodies against Bax, Bcl-2, or PARP. Bands were visualized using hydrogen peroxide/tetrahydrochloride diaminobenzidine or an enhanced chemiluminescent detection kit (Amersham Life Science, Buckinghamshire, UK) and were quantitated with an AlphaImager 2000 (Alpha Innotech, San Leandro, CA).
RESULTS a-PA inhibits Cell Viability Fig. 1A shows that when J5 cells were treated with 10, 30, or 50 mM a-PA for 24 h, cell viability significantly (P < 0.05) decreased compared to the control group. This phenomenon occurred in a dose-dependent manner. In addition, cell viability significantly (P < 0.05) decreased after J5 cells were treated with 30 mM a-PA for 6, 12, 24, or 48 h, compared to the control group (Fig. 1B).
a-PA Had No Effects on Cell Cycle Arrest We used flow cytometry to understand the cell cycle arrest of J5 cells induced by a-PA. Effects of various concentrations of a-PA on the cell cycle distribution of J5 cells are shown in Fig. 2. There were no significant (P < 0.05) changes in the cell cycle distribution when J5 cells were treated with 10, 30, or 50 mM a-PA, respectively, for 24 h compared to same cell phase at various time interval period treatment. (A)
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LDH Leakage, and ATP and Nitrite Level Analysis J5 cells were treated with 0, 10, and 30 mM of a-PA for 24 h. Necrotic cell death was estimated by determining LDH released into the culture medium after 24 h of incubation with a-PA or DMSO as the solvent control. Intra- and extracellular LDH activities were assayed according to the method of Moldeus et al. (18). The intracellular ATP level was measured using a Luminescence ATP Detection Assay with the ATPliteTM kit as previously described (19). Nitrate in media was measured by the Griess assay and was used as an indicator of NO production by cells (20).
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ROS and MMP Assays Cells were incubated with 30 mM a-PA for 0, 6, 12, and 24 h, and changes in ROS production and the MMP were measured. Cells were harvested, washed twice with PBS, then resuspended in 500 ml of DCFH-DA (10 mM) for measurement of ROS levels and DiOC6 (4 mmol/L) for the MMP in a dark room for 30 min at 37 C, and assayed by flow cytometry as previously described (14).
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Statistical Analysis Statistical analyses were performed using SAS software (SAS Institute, Cary, NC); analysis of variance and Duncan’s multiple-range test were used to identify significant differences among the means (P < 0.05).
FIG. 1. Effects of a-phellandrene (a-PA) on the percentage of viable J5 cells. J5 cells (5 £ 105 cells/ml) were treated with 0, 10, 30, and 50 mM a-PA for 24 h (A), or 30 mM a-PA for 0, 6, 12, 24, 24, and 48 h of incubation (B). The 0 mm concentration and 0 h were defined as controls. Total cells were collected and stained with propidium iodide dye. Values are shown as the mean § SD (n D 3).a,b,c Groups with different letters significantly differ by Duncan’s test (P < 0.05).
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FIG. 2. Effects of a-phellandrene (a-PA) on the cell cycle distribution of J5 cells. J5 cells (5 £ 105 cells/ml) were treated with 0 (A), 10 (B), 30 (C), and 50 (D) mM a-PA for 24 h. The 0 mm concentration was defined as the control. Total viable cells were collected and stained with propidium iodide dye. Quantitative analytical results are given in Panel E. Values are presented as the mean § SD (n D 3).a,b,c Groups in a same cell phase with different letters significantly different by Duncan’s test (P < 0.05).
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FIG. 3. Expression levels of Bcl-2, Bax, poly (ADP-ribose) polymerase (PARP) proteins, and caspase-3 activity after a-phellandrene (a-PA) treatment of J5 cells. J5 cells (5 £ 105 cells/ml) were treated with 30 mM a-PA for 0, 6, 12, and 24 h. Levels of Bcl-2, Bax, PARP protein (A) and caspase-3 activity (B) of cells were analyzed by immunoblot assay and flow cytometry, respectively. Analysis of variance and Duncan’s multiple-range test were used to identify significant differences among the means.
Effects of a-PA on Apoptosis-Associated Protein Levels and Caspase-3 Activity Because Bcl-2 family proteins play important roles in the apoptosis program, Bax and Bcl-2 protein expressions were investigated in this study. Fig. 3A shows that Bax and Bcl-2 protein levels did not significantly change when J5 cells were
treated with 30 mM a-PA for 6, 12, or 24 h. In addition, fulllength PARP protein levels also did not change after J5 cells were treated with 30 mM a-PA for 6, 12, or 24 h; and caspase3, a cleavage enzyme of PARP in apoptosis process, activity of J5 cells did not change after 30 mM a-PA treated for 6, 12, or 24 h (Fig. 3B).
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a-PA Altered ROS Production and MMP Levels Effects of a-PA on intracellular ROS and MMP levels in J5 cells are shown in Fig. 4. Intracellular ROS levels of J5 cells after 30 mM a-PA treatment for 6, 12, or 24 h significantly (P < 0.05) increased by 21%, 30%, and 33%, respectively, compared to the control group (Fig. 4A). MMPs of J5 cells significantly (P < 0.05) decreased by 43%»57% after being treated with 30 mM a-PA for 6, 12, or 24 h, respectively, compared to the control group (Fig. 4B).
analyzed in this study. After 24 h of incubation, ATP levels significantly (P < 0.05) decreased when J5 cells were treated with 30 mM a-PA (63.2% § 10.1%) compared to the control group (100% § 10.3%) (Fig. 5A). LDH leakage and NO production also significantly (P < 0.05) increased after J5 cells were treated with 30 mM a-PA (Fig. 5B and 5C, respectively).
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Time (h) FIG. 4. Effects of a-phellandrene (a-PA) on intracellular reactive oxygen species (ROS) and mitochondrial membrane potential (MMP) levels in J5 cells. J5 cells (5 £ 105 cells/ml) were treated with 30 mM a-PA for 0, 6, 12, and 24 h. Percentages of ROS (A) and MMP (B) of cells were stained and analyzed by DCFH-DA or DiOC6, respectively. Values are presented as the mean § SD (n D 3).a,b,c Groups with different letters significantly differ by Duncan’s test (P < 0.05).
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FIG. 5. Effects of a-phellandrene (a-PA) on intracellular ATP levels, lactate dehydrogenase (LDH) leakage, and nitric oxide (NO) production in J5 cells. J5 cells (5 £ 105 cells/well; 12-well plates) were incubated in the absence and presence of a-PA (30 mM) for 24 h. ATP levels (A), LDH leakage (B), and NO levels (C) were analyzed after 30 mM a-PA treatment for 24 h. Values are presented as the mean § SD (n D 3).a,b Groups with different letters significantly differ by Duncan’s test (P < 0.05).
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a-PA Increased Necrotic Cell Death Number To further investigate the effect of a-PA on necrosis, necrotic cell numbers were examined, and rates were analyzed by flow cytometry using Annexin V-Fluos staining. From results of the necrotic cell level analysis (Fig. 6), when J5 cells were treated with 30 mM a-PA for 24 h, necrotic cell levels (15.8% § 1.2%) significantly (P < 0.05) increased compared to the control group (1.0% § 1.1%).
DISCUSSION Induction of cancer cell death and inhibition of cancer cell growth are very important processes in suppressing tumor cell proliferation and chemoprevention. Cell death can be divided into nonprogrammed and programmed cell death (8,21). Among cancer therapy studies, some chemotherapeutic drugs induce apoptosis whereas other drugs
cause necrosis (22,23). Many phytochemicals, including organosulfide compounds, flavonoids, capsinoids, and carotenoids, were shown to induce apoptosis through regulating Bcl-2 and caspase family protein expressions, which affect mitochondrion-mediated caspase-dependent apoptosis pathway activation (24–27). However, the anticancer drug, chloroacetaldehyde, significantly decreased MMP levels and increased ATP depletion, thus inhibiting the proliferation of cancer cells through induction of a cell necrosis phenomenon in Saos-2 human myeloma cells (28). In C6 glioma cells, quercetin nanoliposomes significantly induced MMP loss, ATP depletion, and LDH activity but had no effect on cytochrome C release from mitochondria or caspase-3 and caspse-9 activation, which shows that quercetin nanoliposomes stimulated necrotic cell death (29). In this study, a-AP also induced cell death of human liver tumor cells through necrosis, and a process of ATP depletion was involved.
FIG. 6. Effects of a-phellandrene (a-PA) on necrotic cell numbers in J5 cells. J5 cells (5 £ 105 cells/well; 12-well plates) were incubated in the absence and presence of a-PA (30 mM) for 24 h. Values are presented as the mean § SD (n D 3).a,b Groups with different letters significantly differ by Duncan’s test (P < 0.05).
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The antibiotic activities of a-PA and its metabolites were proven by several studies (30). Essential oils of some herbs and traditional medicines are rich in a-PA which has cell toxicity and anticancer effects and has gradually gained the attention of researchers. For instance, essential oils of Schinus molle L. and Schinus terebinthifolius Raddi contain a-PA (46.52% and 34.38%, respectively). Both of these essential oils act against human breast cancer (MCF-7) cells, which may have been due to their antioxidant and radical-scavenging properties (1). In addition, the essential oil of Solanum erianthum leaves has abundant a-PA (17.5%). This essential oil demonstrated potent inhibitory activities against human breast cancer cells (Hs 578T) and prostate tumor cells (PC-3) (3). In this study, we found that a-PA significantly inhibited the viability of J5 cells in dose- and time-dependent manners. a-PA induction of human liver tumor cell (J5) death is first reported in this study. Apoptosis is triggered by extracellular stimuli; then a serial proapoptotic signal is transduced through an extrinsic and/or intrinsic pathway (31). The mitochondrion-mediated caspasedependent apoptosis pathway is one of the major intrinsic pathways that induces apoptosis (32). When cells are stimulated by a cell-stimulating hormone (cytokine) or chemical substances, or DNA is damaged by radiation, this causes a defect in cell growth factors, intracellular ROS levels increase, endoplasmic reticulum calcium ions are released, and intracellular Ca2C ion overloading occurs. These signal molecules then promote proapoptotic factors, e.g., Bax and Bak, and inhibition of the expression of the antiapoptotic factor, Bcl-2, which reduce MMP levels and increase the mitochondrial membrane permeability, resulting in cytochrome C and apoptosis protease activating factor-1 being released from mitochondria, activation of caspase 9, caspase 3 and PARP, lead to DNA fragmentation, and ultimately apoptosis (33). However, according to results of this study, 30 mM a-PA did not affect the cell cycle distribution of J5 cells. No apoptotic cells were detected by flow cytometry. Although cell ROS levels significantly increased and MMP levels significantly decreased when J5 cells were treated with 30 mM a-PA for 6,12, and 24 h (P < 0.05), immunoblotting assay results showed that after 30 mM a-PA treatment for 6,12, and 24 h, protein expressions of Bax, Bcl-2, and full-length PARP did not change. Caspase3 activity were also not changed by 30 mM a-PA, lead to fulllength PARP protein were not cleavage and activation. It seems that a-PA did not induce J5 cell death via the apoptotic pathway. Necrosis, one of the important pathways of cell death, mainly includes damage to membrane lipids, a proinflammatory response, loss of membrane ion pump homeostasis, and depletion of cellular energy (34). Cellular ATP depletion pushes cells into a necrotic fate. ATP depletion is triggered by physical stimuli and chemical damage, and results in the following steps: 1) increased ROS production; 2) decreased MMPs; and 3) ATP depletion, leading to cell membrane
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damage and necrosis (35–37). Previous studies showed when J5 cells treated with chrysophanol will induce cell necrosis through increase cellular ROS levels, decreased MMP and ATP depletion (15). In this study, a-PA not only significantly increased ROS levels and decreased MMP levels, but also increased ATP depletion (37%), NO production (56%), and LDH leakage (39%). The flow cytometric assay showed that a-PA significantly increased necrotic cell numbers by 15%. Previous studies showed that monoterpene compounds can induce ROS and NO generation on different physiological reactions in vitro and in vivo models. Geraniol, an acyclic monoterpene, can upregulate NADPH oxidase and increase ROS levels in tomato plants (38). Borneol, a monoterpenoid component presented in the essential oils of numerous medicinal plants, will trigger intracellular ROS generation in HepG2 cells and induce apoptosis (39). D-limonene, a common monoterpene, was demonstrated to have antiproliferative action and to increase NO production on a lymphoma cell line (BW5147) (40). Besides, carvacrol promoted a marked gastroprotection on experimentally induced gastric lesions in rodents. It is possibly mediated by increase of mucus NO production and NO synthase activation, and antioxidant properties (41). We are the first study to find that a-PA could increase ROS and NO production. It provides an important evidence in cell necrosis induction. Because the structure of a-PA is similar to geraniol, borneol, D-limonene, and carvacrolall, they may have similar mechanisms to trigger the release of ROS and NO. Further examination is required to clarify how a-PA triggers the release of ROS and NO. Considering the regulatory mechanism, the difference between necrosis and apoptosis is mainly due to that a number of regulatory molecules have different expression levels of cell cycle. Necrosis with cell membrane disruption may result in hypoxia-induced ATP depletion, cell swelling, or rupture leading to inflammation (8,11). However, apoptosis with cell membrane blebbing may result in nuclear collapse (i.e., nuclear fragmentation, chromatin condensation, and chromosomal DNA fragmentation), or appoptopic body formation (8,11). In this study, when J5 cells were treated with 30 mM a-PA, the cell membrane blebbing was not induced, but they ruptured (data not shown). The results evidenced that a-PA leads to increase of ATP depletion and NO production, and it does not activate cellular PARP (DNA fragmentation activator) protein levels in J5 cells. These phenomenon indicated that a-PA can induce necrosis rather than apoptosis. In conclusion, the present study documents that a-PA at a dose of 30 mM exhibited significant inhibitory effects on the viability of human liver tumor cells (J5). As shown in Fig. 7, a-PA induced necrosis of human liver tumor cells (J5) through increased ATP depletion via enhanced NO and ROS production and LDH leakage. It suggested that a-PA has chemopreventive and cancer therapeutic potential.
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FIG. 7. Proposed model of necrosis mechanism signaling induced by a-phellandrene (a-PA) in J5 cells. a-PA induced intracellular nitric oxide (NO), reactive oxygen species (ROS), lactate dehydrogenase (LDH) leakage, and decreased mitochondrial membrane potential (MMP) levels. Furthermore, ATP depletion could lead to the occurrence of necrosis.
FUNDING This study was supported by a grant (NSC99-2320-B-309 -003) from the National Science Council, Taiwan.
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36. Nishikawa K, Satoh H, Hirai A, and Suzuzki K: alpha-Tocopheryloxybutyric acid enhances necrotic cell death in breast cancer cells treated with chemotherapy agent. Cancer Lett 201, 51–56, 2003. 37. Waring P: Redox active calcium ion channels and cell death. Arch. Biochem Biophys 434, 33–42, 2005. 38. Ghosh S, Singh UK, Meli VS, Kumar V, Kumar A, et al: Induction of senescence and identification of differentially expressed genes in tomato in response to monoterpene. PLoS One 8, e76029, 2013. doi:10.1371/ journal.pone.0076029 39. Su J, Lai H, Chen J, Li L, Wong YS, et al.: Natural borneol, a monoterpenoid compound, potentiates selenocystine-induced apoptosis in human hepatocellular carcinoma cells by enhancement of cellular uptake and activation of ROS-mediated DNA damage. PLoS One 8, e63502, 2013. doi:10.1371/journal.pone.0063502 40. Manuele MG, Arcos ML, Davicino R, Ferraro G, Cremaschi G, et al.: Mechanism of the antiproliferative action of limonene on a lymphoma cell line: participation of nitric oxide. antiproliferative action of limonene on a lymphoma cell line. Phytother Res 23, 1011–1017, 2009. 41. Oliveira IS, da Silva FV, Viana AF, dos Santos MR, Quintans-Junior LJ, et al.: Gastroprotective activity of carvacrol on experimentally induced gastric lesions in rodents. Naunyn Schmiedebergs Arch Pharmacol 385, 899–908, 2012.
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Induction of Necrosis in Human Liver Tumor Cells by αPhellandrene a
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Shu-Ling Hsieh , Yi-Chen Li , Weng-Cheng Chang , Jing-Gung Chung , Lan-Chi Hsieh & ChihChung Wu
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Department of Seafood Sciences, National Kaohsiung Marine University, Kaohsiung, Taiwan
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Graduate Institute of Medical Sciences, Chang Jung Christian University, Tainan, Taiwan
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Department of Biological Science and Technology, China Medical University, Taichung, Taiwan d
Department of Dietetics, Kaohsiung Municipal United Hospital, Kaohsiung, Taiwan
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Department of Nutrition and Health Sciences, Chang Jung Christian University, Tainan, Taiwan Published online: 31 Jul 2014.
To cite this article: Shu-Ling Hsieh, Yi-Chen Li, Weng-Cheng Chang, Jing-Gung Chung, Lan-Chi Hsieh & Chih-Chung Wu (2014) Induction of Necrosis in Human Liver Tumor Cells by α-Phellandrene, Nutrition and Cancer, 66:6, 970-979, DOI: 10.1080/01635581.2014.936946 To link to this article: http://dx.doi.org/10.1080/01635581.2014.936946
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Nutrition and Cancer, 66(6), 970–979 Copyright Ó 2014, Taylor & Francis Group, LLC ISSN: 0163-5581 print / 1532-7914 online DOI: 10.1080/01635581.2014.936946
Induction of Necrosis in Human Liver Tumor Cells by a-Phellandrene Shu-Ling Hsieh Department of Seafood Sciences, National Kaohsiung Marine University, Kaohsiung, Taiwan
Yi-Chen Li and Weng-Cheng Chang Graduate Institute of Medical Sciences, Chang Jung Christian University, Tainan, Taiwan
Jing-Gung Chung Downloaded by [Michigan State University] at 13:48 16 February 2015
Department of Biological Science and Technology, China Medical University, Taichung, Taiwan
Lan-Chi Hsieh Department of Dietetics, Kaohsiung Municipal United Hospital, Kaohsiung, Taiwan
Chih-Chung Wu Department of Nutrition and Health Sciences, Chang Jung Christian University, Tainan, Taiwan
a-Phellandrene (a-PA) is a component of dietary spices and herbs. The effect of a-PA on anticancer is unclear. This study aims to investigate the effects of a-PA on liver tumor cell death fate. Human liver tumor (J5) cells were incubated with a-PA and analyzed for cell cycle distribution, expression of Bax, Bcl-2, poly (ADP-ribose) polymerase (PARP) protein, and caspase-3 activity of J5 cells, and levels of nitric oxide (NO) production, lactate dehydrogenase (LDH) leakage, and ATP depletion were also analyzed in this study. Results found that a-PA significantly (P < 0.05) decreased the cell viability of J5 cells after 24-h treatment. The cell cycle distribution, Bax, Bcl-2, PARP protein levels, and caspase-3 activity of J5 cells did not change for 24 h after treatment with 30 mM a-PA. Reactive oxygen species levels significantly increased, mitochondrial membrane potential levels significantly decreased when J5 cells were treated with 30 mM a-PA for 24 h (P < 0.05). Thirty mM a-PA significantly (P < 0.05) increased the necrotic cell number, NO production, LDH leakage, and ATP depletion after 24 h of incubation. These results suggest that a-PA induced J5 cell necrosis but not apoptosis, and a-PAinduced necrosis possibly involved ATP depletion.
Submitted 13 June 2013; accepted in final from 10 March 2014. Address correspondence to Chih-Chung Wu, Department of Nutrition and Health Sciences, Chang Jung Christian University, No. 1, Changda Road, Guiren Dist. Tainan 71101, Taiwan. Tel: 886-62785123 ext. 3304. Fax: 886-7-3640634. E-mail: [email protected]. edu.tw Color versions of one or more of the figures in the article can be found online at http://www.tandfonline.com/hnuc.
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INTRODUCTION a-Phellandrene (a-PA; 5-isopropyl-2-methyl-1,3-cyclohexadiene) is a compound from the essential oil of many dietary species and herbs, such as the essential oil of pepper (ex. Schinus molle L. and S. terebinthifolius Raddi.), salvadora (Solanum erianthum D.), artemisia (Artemisia feddei), and ginger (Zingiber officinale Roscoe). These essential oils have antibacterial, lipid-lowering, and anticancer functions (1–5). However, a-PA-related research in cancer prevention and application is limited. Hepatocellular carcinoma (HCC) is the sixth most common malignancy worldwide. It is the third most common cause of death from cancer, after lung and stomach cancers. More than 600,000 people die of HCC each year worldwide. HCC is also one of the major causes of death from cancer in several regions of Africa and Asia (6). Typical treatment approaches for liver cancer include surgery, radiotherapy, chemotherapy, and transplantation (7), but therapeutic efficacies are not satisfactory. From a chemopreventive view, use of phytochemicals and/or chemotherapeutic agents to induce cell death may be one of the best strategies for killing tumor cells. As currently understood, cell death can be divided into two models: nonprogrammed cell death, also known as necrosis, and programmed cell death, also known as apoptosis and autophagy (8). Necrosis is triggered via many unfavorable chemical or physical conditions such as hypothermia, hypoxia, radiation, low pH, and cell trauma. Necrosis will cause cellular
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damage and disfunction of cell membrane. Necrotic cells exhibit swelling and swollen organelles, leading to membrane rupture and cracking, and cells may eventually cause inflammation due to this phenomenon (9,10). Programmed cell death is usually triggered when cells respond to environmental stimuli. Programmed cell death can be divided into the first type of death program (Type I cell death), also known as apoptosis, and a second type of program (Type II cell death), also known as autophagy (11–13). If we can promote tumor cell death, whether nonprogrammed or programmed cell death, it would be quite useful for cancer prevention and therapy. The aim of this study was to determine if a-PA has cytotoxic effects, if such an effect involves necrotic or apoptotic cell death in human liver tumor cells. To understand whether or not the effect of a-PA on the death of J5 cells is through an apoptotic pathway, the cell cycle distribution and protein expressions of Bax, Bcl-2 and poly (ADP-ribose) polymerase (PARP) and caspase-3 activity were investigated. On the other hand, levels of necrotic cell number, reactive oxygen species (ROS), the mitochondrial membrane potential (MMP), and ATP and LDH leakage after J5 cells were treated with a-PA were also investigated. It will be helpful to understand whether the effect of a-PA on the death of J5 cells is through the necrosis pathway. These experiments will be help determine how a-PA induces human liver tumor cell death fate, and its potential effects on chemoprevention and/or cancer therapy.
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All a-PA was diluted in dimethyl sulfoxide (DMSO). Cells treated with DMSO alone were regarded as the control.
Cell Viability Examination After various time intervals treated by various concentrations of a-PA, the J5 cells (2.5 £ 106 cell/plate) were suspended by trypsin and washed by 2 mL phosphate buffer saline (PBS) containing 9 mM Na2HPO4, 140 mM NaCl, and 1 mM NaH2PO4 (pH 7.4). After centrifuging 300 £ g for 5 min and removing supernatant, cells were stained by 0.5 mL PI (containing 40 mg/mL PI) and analyzed on a flow cytometry (Becton-Dickinson, San Jose, CA) equipped with an argon ion laser at 488 nm. Then the viable cell number in total analyzed cells was determined (14).
Chemical and Reagents a-PA and propidium iodide (PI) was obtained from Sigma (St. Louis, MO). DCFH-DA and DiOC6 were purchased from Molecular Probes (Invitrogen, Carlsbad, CA). Annexin VFluos staining Kit was obtained from Roche Diagnostics GmbH Co. (Mannheim, Germany). The ATP detection kit was part of the Luminescence ATP Detection Assay of the ATPliteTM kit (PerkinElmer, Waltham, MA).
Cell Cycle Distribution and Cell Death Analysis J5 cells were treated with various concentrations of a-PA for various time intervals. The cell cycle distribution analysis was performed on total cells. Cells were washed in PBS then treated with trypsin and harvested after removal of trypsin. After centrifugation (300 £ g, 10 min), the pelleted cells were fixed in 70% ethanol overnight at 20 C. The cells were centrifuged, washed with PBS, and resuspended in PBS containing 40 mg/mL PI and 0.1 mg/mL RNase (Sigma, St. Louis, MO). After 30 min at 37 C, 1 £ 105 cells were analyzed on a flow cytometry (Becton-Dickinson, San Jose, CA) equipped with an argon ion laser at 488 nm. Then the cell cycle was determined. In addition, necrosis was examined, and rates were analyzed by flow cytometry using an Annexin V-Fluos staining Kit. In this experimental, Annexin-V-FLUOS and Annexin-V-Alexa 568 serves as a fluorescent probe for apoptotic cells. They will not bind normal, intact cells. PI and BOBO-1 are excluded from apoptotic and normal cells but are taken up by necrotic cells (14,15).
Cell Culture and a-PA Treatment The human liver cancer (J5) cells, also known as a human hepatocellular carcinoma (HCC) HepJ5 or J5 cells, was obtained from the Food Industry Research and Development Institute (Hsinchu, Taiwan) (14). J5 cells (2.5 £ 106) were plated on 60-mm plastic tissue culture dishes (Becton Dickinson Labware, Franklin Lakes, NJ) and incubated at 37 C (in humidified 5% CO2 and 95% air at 1 atm) in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum, 1% penicillin and streptomycin (100 U/ml), and 1% L-glutamine (0.33%, w/v). After being plated for 24 h, cells were treated with various concentrations (0, 10, 30, or 50 mM) of a-PA for various times (0, 6, 12, 24, or 48 h).
Caspase-3 Activity Assay To measure the enzymatic activity of caspase-3, J5 cells were grown and treated with a-PA (30 mM). Cells were harvested by centrifugation and the mediums were removed. Then, 50 mL of 10 mM substrate solution (PhiPhilux is a unique class of substrates for caspase-3) was added to cell pellet (1 £ 105 cells per sample). Cells then were incubated at 37 C for 60 min. Then the cells were washed once by adding 1 mL of ice cold PBS and resuspended in fresh 1 mL. Cells were analyzed with a flow cytometry (Becton Dickinson FACS Calibur) equipped with an argonion laser at 488 nm wavelength. Then the caspase-3 activity was determined and analyzed (16).
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Cytosolic Bax, Bcl-2, and PARP Protein Expression Analysis Cytosolic Bax, Bcl-2, and PARP expressions were determined by sodium dodecylsulfate polyacrylamide gel electrophoresis and immunoblot assay. J5 cells were treated with 30 mM of a-PA for 0, 6, 12, and 24 h. Total proteins were collected and prepared from cells. Protein concentrations of cells were assayed by the method of Lowry et al. (17). Equal amounts of cytosol proteins were subjected to 10% polyacrylamide gels. Following electrophoresis, proteins separated on SDS–polyacrylamide gels were transferred to polyvinyldiene difluoride membranes. To block the nonspecific binding, membranes were incubated at 4 C overnight with 5% skim milk, followed by a 30 min incubation at 37 C with a antiserum containing antibodies against Bax, Bcl-2, or PARP. Bands were visualized using hydrogen peroxide/tetrahydrochloride diaminobenzidine or an enhanced chemiluminescent detection kit (Amersham Life Science, Buckinghamshire, UK) and were quantitated with an AlphaImager 2000 (Alpha Innotech, San Leandro, CA).
RESULTS a-PA inhibits Cell Viability Fig. 1A shows that when J5 cells were treated with 10, 30, or 50 mM a-PA for 24 h, cell viability significantly (P < 0.05) decreased compared to the control group. This phenomenon occurred in a dose-dependent manner. In addition, cell viability significantly (P < 0.05) decreased after J5 cells were treated with 30 mM a-PA for 6, 12, 24, or 48 h, compared to the control group (Fig. 1B).
a-PA Had No Effects on Cell Cycle Arrest We used flow cytometry to understand the cell cycle arrest of J5 cells induced by a-PA. Effects of various concentrations of a-PA on the cell cycle distribution of J5 cells are shown in Fig. 2. There were no significant (P < 0.05) changes in the cell cycle distribution when J5 cells were treated with 10, 30, or 50 mM a-PA, respectively, for 24 h compared to same cell phase at various time interval period treatment. (A)
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LDH Leakage, and ATP and Nitrite Level Analysis J5 cells were treated with 0, 10, and 30 mM of a-PA for 24 h. Necrotic cell death was estimated by determining LDH released into the culture medium after 24 h of incubation with a-PA or DMSO as the solvent control. Intra- and extracellular LDH activities were assayed according to the method of Moldeus et al. (18). The intracellular ATP level was measured using a Luminescence ATP Detection Assay with the ATPliteTM kit as previously described (19). Nitrate in media was measured by the Griess assay and was used as an indicator of NO production by cells (20).
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ROS and MMP Assays Cells were incubated with 30 mM a-PA for 0, 6, 12, and 24 h, and changes in ROS production and the MMP were measured. Cells were harvested, washed twice with PBS, then resuspended in 500 ml of DCFH-DA (10 mM) for measurement of ROS levels and DiOC6 (4 mmol/L) for the MMP in a dark room for 30 min at 37 C, and assayed by flow cytometry as previously described (14).
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Statistical Analysis Statistical analyses were performed using SAS software (SAS Institute, Cary, NC); analysis of variance and Duncan’s multiple-range test were used to identify significant differences among the means (P < 0.05).
FIG. 1. Effects of a-phellandrene (a-PA) on the percentage of viable J5 cells. J5 cells (5 £ 105 cells/ml) were treated with 0, 10, 30, and 50 mM a-PA for 24 h (A), or 30 mM a-PA for 0, 6, 12, 24, 24, and 48 h of incubation (B). The 0 mm concentration and 0 h were defined as controls. Total cells were collected and stained with propidium iodide dye. Values are shown as the mean § SD (n D 3).a,b,c Groups with different letters significantly differ by Duncan’s test (P < 0.05).
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FIG. 2. Effects of a-phellandrene (a-PA) on the cell cycle distribution of J5 cells. J5 cells (5 £ 105 cells/ml) were treated with 0 (A), 10 (B), 30 (C), and 50 (D) mM a-PA for 24 h. The 0 mm concentration was defined as the control. Total viable cells were collected and stained with propidium iodide dye. Quantitative analytical results are given in Panel E. Values are presented as the mean § SD (n D 3).a,b,c Groups in a same cell phase with different letters significantly different by Duncan’s test (P < 0.05).
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FIG. 3. Expression levels of Bcl-2, Bax, poly (ADP-ribose) polymerase (PARP) proteins, and caspase-3 activity after a-phellandrene (a-PA) treatment of J5 cells. J5 cells (5 £ 105 cells/ml) were treated with 30 mM a-PA for 0, 6, 12, and 24 h. Levels of Bcl-2, Bax, PARP protein (A) and caspase-3 activity (B) of cells were analyzed by immunoblot assay and flow cytometry, respectively. Analysis of variance and Duncan’s multiple-range test were used to identify significant differences among the means.
Effects of a-PA on Apoptosis-Associated Protein Levels and Caspase-3 Activity Because Bcl-2 family proteins play important roles in the apoptosis program, Bax and Bcl-2 protein expressions were investigated in this study. Fig. 3A shows that Bax and Bcl-2 protein levels did not significantly change when J5 cells were
treated with 30 mM a-PA for 6, 12, or 24 h. In addition, fulllength PARP protein levels also did not change after J5 cells were treated with 30 mM a-PA for 6, 12, or 24 h; and caspase3, a cleavage enzyme of PARP in apoptosis process, activity of J5 cells did not change after 30 mM a-PA treated for 6, 12, or 24 h (Fig. 3B).
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a-PA Altered ROS Production and MMP Levels Effects of a-PA on intracellular ROS and MMP levels in J5 cells are shown in Fig. 4. Intracellular ROS levels of J5 cells after 30 mM a-PA treatment for 6, 12, or 24 h significantly (P < 0.05) increased by 21%, 30%, and 33%, respectively, compared to the control group (Fig. 4A). MMPs of J5 cells significantly (P < 0.05) decreased by 43%»57% after being treated with 30 mM a-PA for 6, 12, or 24 h, respectively, compared to the control group (Fig. 4B).
analyzed in this study. After 24 h of incubation, ATP levels significantly (P < 0.05) decreased when J5 cells were treated with 30 mM a-PA (63.2% § 10.1%) compared to the control group (100% § 10.3%) (Fig. 5A). LDH leakage and NO production also significantly (P < 0.05) increased after J5 cells were treated with 30 mM a-PA (Fig. 5B and 5C, respectively).
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FIG. 5. Effects of a-phellandrene (a-PA) on intracellular ATP levels, lactate dehydrogenase (LDH) leakage, and nitric oxide (NO) production in J5 cells. J5 cells (5 £ 105 cells/well; 12-well plates) were incubated in the absence and presence of a-PA (30 mM) for 24 h. ATP levels (A), LDH leakage (B), and NO levels (C) were analyzed after 30 mM a-PA treatment for 24 h. Values are presented as the mean § SD (n D 3).a,b Groups with different letters significantly differ by Duncan’s test (P < 0.05).
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a-PA Increased Necrotic Cell Death Number To further investigate the effect of a-PA on necrosis, necrotic cell numbers were examined, and rates were analyzed by flow cytometry using Annexin V-Fluos staining. From results of the necrotic cell level analysis (Fig. 6), when J5 cells were treated with 30 mM a-PA for 24 h, necrotic cell levels (15.8% § 1.2%) significantly (P < 0.05) increased compared to the control group (1.0% § 1.1%).
DISCUSSION Induction of cancer cell death and inhibition of cancer cell growth are very important processes in suppressing tumor cell proliferation and chemoprevention. Cell death can be divided into nonprogrammed and programmed cell death (8,21). Among cancer therapy studies, some chemotherapeutic drugs induce apoptosis whereas other drugs
cause necrosis (22,23). Many phytochemicals, including organosulfide compounds, flavonoids, capsinoids, and carotenoids, were shown to induce apoptosis through regulating Bcl-2 and caspase family protein expressions, which affect mitochondrion-mediated caspase-dependent apoptosis pathway activation (24–27). However, the anticancer drug, chloroacetaldehyde, significantly decreased MMP levels and increased ATP depletion, thus inhibiting the proliferation of cancer cells through induction of a cell necrosis phenomenon in Saos-2 human myeloma cells (28). In C6 glioma cells, quercetin nanoliposomes significantly induced MMP loss, ATP depletion, and LDH activity but had no effect on cytochrome C release from mitochondria or caspase-3 and caspse-9 activation, which shows that quercetin nanoliposomes stimulated necrotic cell death (29). In this study, a-AP also induced cell death of human liver tumor cells through necrosis, and a process of ATP depletion was involved.
FIG. 6. Effects of a-phellandrene (a-PA) on necrotic cell numbers in J5 cells. J5 cells (5 £ 105 cells/well; 12-well plates) were incubated in the absence and presence of a-PA (30 mM) for 24 h. Values are presented as the mean § SD (n D 3).a,b Groups with different letters significantly differ by Duncan’s test (P < 0.05).
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The antibiotic activities of a-PA and its metabolites were proven by several studies (30). Essential oils of some herbs and traditional medicines are rich in a-PA which has cell toxicity and anticancer effects and has gradually gained the attention of researchers. For instance, essential oils of Schinus molle L. and Schinus terebinthifolius Raddi contain a-PA (46.52% and 34.38%, respectively). Both of these essential oils act against human breast cancer (MCF-7) cells, which may have been due to their antioxidant and radical-scavenging properties (1). In addition, the essential oil of Solanum erianthum leaves has abundant a-PA (17.5%). This essential oil demonstrated potent inhibitory activities against human breast cancer cells (Hs 578T) and prostate tumor cells (PC-3) (3). In this study, we found that a-PA significantly inhibited the viability of J5 cells in dose- and time-dependent manners. a-PA induction of human liver tumor cell (J5) death is first reported in this study. Apoptosis is triggered by extracellular stimuli; then a serial proapoptotic signal is transduced through an extrinsic and/or intrinsic pathway (31). The mitochondrion-mediated caspasedependent apoptosis pathway is one of the major intrinsic pathways that induces apoptosis (32). When cells are stimulated by a cell-stimulating hormone (cytokine) or chemical substances, or DNA is damaged by radiation, this causes a defect in cell growth factors, intracellular ROS levels increase, endoplasmic reticulum calcium ions are released, and intracellular Ca2C ion overloading occurs. These signal molecules then promote proapoptotic factors, e.g., Bax and Bak, and inhibition of the expression of the antiapoptotic factor, Bcl-2, which reduce MMP levels and increase the mitochondrial membrane permeability, resulting in cytochrome C and apoptosis protease activating factor-1 being released from mitochondria, activation of caspase 9, caspase 3 and PARP, lead to DNA fragmentation, and ultimately apoptosis (33). However, according to results of this study, 30 mM a-PA did not affect the cell cycle distribution of J5 cells. No apoptotic cells were detected by flow cytometry. Although cell ROS levels significantly increased and MMP levels significantly decreased when J5 cells were treated with 30 mM a-PA for 6,12, and 24 h (P < 0.05), immunoblotting assay results showed that after 30 mM a-PA treatment for 6,12, and 24 h, protein expressions of Bax, Bcl-2, and full-length PARP did not change. Caspase3 activity were also not changed by 30 mM a-PA, lead to fulllength PARP protein were not cleavage and activation. It seems that a-PA did not induce J5 cell death via the apoptotic pathway. Necrosis, one of the important pathways of cell death, mainly includes damage to membrane lipids, a proinflammatory response, loss of membrane ion pump homeostasis, and depletion of cellular energy (34). Cellular ATP depletion pushes cells into a necrotic fate. ATP depletion is triggered by physical stimuli and chemical damage, and results in the following steps: 1) increased ROS production; 2) decreased MMPs; and 3) ATP depletion, leading to cell membrane
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damage and necrosis (35–37). Previous studies showed when J5 cells treated with chrysophanol will induce cell necrosis through increase cellular ROS levels, decreased MMP and ATP depletion (15). In this study, a-PA not only significantly increased ROS levels and decreased MMP levels, but also increased ATP depletion (37%), NO production (56%), and LDH leakage (39%). The flow cytometric assay showed that a-PA significantly increased necrotic cell numbers by 15%. Previous studies showed that monoterpene compounds can induce ROS and NO generation on different physiological reactions in vitro and in vivo models. Geraniol, an acyclic monoterpene, can upregulate NADPH oxidase and increase ROS levels in tomato plants (38). Borneol, a monoterpenoid component presented in the essential oils of numerous medicinal plants, will trigger intracellular ROS generation in HepG2 cells and induce apoptosis (39). D-limonene, a common monoterpene, was demonstrated to have antiproliferative action and to increase NO production on a lymphoma cell line (BW5147) (40). Besides, carvacrol promoted a marked gastroprotection on experimentally induced gastric lesions in rodents. It is possibly mediated by increase of mucus NO production and NO synthase activation, and antioxidant properties (41). We are the first study to find that a-PA could increase ROS and NO production. It provides an important evidence in cell necrosis induction. Because the structure of a-PA is similar to geraniol, borneol, D-limonene, and carvacrolall, they may have similar mechanisms to trigger the release of ROS and NO. Further examination is required to clarify how a-PA triggers the release of ROS and NO. Considering the regulatory mechanism, the difference between necrosis and apoptosis is mainly due to that a number of regulatory molecules have different expression levels of cell cycle. Necrosis with cell membrane disruption may result in hypoxia-induced ATP depletion, cell swelling, or rupture leading to inflammation (8,11). However, apoptosis with cell membrane blebbing may result in nuclear collapse (i.e., nuclear fragmentation, chromatin condensation, and chromosomal DNA fragmentation), or appoptopic body formation (8,11). In this study, when J5 cells were treated with 30 mM a-PA, the cell membrane blebbing was not induced, but they ruptured (data not shown). The results evidenced that a-PA leads to increase of ATP depletion and NO production, and it does not activate cellular PARP (DNA fragmentation activator) protein levels in J5 cells. These phenomenon indicated that a-PA can induce necrosis rather than apoptosis. In conclusion, the present study documents that a-PA at a dose of 30 mM exhibited significant inhibitory effects on the viability of human liver tumor cells (J5). As shown in Fig. 7, a-PA induced necrosis of human liver tumor cells (J5) through increased ATP depletion via enhanced NO and ROS production and LDH leakage. It suggested that a-PA has chemopreventive and cancer therapeutic potential.
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FIG. 7. Proposed model of necrosis mechanism signaling induced by a-phellandrene (a-PA) in J5 cells. a-PA induced intracellular nitric oxide (NO), reactive oxygen species (ROS), lactate dehydrogenase (LDH) leakage, and decreased mitochondrial membrane potential (MMP) levels. Furthermore, ATP depletion could lead to the occurrence of necrosis.
FUNDING This study was supported by a grant (NSC99-2320-B-309 -003) from the National Science Council, Taiwan.
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